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The contribution of ephaptic interactions to recruitment and synchronization of neuronal discharge during… Richardson, Thomas Lewellyn 1988

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THE CONTRIBUTION OF E P H A P T I C INTERACTIONS TO R E C R U I T M E N T AND SYNCHRONIZATION OF NEURONAL DISCHARGE DURING EVOKED P O T E N T I A L S IN THE H I P P O C A M P A L FORMATION b y THOMAS L . RICHARDSON B . S C , M . S . , M.D. , U n i v e r s i t y of B r i t i s h Columbia A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE F A C U L T Y OF GRADUATE STUDIES (Department of P h y s i o l o g y ) We accept t h i s thes i s as conforming to the r e q u i r e d s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H C O L U M B I A May, 1988 (Q) Thomas L e w e l l y n R i c h a r d s o n 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada — Department of DE-6 (2/88) i i A B S T R A C T The mechanisms u n d e r l y i n g the genera t ion and sp read of s e i zu re a c t i v i t y have remained e lu s ive desp i te a cons ide rab le r e s e a r c h effor t o v e r the las t two decades. Most of th i s work has concen t r a t ed on the c h a r a c t e r i s t i c s of neu rona l e x c i t a b i l i t y and b u r s t d i s c h a r g e at the s ing le c e l l l e v e l . These s tud ies have p r o v i d e d some u n d e r s t a n d i n g of the poss ib le abnormal i t ies of neu rons w i t h i n an ep i l ep t i c focus , bu t l i t t l e d i r e c t i n s i g h t in to the fac tors r e spons ib l e for the s t r i k i n g s y n c h r o n i z a t i o n of ac t ion poten t ia l s d u r i n g i n t e r i c t a l d i s c h a r g e o r i n the sp read of s y n c h r o n o u s a c t i v i t y ac ross a p p a r e n t l y normal b r a i n t i s sue . A l t h o u g h s y n a p t i c a c t i va t i on p r o b a b l y p l a y s a ro le i n the genera t ion of s e i zu re a c t i v i t y , r ecen t ev idence ind ica tes that s e i z u r e - l i k e d i s c h a r g e can o c c u r d u r i n g chemica l b lockade of s y n a p t i c t r ansmis s ion ( Je f fe rys and Haas 1982; T a y l o r and Dudek 1982). T h i s r a t h e r s u r p r i s i n g r e s u l t emphasizes the impor tance of c o n s i d e r i n g n o n - s y n a p t i c mechanisms for bo th the s y n c h r o n i z a t i o n and s p r e a d of abnormal n e u r o n a l a c t i v i t y i n the c e n t r a l n e r v o u s sys tem. One impor tan t n o n - s y n a p t i c mechanism to cons ide r i s ephapt ic i n t e r ac t i ons . T h i s term re fe r s to the d i r e c t e l e c t r i c a l in f luence of ex t r ace l l u l a r f i e ld po ten t ia l s on neu rona l e x c i t a b i l i t y . It i s poss ib le that ephap t ic i n t e r ac t ions , genera ted d u r i n g s e i zu re a c t i v i t y , s imul taneous ly depo la r i ze an en t i r e popu la t ion of neurons l e ad ing to both r ec ru i tmen t and s y n c h r o n i z a t i o n of ac t ion po ten t ia l d i s c h a r g e . T h i s thes i s i nves t i ga t e s ephapt ic in t e rac t ions d u r i n g evoked poten t ia l s i n the h ippocampal format ion. The h ippocampus i s one of the most s e i z u r e - p r o n e r eg ions of the b r a i n and i t s anatomical s t r u c t u r e i s i dea l for the genera t ion of f i e ld effects . E v o k e d potent ia ls were used as iii "models" of synchronous neuronal discharge since they are more reproducible, easier to control, and better understood than seizure activity. This initial investigation of ephaptic interactions lays the foundation for further studies involving the complexities of epileptic activity. The first phase of this project examined the spatial characteristics of field potentials evoked in the hippocampus and the dentate gyrus. Current source density (CSD) analysis and voltage gradient determinations obtained from these fields were used to characterize the pattern of current flow within the neuropil and to predict the polarity and relative intensity of ephaptic influences on neuronal excitability. The detailed characteristics of extracellular voltage gradients varied between CAl and the dentate gyrus, and also between anti- and orthodromic responses. In general, voltage gradients during the positive components of a somatic population spike predicted ephaptic hyperpolarization of the neuronal population, whereas gradients observed during the negative component predicted depolarization. They were often an order of magnitude greater than the smallest gradient known to influence granule cell activity. An exception to this rule was the minimal gradient observed during the negative component of the dentate response. In the second phase of the study, extracellular voltage gradients were experimentally applied to the dentate gyrus to determine the sensitivity of granule cells to ephaptic interactions. The magnitude of the applied gradients were in the range observed during the evoked potentials studied in the first phase. These experiments demonstrated a remarkable sensitivity of granule cells to the applied fields. The fields could alter the population spike from near minimal to near maximal. Surprisingly, even antidromic potentials were influenced by the gradients. On the other hand, i v the E P S P phase of the popu la t ion sp ike was not i n f l u e n c e d . These f i n d i n g s e s t ab l i shed that ex t r ace l lu l a r c u r r e n t s can in f luence the e x c i t a b i l i t y w i t h i n a neu rona l popu la t ion wi thout a l t e r i n g s y n a p t i c d r i v e . The f i n a l phase of the p ro jec t i n v e s t i g a t e d the t ransmembrane po ten t i a l (TMP) of p y r a m i d a l and g ranu le ce l l s d u r i n g app l i ed f i e lds and e v o k e d poten t ia l s . The TMP was ca lcu la ted b y s u b t r a c t i n g the ex t r ace l lu l a r from the i n t r a c e l l u l a r r e sponse . T h i s po ten t i a l u l t imate ly determines the vo l tage dependent behav io r of a n e u r o n and g ive s a d i r e c t measure of a n y ephap t ic i n t e r ac t i ons . I n o r d e r to measure the i n t r a c e l l u l a r in f luences of app l i ed f i e ld s , the TMP was moni tored whi le the impaled c e l l was exposed to ex t r ace l lu l a r vo l tage g r a d i e n t s s p a n n i n g the same range as u s e d i n phase two of the p ro jec t . The TMP sh i f ted b y as much as p l u s or minus 5 mV, d e p e n d i n g on the ampl i tude and p o l a r i t y of the g r ad i en t . T h i s l a rge shi f t i n TMP accounts for the o b s e r v e d in f luence of the app l i ed f i e ld po ten t ia l s , and sugges t s that the vo l tage g r a d i e n t s associa ted w i t h e v o k e d poten t ia l s shou ld also have a marked effect on the T M P . A d e p o l a r i z i n g wave of the TMP o c c u r r e d d u r i n g the nega t ive component of a n t i - and o r thodromic CA1 r e sponses . T h i s depo la r i za t ion was capable of i n i t i a t i n g ac t ion poten t ia l s , and decreased the l a t ency to d i s c h a r g e d u r i n g o r thodromic responses . D u r i n g ep i lep t i fo rm d i s cha rge , a s imi la r d e p o l a r i z i n g wave was associa ted w i t h each nega t ive component of the b u r s t . These depo la r i za t ions r e c r u i t and s y n c h r o n i z e neu rona l d i s c h a r g e b y s imul taneous ly i n c r e a s i n g the e x c i t a b i l i t y w i t h i n an en t i re popu la t ion of ce l l s . V These data support the hypothesis that ephaptic interactions in the hippocampal formation influence the pattern of cell discharge during evoked potentials. It is postulated that similar ephaptic interactions may contribute to recruitment and synchronization during seizure activity. v i TABLEI.. OP Certificate of Examination i Abstract • i i Table of Contents v i Table of Figures ix Acknowledgements x i i i CHAPTER 1: INTRODUCTION . 1 1.1 The Hippocampal Formation 4 A) Anatomy Of The Hippocampal Formation 5 B) Anatomy Of The Transverse Hippocampal Slice 6 C) Principle Cells - Hippocampus 9 C) Intrinsic Cells - Hippocampus 11 D) Inputs - Hippocampus 12 D) Principle Cells - Dentate Gyrus 12 E) Intrinsic Cells - Dentate Gyrus 13 F) Inputs - Dentate Gyrus 13 1.2 Epilepsy 14 A) Pathophysiology Of Epilepsy 15 B) Neurological States of Epilepsy 15 C) Interictal Discharge 16 Penicillin Induced Interictals 17 D) Advantages Of The Hippocampusln Epilepsy Research 18 E) Intrinsic Versus Extrinsic Hypothesis Of Epilepsy 19 Intrinsic - FFP's 20 Intrinsic - Bursting 22 Extrinsic - Giant EPSP 23 The Combined Theory 24 F) The Role Of Inhibition In Epileptogenesis 25 G) Mechanisms Contributing To Synchronous Discharge 26 Synaptic Mechanisms 26 Non Synaptic Mechanisms 28 The Role Of Electrotonic Synapses 30 The Role Of Extracellular Ions 32 The Role Of Ephaptic Interactions 34 1.3 The Present Study 41 CHAPTER 2: METHODS 44 2.1 The Slice Chamber 44 2.2 Artificial Cerebral Spinal Fluid 45 2.3 Surgical Preparation 46 2.4 Stimulating Electrodes 48 2.5 Stimulating Techniques 48 2.6 Recording Electrodes .....49 2.7 Computer Facilities 52 CHAPTER 3: VOLTAGE GRADIENT ANALYSIS 53 3.1 Theoretical Framework 53 v i i A) The Open F i e l d Concept 54 B) C u r r e n t F low D u r i n g S y n c h r o n o u s Discha rge 55 C) The Genera t ion Of Ephap t i c C u r r e n t s 63 D) Expe r imen ta l P l a n 64 3.2 Spec i f i c Methods 67 A) R e c o r d i n g Laminar P ro f i l e s 67 B) C u r r e n t - S o u r c e Dens i ty Ca lcu la t ions 69 C) Vol tage Grad ien t Ca lcu la t ions 72 D) Measurement Of The E v o k e d Po ten t i a l Waveform 73 3.3 Resu l t s 73 A) A n t i d r o m i c Dentate Response 76 Laminar P ro f i l e s 76 Vol tage Grad ien t s 77 B) Or thodromic Dentate Response 87 Laminar P ro f i l e s 87 Vol tage Grad ien t s 100 P I Po ten t i a l 101 NI Po ten t i a l 102 P2 Po ten t i a l 104 Summary of P I NI P2 105 C) A n t i d r o m i c C A l Response 105 Laminar P ro f i l e s 105 Vol tage Grad ien t s 106 D) Or thodromic C A l Response 113 Laminar P ro f i l e s 114 Vol tage Grad ien t s 115 P I Po ten t i a l 115 NI Po ten t i a l 128 P2 Po ten t i a l 129 Summary P I NI P2 129 3.4 D i s c u s s i o n 130 A) Si te of A c t i o n Po ten t i a l In i t i a t i on 130 B) Vol tage Grad ien t s 131 Re la t ionsh ip To Other S tud ie s 136 CH A P TER 4: A P P L I E D F I E L D S 138 4.1 Spec i f i c Methods 139 A) C u r r e n t P a s s i n g E lec t rodes 140 4.2 Resu l t s 142 A) Or thodromic Responses 145 B) A n t i d r o m i c Responses 160 C) M u l t i - s p i k e and B u r s t D i scha rge 165 4.3 D i s c u s s i o n 169 A) Or thodromic Potent ia l s 169 B) A n t i d r o m i c Po ten t ia l s 170 C) M u l t i - s p i k e Di scha rge 171 D) Conc lus ions 172 v i i i C H A P T E R 5: TRANSMEMBRANE P O T E N T I A L 173 5.1 Theo re t i ca l Concept - The Transmembrane Po ten t i a l 174 5.2 Spec i f i c Methods 179 5.3 Resu l t s 183 A) C A l - Or thodromic r e sponse 184 B) C A l - A n t i d r o m i c Response 191 C) Spec i f i c D i s c u s s i o n 192 TMP V e r s u s C e l l D i scha rge 192 D e p o l a r i z i n g Wave V e r s u s Ephap t i c In te rac t ions 195 E p h a p t i c In te rac t ions V e r s u s P a t t e r n of D i scha rge 197 D) C A l - Po ten t ia t ion 198 E) C A l - Ep i l ep t i fo rm A c t i v i t y 199 F) Spec i f i c D i s c u s s i o n 204 G) Dentate G y r u s - TMP and A p p l i e d F i e l d s 208 H) Spec i f i c D i s c u s s i o n 216 Dentate V e r s u s C A l 219 S teady State Sh i f t s In The TMP 220 5.4 C o n c l u s i o n 220 C H A P T E R 6: G E N E R A L DISCUSSION 221 6.1 Summary 221 6.2 Impl ica t ions 223 A) Exper imen ta l 223 B) P h y s i o l o g i c a l 224 C) Pa tho log ica l 225 6.3 C o n c l u s i o n 226 R E F E R E N C E S 227 ix TABLE OF FIGURES FIG. 1.1 Schematic diagram showing the anatomical organization of the hippocampal slice 7 FIG. 3.1 Illustrates the generation of extracellular field potentials during synchronous antidromic activation of a neuronal population 56 FIG. 3.2 Illustrates the generation of extracellular field potentials during synchronous orthodromic activation of a neuronal population (EPSP phase) 59 FIG. 3.3 Illustrates the generation of extracellular field potentials during synchronous orthodromic activation of a neuronal population (discharge phase) 61 FIG. 3.4 Illustrates the concept of field induced alterations of neuronal excitability 65 FIG. 3.5 Illustrates the method used to measure various components of a population potential 74 FIG. 3.6 Laminar profile and CSD analysis of antidromically evoked potentials in the dentate gyrus 79 FIG. 3.7 Voltage gradient present during antidromic activation of the dentate gyrus 81 FIG. 3.8 Top: spatial distribution of extracellular potential during antidromic activation of the dentate gyrus. Bottom: regression analysis of voltage gradient versus NI amplitude 83 FIG. 3.9 Laminar profile and CSD analysis of orthodromically evoked potentials in the dentate gyrus 88 FIG. 3.10 Voltage gradient present during orthodromic activation of the dentate gyrus 90 FIG. 3.11 PI potential Top: spatial distribution of extracellular potential during orthodromic activation of the dentate gyrus. Bottom: regression analysis of voltage gradient versus PI amplitude 92 FIG. 3.12 NI potential Top: spatial distribution of extracellular potential during orthodromic activation of the dentate gyrus. Bottom: regression analysis of voltage gradient versus NI amplitude 94 X FIG. 3.13 P2 potential Top: spatial distribution of extraceilular potential during orthodromic activation of the dentate gyrus. Bottom: regression analysis of voltage gradient versus P2 amplitude 96 FIG. 3.14 Compares the voltage distribution during the three phases of an orthodromically evoked potential in the dentate gyrus ( P l , NI and P2) 98 FIG. 3.15 Laminar profile and CSD analysis of antidromically evoked potentials in the hippocampal CA1 region 107 FIG. 3.16 Voltage gradient present during antidromic activation of the hippocampal CAl region 109 FIG. 3.17 Top: spatial distribution of extracellular potential during antidromic activation of the hippocampal CAl area. Bottom: regression analysis of voltage gradient versus NI amplitude I l l FIG. 3.18 Laminar profile and CSD analysis of orthodromically evoked potentials in the hippocampal CAl region 116 FIG. 3.19 Voltage gradient present during orthodromic activation of the hippocampal CAl region 118 FIG. 3.20 PAJPjolentiW Top: spatial distribution of extracellular potential during orthodromic activation of the hippocampal CAl area. Bottom: regression analysis of voltage gradient versus P l amplitude 120 FIG. 3.21 NI Potential Top: spatial distribution of extraceilular potential during orthodromic activation of the hippocampal CAl area. Bottom: regression analysis of voltage gradient versus NI amplitude 122 FIG. 3.22 P2 Potential Top:" spatial distribution of extracellular potential during orthodromic activation of the hippocampal CAl area. Bottom: regression analysis of voltage gradient versus P2 amplitude 124 FIG. 3.23 Compares the voltage distribution during the three phases of an orthodromically evoked potential in the hippocampal CAl region ( P l , NI and P2).... 126 FIG. 3.24 Compares the generation of voltage gradients in the dentate gyrus and hippocampal CAl region 133 FIG. 4.1 Extracellular voltage gradients generated during experimentally applied currents 143 xi F I G . 4.2 Inf luence of a r t i f i c i a l l y genera ted vol tage g r a d i e n t s on the o r thod romica l l y evoked po ten t i a l i n the dentate g y r u s 146 F I G . 4.3 Compares the change i n ampl i tude of P I , NI and P2 d u r i n g ex t e rna l l y a p p l i e d vo l tage g r a d i e n t s 148 F I G . 4.4 C h a r a c t e r i s t i c s of the o r t h o d r o m i c a l l y evoked dentate po ten t i a l at th ree d i f fe ren t i n t ens i t i e s d u r i n g app l i ed vo l tage g r a d i e n t s 151 F I G . 4.5 Compares the change i n ampl i tude of N I and P2 of the o r t h o d r o m i c a l l y e v o k e d dentate po ten t i a l d u r i n g d i f fe ren t app l i ed vo l tage g r a d i e n t s and d i f fe ren t s t imulus in t ens i t i e s 153 F I G . 4.6 F u r t h e r i l l u s t r a t e s the the in f luence of app l i ed vo l tage g r a d i e n t s on the c h a r a c t e r i s t i c s of P I , NI and P2 of the o r thod romica l l y e v o k e d dentate r e sponse 156 F I G . 4.7 Compares the e x t r a d e n d r i t i c to the extrasomatic po ten t i a l o r t h o d r o m i c a l l y evoked i n the dentate g y r u s d u r i n g app l i ed vo l tage g r a d i e n t s 158 F I G . 4.8 Inf luence of app l i ed vo l tage g r a d i e n t s on the an t i d romica l l y e v o k e d dentate response 161 FIG. 4.9 F u r t h e r c h a r a c t e r i z e s the in f luence of app l i ed vo l t age g r a d i e n t s on the an t i d romica l l y evoked dentate response 163 F I G . 4.10 B u r s t of popu la t ion r e sponses r e c o r d e d i n the c e l l l a y e r of C A l d u r i n g an ex t e rna l l y app l i ed vo l tage g r a d i e n t 167 F I G . 5.1 I l l u s t r a t e s the normal a r rangement for r e c o r d i n g i n t r a c e l l u l a r potent ia ls i n e l e c t r o p h y s i o l o g i c a l exper iments 175 FIG. 5.2 I l l u s t r a t e s the two e lec t rode a r rangement for r e c o r d i n g the t ransmembrane po ten t i a l 177 F I G . 5.3 I l l u s t r a t e s the concept of t ransmembrane po ten t i a l (TMP) 180 FIG. 5.4 Compares the i n t r a c e l l u l a r , ex t r ace l lu l a r and t ransmembrane po ten t i a l of an o r thod romica l l y evoked response i n C A l 185 FIG. 5.5 Shows the in f luence of i n c r e a s i n g the o r thodromic s t imulus i n t e n s i t y on the t ransmembrane po ten t ia l r e c o r d e d from C A l p y r a m i d a l neurons 189 xii FIG. 5.6 Relationship between the transmembrane potential and action potential discharge during antidromic stimulation of CAl 193 FIG. 5.7 Compares the intracellular and transmembrane potentials of a pyramidal cell during increasing intensities of paired pulse stimulation 200 FIG. 5.8 Compares the intracellular and transmembrane potentials of pyramidal cell during orthodromic stimulation at 10 Hz 202 FIG. 5.9 Compares the intracellular and transmembrane potentials of a pyramidal cell in a slice demonstrating multiple population spikes following orthodromic stimulation 205 FIG. 5.10 Intracellular responses of dentate granule cells to orthodromic stimulation 209 FIG. 5.11 TMP during an orthodromic population potential i n the dentate gyrus 211 FIG. 5.12 Influence of applied voltage gradients on the intracellular and transmembrane potentials of a dentate granule cell 213 FIG. 5.13 Shows how applied voltage gradients alter the relationship between ground referenced and transmembrane potentials during orthodromically evoked responses in the dentate gyrus 217 xiii The ques t for sc ien t i f i c knowledge a lways benef i ts from a close i n t e r a c t i o n between a g r o u p of ded ica ted i n d i v i d u a l s . T h i s team app roach has been i n c r e d i b l y f r u i t f u l for me d u r i n g my yea r s of sc ien t i f i c endeavor at U B C . S e v e r a l pounds of cha lk have t u r n e d to dus t o v e r the l ong d i s c u s s i o n s w i t h col leagues r e g a r d i n g e v e r y t h i n g from ionic channe ls to t r a p door func t ions i n cogn i t i on . I feel p r i v i l e g e d to have p a r t i c i p a t e d i n s u c h a r i c h i n t e l l e c tua l env i ronment . The close w o r k i n g r e l a t i onsh ip between Ray T u r n e r and myself , the end less d i s c u s s i o n s w i t h J im M i l l e r , Pe te r V a u g h a n and Dave Mathe r s , as we l l as the companionsh ip and s u p p o r t of Mike O l i v e r , S teve M o d y , Pat L e u n g and Ken Ba imbr idge a l l made an immeasurable impact on the q u a l i t y of t h i s p ro jec t . On the p e r s o n a l s ide , J im M i l l e r has c o n t r i b u t e d tp my g r o w t h as a sc i en t i f i c i n v e s t i g a t o r b y h is con t inuous be l ie f i n my basic ab i l i t i e s , h i s v e r y l i b e r a l p r o v i s i o n of i n t e l l ec tua l freedom and h is to lerance of my need for se l f d i r e c t i o n . We remain f r i e n d s desp i te yea r s of the u s u a l f i e r y compet i t ion between s u p e r v i s o r and s tuden t . F i n a l l y , the home f ron t . Can y o u imagine t o l e r a t i ng the mu l t i d i r ec t i ona l , a lways t angen t i a l and of ten u n d i s c i p l i n e d p u r s u i t s of a l i fe l o n g s tuden t who fa i l s to f i n d an a p p r o p r i a t e n iche w i t h i n the scheme of t h i n g s ? T h a n k y o u family and f r i e n d s . The wors t i s yet to come. 1 CHAFER Over the past few decades neuroscience has made remarkable progress towards understanding both the anatomy and physiology of the central nervous system. Investigators have explored an extensive range of problems from the level of gross anatomy and whole brain function to the details of fine structure, biochemical processes and electrophysiology of single cells. At present our knowledge is concentrated at the extreme poles of this continuum. At one extreme we know a great deal about the major nuclei of the brain, including their interconnections, primary cell types and probable functional significance. At the opposite extreme we have a very detailed understanding of the structure and function of the neuron, the elementary unit of brain tissue. However, relatively little is known about the principles underlying the complex interactions of even simple neuronal networks. Two major problems block the path towards a better understanding of the principles underlying the activity within a population of neurons. First, technical difficulties limit our ability to record the discrete activity from more than a few neurons at the same time. Secondly, the firing pattern of individual neurons is often complex and therefore difficult to analyze in a meaningful way. This complexity is multiplied many fold when one attempts to analyze the relationship between activity recorded from several neurons. However, under certain conditions, the pattern of activity in a neuronal network is reasonably simple and there is some hope of understanding the mechanisms underlying the activity within the population using conventional techniques. The simplest of these patterns occurs 2 during synchronous activity in which many neurons fire more or less at the same time. Since simultaneous neuronal discharge often generates large field potentials, one can use well established extracellular recording techniques to derive a great deal of information about the underlying cellular activity. The importance of understanding the mechanisms responsible for even this very simple form of cell discharge is greater than one might f i r s t suspect. In fact, synchronous neuronal activity is the fundamental pattern of discharge observed under a number of important physiological, pathological and experimental conditions. Loosely synchronized action potentials are often present in cortical regions during normal behavioral states. This activity generates the rhythmical and almost sinusoidal waves seen on EEG recordings under a variety of circumstances. For example, theta rhythm (4-8 hertz) is generated by the synchronous activity of neurons in the hippocampal formation during voluntary movement or movement in response to environmental stimuli (Vanderwolf 1969; Feder and Ranck Jr. 1973; Rudell et al 1979). Cortical activity during sleep is another example of synchronized activity under physiological conditions. Sleep spindles and high amplitude delta waves (1-2 hertz) are generated by the rhythmical discharge of cortical neurons while periodic bursts of action potentials i n the pontine, lateral geniculate and occipital cortex generate field potentials referred to as PGO waves (Kelly 1981). In the laboratory, synchronous activation of large populations of neurons is an extensively used experimental technique. For example, cortical evoked potentials seen on EEG recordings following auditory, visual or other sensory stimuli are important diagnostic and investigative tools in clinical medicine. Similarly, the field potentials recorded from within the 3 b r a i n subs tance fo l lowing d i sc re t e a c t i va t i on of spec i f ic n u c l e i o r f i be r bund l e s p r o v i d e a r i c h source of in format ion for the n e u r o p h y s i o l o g i s t . S y n c h r o n y i s also a f r equen t component of pa tho log ica l neu rona l a c t i v i t y , of ten c a u s i n g h i g h l y v i s i b l e symptoms i n affected i n d i v i d u a l s . The most s t r i k i n g example o c c u r s i n e p i l e p s y when l a rge numbers of c o r t i c a l neu rons r e p e t i t i v e l y f i r e i n a s y n c h r o n o u s manner d u r i n g a genera l i zed s e i zu re . The p resence of s y n c h r o n o u s d i s c h a r g e i n d i f fe ren t anatomical locat ions u n d e r w i d e l y v a r y i n g cond i t ions u n d e r l i n e s the impor tance of u n d e r s t a n d i n g t h i s phenomenon. C l e a r l y , a number of spec i f i c mechanisms are l i k e l y to c o n t r i b u t e to s y n c h r o n y d e p e n d i n g on the p rec i se cond i t ions and s t r u c t u r e s i n v o l v e d . However , c e r t a i n mechanisms may be fundamenta l i n na tu re and c o n t r i b u t e to s y n c h r o n y u n d e r a v a r i e t y of cond i t i ons . To u n d e r s t a n d these gene ra l mechanisms wou ld be an i n v a l u a b l e i n i t i a l s tep towards a n a l y z i n g more complex in t e r ac t i ons w i t h i n neu rona l n e t w o r k s and c o u l d p r o v i d e cons ide rab le i n s i g h t in to the p a t h o p h y s i o l o g y of e p i l e p s y . Our p r e sen t knowledge of mechanisms u n d e r l y i n g s y n c h r o n o u s neu rona l d i s c h a r g e i s v e r y l imi ted . I nves t i ga t i ons in to the na tu re of s e i zu re a c t i v i t y have been the main source of in format ion bu t most of these have concen t r a t ed on the behav io r of i n d i v i d u a l neu rons . U n f o r t u n a t e l y these s tud ies p r o v i d e compara t ive ly l i t t l e in format ion about the mechanisms u n d e r l y i n g s y n c h r o n i z a t i o n of d i s c h a r g e w i t h i n an ep i l ep t i c focus or the s p r e a d of s e i zu re a c t i v i t y to s u r r o u n d i n g b r a i n t i s sue . One mechanism that has of ten been d i s c u s s e d w i t h r e spec t to the s y n c h r o n i z a t i o n and sp read of s e i z u r e a c t i v i t y i s the d i r e c t e l e c t r i c a l in f luence of f i e ld po ten t ia l s on the e x c i t a b i l i t y of neu rons . These f i e ld i n d u c e d or "ephap t ic" i n t e r ac t i ons are 4 likely to play a role in epilepsy since very high amplitude field potentials are characteristic of seizure activity. This thesis investigates the role of ephaptic interactions in the generation of evoked population responses in the hippocampus. By avoiding the complexities of the epileptic state, well controlled and detailed studies were possible. However, the role of ephaptic interactions in seizure activity remains a major focus of interest. In fact the evoked potentials studied in this thesis could be considered as models of the highly synchronized neuronal discharge often present in an epileptic focus. In this context, most of the information obtained from these studies is directly relevant to improving our understanding of the mechanisms underlying synchronization and spread of seizure activity in the cortex. The hippocampus is an ideal structure for this project since it is highly susceptible to seizure discharge and is a common site of human epileptic foci (Jung and Kornmuller 1938). Furthermore, its anatomical organization into specific dendritic and somatic layers is optimal for generating high amplitude field potentials as well as ephaptic interactions (see chapter 3, theoretical concepts). The following section provides a general overview of the hippocampal formation in preparation for a more detailed discussion of the existing literature on the generation and spread of synchronous activity in the cortex and the possible role of ephaptic interactions in these phenomena. 1.1 THE M??9C^PJl^.FORMATION The hippocampal formation is a member of a loosely associated group of cortical structures referred to as the limbic system (Broca 1878). Although the precise function of the hippocampal formation remains 5 unknown, clinical data suggest that it has an important role in memory. Following bilateral destruction of the hippocampus for control of severe temporal lobe epilepsy, Milner (1966) found that patients developed a specific memory deficit. They had no memory for events which occurred after surgery except during the few seconds to minutes immediately . following an event. In dramatic contrast to this deficit, they had normal recall for the period prior to hippocampal ablation. These findings suggest that the hippocampus is not a storage area for memory traces but rather is involved i n either the laying down or the recall of long term memories. The fact that electrical stimulation of temporal lobe cortex can evoke vi v i d memories in awake patients (Penfield 1958) further suggests a role for the hippocampus and related structures in memory recall. A) Anatomy Of The Hippocampal Formation The hippocampal formation is a bilateral region of specialized cortex within the temporal lobes. It consists of a group of closely associated cortical structures including the dentate gyrus, cornu ammonis, subiculum and subicular region of the parahippocampal gyrus. It forms the archipallium, a phylogenetically old part of the cortex placed midway in development between the paleopallium or the olfactory cortex, and the more recently developed neopallium. The dentate gyrus and cornu ammonis are generally regarded as having a primitive trilaminar structure while the subiculum varies from a four to a five and finally to a modified six layered structure where it merges with the surrounding neocortex. In lower vertebrates the hippocampal formation sits in a dorsal position in close proximity to the septum and hypothalamus but during the course of evolutionary development it has been "dragged" to a more posterior and ventral position within the cerebral hemispheres. Its massive 6 interconnections with septum and hypothalamus are retained via the fornix, one of the most impressive fiber tracts in the brain. The elongated curving shape of the hippocampal formation led early anatomists to use the term cornu ammonis or "ram's horn" to refer to the central core of this structure. Perhaps the easiest way to visualize the outline of the cornu ammonis is to think of a long cylinder with a longitudinal crack along its entire length. On transverse section this cylinder becomes a "C" with the longitudinal slit corresponding to the open side of the C. The dentate gyrus has the form of a smaller reverse "C" hanging from its lower curvature and the subiculum is formed by a continuation of its upper pole curving backwards to form a reverse "s" shape (figure 1.1). The curved appearance of this group of structures, when viewed on a cross section, lead to the term hippocampus or "sea horse". However, modern usage of the term "hippocampus" is used to refer specifically to the cornu ammonis whereas the whole group of structures remain the "hippocampal formation". The hippocampus (cornu ammonis) has a highly stereotyped internal structure consisting of a single sheet of neurons a few cells thick with dendritic arborizations projecting at right angles from both its surfaces. This sheet of neurons is rolled into the curvature of a "C", as previously described, and extends the whole longitudinal length of the structure. (Cajal 1911; Lorente de No 1934). B) Anatomy Of The Transverse Hippocampal Slice The overall organization of microcircuitry within the hippocampal formation suggests that i t may be useful to view its structure in terms of a longitudinal series of transverse units. A given unit contains a 7 FIGURE 1.1 is a schematic diagram showing the anatomical organization of the hippocampal slice. The principal neurons of the hippocampus, the pyramidal cells, form a well defined cell layer extending in a C shaped fashion from CAl to CA4. These cells have both apical and basal dendritic arborizations projecting into the surrounding layers. The dentate gyrus hangs like an inverted C from the CA4 region of the hippocampus. Its principal neuron, the granule cell, is well localized to the granule cell layer. It has a single dendritic system which arborizes within the molecular layer. Notations: S. ORI S. PYR S. RAD S. LAC S. MOL GRAN. L MOL. L S. C. B. C. stratum oriens stratum pyramidale stratum radiatum stratum lacunosum stratum moleculare granule cell layer molecular layer Schaffer collaterals basket cell C A I (Regio Superior Area Dentata 9 population of richly interconnected hippocampal, dentate and subicular neurons as well as the specific afferent and efferent fibers connecting these cells to distant regions of the brain. The units are not discrete but form a continuum throughout the length of the hippocampus. Neurons within any region have local synaptic interactions with neurons in adjacent regions and specific longitudinal association fibers connect more widely separated regions of hippocampus. This concept of internal organization has proven very useful in the investigation of hippocampal physiology. For example in the in vitro slice technique a transverse section of hippocampal tissue with its associated dentate and subicular cortex can be studied as a reasonably complete functional unit of neuronal tissue (Skrede and Westgaard 1971). C) Principal Cells - Hippocampus The principal- cell type of the hippocampus is the pyramidal cell. These large cells are organized into a discrete layer about 3 or 4 cell bodies thick called the stratum pyramidale. The cell layer follows the outline of the C shaped hippocampus as it curves around to meet the dentate gyrus. Each pyramidal cell has two sets of well developed dendritic arborizations. The basal dendrites arise from the soma as two or more trunks that branch frequently throughout their length as they spread towards the surface of the hippocampus. This dense network of overlapping processes forms a distinct layer referred to as the stratum oriens (Cajal 1911; Lorente de No 1934). On the opposite side of the pyramidal cell body the apical dendrite arises as a single large diameter shaft passing into the stratum radiatum in a direction perpendicular to the cell layer. After traveling a variable 10 distance the shaft forms two or more secondary branches within the distal stratum radiatum with further branching occurring throughout the stratum lacunosum. The general direction of the dendritic arborization remains perpendicular to the cell line until the dendrites reach the distal regions of the stratum moleculare, near the hippocampal fissure, where they exhibit a diffuse lateral branching. Dendritic spines are relatively sparse on the main shaft of the apical dendrite but are very profuse throughout the branches of both the apical and basal arborizations (Cajal 1911; Lorente de No 1934; Westrum and Blackstad 1962). The details of pyramidal cell structure vary continuously along the transverse length of the cell layer. This led Cajal (1911) to divide the hippocampus into the regio superior, continuous with the subiculum and the regio inferior, which runs towards the dentate gyrus. Lorente de No (1934) further divided the cell layer into CA (cornu ammonis) regions 1 to 4. Regio superior and CAl both refer to approximately the same dorsal region whereas regio superior and CA2 to CA4 refer to the medial and ventral hippocampus. As one moves along the cell layer from CAl towards CA2 and CA3 the cell bodies become larger and the apical dendrites become shorter, thicker and more profuse in their branching patterns. The largest pyramidal cells are found in CA3, in the curvature between the dorsal and ventral hippocampus. The CA4 region of the hippocampus forms the distal extent of the inferior limb of the C shaped curve of the hippocampus and extends into the hylar region of the dentate gyrus. The pyramidal cells in this region are pleomorphic and the cell layer is not as well formed as other CA regions. 11 The axon of the p y r a m i d a l c e l l leaves one of the basa l d e n d r i t e s near the soma and cour ses t h r o u g h o u t the s t ra tum o r i ens towards the v e n t r i c u l a r su r face of the h ippocampus . A s the axons leave the s t r a tum o r i e n s they t u r n l a t e r a l l y to form a sheet of f i be r s o v e r the sur face of the h ippocampus , r e f e r r e d to as the a lveus . The f i be r s t hen g r o u p toge ther a long the l a t e r a l aspect of the h ippocampus as the f imbr i a and f i n a l l y leave i n the fo rn ix , a major f i be r bund le p r o j e c t i n g to mammillary bodies of the hypotha lamus , the a n t e r i o r nuc lea r g r o u p of the thalamus a n d the sep ta l r e g i o n (Lorente de No 1934). The axons of p y r a m i d a l ce l l s w i t h i n the CA3 r e g i o n also g ive off a major co l l a t e ra l f i b e r before p a s s i n g in to the f imbr i a . These Schaf fe r co l la te ra l s t r a v e l back t h r o u g h s t r a tum o r i ens and s t r a tum pyramida le and f i n a l l y t u r n to cour se t h r o u g h the s t r a tum rad ia tum of C A l . A l o n g the way they make mul t ip le en passent exc i t a to ry synapses w i t h the a p i c a l den t r i t e s of the p y r a m i d a l ce l l s i n th i s r e g i o n (Schaffer 1892). C) Intrinsic The major i n t r i n s i c n e u r o n of the h ippocampus i s the baske t c e l l . S e v e r a l s u b t y p e s have been d e s c r i b e d (Lorente de No 1934) bu t most baske t ce l l s have a number of c h a r a c t e r i s t i c s i n common. T h e y have l a rge p o l y m o r p h i c somata s i tua ted e i the r i n the p rox imal s t r a tum o r i ens or w i t h i n the s t r a tum pyramida le and have s e v e r a l t h i c k den t r i t e s that b r a n c h s p a r i n g l y and are r e l a t i v e l y d e v o i d of sp ines . A l t h o u g h the o r i en t a t i on of d e n d r i t i c b r anches i s u s u a l l y i r r e g u l a r , some baske t ce l l s have d e n d r i t i c a r b o r i z a t i o n s that p a r a l l e l the a p i c a l d e n d r i t e s of s u r r o u n d i n g p y r a m i d a l neu rons . 12 The axons from baske t ce l l s course u p t h r o u g h the s t r a tum rad ia tum and then form a number of co l l a te ra l s . Some terminate l oca l l y bu t many r e t u r n to the s t r a tum pyramida le to form l a r g e c l u s t e r s of axosomatic contac ts onto p y r a m i d a l ce l l s . These contac ts are t hough t to be r e spons ib l e for the in tense r e c u r r e n t i n h i b i t i o n w i t h i n the h ippocampus (Al len et a l 1977; A n d e r s e n et a l 1964, 1969; Ding led ine and Langmoen 1980; Knowles and S c h w a r t z k r o i n 1981; Tombol et a l 1979). D) Inputs - Hippocampus A major i n p u t to the h ippocampus comes from the e n t o r h i n a l cor tex b y way of two pa thways . The f i r s t i s r e f e r r e d to as the pe r fo ran t pa th s ince i t passes t h r o u g h o r "per fora tes" the s u b i c u l u m a long i t s way . These f i b e r s make exc i t a to ry contac ts w i t h the s u p e r f i c i a l b r anches of the a p i c a l d e n d r i t e s of C A l , CA2 a n d CA3. The h ippocampus also r ece ives i n p u t s from v a r i o u s o the r r eg ions of the b r a i n i n c l u d i n g the septum and the CA3 r e g i o n of the con t r a l a t e r a l h ippocampus . These f i b e r s en te r the h ippocampus v i a the fo rn ix and make exc i t a to ry contac ts w i t h the p y r a m i d a l c e l l d e n d r i t e s ( A n d e r s e n et a l 1971b; Raisman et a l 1965). D) Principal Cells - Dentate Gyrus The p r i n c i p a l c e l l of the dentate g y r u s i s the g r anu l e c e l l . L i k e the p y r a m i d a l ce l l s of the h ippocampus these smaller s p h e r i c a l ce l l s also form a d i s t i n c t c e l l l a y e r . However , t h i s l a y e r i s many ce l l s t h i c k and the somata are v e r y d e n s e l y p a c k e d . U n l i k e p y r a m i d a l ce l l s , the g r anu l e c e l l has o n l y one d e n d r i t i c a r b o r i z a t i o n . S e v e r a l t r u n k s a r i se near the soma and cour se t h r o u g h the molecular l a y e r towards the sur face of the dentate g y r u s . These d e n d r i t e s are r i c h l y i n v e s t e d w i t h sp ines . 13 The axons of g ranu le ce l l s form the h i lu s as t hey t r a v e l deep w i t h i n the dentate g y r u s i n a d i r e c t i o n oppos i te to that of the d e n d r i t i c a r b o r i z a t i o n . These axons c o n v e r g e i n the h ippocampus as a t h i n sheet of f i b e r s p a s s i n g j u s t s u p e r i o r to the CA4, CA3 and CA2 c e l l l a y e r . These f i b e r s have mul t ip le v a r i c o s i t i e s a long t h e i r l e n g t h and as a r e s u l t a re r e f e r r e d to as mossy f i b e r s . These f i b e r s make mul t ip le exc i t a to ry axodendr i t i c synapses w i t h p y r a m i d a l ce l l s a long the way (Lorente de No 1934). E) Intrinsic Cells - Dentate Gyrus The p r i m a r y i n t r i n s i c ce l l s of the dentate g y r u s are also r e f e r r e d to as baske t ce l l s s ince t h e i r axons form mul t ip le i n h i b i t o r y synapses onto the somata of p r i n c i p a l neu rons w i t h i n the r e g i o n . These i n h i b i t o r y contac ts are be l i eved to mediate the in tense r e c u r r e n t i n h i b i t i o n p r e sen t w i t h i n the dentate g y r u s (Ande r sen et a l 1966; F r i c k e and P r i n c e 1984; L^mo 1971b). Baske t ce l l s a re u s u a l l y loca ted i n the p rox imal r eg ions of the molecular l a y e r o r w i t h i n the l a y e r of g ranu le ce l l s and are s imi lar i n shape a n d s ize to t h e i r c o u n t e r p a r t s i n the h ippocampus (Lorente de No 1934; Seress and P o k o r n y 1981). F) Inputs - Dentate Gyrus The major i n p u t to the dentate g y r u s comes from the e n t o r h i n a l co r t ex v i a the pe r fo ran t pa th . A d d i t i o n a l i n p u t s are r e c e i v e d from the c o n t r a l a t e r a l CA3 r e g i o n of the h ippocampus as we l l as the septum b y way of the f o r n i x . I n each case the te rmina ls form exc i t a to ry s y n a p t i c contac ts onto the d e n d r i t e s w i t h i n the molecular l a y e r (Hj^ r th -S imonsen a n d Jeune 1972; S t ew ard , 1976; Wyss 1981). 14 E p i l e p s y i s a d i s a b l i n g d i s o r d e r of the c e n t r a l n e r v o u s sys tem a f fec t ing as many as 2% of the popu la t ion . It i s c h a r a c t e r i z e d b y pe r iods of normal b r a i n f u n c t i o n i n t e r r u p t e d unexpec t ed ly b y motor, s e n s o r y , autonomic or p s y c h i c symptoms associa ted i n many cases b y the p r e c i p i t o u s onset of unconsc iousnes s and b e h a v i o r a l c o n v u l s i o n s . It has been a r e c u r r i n g c u r s e to man s ince the ea r l i es t t imes when " f i t s " were r e g a r d e d as s o u l r a c k i n g v i s i t a t i o n s from a d i v i n i t y . E v e n d u r i n g these e a r l y phases of man's development the dramat ic and fear i n d u c i n g appearance of a c o n v u l s i o n d r o v e medical p r a c t i t i o n e r s to s ea rch for a n ef fec t ive t reatment for t h i s malady. The p r ac t i c e of t r epana t ion ( b u r r holes i n the s k u l l ) may have been , at leas t i n some ins t ances , a r a d i c a l t reatment i n t e n d e d to release the demons from the s k u l l of un fo r tuna te ep i l ep t i c s (Penf ie ld and E r i c k s o n 1941). A l t h o u g h e a r l y at tempts to u n d e r s t a n d the e t io logy of e p i l e p s y were not v e r y f r u i t f u l , e a r l y w r i t i n g s demonstra te a r e m a r k a b l y c lea r u n d e r s t a n d i n g of the c o n v u l s i o n . F o r example the poet L u c r e t i u s the E p i c u r e a n (95-55 B.C.) e loquen t ly d e s c r i b e d the sequen t i a l symptomatology of a c o n v u l s i o n i n c l u d i n g the p e r i o d of pos t i c t a l d e p r e s s i o n when he wrote : Oft too some w r e t c h , before o u r s t a r t l e d s igh t , S t r u c k as w i t h l i g h t n i n g , b y some keen disease Drops s u d d e n : - b y the d r e a d a t tack o ' e rpowered He foams, he g roans , he t rembles , and he fa in ts ; Now r i g i d , now c o n v u l s e d , h i s l a b o r i n g l u n g s Heave q u i c k , and q u i v e r s each exhaus ted l imb, S p r e a d t h r o u g h the frame, so deep the d i r e disease P e r t u r b s h i s s p i r i t : as the b r i n y main Foams t h r o u g h each wave beneath the tempest 's i r e . 15 But when, at length, the morbid cause declines, And the fermenting humors from the heart Flow back-with staggering foot the man f i r s t treads, Led gradual on to intellect and strength. ( I l l , 487, Good's translation.) (from Penfield and Erickson 1941) We now know that an epileptic convulsion is the behavioral expression of an excessive discharge within a population of hyperexcitable neurons (Jackson 1870; Ajmone Marsan 1969). In its present usage the word seizure refers specifically to this abnormal discharge of central neurons (Gastaut and Broughton 1973). A) Pathophysiology Of Epilepsy Hughlings Jackson (1870) f i r s t established the basic concept of the epileptic focus as a large aggregate of highly unstable neurons in a "hyperphy B i o l o g i c a l " state. He described how a sudden and excessive discharge of these cells could result in a spasm of muscle contraction referred to as a focal seizure, and how the spread of this abnormal discharge to surrounding brain tissue would result in both a generalized seizure and a behavioral convulsion. M).._Neuroiogical Stetefli of„Ej>Uej>ay Before any useful association of neurophysiological data to the general problem of seizure disorders is possible, it is f i r s t necessary to clearly identify the different neurological states present during the interictal (between seizures) and ictal (seizure) phases of epilepsy. During the interictal period of normal behavior there is seldom any detectable neurological deficit and EEG recordings indicate that the majority of the 16 b r a i n i s e s sen t i a l l y normal . However , a small r e g i o n of b r a i n t i s sue , the focus , w i l l i n t e r m i t t e n t l y genera te s p i k e and wave E E G abnormal i t i es , r e f e r r e d to as i n t e r i c t a l s p i k e s . T h e y are seldom assoc ia ted w i t h b e h a v i o r a l changes bu t do ind ica te that some e l e c t r o p h y s i o l o g i c a l abnormal i ty ex is t s w i t h i n a p a r t i c u l a r g r o u p of ce l l s . F rom the c l i n i c a l po in t of v i ew an ep i l ep t i c i n d i v i d u a l i s u s u a l l y normal d u r i n g th i s p e r i o d . A t the onset of a focal s e i zu re the focus exh ib i t s a h i g h l y s y n c h r o n o u s and r e p e t i t i v e d i s c h a r g e . T h i s d i s c h a r g e i s of ten associa ted w i t h spec i f i c p s y c h o l o g i c a l , s e n s o r y , o r motor symptoms, r e f e r r e d to as a focal s e i zu re . Over the next few seconds the s u r r o u n d i n g and a p p a r e n t l y normal b r a i n t i s sue may b e g i n to exh ib i t s y n c h r o n o u s d i s c h a r g e and e v e n t u a l l y th i s a c t i v i t y may sp read to i n v o l v e the whole cor tex , cu lmina t ing i n a tonic c lon ic c o n v u l s i o n . The s t r i k i n g fea ture of a gene ra l i z ed se i zu re i s t h i s r a p i d s p r e a d of a c t i v i t y to a p p a r e n t l y normal neu rons . The mechanism u n d e r l y i n g the s p r e a d of s e i zu re a c t i v i t y i s c l e a r l y of g rea t i n t e r e s t s ince the s i gn i f i c an t symptom of ep i l ep sy , the c o n v u l s i o n , o n l y o c c u r s d u r i n g and immediately fo l lowing t h i s phase of the d i s o r d e r . C) Interictal Discharge Since the . time of J a c k s o n ' s i n i t i a l w r i t i n g s a grea t dea l of effor t has been app l i ed to the i n v e s t i g a t i o n of e p i l e p s y . Most of th i s work has concen t r a t ed at the l e v e l of the i n d i v i d u a l n e u r o n i n an attempt to u n d e r s t a n d the i n t e r i c t a l pa roxysmal d i s c h a r g e , the p r i m a r y manifesta t ion of abnormal i ty w i t h i n an ep i l ep t i c focus . When r e c o r d e d w i t h sur face EEG e lec t rodes an i n t e r i c t a l d i s c h a r g e i s seen as a s h a r p pos i t i ve sp ike of h i g h ampl i tude fol lowed b y a l onge r d u r a t i o n nega t ive po ten t ia l . D u r i n g th i s E E G event , c o r t i c a l ce l l s w i t h i n 17 the focus u n d e r g o a b u r s t of d i s c h a r g e fol lowed b y a s i l en t p e r i o d (Ajmone M a r s a n 1969; P r i n c e 1982). I n v e s t i g a t o r s have found the i n t e r i c t a l s p i k e a v e r y a t t r a c t i v e t a rge t fo r s t u d y s ince i t i s so c h a r a c t e r i s t i c of e p i l e p s y a n d i s a r epea t i ng , ea s i l y de f ined e l e c t r i c a l event . The hope i s that a be t te r u n d e r s t a n d i n g of the i n t e r i c t a l s p i k e w i l l he lp de termine the mechanism u n d e r l y i n g gene ra l i zed s e i zu re a c t i v i t y . P e n i c i l l i n I n d u c e d In t e r i c t a l s The p e n i c i l l i n focus has p r o v e d v e r y p r o d u c t i v e fo r the i n v e s t i g a t i o n of i n t e r i c t a l a c t i v i t y (Matsumoto and Ajmone M a r s a n 1964ab; D ich te r a n d Spence r 1969ab; D ich te r et a l 1973). Wi th in about 5 minutes fo l lowing the s u p e r f i c i a l app l i c a t i on of p e n i c i l l i n to the cor tex , s h a r p pos i t i ve s p i k e s a re seen on sur face E E G r e c o r d i n g s . Over the next 30 minutes to an hour fch§§© § g i k § § develop a §©e©ndary, longer d u r a t i e n negative component , and t h u s take on the f u l l appearance of i n t e r i c t a l pa roxysmal d i s c h a r g e s seen i n n a t u r a l f o c i . A l t h o u g h these i n t e r i c t a l s o c c u r spon taneous ly t h e y can also be t r i g g e r e d b y s t imula t ing af ferent pa thways to the cor tex mak ing e l e c t r o p h y s i o l o g i c a l i n v e s t i g a t i o n much more conven ien t ( S c h w a r t z k r o i n and P r i n c e 1977; S c h w a r t z k r o i n and Sta fs t rom 1980). A s time goes on , the nega t ive wave of the i n t e r i c t a l may deve lop a se r ies of low vol tage def lec t ions r e sembl ing a b r i e f af ter d i s c h a r g e . F i n a l l y , one p a r t i c u l a r af ter d i s c h a r g e may con t inue on in to a gene ra l i zed se i zu re (Ayala et a l 1973; Ra l s ton 1958). Matsumoto and Ajmone M a r s a n (1964ab) made a fundamenta l c o n t r i b u t i o n to o u r u n d e r s t a n d i n g of e p i l e p s y when they c l e a r l y e s t ab l i shed the i n t r a c e l l u l a r co r re la te of the pa roxysmal i n t e r i c t a l d i s c h a r g e . T h e y 18 found that the s p i k e and wave r e c o r d e d from the sur face of a p e n i c i l l i n focus was associa ted w i t h a depo la r i za t ion of c o r t i c a l neu rons fol lowed b y a l o n g l a s t i n g h y p e r p o l a r i z i n g after po ten t i a l . The depo la r i za t ion , r e f e r r e d to as a pa roxysmal d e p o l a r i z i n g sh i f t (PDS), has a g rea te r ampl i tude and d u r a t i o n than a simple E P S P a n d has s e v e r a l super imposed ac t ion po ten t ia l s . The PDS i s the common e l e c t r o p h y s i o l o g i c a l p r o p e r t y u n d e r l y i n g a l l focal ep i l ep t i c a c t i v i t y (Ayala et a l 1970, 1973; A i r d et a l 1984; Dich te r and Spence r 1969ab; P r i n c e 1966, 1968) and has remained of c e n t r a l i n t e re s t to i n v e s t i g a t i o n s in to the mechanisms u n d e r l y i n g e p i l e p s y . D) A d v a n t a g e s Of The Hippocampus I n E p i l e p s y Resea rch M u c h of the more recen t w o r k on e p i l e p s y has u s e d the h ippocampus as the p r i m a r y t i s sue for i n v e s t i g a t i o n (Dichter et a l 1973; D ich te r and Spence r 1969ab; J o h n s t o n and B r o w n 1984; S c h w a r t z k r o i n and P r i n c e 1977, 1978). T h i s has come about for two reasons . F i r s t of a l l , i t i s w e l l k n o w n that the h ippocampus has one of the lowest t h r e s h o l d s fo r s e i zu re gene ra t ion of a n y s t r u c t u r e i n the c e n t r a l n e r v o u s sys tem. ( J u n g and Kornmul l e r 1938). F o r example, psychomotor o r temporal lobe foc i a re common i n c l i n i c a l e p i l e p s y and u n d e r exper imenta l cond i t ions h ippocampal p y r a m i d a l ce l l s d i s p l a y we l l deve loped pa roxysmal d e p o l a r i z i n g sh i f t s fo l lowing p e n i c i l l i n app l i ca t ions . ( S c h w a r t z k r o i n and P r i n c e 1977; Gje r s t ad et a l 1978) S e c o n d l y , the i n v i t r o h ippocampal s l ice has p r o v e n to be a v e r y u s e f u l p r e p a r a t i o n for e l e c t r o p h y s i o l o g i c a l i n v e s t i g a t i o n . (Skrede and Westgaard , 1971; Yamamoto 1972) In th i s t echn ique 300-500 mic ron t h i c k s l ices of the h ippocampal format ion are sec t ioned i n a plane normal to i t s 19 l o n g i t u d i n a l axis and p laced i n a spec ia l i zed r e c o r d i n g chamber for s t u d y . I n t h i s way the p y r a m i d a l c e l l d e n d r i t e s are l a r g e l y in t ac t and many of the af ferent and efferent t r ac t s are also p resen t . T h i s t echn ique has a number of d i s t i n c t advan tages o v e r s t a n d a r d s tereotaxic s tud ies of in t ac t b r a i n . S ince the h ippocampal s l ice i s p laced u n d e r a microscope w i t h back l i g h t i n g , s t imu la t ing and r e c o r d i n g e lec t rodes can be p laced v e r y a c c u r a t e l y u n d e r d i r e c t v i s u a l c o n t r o l . F u r t h e r m o r e , the s l i ce can be mic rod i s sec ted to isolate small popu la t ions of neu rons o r to separa te the h ippocampus in to i t s spec i f ic anatomical s u b s t r u c t u r e s . F i n a l l y the ex t r ace l l u l a r env i ronment of the s l ice i s u n d e r d i r e c t exper imenta l c o n t r o l . There fore the i n v e s t i g a t o r can c o n v e n i e n t l y and r a p i d l y a l t e r the ion ic and pharmacolog ica l env i ronment of the neu rona l t i s sue . Of p a r t i c u l a r s ign i f i cance to the s t u d y of e p i l e p s y i s the fact that a d d i t i o n of p e n i c i l l i n to the media p e r f u s i n g a s l ice of h ippocampus t r ans fo rms the normal p y r a m i d a l c e l l r e sponse from a simple E P S P w i t h a s ing le ac t ion po ten t i a l , to a l ong l a s t i n g depo la r i za t ion w i t h mul t ip le o v e r r i d i n g s p i k e s ( S c h w a r t z k r o i n and P r i n c e 1977, 1978). S ince t h i s r e sponse i s v e r y s imi la r to the c l a s s i c a l pa roxysmal d e p o l a r i z i n g shi f t seen i n the in tac t b r a i n (see D ich te r et a l 1973) the p e n i c i l l i n focus of the h ippocampal s l ice has become the main p r e p a r a t i o n for e p i l e p s y r e s e a r c h . E) I n t r i n s i c V e r s u s E x t r i n s i c Hypo thes i s Of E p i l e p s y Two hypo theses were pu t f o r w a r d b y P r i n c e i n 1966 to exp la in the pa roxysma l depo la r i za t ion associa ted w i t h the p resence of p e n i c i l l i n . A c c o r d i n g to h i s f i r s t h y p o t h e s i s , an a l t e ra t ion i n the " i n t r i n s i c " p r o p e r t i e s of neu rons w i t h i n a focus may cause these abnormal o r "ep i l ep t i c neu rons" 20 to f i r e w i t h b u r s t s of ac t ion potent ia ls (P r ince 1967, 1968). The a l te rna te v i e w sugges t s that " e x t r i n s i c " fac tors a l t e r the dynamic i n t e r ac t i ons of ce l l s w i t h i n an ep i l ep t i c aggrega te and that the PDS i s s imply a "g iant E P S P " i n response to a h y p e r s y n c h r o n o u s i n p u t (Aya la et a l 1973). These two hypo theses have dominated the f i e ld of e p i l e p s y r e s e a r c h o v e r many y e a r s . A l t h o u g h these two theor ies are often t r ea ted as i f t hey are mutua l ly e x c l u s i v e , there are a c tua l l y a number of poss ib le o v e r l a p p i n g mechanisms that c o u l d generate a P D S . Cons ide r what wou ld o c c u r i f an ep i l ep t i c agent d ramat ica l ly i nc r ea sed the s e n s i t i v i t y of a n e u r o n a l popu la t ion to neu ro t r ansmi t t e r . U n d e r these c i r cums tances a normal i n p u t c o u l d genera te an u n u s u a l l y l a rge response . That i s , a n " i n t r i n s i c p r o p e r t y " of the n e u r o n led to a "g ian t E P S P " . Ove r the yea r s a more spec i f ic form of the two hypo theses has e v o l v e d . The " i n t r i n s i c " hypo the s i s i s r e s t r i c t e d to a l t e ra t ions of spec i f i c n e u r o n a l c h a r a c t e r i s t i c s associa ted w i t h new modes of s p i k e genera t ion r e s u l t i n g i n the i n t r i n s i c a b i l i t y to genera te b u r s t s of ac t ion poten t ia l s . S ince each n e u r o n becomes capable of g e n e r a t i n g a l t e red pa t t e rns of a c t i v i t y i t may be c o n s i d e r e d an "ep i l ep t i c n e u r o n " . The " e x t r i n s i c " h y p o t h e s i s i s r e s t r i c t e d to quan t i t a t i ve changes i n c e l l u l a r p r o p e r t i e s o r i n the i n t e r ac t i ons of neu rons w i t h i n a focus that u l t imate ly lead to the gene ra t ion of "g iant E P S P s " (Ayala et a l 1973). I n t r i n s i c - F F P ' s Matsumoto and Ajmone M a r s a n (1964a) were ac tua l l y the f i r s t to c l e a r l y state the i n t r i n s i c hypo the s i s when they specula ted that " . . . excess ive , o v e r s u s t a i n e d response to r e l a t i v e l y u n a l t e r e d s y n a p t i c 21 influences ...[may be]... determined by intrinsic alterations within the cell itself". Indirect evidence for the "epileptic neuron" was provided by the observation that fast prepotentials (PPPs) were commonly observed during epileptic activity in a penicillin focus (Prince 1967; Schwartzkroin 1975, 1977). Previously, Spencer and Kandel (1961) had suggested that these low amplitude, all or none events, were somatic reflections of "dendritic" action potentials following electrotonic decay along the dendritic arborization. On this basis Prince (1968) postulated that the dendrites of neurons within the focus develop abnormal patterns of discharge and the resulting FPPs then discharge the soma leading to bursts of action potentials. A few electrotonically distant dendritic spikes may fail to discharge the soma and would appear as FPPs on somatic records. This interpretation was supported by the observation that experimentally induced hyperpolarization of the soma during a PDS often uncovered FPPs previously hidden within somatic action potentials (Schwartzkroin and Prince 1978). Furthermore, somal spikes during a PDS often arise abruptly from a voltage below the apparent threshold for action potential generation and can even arise from a level below the resting membrane potential. This lack of correlation between somal potential and spike generation suggests that the action potentials may be initiated at a site distant from the recording location, lending further support to the possible role of dendritic spikes in seizure discharge. However, several lines of evidence argue against these interpretations. At the present time there is some doubt about the dendritic origin of FPPs since at least some of these potentials are probably the result of electrotonic coupling between cells (Andrew et al 1982; MacVicar and Dudek 22 1980). I n these cases an ac t ion po ten t ia l i n one c e l l of a coup led p a i r w i l l genera te a much smaller a l l o r none event i n the o ther coup led c e l l . T h i s i n t e r p r e t a t i o n of at least some F P P s i s s u p p o r t e d b y the o b s e r v a t i o n of F P P s i n ce l l s w i t h i n a popu la t ion of neurons u n d e r g o i n g an t id romic a c t i v a t i o n (Gutn ick and P r i n c e 1981; T a y l o r and Dudek 1982a,b). I n these cases i t i s v e r y u n l i k e l y that the F P P s cou ld o r ig ina t e from d e n d r i t i c s p i k e s . The most impor tan t l ine of ev idence aga ins t the d e n d r i t i c o r i g i n of F P P s comes from d i r e c t i n t r a d e n d r i t i c r e c o r d i n g s ( T u r n e r 1985; Wong et a l 1979). T u r n e r has r e c o r d e d h i g h ampl i tude ac t ion po ten t ia l s t h r o u g h o u t the d e n d r i t i c t ree fo l lowing s y n a p t i c a c t i va t i on of p y r a m i d a l neu rons a n d found that the ampl i tude of these poten t ia l s i s a c tua l l y grea tes t near the soma. F u r t h e r m o r e , i t appea r s that ac t ion poten t ia l s f i r s t o c c u r near the soma and then p ropaga te ou twards towards the d i s t a l d e n d r i t e s . With r e spec t to the appa ren t l ack of c o r r e l a t i o n between membrane po ten t i a l and s p i k e t h r e s h o l d , i t i s l i k e l y that f i e ld i n d u c e d i n t e r a c t i o n between ce l l s w i t h i n the b u r s t i n g popu la t ion of neurons accounts for much of the o b s e r v e d v a r i a b i l i t y . The j u s t i f i c a t i o n for t h i s statement i s the c e n t r a l theme of t h i s thes i s and w i l l be dealt w i t h at l e n g t h i n l a te r chap te r s . Intrinsic - Bursting A l t h o u g h the impor tance of d e n d r i t i c ac t ion poten t ia l s i n the h ippocampus remains c o n t r o v e r s i a l at the p re sen t time, o the r ev idence sugges t s that i n t r i n s i c p r o p e r t i e s of c o r t i c a l neu rons may be impor tan t i n the gene ra t ion of se i zu re a c t i v i t y . I n p a r t i c u l a r , some c o r t i c a l neurons have the i n t r i n s i c a b i l i t y to genera te b u r s t s of ac t ion poten t ia l s u n d e r a p p r o p r i a t e cond i t i ons . The CA3 p y r a m i d a l neurons have a p a r t i c u l a r p r o p e n s i t y for spontaneous b u r s t i n g at r e g u l a r i n t e r v a l s i n bo th the in tac t animal and h ippocampal s l ice (Habli tz and J o h n s t o n 1981; K a n d e l and 23 Spence r 1961a; Wong and P r i n c e 1978). These ce l l s are also par t icu lar ly-s e n s i t i v e to shor t in jec t ions of d e p o l a r i z i n g c u r r e n t . T h e y r e s p o n d w i th a p r o l o n g e d membrane depo la r i za t ion w i t h mul t ip le ac t ion poten t ia l s i n c l u d i n g sodium as we l l as more s lowly r i s i n g ca lc ium s p i k e s (Johnstone and Habl i tz 1980; Wong and P r i n c e 1978). Spec i f i c subpopu la t ions of neu rons w i t h s imi lar f i r i n g pa t t e rns are also found i n the middle l a y e r s of the neocortex (P r ince and Conner s 1986). A l t h o u g h these c lasses of ce l l s have the i n t r i n s i c a b i l i t y to b u r s t t hey normal ly r e s p o n d w i t h o n l y a sho r t d u r a t i o n E P S P and one to th ree ac t ion poten t ia l s when s y n a p t i c a l l y ac t i va t ed (Wong and P r i n c e 1979). P r e s u m a b l y the s y n a p t i c r e sponse i s cu t sho r t b y the feedback a n d feed f o r w a r d i n h i b i t i o n p r e sen t d u r i n g a n e v o k e d response (Alger and Nico l l 1982; A n d e r s e n et a l 1964). However , fo l lowing the a d d i t i o n of p e n i c i l l i n , the s y n a p t i c response r e v e r t s to a b u r s t of ac t ion poten t ia l s not u n l i k e the c u r r e n t i n d u c e d b u r s t s seen u n d e r c o n t r o l cond i t ions ( S c h w a r t z k r o i n and P r i n c e 1978; Wong a n d T r a u b 1983). T h i s o b s e r v a t i o n s u g g e s t s that p e n i c i l l i n may lead to a n " exp re s s ion" of the i n t r i n s i c a b i l i t y for c e r t a i n c o r t i c a l neurons to genera te b u r s t s of ac t ion poten t ia l s . T h i s hypo the s i s i s f u r t h e r s u p p o r t e d b y the o b s e r v a t i o n that ce l l s w i t h the i n t r i n s i c a b i l i t y to b u r s t a re a lso the most s ens i t i ve to ep i lep togenic agents s u c h as p e n i c i l l i n ( S c h w a r t z k r o i n and P r i n c e 1978; Wong and T r a u b 1983). E x t r i n s i c - Gian t E P S P The ea r l i es t ev idence to s u p p o r t the e x t r i n s i c o r g ian t E P S P h y p o t h e s i s came from the work of K a n d e l and Spence r (1961b). They o b s e r v e d ep i l ep t i fo rm d i s c h a r g e of p y r a m i d a l neu rons fo l lowing te tanic s t imula t ion of h ippocampal a f ferents i n the in t ac t cat . These d i s c h a r g e s 24 had a form similar to the penicillin induced P D S with multiple action potentials overriding a prolonged depolarization . Since the cells had not been treated with drugs and were synaptically driven, it was very reasonable to postulate that the depolarizing potential was indeed a giant EPSP. Intracellular investigations have lent further support to the extrinsic hypothesis. For example, the passive membrane properties of pyramidal cells, such as membrane conductance and resting potential, as well as the characteristics of action potentials and the response to current injection are unaffected by penicillin (Schwartzkroin and Prince 1978, 1980; Andersen 1983). Recently, the intracellular characteristic of the PDS were rigorously tested by Johnston and Brown (1981). They predicted that if the PDS is indeed a giant EPSP then 1) membrane hyperpolarization should not alter the frequency of PDS generation, 2) PDS amplitude should be a monotonically decreasing function of membrane potential, 3) the PDS should have a reversal potential and 4) the membrane current associated with the PDS should be large compared to a normal EPSP. In fact they were able to demonstrate all of these predictions for the PDS observebed in CA3 pyramidal cells following penicillin application. The Combined Theory Considering the complex physiology of seizure activity it seems unlikely that a purely intrinsic or extrinsic mechanism underlies the PDS. In fact a number of interacting factors are probably important in epileptogenesis. Prince and Conners (1986) now propose that the interaction between three general factors determines cortical susceptibility to epilepsy. The factors are 1) the intrinsic ability of some cells to 25 genera te b u r s t s of ac t ion poten t ia l s , 2) the e f f icacy of loca l i n h i b i t o r y mechanisms s u p p r e s s i n g b u r s t gene ra t ion i n suscep t ib l e popu la t ions of n e u r o n s , and 3) the e f f i cacy of exc i t a to ry s y n a p t i c connec t ions and o the r mechanisms l e a d i n g to pos i t i ve feedback w i t h i n a neu rona l popu la t ion . F) The Role Of Inhibition In Epileptogenesis Ul t ima te ly the balance between exc i ta t ion and i n h i b i t i o n i s p r o b a b l y the most impor tan t fac tor i n the gene ra t ion of ep i l ep t i c phenomenon (Futamachi and P r i n c e 1975; A n d e r s e n et a l 1978; Noebels a n d P r i n c e 1978; Wong a n d P r i n c e 1978, 1979; A n d e r s e n a n d Rut ledge 1979; C u t l e r a n d Y o u n g 1979; D ing led ine and G je r s t ad 1980). Wi th in the h ippocampus , i n h i b i t i o n i s mediated b y i n t e r n e u r o n s (Ande r sen et a l 1978) w h i c h use GABA as t he i r neu ro t r ansmi t t e r ( C u r t i s et a l 1970; R iback et a l 1978). A d i s r u p t i o n of th i s i n h i b i t i o n b y compounds s u c h as b i c u c u l l i n e o r p i c r o t o x i n , w h i c h are k n o w n to b lock the ac t ions of GABA, can lead to s e i zu re a c t i v i t y (Alger and N i c o l l 1980; Habl i t z 1981). Of p a r t i c u l a r i n t e r e s t i s the recen t f i n d i n g that p e n i c i l l i n compe t i t i ve ly b l o c k s the ac t ion of GABA (Dingledine and Gje r s t ad 1979; McDonald and B a r k e r 1977; Mesher and S c h w a r t z k r o i n 1980). There fore i t seems l i k e l y that at least one prominent fac tor u n d e r l y i n g p e n i c i l l i n i n d u c e d ep i lep togenes i s i s a d i s r u p t i o n of i n h i b i t o r y p rocesses . However , not a l l the data s u p p o r t a loss of i n h i b i t i o n as the p r i m a r y def ic i t i n e p i l e p s y . Fo r example, k i n d l e d animals have a r emarkab le enhancement of i n h i b i t o r y p rocesses w i t h i n the dentate g y r u s (McNamara et a l 1980; N i z n i k et a l 1984; O l ive r and M i l l e r 1985; T u f f et a l 1983b) assoc ia ted w i t h an i n c r e a s e d concen t r a t i on of benzodiazep ine r ecep to r s (McNamara et a l 1980; N i z n i k et a l 1984; T u f f et a l 1983b). Ra ther s u r p r i s i n g l y , th i s i n h i b i t i o n g r a d u a l l y inc reases d u r i n g the k i n d l i n g 26 p roces s , i n p a r a l l e l w i t h the development of ep i l ep t i fo rm a c t i v i t y i n the denta te g y r u s (Ol iver and M i l l e r 1985). V a r i o u s i n v e s t i g a t o r s have a r g u e d that th i s enhanced i n h i b i t i o n w i t h i n the dentate g y r u s may ac tua l l y p l a y a p r o t e c t i v e role b y damping the s y n a p t i c i n p u t to the r e s t of the h ippocampus v i a the mossy f i be r s (Ol iver et a l 1985; S c h w a r t z k r o i n 1986). However , t h i s h y p o t h e s i s i s s t r i c t l y specu la t ive at the p r e sen t time and the ro le of i n h i b i t i o n i n k i n d l e d e p i l e p s y i s fa r from c lea r . G) MechanismB Contributing To Synchronization Of Seizure Discharge A l t h o u g h these s tud ies have made a s i gn i f i c an t c o n t r i b u t i o n to o u r u n d e r s t a n d i n g of the p r o p e r t i e s of neu rons w i t h i n a focus , t hey p r o v i d e v e r y l i t t l e d i r e c t i n s i g h t in to the fac to r s r e spons ib l e for the s t r i k i n g s y n c h r o n i z a t i o n of ac t ion potent ia ls d u r i n g i n t e r i c t a l d i s c h a r g e o r i n the sp read of s y n c h r o n o u s a c t i v i t y ac ros s a p p a r e n t l y normal b r a i n t i s sue d u r i n g the onset of a gene ra l i zed s e i zu re . It seems p robab le that a number of d i f f e ren t p rocesses operate toge ther to genera te these c h a r a c t e r i s t i c s of ep i l ep t i fo rm a c t i v i t y . Synaptic Mechanisms C e r t a i n l y s y n a p t i c i n t e r ac t i ons are l i k e l y to p l a y an impor tan t ro le i n these p rocesses (Ayala et a l 1973). F o r example, a c t i v i t y i n f i b e r t r ac t s p r o j e c t i n g from an ac t ive se i zu re focus cou ld r e c r u i t s y n c h r o n o u s d i s c h a r g e i n d i s t an t t a rge t ce l l s , t h u s a c c o u n t i n g for s y n c h r o n i z a t i o n between w i d e l y separate loca t ions . A s imi lar p rocess may o c c u r on a smaller scale w i t h i n loca l i zed r eg ions of the CNS. In the h ippocampal s l ice p r e p a r a t i o n , p e n i c i l l i n i n d u c e d i n t e r i c t a l d i s c h a r g e s o r ig ina t e i n CA2-3 and then r a p i d l y sp read in to the adjacent 27 C A l r e g i o n ( S c h w a r t z k r o i n and P r i n c e 1978). Th i s t r a n s f e r of s e i zu re a c t i v i t y to C A l i s t hough t to r e l y on the Schaf fe r co l la te ra l s s ince d i s r u p t i o n of t h i s exc i t a to ry pa thway w i t h a kn i fe cu t s tops a l l s e i zu re a c t i v i t y i n C A l ( S c h w a r t z k r o i n and P r i n c e 1978). H i g h concen t r a t ions of p e n i c i l l i n w i l l occas iona l ly genera te s y n c h r o n o u s b u r s t a c t i v i t y i n the i so la ted C A l r e g i o n bu t i t i s v e r y r e s i s t an t to p e n i c i l l i n i n d u c e d i n t e r i c t a l s . T h i s i s i n marked con t r a s t to the CA3 r e g i o n w h i c h con t inues to genera te spontaneous s y n c h r o n o u s d i s c h a r g e s af ter the kn i fe cu t . I n fact e v e n small i s l e t s of CA3 r e g i o n d i s sec t ed away from s u r r o u n d i n g t i s sue can con t inue to p r o d u c e ep i l ep t i fo rm a c t i v i t y (Miles et a l 1984). T h i s d i f fe rence be tween C A l and CA3 may be based i n p a r t on the i n t r i n s i c a b i l i t y of CA3 p y r a m i d a l neu rons to genera te b u r s t s of ac t ion po ten t ia l s . However , loca l exc i t a to ry i n t e r ac t i ons w i t h i n CA3 may be an impor tan t s y n c h r o n i z i n g fac tor . Recent p h y s i o l o g i c a l ev idence for r e c u r r e n t exc i ta t ion w i t h i n CA3 s u g g e s t s that some of these s y n a p t i c i n t e rac t ions may be qui te potent . Based on s imul taneous r e c o r d s from p a i r s of CA3 p y r a m i d a l neu rons , a b u r s t i n a s ing le c e l l can have su f f i c i en t s y n a p t i c d r i v e to d i s c h a r g e a second c e l l (Wong et a l 1986). F u r t h e r m o r e , a c u r r e n t - i n d u c e d b u r s t i n one c e l l c an sometimes generate s y n c h r o n o u s a c t i v i t y w i t h i n a popu la t ion of CA3 neu rons (Miles and Wong 1983). E v e n t h o u g h ce l l s w i th s u c h i m p r e s s i v e s y n a p t i c in f luences are r a r e (Wong 1986) a computer s imula t ion b y T r a u b and Wong (1982, 1983) has shown that e v e n a v e r y low rate of s y n a p t i c c o n n e c t i v i t y between CA3 neu rons c o u l d account for the se i zu res o b s e r v e d d u r i n g p e n i c i l l i n - i n d u c e d i n t e r i c t a l d i s c h a r g e i n CA3. The loss of p e n i c i l l i n - i n d u c e d se i zu re a c t i v i t y i n the h ippocampus fo l lowing d e p r e s s i o n of s y n a p t i c t r ansmiss ion b y 28 pharmacolog ica l t echn iques f u r t h e r s u p p o r t s the ro le of s y n a p t i c i n t e r ac t i ons i n ep i lep togenes i s ( S c h w a r t z k r o i n and P r i n c e 1978). Non S y n a p t i c Mechanisms However , i t appea r s that s y n a p t i c t r ansmis s ion i s not an absolu te r equ i remen t fo r the genera t ion and sp read of s e i zu re a c t i v i t y . Recen t ly , the re have been a number of r e p o r t s of a n ep i l ep t i c phenomenon w h i c h p e r s i s t s i n the face of a complete b lockade of s y n a p t i c t r ansmis s ion ( Je f fe rys and Haas 1982; T a y l o r and Dudek 1982, 1984a,b; Y a a r i et a l 1983; Haas a n d J e f f e r y s 1984) It i s we l l k n o w n that C a + + i n f l u x in to p r e s y n a p t i c te rmina ls i s a p r e r e q u i s i t e for s y n a p t i c t r ansmis s ion (Katz 1969). There fore a l l s y n a p t i c t r a n s m i s s i o n i s b l o c k e d i f neu rona l t i s sue i s exposed to a n ex t r ace l l u l a r f l u i d w i t h l i t t l e o r no Ca++. T h i s i s not p r a c t i c a l i n the i n t ac t animal bu t i n the h ippocampal s l ice the ex t r ace l l u l a r env i ronmen t i s eas i ly manipu la ted . J e f f e r y s and Haas (1982) and T a y l o r and Dudek (1982) i n d e p e n d e n t l y d i s c o v e r e d that h ippocampal s l ices exposed to a pe r fu s ion medium low i n C a + + deve lop a r h y t h m i c a l spontaneous b u r s t i n g a c t i v i t y of C A l p y r a m i d a l neu rons . The b u r s t i n g a c t i v i t y o c c u r s o n l y af ter a p r o l o n g e d exposure of more t h a n one hour , at a time when a l l s y n a p t i c t r ansmis s ion i s demons t rab ly b l o c k e d . Each b u r s t cons i s t s of a h i g h f r e q u e n c y t r a i n of popu la t ion d i s c h a r g e s super imposed on a slow nega t ive ex t r ace l lu l a r po ten t ia l . When the b u r s t i n g a c t i v i t y is f u l l y deve loped the slow nega t ive waves c a n be g rea t e r t han 5 mV i n ampl i tude and often o c c u r w i t h grea t r e g u l a r i t y . T h e y u s u a l l y b e g i n i n one loca l ized r e g i o n w i t h i n C A l a n d then sp read i n bo th d i r e c t i o n s a long the p y r a m i d a l c e l l l a y e r at a ra te of about 0.5 mm per second (Yaar i et a l 1983). 29 These sel f sus t a ined b u r s t s of n e u r o n a l f i r i n g have a number of c h a r a c t e r i s t i c s w h i c h are ep i l ep t i fo rm i n na tu re . I n t r a c e l l u l a r r e c o r d i n g s d u r i n g a b u r s t ind ica te that the membrane po ten t ia l of the p y r a m i d a l neu rons unde rgoes a p r o l o n g e d depo la r i za t ion w i t h a super imposed h i g h f r e q u e n c y d i s c h a r g e of ac t ion poten t ia l s . T h i s a c t i v i t y i s s imul taneous ly p r e sen t i n l a rge numbers of ce l l s w i t h i n a popu la t ion and i s p r o b a b l y r e spons ib l e for the ex t r ace l lu l a r n e g a t i v i t y assoc ia ted w i t h the b u r s t ( Je f fe rys and Haas 1982). T h i s p a t t e r n of a c t i v i t y c lo se ly resembles the pa roxysmal d e p o l a r i z i n g sh i f t u n d e r l y i n g i n t e r i c t a l d i s c h a r g e . A n o t h e r r a t h e r s t r i k i n g c h a r a c t e r i s t i c of the low ca lc ium b u r s t i s the p resence of mul t ip le popu la t ion poten t ia l s d u r i n g the slow wave of ex t r ace l l u l a r n e g a t i v i t y . It i s h i g h l y l i k e l y that each popu la t ion po ten t i a l r e p r e s e n t s the s imul taneous d i s c h a r g e of a g r o u p of of C A l p y r a m i d a l ce l l s (Ande r sen et a l 1971; J e f f e r y s and Haas 1982; T a y l o r and Dudek 1982). S imi la r t r a i n s of popu la t ion poten t ia l s are t y p i c a l of the d i s c h a r g e r e c o r d e d from c o r t i c a l s t r u c t u r e s d u r i n g a gene ra l i zed se i zu re ( M a d r y g a et a l 1975). On a n i n d i v i d u a l bas i s , each popu la t ion po ten t i a l i s also s imi la r to the i n t e r i c t a l d i s c h a r g e i n d u c e d b y p e n i c i l l i n app l i ca t ion to the h ippocampus ( S c h w a r t z k r o i n and P r i n c e 1978). The p resence of ep i l ep t i fo rm a c t i v i t y d u r i n g s y n a p t i c b lockade emphasizes the impor tance of c o n s i d e r i n g n o n - s y n a p t i c mechanisms for bo th the gene ra t ion and sp read of s y n c h r o n o u s neu rona l a c t i v i t y . Three p o s s i b i l i t i e s shou ld be c o n s i d e r e d : (1) e lec t ro ton ic c o n d u c t i o n ac ros s gap j u n c t i o n s , (2) changes i n ex t r ace l lu l a r ion ic concen t r a t i on a n d (3) f i e ld i n d u c e d o r ephap t ic i n t e r ac t i ons . 30 The Role Of E l ec t ro ton i c S y n a p s e s The gap j u n c t i o n o r e lec t ro ton ic synapse i s a spec ia l i zed connec t ion a l l o w i n g communicat ion between two neu rons wi thout a need for neu ro t r ansmi t t e r subs tance (Loewens te in 1981). T h i s communicat ion o c c u r s as a simple passage of c u r r e n t between two ce l l s t h r o u g h a low res i s t ance pa thway formed b y the gap j u n c t i o n . The re i s n o t h i n g ac t ive about the c u r r e n t t r ans f e r . I t s imply f lows p a s s i v e l y from one c e l l to the o ther a long a n y vo l tage g r a d i e n t that might exis t . E i t h e r i n h i b i t i o n o r exc i ta t ion of the post j u n c t i o n a l c e l l c an o c c u r , d e p e n d i n g on the d i r e c t i o n of pas s ive c u r r e n t f low, bu t the in f luence w i l l a lways be i n the same d i r e c t i o n i n bo th ce l l s . Tha t i s , a n ac t ion po ten t i a l i n one c e l l of a " c o u p l e d " p a i r w i l l a lways cause a depo la r i za t i on of i t s p a r t n e r . T h i s i s u n l i k e chemica l s y n a p t i c j u n c t i o n s w h i c h al low i n v e r s i o n of t h e i r s i g n a l v i a a n i n h i b i t o r y neu ro t r ansmi t t e r . I t i s easy to imagine that the r e c u r r e n t exc i ta t ion genera ted b y a l a rge number of gap j u n c t i o n s w i t h i n a popu la t ion of neu rons c o u l d lead to s y n c h r o n i z a t i o n and sp read of a h i g h f r e q u e n c y d i s c h a r g e o r i g i n a t i n g o n l y i n a few ce l l s . One of the f i r s t examples of func t iona l e lec t ro ton ic c o u p l i n g was found i n the g ian t motor synapse of the c r a y f i s h where n e u r o t r a n s m i s s i o n i s v i a r e c t i f i e d c u r r e n t flow ac ross gap j u n c t i o n s j o i n i n g the p re and post s y n a p t i c c e l l ( F u r s h p a n and Po t te r 1959). L a t e r , e lec t ro ton ic c o u p l i n g of neu rons was d i s c o v e r e d i n the f i s h s p i n a l c o r d and b r a i n stem (Bennett et a l 1967abc) as we l l as mammalian b r a i n stem (Ba rke r and L l i a n a s ' 1971; K o r n et a l 1973, Sotelo et a l 1974).. It i s o n l y more r e c e n t l y that s tud ie s of e lec t ro ton ic c o u p l i n g have been c a r r i e d out i n the cor tex . 31 M u c h of the ev idence for e lec t ro ton ic c o u p l i n g i n the h ippocampus comes from s tud ies of d y e c o u p l i n g i n w h i c h L u c i f e r ye l low i s in j ec ted in to the soma of a c e l l a n d h i s to log i ca l sec t ions are c h e c k e d l a t e r for the p resence of s t a i n i n g (Stewart 1978). S t a i n i n g i n two o r more ce l l s sugges t s that these ce l l s may have been connec ted b y a gap j u n c t i o n . U s i n g t h i s t echn ique , dye c o u p l i n g has been de tec ted i n many ce l l s i n the neocortex, h ippocampus and dentate g y r u s (Andrew et a l 1982; G u t n i c k and P r i n c e 1981; MacVica r and Dudek 1980, 1982). E l e c t r o p h y s i o l o g i c a l ev idence fo r func t iona l c o u p l i n g has also been ob ta ined for these r eg ions of cor tex . Most of the ev idence comes from i n t r a c e l l u l a r s tud ie s d u r i n g an t id romic ac t i va t i on of a popu la t ion of neu rons . The ra t ionale beh ind these s tud ies i s based on the assumpt ion that , at a c e r t a i n s t imulus i n t e n s i t y , o n l y one c e l l of a coup led pa i r w i l l be above t h r e s h o l d for a c t i v a t i o n . I f the r e c o r d i n g , e lec t rode happens to be i n the s u b t h r e s h o l d n e u r o n a shor t l a t ency , a l l o r none depo la r i za t ion (SLD) , w i l l o c c u r as c u r r e n t passes between the ce l l s . U s i n g t h i s t echn ique , about 27% of neocor t i ca l neu rons (Gutn ick and P r i n c e 1981) and 5% of C A l p y r a m i d a l ce l l s (Tay lo r and Dudek 1982a) r e sponded w i t h a S L D , s u g g e s t i n g that at least some ce l l s i n these r eg ions are e l ec t ro ton ica l ly coup l ed . A more d i r e c t bu t much more d i f f i c u l t test fo r c o u p l i n g uses d u a l i n t r a c e l l u l a r r e c o r d i n g s . If the two impaled ce l l s are e l ec t ro ton ica l ly coup l ed , c u r r e n t passed in to one w i l l cause a sh i f t i n membrane po ten t i a l of the o the r as c u r r e n t passes t h r o u g h the gap j u n c t i o n . T h i s t echn ique de tec ts weak c o u p l i n g i n about 5-7% of CA3 p y r a m i d a l and dentate g r anu l e ce l l s (McVicar and Dudek 1981). However , Knowles and S c h w a r t z k r o i n (1981) were unable to f i n d a n y examples of weak e lec t ro tonic c o u p l i n g i n the C A l r e g i o n of the h ippocampus . 32 The i n t e r p r e t a t i o n of these e l e c t r o p h y s i o l o g i c a l exper iments , and the s ign i f i cance of dye c o u p l i n g are c o n t r o v e r s i a l at the p r e sen t time. C r i t i c i s m of the dye c o u p l i n g t echn ique stems mainly from the fact that mul t ip le s t a i n i n g cou ld r e s u l t from d y e leakage i n the v i c i n i t y of s e v e r a l neu rons damaged b y the microe lec t rode , o r b y s imul taneous impalements of two o r more ce l l s (Knowles et a l 1982; A l g e r et a l 1983). A f u r t h e r c o n c e r n i s that most of these s tud ies have been per formed on c o r t i c a l s l i ces . Neurona l p rocesses damaged d u r i n g p r e p a r a t i o n of the s l i ce may form new gap j u n c t i o n s to adjacent neu rona l membranes (Bul lock and Kate r 1981). The re i s also a marked d i s p a r i t y between r e s u l t s d e r i v e d from the v a r i o u s t echn iques ava i l ab le to detect c o u p l i n g . F o r example, as many as 70% of C A l p y r a m i d a l neu rons show ev idence of d y e c o u p l i n g (Andrew et a l 1982) ye t o n l y 5% of these ce l l s show S L D s u s i n g the an t id romic tes t (Taylor and Dudek 1982a) a n d v i r t u a l l y no p a i r s of d u a l impaled ce l l s show ev idence of weak c o u p l i n g (Knowles and S c h w a r t z k r o i n 1981). F i n a l l y the e l ec t ron mic roscop ic s tud i e s of gap j u n c t i o n s i n the h ippocampus ind i ca t e that gap j u n c t i o n s are i n f r e q u e n t i n CA3 and are p a r t i c u l a r l y r a r e on p y r a m i d a l neu rons i n C A l . Based on t h i s data , i t seems u n l i k e l y that c o r t i c a l neu rons have the k i n d of " s y n c y t i a l " c o u p l i n g one wou ld expect i f e lec t ro ton ic c o u p l i n g was a major fac tor i n low ca lc ium b u r s t i n g , o r i n the s y n c h r o n i z a t i o n o r p ropaga t ion of o ther forms of s e i zu re a c t i v i t y i n the cor tex . The Role Of E x t r a c e l l u l a r Ions A l t e r e d concen t r a t ions of e x t r a c e l l u l a r ions may p l a y an impor tant ro le i n the gene ra t ion and sp read of s e i zu re a c t i v i t y . I t i s we l l k n o w n that ep i l ep t i fo rm a c t i v i t y i s assoc ia ted w i t h an inc rease i n the ex t r ace l lu l a r 33 concen t r a t i on of K* (Hotson et a l 1973; P r i n c e et a l 1973) and C l " (Dietzel et a l 1980, 1982ab) as w e l l as a decreased ex t r ace l l u l a r c o n c e n t r a t i o n of Na* (Dietzel et a l 1982ab; Lewi s and Schuet te 1975) and C a + + (Heinemann and L u x 1983; Heinemann et a l 1977, 1978). These changes i n K+, C l " a n d Ca+* c o u l d lead to an i n c r e a s e d neu rona l e x c i t a b i l i t y w i t h i n the loca l r e g i o n of the ep i l ep t i c a c t i v i t y . Fo r example, e leva ted ex t r ace l l u l a r K + concen t ra t ions c o u l d depola r ize the r e s t i n g membrane po ten t i a l as w e l l as de p r e s s the effect of h y p e r p o l a r i z i n g K+ c u r r e n t s re la ted to ac t ion po ten t ia l r e p o l a r i z a t i o n and to C a + + a c t i va t ed K + conduc tances . A low ex t r ace l l u l a r C a + + concen t r a t i on c o u l d f u r t h e r r educe C a + + a c t i va t ed K + c u r r e n t s and may also enhance neu rona l exc i t ab i l i t i e s b y d i r e c t l y a l t e r i n g the cha rge c h a r a c t e r i s t i c s of the n e u r o n a l membrane. F i n a l l y a r e d u c t i o n of ex t r ace l l u l a r C l - concen t r a t i on may sh i f t the c h l o r i d e e q u i l i b r i u m po ten t ia l i n a d e p o l a r i z i n g d i r e c t i o n t h e r e b y r e d u c i n g the e f f icacy of C l - mediated i n h i b i t i o n . The pos i t i ve feed back of these in f luences on neu rona l e x c i t a b i l i t y cou ld mainta in o r e v e n inc rease s e i zu re a c t i v i t y once i n i t i a t e d . Two impor tan t ion ic mechanisms tend to l imi t these e x c i t a b i l i t y ef fects . F i r s t of a l l , accumula t ion of i n t r a c e l l u l a r Na* a n d ex t r ace l l u l a r K* may ac t iva te a h y p e r p o l a r i z i n g e lec t rogen ic Na + /K+ pump, l e a d i n g to a d e p r e s s i o n of s e i zu re a c t i v i t y (Heinemann and G u t n i c k 1979; Ito and Oshima 1964; K o r k e et a l 1972), and second , the r a p i d spa t i a l b u f f e r i n g of K* b y g l i a l p rocesses ( G a r d n e r - M e d w i n 1983; G a r d n e r - M e d w i n et a l 1981) l imi ts the loca l accumula t ion of K + w i t h i n a s e i zu re focus . Ionic mechanisms may also p l a y an impor tan t role i n the sp read of s e i zu re a c t i v i t y ac ross the cor tex . A s K + d i f fuses away from an ep i lep t ic focus in to the s u r r o u n d i n g n e u r o p i l i t cou ld depola r ize n e i g h b o r i n g ce l l s 34 and cause them to join the epileptic activity within the focus. The rapid redistribution of K+ by glial cells may actually enhance this process. Several lines of evidence suggest that ionic mechanisms may be important i n the low Ca+* model of epilepsy. For example, extracellular K* levels show a marked elevation during each low Ca** burst, and the K* elevation precedes the appearance of a burst at a given location along the CAl cell layer (Konnerth et al 1984; Yaari et al 1983). Furthermore, the spreading epileptic activity propagates along the cell layer with a rate similar to the rate of K + redistribution. Finally, pressure injection of potassium chloride, but not sodium chloride can initiate a burst at the site of injection (Konnerth et al 1984). Clearly, altered levels of K+ and other extracellular ions could contribute to the enhanced excitability within an epileptic focus and could participate in the spread of seizure activity. However, altered ionic concentrations do not explain the tightly synchronized neuronal activity observed during an interictal discharge or during the spontaneous "population spikes" characteristic of low Ca++ bursts, after-discharge, and other forms of epileptic activity. These events occur over a period of a few milliseconds, a time scale far too short for significant redistribution of K+ (Gardner-Medwin 1983). On the other hand, electrical interactions are extremely rapid (Taylor and Dudek 1984b) and may well underlie the synchronization of many of these epileptic phenomena. The Role Of Ephaptic Interactions The concept of electrical, non-synaptic interactions between neurons is certainly not new. As early as 1930, Adrian suggested that synchronization of action potentials within the isolated phrenic nerve came 35 about because "an ac t ive f i b e r can cause a s l i g h t momentary inc rease i n the s t imulus to o the r f i b e r s and i t can do so owing to the ac t ion c u r r e n t w h i c h p roduces i t . " S i m i l a r l y , Gasser (1938) conc luded that p u r e l y e l e c t r i c a l in f luences r e c r u i t e d a c t i v i t y i n p r e v i o u s l y qu iescen t axons exposed to r h y t h m i c a l a c t i v i t y i n n e a r b y f i b e r s (Gasser 1938). Ka tz and Schmi t t (1940) c a r r i e d out one of the f i r s t quan t i t a t ive i n v e s t i g a t i o n s of e l e c t r i c a l i n t e r ac t i ons between two adjacent n e r v e f i b e r s . T h e y found that the passage of a n impulse i n one axon was associa ted w i t h a t r i p h a s i c a l t e ra t ion i n the e x c i t a b i l i t y of a n adjacent f i b e r and that the p o l a r i t y and i n t e n s i t y of these changes were p r e d i c t e d b y the c h a r a c t e r i s t i c s of the membrane c u r r e n t s of the ac t ive n e r v e . T h e y a lso demonst ra ted tha t i f ac t ion poten t ia l s are t r a v e l l i n g down two adjacent n e r v e s "a mutual i n t e r a c t i o n takes place p r o d u c i n g v a r i o u s combinat ions of speed ing a n d s l o w i n g " of c o n d u c t i v e v e l o c i t y . I n some p a i r s of axons t h i s i n t e r a c t i o n leads to a " s y n c h r o n i z a t i o n and equa l i za t ion of speeds of the impulses" so that t h e y t r a v e l l e d i n a time locked fash ion . The mutua l i n t e r a c t i o n of axons was c l e a r l y enhanced b y i n c r e a s i n g the res i s t ance of the ex t r ace l l u l a r f l u i d , l e n d i n g f u r t h e r s u p p o r t to the ro le of loca l c u r r e n t s i n t h i s phenomenon. J a s p e r a n d Monn ie r (1938) were f i r s t to c l e a r l y demonstra te the t r a n s m i s s i o n of an ac t ion po ten t i a l f rom one axon to ano ther t h r o u g h a po in t of c lose contac t . They r e f e r r e d to th i s p h y s i c a l contac t as an " a r t i f i c i a l s y n a p s e " and c o n c l u d e d that a p u r e l y e l e c t r i c a l form of t r a n s m i s s i o n must have o c c u r r e d between the axons. However , de ta i led a n a l y s i s of th i s e l e c t r i c a l i n t e r ac t i on was not poss ib le s ince whole n e r v e p r e p a r a t i o n s were u s e d . They had no way of k n o w i n g the number of n e r v e f i b e r s i n v o l v e d , the c h a r a c t e r i s t i c s of p h y s i c a l contact between f i be r s , and 36 no way of measu r ing the e l e c t r i c a l c h a r a c t e r i s t i c s of the i n t e r ac t i ons at a s i ng l e axon l e v e l . These d i f f i cu l t i e s were r e d u c e d when A r v a n i t a k i (1942) c a r r i e d out s imi la r s tud ies u s i n g the s q u i d g ian t axon. B y s imul taneous ly r e c o r d i n g from two axons she was able to demonstra te t r ansmis s ion of a n ac t ion po ten t i a l ac ross a we l l def ined po in t of contac t between a p a i r of f i b e r s . A r v a n i t a k i a r g u e d that these po in t s of close p h y s i c a l approx imat ion shou ld not be r e f e r r e d to as a r t i f i c i a l synapses s ince the e s t ab l i shed usage of the w o r d " synapse" was spec i f i c a l l y for spec i a l i zed po in t s of contac t w i t h loca l d i f f e ren t i a t ion of c e l l membranes. Ins tead she sugges t ed the w o r d ephapse to des igna te "the contac t or c lose v i c i n i t y of two ac t ive func t i ona l su r faces , whe the r t h i s contac t be exper imenta l o r b r o u g h t about b y n a t u r a l means". She conc luded that loca l e l ec t r i c c u r r e n t s genera ted b y the " p r e - e p h a p t i c " axon i n d u c e d loca l changes i n the membrane po ten t i a l of the "pos tephap t i c" axon, t hus a l t e r i n g i t s e x c i t a b i l i t y . D u r i n g th i s same pe r iod s e v e r a l o the r i n v e s t i g a t o r s demonst ra ted s imi lar e l e c t r i c a l i n t e r ac t i ons between myel ina ted a x o n s . (Bla i r and E r l a n g e r 1940; Renshaw and Therman 1941; Rosenb lue th 1941) and a sys temat ic a n a l y s i s of the ephap t ic in f luence of n e i g h b o r i n g n e r v e f i b e r s b y M a r r a z z i and Loren te de No (1944) c l e a r l y e s t ab l i shed loca l c u r r e n t flow from ac t ive to pas s ive axon as the mechanism of i n t e r a c t i o n . I t i s i n t e r e s t i n g from the h i s t o r i c a l p e r s p e c t i v e that these i n v e s t i g a t i o n s were o c c u r r i n g at a time when there was grea t debate as to the e l e c t r i c a l o r chemica l na tu re of c o n v e n t i o n a l s y n a p t i c t r ansmis s ion . The p h y s i o l o g i s t s at that time were s k e p t i c a l of the chemical hypo the s i s and fel t tha t spec i a l i zed s y n a p t i c contac ts func t ion b y i n j e c t i n g loca l c u r r e n t s in to the pos t s y n a p t i c c e l l . T h i s accounts for A r v a n i t a k i ' s comment (1942) that 37 "the mechanisms of t r ansmis s ion are doub t less s imi lar i n the two cases (ephapt ic and synap t i c ) bu t not i d e n t i c a l " . Ove r the next few yea r s chemical t r ansmis s ion was c l e a r l y e s t ab l i shed as the predominant form of c e l l to c e l l communicat ion and i n t e r e s t i n ephap t i c i n t e r a c t i o n as a form of neu rona l t r ansmis s ion decreased . However , more gene ra l e l e c t r i c a l i n t e r ac t i ons p r o d u c e d b y d i f fuse c u r r e n t f low i n the ex t r ace l l u l a r space of the CNS became the focus of cons ide rab le in t e re s t . B r o o k h a r t and B l a c h l y (1952) found that t hey c o u l d modulate the f i r i n g f r e q u e n c y of s ing le p u r k i n j e ce l l s i n the cat b y a p p l y i n g a s teady state c u r r e n t ac ros s the ce rebe l l a r cor tex . Wi th in the ce rebe l lum, the P u r k i n j e ce l l s a re o r g a n i z e d i n a h i g h l y r e g u l a r manner w i t h t h e i r d e n d r i t e s p r o j e c t i n g towards the b r a i n sur face and a l l of the somata i n a s ing le we l l def ined l a y e r . The c u r r e n t was most e f fec t ive i f a p p l i e d a long the dendrosomat ic axis of the P u r k i n j e ce l l s and was ine f fec tua l when a p p l i e d i n the p lane of the c e l l l a y e r . U n i t s i n c r e a s e d t h e i r f i r i n g f r e q u e n c y when the c u r r e n t made the po ten t i a l i n the s u p e r f i c i a l d e n d r i t i c l a y e r more pos i t i ve than the deeper c e l l l a y e r , and c e l l f i r i n g was dep re s sed when the po ten t i a l between d e n d r i t e s and somata was r e v e r s e d . S imi la r changes i n neu rona l a c t i v i t y are o b s e r v e d i n the c e r e b r a l co r t ex d u r i n g app l i ca t i on of ex t r ace l lu l a r c u r r e n t s (Creu tz fe ld t et a l 1962; B indman et a l 1964). I n the cor tex , the major ap i ca l d e n d r i t e of p y r a m i d a l ce l l s reaches towards the c o r t i c a l sur face from deeper l a y e r s , i n a manner s imi lar to the s ing le d e n d r i t i c a r b o r i z a t i o n of the P u r k i n j e c e l l . P y r a m i d a l c e l l a c t i v i t y a n d the ampl i tude of e v o k e d potent ia ls were bo th enhanced d u r i n g c u r r e n t s associa ted w i t h sur face p o s i t i v i t y , and d e p r e s s e d when the vo l tage g r a d i e n t ac ross the ap i ca l d e n d r i t e was r e v e r s e d . 38 More r e c e n t l y J e f f e r y s (1981) s t ud i ed the in f luence of a r t i f i c i a l l y a p p l i e d c u r r e n t on the e x c i t a b i l i t y of dentate g ranu le ce l l s b y o b s e r v i n g the in f luence of app l i ed c u r r e n t on the popu la t ion response to af ferent s t imula t ion . A g a i n , he found that d r i v i n g the e x t r a d e n d r i t i c space to a vo l tage more pos i t i ve than the extrasomatic space enhanced the e x c i t a b i l i t y of g r a n u l e ce l l s and potent ia ted the s y n a p t i c r esponse . I n each of these s tud ies , the au tho r s conc luded that the a l t e r ed n e u r o n a l e x c i t a b i l i t y p r o b a b l y o c c u r r e d because a f r a c t i on of the app l i ed c u r r e n t en te red the i n t r a c e l l u l a r space and sh i f t ed the membrane po ten t i a l of the affected ce l l s . P u r p u r a and M c M u r t r y (1965) ob ta ined ev idence i n s u p p o r t of th i s hypo the s i s when they r e c o r d e d the i n t r a c e l l u l a r effects of a r t i f i c i a l l y a p p l i e d f i e ld s . T h e y found that app l i ed f i e lds a l t e red the i n t r a c e l l u l a r waveform of E P S P ' s and ac t ion poten t ia l s i n a manner cons i s t en t w i t h a d i r e c t modif ica t ion of membrane po ten t ia l . U n f o r t u n a t e l y t h e i r exper imenta l pa rad igm made a d i r e c t measurement of the membrane po ten t i a l i m p r a c t i c a l . These i n v e s t i g a t i o n s c l e a r l y ind ica te that , u n d e r the c o r r e c t cond i t i ons , a r t i f i c i a l l y genera ted ex t r ace l l u l a r f i e lds can modify neu rona l a c t i v i t y . B u t i s neu rona l e x c i t a b i l i t y also modified b y the e l ec t r i c c u r r e n t s associa ted w i t h s y n a p t i c t r ansmis s ion , c e l l d i s c h a r g e and o ther phenomena i n t r i n s i c to the f u n c t i o n i n g b r a in? F u r u k a w a and F u r s h p a n (1963) demonst ra ted a d i r e c t e l e c t r i c a l i n h i b i t i o n of the Mau thne r c e l l of the g o l d f i s h genera ted b y p h y s i o l o g i c a l a c t i v i t y i n n e a r b y n e r v e te rminals . The Mau thne r c e l l has a n u n u s u a l anatomical fea ture ca l led the axon cap located o v e r the t r i g g e r zone of the c e l l . Termina l s a r i s i n g from co l la te ra l s of the c o n t r a l a t e r a l Mau thne r c e l l s p i r a l a r o u n d the t r i g g e r zone w i t h i n th i s spec ia l i zed cap . A p p a r e n t l y , ac t ion potent ia ls from these co l la te ra l 39 axons fail to invade the terminal. As a result, current flows outward across the spiralling membranes of the terminal, causing a short duration but potent depression of Mauthner cell activity. A second form of ephaptic inhibition mediated by physiological currents is also seen in the goldfish CNS. Korn and Faber (1975) found that the field potential associated with discharge of the Mauthner cell inhibited nearby medullary neurons. These medullary neurons have short axons which synapse onto the Mauthner cell within the axon cap. The current sink generated by the mauthner cell in the immediate vicinity of these terminals induces an intracellular current flow and somatic hyperpolarization of the medullary neurons. It is interesting that the axon cap is involved in both of these examples of ephaptic inhibition. Korn and Farber (1975) postulated that this anatomical specialization may have properties which enhance electrical interactions. In fact they found a marked increase in extracellular resistance within the cap as compared to the surrounding neurons. They discussed how this property should enhance ephaptic interactions by increasing the intracellular component of currents associated with field potentials. An anatomical specialization similar to the axon cap is also present in the mammalian CNS. Within the cerebellum, basket cell terminals form tight meshworks around the initial segments of individual Purkinje cells. These terminals are intermingled with glial processes and entirely encapsulate the axon as it emerges from the soma in a manner reminiscent of the axon cap (Palay and Chan-Palay 1974). Korn and Axelrad (1980) demonstrated that basket cell activity generates a direct electrical depression of Purkinje cell excitability immediately preceding the expected synaptic inhibition. The 40 s i m i l a r i t y of th i s sequence of even ts to that seen fo l lowing ac t i va t i on of a f fe ren ts to the go ld f i sh Mau thne r c e l l i s r a t h e r s t r i k i n g . Not a l l examples of ephapt ic i n t e r ac t i ons i n the f u n c t i o n i n g CNS i n v o l v e anatomical spec ia l i za t ions . The p o s s i b i l i t y of ephap t ic i n t e r a c t i o n is p r e sen t a n y w h e r e neu rona l a c t i v i t y genera tes s u b s t a n t i a l f i e ld po ten t ia l s , and most r eg ions of the CNS can generate f i e ld po ten t ia l s u n d e r the c o r r e c t cond i t i ons . I n fact e v e n the d i s c h a r g e of a s ing le n e u r o n i n the s p i n a l c o r d of the cat can genera te an ex t r ace l l u l a r f i e ld of a few mi l l ivo l t s ex t end ing for u p to 500 microns in to the s u r r o u n d i n g n e u r o p i l (Nelson and F r a n k 1964). However , f i e ld po ten t ia l s of su f f i c ien t ampl i tude to genera te ephap t ic in f luences are more l i k e l y to be associa ted w i t h s y n c h r o n o u s a c t i v i t y w i t h i n a popu la t ion of neu rons . F o r example, the f i e ld po ten t i a l i n the s p i n a l c o r d fo l lowing v e n t r a l root s t imula t ion can genera te a potent ephap t i c f ac i l i t a t ion of uns t imula ted motor neu rons , a c c o u n t i n g for as much as a 30% r e d u c t i o n i n t he i r f i r i n g t h r e s h o l d (Nelson 1966). More recen t i n t e r e s t i n ephap t ic i n t e r ac t i ons has cen te red a r o u n d ex t r ace l l u l a r po ten t ia l s genera ted w i t h i n the h ippocampal format ion. Many i n v e s t i g a t o r s have sugges t ed that ephapt ic i n t e r ac t i ons are l i k e l y to o c c u r i n the h ippocampus and o ther c o r t i c a l a reas because of t h e i r h i g h l y o r g a n i z e d laminar s t r u c t u r e s , and p r o p e n s i t y for g e n e r a t i n g h i g h ampl i tude f i e ld po ten t ia l s ( Jasper 1969, P u r p u r a et a l 1966; Nelson 1965). However , the d i s c o v e r y of low ca lc ium b u r s t i n g s t imula ted a d i r e c t i n v e s t i g a t i o n of e l e c t r i c a l , non s y n a p t i c i n t e r ac t i ons w i t h i n the h ippocampus . T a y l o r and Dudek (1982, 1984a,b) demonst ra ted that each i n d i v i d u a l ex t r ace l l u l a r "popu la t ion s p i k e " d u r i n g a low calc ium b u r s t i s associa ted w i t h a t ransmembrane depo la r i za t ion of C A l p y r a m i d a l neu rons . S ince th i s depo la r i za t i on o c c u r r e d d u r i n g s y n a p t i c b lockade , and i t s c h a r a c t e r i s t i c s 41 were not cons i s t en t w i t h e lec t ro ton ic o r ion ic mechanism, these au tho r s c o n c l u d e d that a d i r e c t e l e c t r i c a l effect must have o c c u r r e d . They pos tu la t ed that these e l e c t r i c a l in f luences p l a y a s i gn i f i c an t ro le i n s y n c h r o n i z i n g i n d i v i d u a l ac t ion poten t ia l s ac ross many p y r a m i d a l neu rons . 1.3 THE P R E S E N T S T U D Y Despi te sus t a ined and ene rge t i c r e s e a r c h b y many i n v e s t i g a t o r s e p i l e p s y remains a se r ious neu ro log i ca l d i s o r d e r w i t h t r a g i c consequences fo r a s u b s t a n t i a l p r o p o r t i o n of the popu la t ion . E l e c t r o p h y s i o l o g i c a l s tud ie s at the s ing le c e l l l e v e l have iden t i f i ed a number of impor tan t fac tors c o n t r i b u t i n g to s e i zu re a c t i v i t y bu t have fa i led to p r o v i d e a n i n s i g h t in to s y n c h r o n i z a t i o n of d i s c h a r g e w i t h i n a n ep i l ep t i c focus o r the sp read of s e i zu re a c t i v i t y to s u r r o u n d i n g b r a i n t i s sue . Recent e f for t s to e luc ida te the mechanisms u n d e r l y i n g those two fundamenta l c h a r a c t e r i s t i c s of e p i l e p s y have focused on the i n t e r ac t i ons of neu rons w i t h i n loca l i zed r eg ions of cor tex . S y n a p t i c t r ansmis s ion c l e a r l y pa r t i c ipa t e s i n ep i lep togenes i s bu t the d i s c o v e r y of low ca lc ium b u r s t i n g i n the h ippocampus sugges t s that ion ic and e l e c t r i c a l mechanisms may be p a r t i c u l a r l y impor tan t i n the s y n c h r o n i z a t i o n and sp read of s e i zu re a c t i v i t y . I n v e s t i g a t o r s have l ong suspec ted that ephapt ic i n t e rac t ions may c o n t r i b u t e to s e i zu re a c t i v i t y i n the h ippocampus s ince t h i s s t r u c t u r e has t i g h t l y p a c k e d d e n d r i t i c and somatic l a y e r s capable of g e n e r a t i n g s u b s t a n t i a l f i e ld po ten t ia l s . Recent ev idence i n s u p p o r t of th i s hypo the s i s sugges t s that ephap t ic in f luences may r e c r u i t and s y n c h r o n i z e a c t i v i t y d u r i n g the " s p i k e l i k e " even t s of a low ca lc ium b u r s t . These low calc ium s p i k e s are s imi la r to s t imulus evoked poten t ia l s , as we l l as v a r i o u s forms of 42 ep i lep t i fo rm a c t i v i t y i n c l u d i n g i n t e r i c t a l d i s c h a r g e . T h i s s i m i l a r i t y sugges t s that ephap t ic i n t e rac t ions may be an impor tan t component of h ippocampal p h y s i o l o g y and may make s ign i f i can t c o n t r i b u t i o n s to the r ec ru i tmen t , s y n c h r o n i z a t i o n and sp read of many forms of c o r t i c a l a c t i v i t y assoc ia ted w i t h h i g h ampl i tude f i e ld po ten t ia l s . The p re sen t s t u d y inves t i ga t e s t h i s hypo the s i s b y c o r r e l a t i n g the e x c i t a b i l i t y of i n d i v i d u a l p y r a m i d a l and g ranu le ce l l s w i t h the de ta i led c h a r a c t e r i s t i c s of s t imulus e v o k e d f i e ld po ten t ia l s . A l t h o u g h t echn ica l d i f f i cu l t i e s l imi ted ea r l i e r i n v e s t i g a t i o n s of f i e ld effects , i n t r o d u c t i o n of the i n v i t r o s l ice t echn ique and the adven t of power fu l l a b o r a t o r y computers have made s table , l o n g d u r a t i o n i n t r a c e l l u l a r r e c o r d i n g s a n d ex tens ive manipula t ion of i n t r a and ex t r ace l l u l a r da ta much more p r a c t i c a l . The r e s u l t s c o v e r th ree phases of i n v e s t i g a t i o n . Chap te r th ree examines the spa t i a l c h a r a c t e r i s t i c s of f i e ld po ten t ia l s e v o k e d i n the h ippocampus and dentate g y r u s . C u r r e n t - s o u r c e d e n s i t y a n a l y s i s and vo l tage g r a d i e n t de terminat ions ob ta ined from these f i e lds are u s e d to c h a r a c t e r i z e the p a t t e r n of c u r r e n t f low w i t h i n the n e u r o p i l . The p a t t e r n of c u r r e n t flow i s t hen u s e d to p r e d i c t the p o l a r i t y and r e l a t i ve i n t e n s i t y of poss ib le ephap t i c in f luences on neu rona l e x c i t a b i l i t y . Chap te r fou r examines the s e n s i t i v i t y of h ippocampal neurons to a r t i f i c i a l l y app l i ed f i e ld po ten t ia l s . The spa t i a l c h a r a c t e r i s t i c s of the app l i ed f i e lds were chosen to c o r r e s p o n d to those o b s e r v e d d u r i n g the s t imulus e v o k e d poten t ia l s d i s c u s s e d i n chap te r th ree . The r e s u l t s help determine whe the r the vo l tage g r a d i e n t s genera ted d u r i n g evoked potent ia ls are of su f f i c i en t ampl i tude to have a major in f luence on c e l l d i s c h a r g e . Chap te r f i ve examines the t ransmembrane po ten t i a l (TMP) of i n d i v i d u a l p y r a m i d a l and g r anu l e ce l l s d u r i n g e v o k e d f i e ld po ten t ia l s . The TMP is ca lcu la ted b y 43 s u b t r a c t i n g the e x t r a from the i n t r a c e l l u l a r po ten t ia l . I t u l t imate ly de termines the vo l t age -dependen t behav io r of a n e u r o n and g i v e s a d i r e c t measure of a n y ephap t i c in f luences w h i c h might have o c c u r r e d . The r e l a t i o n s h i p of the TMP to t h r e s h o l d for s p i k e genera t ion i s de te rmined d u r i n g a n t i and o r thodromic s t imula t ion and the ro le of ephap t i c i n t e r ac t i ons i n p a i r e d pu l se and f r e q u e n c y po ten t ia t ion i s also examined. 44 C H A P T E R M E T H O D S Male Wistar ra ta w e i g h i n g from 150 to 200 grams (Char les R i v e r , Montrea l ) were u s e d t h r o u g h o u t t h i s p ro jec t . T h i n s l ices of the h ippocampal format ion p r e p a r e d from these animals were mainta ined i n a n i n v i t r o s l ice chamber d u r i n g the e l e c t r o p h y s i o l o g i c a l s tud ies . The fo l lowing sec t ions p r o v i d e de ta i l s c o n c e r n i n g the s l ice p r e p a r a t i o n as we l l as r e c o r d i n g and s t imu la t ing t echn iques . 2.1 T H E S L I C E C H A M B E R The i n v i t r o s l ice chamber i s capable of ma in ta in ing t h i n s l ices of i so la ted neu rona l t i s sue i n a n a p p a r e n t l y hea l thy state for p e r i o d s of ten e x t e n d i n g b e y o n d e igh t h o u r s . It does th i s b y s imu la t ing , as c lose ly as poss ib l e , the cond i t i ons p re sen t w i t h i n the in t ac t b r a i n . I n p a r t i c u l a r , the chamber is de s igned to bathe the s l ice i n a r t i f i c i a l C S F and to expose i t to a warm, o x y g e n r i c h env i ronment . The r e c o r d i n g w e l l of the chamber i s formed b y a shal low d e p r e s s i o n machined i n a t h i c k d i s c of p l ex ig l a s s r e f e r r e d to as the the head. Below the head i s a much l a r g e r c y l i n d r i c a l c a v i t y p a r t i a l l y f i l l e d w i t h d i s t i l l e d wate r . T h i s "outer ba th" con ta ins a hea t ing element as we l l as a gas permeable r i n g of t u b i n g . T h i s tube i s u sed to bubb le a gas mixture c o n s i s t i n g of 95% o x y g e n and 5% c a r b o n dioxide t h r o u g h the d i s t i l l e d water . The warmed, humid i f i ed and oxygena ted gas passes t h r o u g h channe l s i n the head a n d then blows ac ros s the r e c o r d i n g we l l . A s imi lar gas mix ture i s also b u b b l e d t h r o u g h a r e s e r v o i r of a r t i f i c i a l C S F (ACSF) s i t t i n g on a hot plate e levated above the chamber . T h i s 45 warmed o x y g e n r i c h A C S F i s t hen g r a v i t y fed t h r o u g h p l a s t i c t u b i n g to the chamber . A cons tan t flow dev ice (Dia l -a - f lo , So renson Resea rch Co.) main ta ins the flow ra te at about 3 ml pe r minute . The t u b i n g en te r s the ou te r ba th a n d t r a v e l s i t s c i r cumference twice before e n t e r i n g the r e c o r d i n g w e l l . A s A C S F passes a long th i s t u b i n g i t s tempera ture approaches that of the s u r r o u n d i n g f l u i d . A the rmis to r p robe measures the tempera ture i n the r e c o r d i n g we l l and a tempera ture c o n t r o l u n i t modifies the tempera ture i n the ou te r ba th u n t i l the tempera ture i n the r e c o r d i n g w e l l i s 34 +- 0.5 degrees cen t ig r ade . The A C S F t h e n leaves the w e l l v i a a small pore l e a d i n g to a second c a v i t y i n the head where excess C S F i s s u c k e d off and d i s c a r d e d . The s l ices of b r a i n t i s sue are p laced on a h o r i z o n t a l n y l o n mesh w i t h i n the r e c o r d i n g w e l l . The l e v e l of A C S F i s ad ju s t ed s u c h that the s l ice i s s i tua ted at the f l u i d / g a s in te r face . The lower sur face of the s l ice i s c o n t i n u o u s l y ba thed i n A C S F , whi le warmed, humid i f i ed gas from the ou te r ba th i s g e n t l y b lown ac ros s i t s u p p e r sur face . The r e g i o n above the s l ice i s open to al low gas to escape as we l l as to p r o v i d e access for s t imula t ing and r e c o r d i n g e lec t rodes . L i g h t t r ansmi t t ed t h r o u g h the p lex ig la s s bottom of the r e c o r d i n g w e l l and a d i s s e c t i n g microscope mounted above the chamber a s s i s t s accura te placement of e lec t rodes . 2.2 ARTIFICIAL CEREBRAL SPINAL FLUID The A C S F u s e d i n these exper iments was a modif ied R i n g e r s so lu t ion c o n s i s t i n g of 124 mM NaCl , 3.0 mM KC1, 0.75 mM KH2PO4, 1.6 mM CaCla, 1.2 mM M g S 0 4 , 24 mM NaHC0 3 , 10 mM D-g lucose . I n the p resence of C 0 2 i n the gas mix ture the pH was maintained at 7.4. T h i s so lu t ion s imulates o n l y the ion ic components of mammalian c e r e b r a l s p i n a l f l u i d . A l t h o u g h i t conta ins 46 no amino ac ids o r p ro t e in s i t has been shown to mainta in v i ab l e s l ices (Lee et a l , 1981). I n one set of exper iments the MgSO-4 was i n c r e a s e d to (4.0 mM) and the C a C h was decreased to 0.8 mM to b lock release of s y n a p t i c t r ansmi t t e r . Before s t a r t i n g a n y s u r g i c a l p r o c e d u r e s the ra t was b r i e f l y exposed to an e ther r i c h env i ronment i n a l a rge g lass chamber . Af t e r loss of consc iousness the animal was decapi ta ted , the soft t i s sue of the s k u l l was s c r a p e d away u s i n g a sca lpe l , and the t h i n c r a n i a l bones were removed w i t h the a id of r o n g e u r s . The o v e r l y i n g d u r a was then g e n t l y cu t and peeled away. A s soon as the b r a i n was exposed, cooled (4 °C) o x y g e n r i c h A C S F was p o u r e d o v e r the c o r t i c a l su r face to cool the t i s sue as q u i c k l y as poss ib l e . T h i s coo l ing i s t hough t to slow the metabolic ra te and help p ro tec t the b r a i n aga ins t anoxic damage. A t r a n s v e r s e sca lpe l c u t ac ross the a n t e r i o r f o r e b r a i n a n d between the ce rebe l lum and the c e r e b r a l cor tex he lped isolate the r ema in ing b r a i n from s u r r o u n d i n g t i s sue . T h i s sec t ion of b r a i n was then g e n t l y l i f t ed from the c r a n i a l v a u l t u s i n g the sca lpe l handle . A l l f u r t h e r s u r g i c a l p r o c e d u r e s were per formed on a co ld A C S F soaked su r face . T h i s sur face was set u p i n the fo l lowing manner . A flat bottomed g lass d i s h was f i l l e d w i t h c r u s h e d ice and t u r n e d u p s i d e down i n a p l a s t i c t r a y . A piece of f i l t e r paper was p laced on the u p t u r n e d bottom sur face and the paper was sa tu ra ted w i t h o x y g e n r i c h A C S F . T h i s t echn ique not o n l y kep t the t i s sue cool d u r i n g s u r g e r y bu t also al lowed the sur face to be eas i ly ro ta ted to improve the ope ra to r ' s w o r k i n g pos i t ion . The two c o r t i c a l su r faces were then separa ted b y a l o n g i t u d i n a l cu t t h r o u g h the c o r p u s col losum and b r a i n stem, and hal f of the b r a i n was 47 t e m p o r a r i l y s to red i n a beaker of co ld oxygena ted A C S F . The r ema in ing ha l f was p laced on i t s a n t e r i o r cu t sur face and the b ra ins t em was peeled away from the o v e r l y i n g cor tex b y a b lun t i n s t rumen t . T h i s p r o c e d u r e exposed the ven t romed ia l su r face of the h ippocampus . The b l u n t i n s t rumen t was p laced beneath the c u r v a t u r e of the fo rn ix and the h ippocampus was c a r e f u l l y peeled away from the associa ted cor tex . A n y r ema in ing c o r t i c a l t i s sue was cu t away a long w i t h most of the f o r n i x . The i so la ted h ippocampus was then p laced on the cooled s tage of a S o r v a l l t i s sue c h o p p e r w i t h i t s l o n g i t u d i n a l axis p e r p e n d i c u l a r to the angle of cu t . T h i s angle was chosen s ince i t l eaves a l l of the impor tan t f i b e r t r a c t s i n t e r c o n n e c t i n g the C A l , CA3 and dentate r eg ions in tac t . On ly the l o n g i t u d i n a l a s soc ia t ion f i b e r s are t r ansec t ed to a major degree . Approx ima te ly 6 s l ices of 400 microns t h i c k n e s s were cu t from the mid sec t ion of the h ippocampus . A f t e r each c u t the s l ice was f loated off and g e n t l y s u c k e d u p in to a p ipe t te for t r a n s f e r to the chamber . I n some cases a f ine b r u s h was used to p i c k u p the s l i ces . Af t e r a b r i e f i n t e r im i n a p e t r i d i s h of A C S F the s l ices were p laced on the n y l o n net of the r e c o r d i n g w e l l u s i n g e i the r the p ipe t te o r the b r u s h t echn ique . F o l l o w i n g the 5 to 7 minutes i t took to complete t h i s p r o c e d u r e , s l i ces were cu t from the r ema in ing h ippocampus . Th i s second set of s l i ces was c o n s i s t e n t l y v i a b l e , w i t h no o b v i o u s d i f fe rences from the f i r s t set. P r e s u m a b l y an adequate l e v e l of metabolism was mainta ined i n the co ld oxygena ted beaker of A C S F . F o l l o w i n g a one hour p e r i o d of e q u i l i b r a t i o n i n the chamber , the s l ices were tes ted for v i a b i l i t y . Dunwidd ie (1981) has shown that i n h i b i t o r y systems are most s ens i t i ve to damage d u r i n g p r e p a r a t i o n of the h ippocampal s l i ce . There fo re a n y s l ice showing mul t ip le popu la t ion sp ikes 48 fo l lowing o r thodromic s t imula t ion was re jec ted from f u r t h e r s t u d y . Each s l ice was also d i r e c t l y tes ted for the p resence of p a i r e d pu l se i n h i b i t i o n . The i n h i b i t i o n was tes ted b y p r e s e n t i n g two s t imul i to the af ferent pa thway separa ted b y 20 mS. I n h i b i t o r y mechanisms w i t h i n the s l ice were c o n s i d e r e d v i ab l e i f the popu la t ion s p i k e i n r e sponse to the second s t imulus was d e p r e s s e d . 2.4 S T I M U L A T I N G E L E C T R O D E S S t imu la t ing e lec t rodes were c o n s t r u c t e d from a tw i s t ed pa i r of 62 mic ron n ichrome w i r e . The end of each w i r e was cu t back w i t h a s h a r p sca lpe l blade to c rea te a we l l formed, i n s u l a t i o n free t i p for contac t w i t h n e u r o n a l t i s sue . The e lec t rodes were mounted on a N a r i s h i g e m i c r o -manipula tor and v i s u a l l y p laced on the sur face of the s l i ce . The t iming of s t imulus a p p l i c a t i o n was con t ro l l ed b y a fou r de lay Dig i t imer (Medical Sys tems Corp) a n d c o n t r o l of s t imulus d u r a t i o n and ampl i tude as we l l as e l e c t r i c a l i so la t ion from g r o u n d was p r o v i d e d b y a p a i r of Model DS2 b a t t e r y d r i v e n s t imula tors (Medical Sys tems C o r p ) . Each s t imulus cons i s t ed of a s ing le 0.1 ms square wave pu l se i n the r ange of 1 to 70 v o l t s (approximate ly 10 to 200 microamps) . Excep t d u r i n g p a i r e d pu l se and f r e q u e n c y s t imula t ion the i n t e r v a l between s t imul i was maintained at 7 seconds o r g rea te r to minimize the i n d u c t i o n of a n y f r e q u e n c y re la ted a l t e ra t ions i n neu rona l e x c i t a b i l i t y . 2.5 S T I M U L A T I N G TECHNIQUES In the p r e sen t w o r k , s t imu la t ing e lec t rodes were pos i t ioned at s e v e r a l locat ions t h r o u g h o u t the s l ice d e p e n d i n g on the a rea u n d e r s t u d y . E lec t rodes p laced i n the s t r a tum rad ia tum were u s e d to o r t h o d r o m i c a l l y 49 ac t iva te the ap i ca l d e n d r i t e s of C A l ce l l s v i a the commissura l f i b e r s a n d / o r Schaf fe r co l l a te ra l s . A n t i d r o m i c a c t i v a t i o n of C A l ce l l s was accompl i shed by-s t imula t ion of the a l v e u s , a dense l a y e r of p y r a m i d a l c e l l axons o v e r l y i n g the s t r a tum o r i ens . F i n a l l y , e lec t rodes p laced i n the molecular l a y e r of the dentate g y r u s were u s e d to s y n a p t i c a l l y ac t iva te g r anu l e ce l l s v i a f i b e r s of the pe r fo ran t pa th and an t id romic a c t i v a t i o n of these ce l l s was accompl i shed b y s t imula t ion of the mossy f ibe r bund l e i n the dentate h i l u s . 2.6 RECORDING ELECTRODES E x t r a c e l l u l a r f i e ld potent ia ls were r e c o r d e d w i t h g lass microp ipe t tes ( F r e d e r i c k Haer, Omega Dot t u b i n g , 1.5 mm ou ts ide diameter) p r e p a r e d on a Nar i sh ige o r F r e d e r i c k Haer e lec t rode p u l l e r and back f i l l e d w i t h 2.0 M NaCI. The p u l l e r was ad jus t ed to p roduce e lec t rodes w i t h an impedance of 4 to 6 megohms. The e lec t rodes were pos i t ioned u s i n g a B u r l e i g h Inchworm (PZ-550) pezoe lec t r i c d r i v e mounted on a f ine r e so lu t i on micromanipula tor . T h i s a r rangement a l lowed the p rec i se p o s i t i o n i n g of the r e c o r d i n g e lec t rode . I n t r a c e l l u l a r e lec t rodes were made i n a s imi lar manner except that t he i r impedance r a n g e d from 30 to 80 megohms and they were back f i l l e d w i t h 1 M potass ium acetate. The " inchworm" was a lso u s e d to pos i t i on i n t r a c e l l u l a r e lec t rodes s ince t h i s micro d r i v e has the a b i l i t y to move i n mic ron s teps . Bo th i n t r a and ex t r ace l l u l a r e lec t rodes were connec ted to an impedance match ing ampl i f ie r (WPI model K57 o r M707) r e fe renced to the r e c o r d i n g w e l l g r o u n d ( A g / A g C l w i r e ) . These ampl i f i e r s have a b r i d g e c i r c u i t su i tab le for p a s s i n g c u r r e n t t h r o u g h the e lec t rode t i p for use d u r i n g i n t r a c e l l u l a r s tud ies . A T e k t r o n i x d u a l beam osc i l loscope p r o v i d e d both s i g n a l g a i n and f i l t e r i n g . A l l i n t r a and ex t r ace l lu l a r po ten t ia l s i n th i s study were recorded using only a low pass filter with a cut off frequency of 10 KHz. If baseline offset voltages were not required for a given experiment they could be corrected for by the computer during data analysis. Extracellular evoked potentials were recorded throughout the full extent of the CAl region from the distal tips of the basal dendrites to the distal tips of the apical dendrites. Similar recordings were performed in the dendritic field and cell layer of the dentate gyrus. Further details regarding field potential studies are provided in the appropriate chapters. Intracellular recordings were obtained from the pyramidal cell layer of CAl and the granule cell layer of the dentate gyrus. The procedure for impalement was similar in the two areas, although the granule cells were more difficult to penetrate and maintain. When in search of an impalement the electrode was advanced slowly through the cell layer. As the electrode moved through the tissue a change in the background noise or the presence of a single unit would indicate that a cell was nearby. At that time a brief (0.25 second) burst of oscillating current through the tip of the electrode could, on happy occasions, assist in driving the electrode through the cell membrane. An immediate drop in recorded voltage indicated a successful penetration. Although most penetrations failed after a few seconds the immediate application of hyperpolarizing current from the recording electrode increased the odds of maintaining the cell. The recording was considered stable if the membrane potential remained steady after the hyperpolarizing current was slowly removed. Recordings were often stable for up to several hours. 51 The i n p u t r e s i s t ance of each c e l l was de termined b y o b s e r v i n g the in f luence of i n j ec t ed c u r r e n t on the membrane po ten t i a l . These measurements were done w i th the a id of a dev ice r e f e r r e d to as a "probe d r i v e r " . T h i s dev ice was deve loped b y the au thor to w o r k i n c o n j u n c t i o n w i t h the computer based data a c q u i s i t i o n sys tem to c o n t r o l the c u r r e n t i n j e c t i o n capab i l i t i e s of the i n t r a c e l l u l a r b r i d g e ampl i f ie r . U n d e r c o n t r o l of the "probe d r i v e r " a se r ies of 6 to 12, 100 ms l ong pu l ses were passed t h r o u g h the e lec t rode and the computer ob ta ined an ave rage va lue for the vo l tage def lec t ion of the membrane po ten t ia l . The va lue of the c u r r e n t passed in to the c e l l was v a r i e d from about -0.6 to +0.5 nanoamp. The change i n vo l tage was then p lo t t ed aga ins t in j ec ted c u r r e n t and a r e g r e s s i o n a n a l y s i s o v e r the l inea r p o r t i o n of the r e sponse gave an estimate of the s lope. The slope of the r e g r e s s i o n l ine was t aken as the i n p u t r e s i s t ance of the n e u r o n . Expe r imen ta l l y de te rmined i n t r a c e l l u l a r c h a r a c t e r i s t i c s of ten v a r y s u b s t a n t i a l l y between i n d i v i d u a l neu rons . B io log ica l v a r i a t i o n accounts for at least some of these d i f fe rences bu t n e u r o n a l damage must be c o n s i d e r e d when p a r t i c u l a r l y low va lues are ob ta ined for c e r t a i n membrane c h a r a c t e r i s t i c s . To help minimize the number of damaged ce l l s i n a d v e r t e n t l y i n c l u d e d i n the s t u d y , o n l y pene t ra t ions meeting c e r t a i n c r i t e r i a were accep ted for a n a l y s i s . The c r i t e r i a were a s table r e c o r d i n g for at least 5 minutes , a r e s t i n g membrane po ten t i a l g rea te r t han minus 60 mV, a n i n p u t r e s i s t ance g rea te r t h a n 20 megohms, and ac t ion potent ia ls w i t h ampl i tudes g rea te r t han 80 mV. 2.7 COMPUTER FACILITIES A computer based system was necessary for the detailed analysis of evoked potentials and intracellular data carried out in this project. To meet this requirement, the author developed a data collection and graphical analysis system for use on the PDP-11/23 computer. Wave forms were displayed on a Tektronix 4010 high resolution graphics terminal for on line viewing, or sent to a Tektronix-4052 plotter for the generation of permanent records. Numerical results and statistical analysis were displayed on a standard terminal or sent to a dot matrix printer. Data were analyzed in real time during data collection and/or accessed from permanent storage for analysis at a later date. All waveforms were digitized at resolution of 12 bits and a freguency of 20 KHz. The system was capable of a variety of manipulations of digitized waveforms including averaging, software filtering, baseline correction, summation, subtraction, differentiation and integration. The program automatically detected multiple maximum and minimum points within a waveform and generated both graphs and printed tables of these measurements across a sequence of evoked potentials. More specific extracellular analysis included the generation of current source density, voltage gradient, and current vector plots. Intracellular analysis included generation of current-voltage profiles as well as time constant measurement with the ability to peel multiple exponentials from an intracellular charging curve. The program consisted of approximately 1500 lines of assembly and 6500 lines of fortran code. 53 CHAPTER 3: VOLTAGE GRADIENT ANALYSIS H i g h ampl i tude ex t r ace l lu l a r f i e ld po ten t ia l s are p r e sen t i n the h ippocampal format ion u n d e r bo th p h y s i o l o g i c a l and exper imenta l cond i t i ons . A n a l y s i s of these f i e lds remains a major i n v e s t i g a t i v e tool i n the s t u d y of h ippocampal p h y s i o l o g y (Ande r sen 1960; A n d e r s e n and Lpfaio 1966; A n d e r s e n et a l 1966; Gloor et a l 1963; L e u n g 1979a,b,c; L/Smo 1970; F u j i t a and Saka ta 1962; S c h w a r t z k r o i n and P r i n c e 1978). T r a d i t i o n a l l y i t has been assumed that f i e ld po ten t ia l s e v o k e d i n the h ippocampus are s imply ex t r ace l l u l a r r e f l ec t ions of n e u r o n a l a c t i v i t y a n d are not, i n themselves , impor tan t de te rminants of c e l l d i s c h a r g e . However , the c e n t r a l h y p o t h e s i s of th i s w o r k i s that e x t r a c e l l u l a r c u r r e n t s assoc ia ted w i t h a f i e ld po ten t i a l c o u l d , u n d e r the c o r r e c t cond i t i ons , have a d i r e c t in f luence on n e u r o n a l e x c i t a b i l i t y i n the h ippocampus . T h i s chap t e r examines the mechanisms u n d e r l y i n g the gene ra t ion of f i e l d po ten t ia l s and p r o v i d e s the b a c k g r o u n d for u n d e r s t a n d i n g how these f i e lds c o u l d in f luence c e l l u l a r a c t i v i t y . E x t r a c e l l u l a r f i e ld po ten t ia l s r e c o r d e d i n the C A l and dentate g y r u s are p r e sen t ed a long w i t h the c o r r e s p o n d i n g vo l tage g r a d i e n t and c u r r e n t - s o u r c e d e n s i t y (CSD) ana ly s i s . These data e s t a b l i s h that cond i t i ons a p p r o p r i a t e for ephap t i c i n t e r ac t i ons are i ndeed p r e sen t d u r i n g both o r t ho and an t id romic evoked a c t i v i t y i n the h ippocampal format ion . M^THEORETIC Rigorous t reatment of f i e ld po ten t i a l t h e o r y i s ava i lab le i n the l i t e r a t u r e (Cla rk and P lonsey , 1966; Woodbury 1960). However , a quan t i t a t i ve a n a l y s i s of ex t r ace l l u l a r f i e lds i s not p r a c t i c a l i n the 54 hippocampus s ince we lack the p r ec i s e knowledge of the membrane c h a r a c t e r i s t i c s of the ce l l s and the re i s a l a rge v a r i a t i o n i n the b r a n c h i n g pa t t e rn s of i n d i v i d u a l d e n d r i t i c a r b o r i z a t i o n s . F u r t h e r m o r e , app l i c a t i on of these theor ies u s u a l l y r e q u i r e s 2 major assumpt ions . These are : 1) that n e r v o u s t i s sue ac ts l i k e a pe r fec t ohmic r e s i s t o r , and 2) that the r e s i s t ance of the n e u r o p i l i s e q u a l i n a l l d i r e c t i o n s . The f i r s t a s sumpt ion may be close to t r ue bu t i t i s we l l k n o w n that r e s i s t i v i t y w i l l v a r y d e p e n d i n g on the t y p e of n e u r o n a l elements p re sen t and the d i r e c t i o n of c u r r e n t f low. F o r example, the t r a n s v e r s e r e s i s t ance ac ross a f i be r t r a c t c a n be many times h i g h e r t h a n the l o n g i t u d i n a l r e s i s t ance (Rush ton 1937; T a s a k i 1964) and i n C A l , the r e s i s t ance may v a r y b y a fac tor of two between the somatic and d e n d r i t i c l a y e r s (Holsheimer 1987). A l t h o u g h more recen t s tud ies have t a k e n some of these asymmetr ies in to c o n s i d e r a t i o n (Nicholson and L l i n a s 1971), a much more g e n e r a l a p p r o a c h best s e r v e s the pu rpose of the p r e sen t s t u d y . A) The Open F i e l d Concep t Lo ren t e de N o ' (1947) deve loped a u se fu l way to p r e d i c t the ex t r ace l l u l a r po ten t ia l s fo l lowing s y n c h r o n o u s ac t i va t i on of a popu la t ion of neu rons . I f the neurons w i t h i n a popu la t ion are o r g a n i z e d in to a symmet r i ca l p a t t e r n the ensemble can be r e d u c e d to a s ing le idea l neu rona l element. The ex t r ace l l u l a r f ie lds p r e d i c t e d for th i s i dea l element w i l l be s imi lar to the f ie lds p r o d u c e d b y the whole popu la t ion . The C A l r e g i o n a n d the denta te g y r u s f a l l i n to the "open f i e l d " c a t ego ry . T h e y have a n o r d e r l y o r g a n i z a t i o n of t he i r neu rons w i t h somata l imi ted to one l a y e r or "p lane" , and d e n d r i t e s r u n n i n g p e r p e n d i c u l a r to the plane i n a h i g h l y o r g a n i z e d f a sh ion . 55 I f one assumes that a sheet of ce l l s i n an open f i e ld con t inues i n d e f i n i t e l y , and that a l l the ce l l s are ac t i va t ed s imul taneous ly , then a n y c u r r e n t p a s s i n g i n the plane of the c e l l l a y e r w i l l be cance led b y a s imi la r o p p o s i n g c u r r e n t from n e i g h b o r i n g neu rons . I n t h i s way a l l l a t e r a l c u r r e n t s are cance led and vol tage d i f fe rences are o n l y genera ted a long the dendrosomat ic ax i s , p e r p e n d i c u l a r to the c e l l l a y e r . There fo re , w i t h i n an open f i e l d , the ex t r ace l l u l a r c u r r e n t s can be p r e d i c t e d b y o b s e r v i n g the a c t i v i t y of an " idea l element" i n the form of a soma w i t h a s ing le s t r a i g h t c y l i n d r i c a l d e n d r i t i c p rocess . What fol lows i s an i n t u i t i v e , n o n - r i g o r o u s d e s c r i p t i o n of the sequence of even t s expected d u r i n g a c t i v a t i o n of a n idea l element w i t h i n the h ippocampus . The i n t en t ion i s to l ay a founda t ion for a d i s c u s s i o n of the theore t i ca l mechanism u n d e r l y i n g the i n d u c t i o n of ephap t ic i n t e r ac t i ons . B) C u r r e n t F low D u r i n g S y n c h r o n o u s D i scha rge A schematic r ep re sen t a t i on of a h y p o t h e t i c a l c e l l from a n open f i e ld i s shown i n f i g u r e 3.1. T h i s c e l l r e p r e s e n t s a popu la t ion of C A l neu rons u n d e r g o i n g s imul taneous an t id romic ac t i va t i on . To the r i g h t are the expected f i e ld po ten t ia l s for the d e n d r i t i c and somatic l a y e r s . A l t h o u g h these are ac tua l an t id romic poten t ia l s r e c o r d e d from C A l , t h e y are i n t e n d e d o n l y as h y p o t h e t i c a l r e sponses to a s s i s t i n the theore t i ca l d i s c u s s i o n . D u r i n g an t id romic i n v a s i o n , somal conduc tance inc reases s h a r p l y and c u r r e n t f lows from the ex t r ace l lu l a r space in to the c e l l . T h i s movement of i n w a r d c u r r e n t , or s i n k , accounts for the r i s i n g edge of the ac t ion po ten t i a l and i s assoc ia ted w i t h a r e l a t i ve n e g a t i v i t y i n the immediate ex t r ace l l u l a r space. Some of the c u r r e n t e n t e r i n g the soma sp reads e l ec t ro ton i ca l l y u p the d e n d r i t i c t ree and f i n a l l y ou tward ac ross the 56 FIGURE 3.1 i l l u s t r a t e s the gene ra t ion of ex t r ace l l u l a r f i e ld potent ia ls d u r i n g s y n c h r o n o u s an t id romic a c t i v a t i o n of a neu rona l popu la t ion . The schematic n e u r o n r ep re sen t s a popu la t ion of C A l p y r a m i d a l ce l l s and to the r i g h t are the expected somatic and d e n d r i t i c f i e ld potent ia l s . The dot ind ica tes the time at the peak n e g a t i v i t y of the extrasomatic po ten t ia l . A l t h o u g h these are ac tua l potent ia ls r e c o r d e d from the h ippocampus , t hey are i n t e n d e d as hypo the t i ca l r e sponses for the p u r p o s e s of th i s f i g u r e . The + and -symbols r ep re sen t the o v e r a l l po ten t i a l i n the ex t r ace l lu l a r space at the time i nd i ca t ed b y the dot. The a r r o w s ind ica te the d i r e c t i o n of ex t r ace l lu l a r c u r r e n t f low. 57 ANTIDROMIC STIMULATION 58 resistance of the dendritic membrane. The source generated by this outward current produces a relative positivity in the distal dendritic regions. Extracellular current flow from the positive extradendritic regions towards the negative extrasomatic space completes the necessary current path. An important point to emphasize here is that extracellular current may flow any time there is an active conductance change on a localized region of a neuron. In neurons possessing long dendrites heading off more or less in the same direction, such as pyramidal and granule cells, the major current flow will be along the dendrosomatic axis of the cell. In effect, the conductance change functions like a battery to drive current around a local circuit involving both the intra and extracellular space. Since the representative cell predicts the field potential and current flow for the whole population, we expect that during a real antidromic population response current will flow along the dendrosomatic axis of the population in a manner similar to that shown in the schematic. The situation becomes somewhat more complicated when one considers the sequence of events following synaptic activation of the dendritic region of a cell. Figures 3.2 and 3.3 show a similar representative neuron from an open field undergoing synchronous orthodromic activation. Axons from the afferent pathway are shown terminating on the dendrites and to the right are the field potentials predicted for the population. Figure 3.2 represents phase one of the response. As the dendrite is synaptically activated a local inward current (sink) generates an intracellular depolarization and a relative extracellular negativity. The inward current electrotonically propagates towards the soma generating a source and extracellular positivity in the extracellular region around the soma. These changes are 59 F I G U R E 3.2 i l l u s t r a t e s the genera t ion of ex t r ace l lu l a r f ie ld potent ia ls d u r i n g s y n c h r o n o u s o r thodromic ac t iva t ion of a neurona l popula t ion (PART ONE -E P S P phase) . The schematic n e u r o n r ep re sen t s a popu la t ion of C A l p y r a m i d a l ce l l s and to the r i g h t are the expected somatic and d e n d r i t i c f i e ld potent ia l s . The dot ind ica tes the time at the peak p o s i t i v i t y ( E P S P phase) of the extrasomatic po ten t ia l . A l t h o u g h these are ac tua l po ten t ia l s r e co rded from the h ippocampus , they are in t ended as h y p o t h e t i c a l r esponses for the pu rposes of th i s f i g u r e . The + and - symbols r e p r e s e n t the o v e r a l l po ten t ia l i n the ex t r ace l lu l a r space at the time i nd i ca t ed b y the dot. The a r r o w s ind ica te the d i r e c t i o n of ex t r ace l lu l a r c u r r e n t f low. The l ines e n t e r i n g from the left of the f i gu re ind ica te af ferent axons s y n a p s i n g onto the ap i ca l d e n d r i t e s . 60 O R T H O D R O M I C STIMI1I A T I O N (EPSP PHASE) 61 F I G U R E 3.3 i l l u s t r a t e s the gene ra t ion of ex t r ace l l u l a r f i e ld po ten t ia l s d u r i n g s y n c h r o n o u s o r thodromic a c t i v a t i o n of a neu rona l popu la t ion (PART TWO -d i s c h a r g e phase) . The schematic n e u r o n r ep re sen t s a popu la t ion of C A l p y r a m i d a l ce l l s and to the r i g h t are the expected somatic and d e n d r i t i c f ie ld po ten t ia l s . The dot i nd ica t e s the time at the peak n e g a t i v i t y (d i scharge phase) of the extrasomatic po ten t ia l . A l t h o u g h these are ac tua l potent ia ls r e c o r d e d from the h ippocampus , t hey are i n t e n d e d as h y p o t h e t i c a l r e sponses for the p u r p o s e s of th i s f i g u r e . The + and -symbols r e p r e s e n t the o v e r a l l po ten t i a l i n the ex t r ace l l u l a r space at the time i n d i c a t e d b y the dot . The a r r o w s ind ica te the d i r e c t i o n of ex t r ace l lu l a r c u r r e n t f low. The l i ne s e n t e r i n g from the left of the f i gu re ind ica te af ferent axons s y n a p s i n g onto the ap i ca l d e n d r i t e s . 62 ORTHODROMIC STIMULATION (ACTION POTENTIAL) 63 assoc ia ted w i t h an ex t r ace l lu l a r c u r r e n t flow from the somatic, towards the d e n d r i t i c r e g i o n . S h o r t l y af ter th i s phase of the r e sponse has s t a r t ed the c e l l reaches t h r e s h o l d and an ac t ion po ten t i a l i s i n i t i a t ed near the soma. The next sequence of even t s s h o u l d be s imi la r to those d i s c u s s e d for the an t id romic r e sponse i n f i g u r e 3.1. However , the s y n a p t i c s i n k may con t inue to o c c u r i n the d e n d r i t e s . The r e s u l t w i l l be a combinat ion of the c u r r e n t s and po ten t ia l s associa ted w i th sp ike d i s c h a r g e ( f igure 3.3) and those associa ted w i t h s y n a p t i c d r i v e ( f igure 3.2). D u r i n g the r i s i n g edge of the ac t ion po ten t i a l the somatic ex t r ace l l u l a r space shou ld sh i f t i n the nega t ive d i r e c t i o n and the e x t r a d e n d r i t i c space shou ld become r e l a t i v e l y more pos i t i ve . The f i n a l po ten t i a l i n these r eg ions w i l l depend on the r e l a t i ve magni tude of the s i n k s a n d sources associa ted w i t h the s y n a p t i c d r i v e v e r s u s those associa ted w i th c e l l d i s c h a r g e . The d i r e c t i o n of c u r r e n t flow d u r i n g s p i k e d i s c h a r g e u l t imate ly depends o n l y on the d i f fe rence i n ex t r ace l lu l a r po ten t i a l between the d e n d r i t e s and soma. If the somatic s i n k associa ted w i t h ac t ion po ten t i a l gene ra t ion i s dominant , then c u r r e n t w i l l f low from the e x t r a d e n d r i t i c to the extrasomatic space as shown i n f i g u r e 3.3. C) The Genera t ion Of E p h a p t i c C u r r e n t s The p r e v i o u s sec t ion e s t ab l i shed , on i n t u i t i v e g r o u n d s , that ex t r ace l l u l a r c u r r e n t shou ld flow a long the dendro-somat ic axis of a c e l l w i t h i n an open f i e ld wheneve r the popu la t ion i s s y n c h r o n o u s l y ac t i va t ed . Bu t how might th i s ex t r ace l l u l a r c u r r e n t in f luence the a c t i v i t y of a neuron? Is t h i s , i n fact , the t ype of ex t r ace l lu l a r c u r r e n t flow that i s r e spons ib l e for ephap t i c i n t e r ac t i ons? 64 Wi th in a n e u r o n a l popu la t ion the somal and d e n d r i t i c membranes, as w e l l as the i n t r a - and ex t r ace l lu l a r f l u i d , form a complex r e s i s t i v e ne twork . The K i r c h o f f laws of c u r r e n t flow state that c u r r e n t i s sha red b y a l l the r e s i s t i v e elements i n a ne twork s u c h that the to ta l e f fec t ive res i s t ance i s minimized . There fo re , a p r o p o r t i o n of a n y c u r r e n t f l owing w i t h i n the n e u r o p i l w i l l pass t h r o u g h the i n t r a c e l l u l a r compartment . F i g u r e 3.4 dep ic t s the c u r r e n t f low i n d u c e d i n a popu la t ion of uns t imula ted neurons ("pass ive ce l l " ) exposed to a f i e ld po ten t i a l genera ted b y an t id romic a c t i v i t y i n a n e a r b y popu la t ion of neu rons ("act ive c e l l " ) . A s i nd i ca t ed b y the a r r o w s , some of the c u r r e n t f l owing a long the ex t r ace l l u l a r vo l tage g r a d i e n t w i l l pass t h r o u g h the d e n d r i t i c membrane of t h i s "pa s s ive" c e l l , t r a v e l down the ax ia l r e s i s t ance of the d e n d r i t i c cy top lasm, and leave ac ros s the somal membrane towards the ex t r ace l lu l a r n e g a t i v i t y . T h i s pa s s ive flow of c u r r e n t between the d e n d r i t e s and soma i s the bas is of ephap t ic i n t e r ac t i ons . Depend ing on the d i r e c t i o n of c u r r e n t f low, the somal membrane po ten t i a l w i l l be e i the r depo la r i zed o r h y p e r p o l a r i z e d , l e a d i n g to e i the r a n enhancement o r d e p r e s s i o n of neu rona l e x c i t a b i l i t y . D) Experimental Plan Based on the p r e c e d i n g d i s c u s s i o n i t becomes o b v i o u s that the simple p resence of a h i g h ampl i tude f i e ld po ten t i a l i n the v i c i n i t y of the soma i s not , i n i t se l f , h e l p f u l i n de t e rmin ing the l i k e l i h o o d of s i gn i f i c an t ephapt ic i n t e r ac t i ons w i t h i n a popu la t ion of neu rons . I n o r d e r to determine the c h a r a c t e r i s t i c s of ax ia l c u r r e n t flow d u r i n g e v o k e d po ten t ia l s i n the h ippocampal format ion, a laminar a n a l y s i s of a n t i and o r thodromic r e sponses was c a r r i e d out for both C A l and the dentate g y r u s . C u r r e n t source d e n s i t y (CSD) and vo l tage g rad i en t in format ion 65 F I G U R E 3.4 i l l u s t r a t e s the concep t of f i e ld i n d u c e d a l te ra t ions of neurona l e x c i t a b i l i t y . The lower schematic c e l l r ep r e sen t s a subpopu la t ion of neu rons u n d e r g o i n g s y n c h r o n o u s an t idromic a c t i v a t i o n . The u p p e r c e l l r e p r e s e n t s o ther neurons w i t h i n the popu la t ion not d i r e c t l y ac t iva t ed by the s t imulus . The two t races on the bottom of the f i g u r e show the h y p o t h e t i c a l ex t r ace l lu l a r po ten t i a l w i t h i n the c e l l l a y e r (left) and the d e n d r i t i c r e g i o n ( r i gh t ) . A c t i o n po ten t ia l c u r r e n t s at the somatic l eve l ( th ick a r row) create an extrasomatic n e g a t i v i t y (-) and ou tward d e n d r i t i c c u r r e n t s create an e x t r a d e n d r i t i c p o s i t i v i t y (+). These sh i f t s i n e x t r a c e l l u l a r po ten t ia l create a vol tage g r a d i e n t as i nd i ca t ed b y the slope of the l ine j o i n i n g the two e v o k e d potent ia ls on the bottom of the f i g u r e . A c c o r d i n g to ohms law (voltage = c u r r e n t * res i s tance) c u r r e n t w i l l flow " d o w n " th i s g r ad i en t i n the d i r e c t i o n of the ho r i zon t a l a r r o w . A c c o r d i n g to the K i r c h o f f laws c u r r e n t w i l l be sha red b y a l l elements w i t h i n the n e u r o p i l . T h u s some of the c u r r e n t w i l l t r a v e l w i t h i n the i n t r a c e l l u ^ space of the neurona l popu la t ion (ar rows i n v o l v i n g i n a c t i v e ce l l ) . The end r e s u l t is a movement of i n t r a c e l l u l a r c u r r e n t from the dend r i t e s towards the somata and a r e l a t ive depo la r i za t i on of the somatic membrane po ten t ia l . T h i s somatic depo la r i za t ion w i l l i nc rease the e x c i t a b i l i t y of the neurona l popu la t ion exposed to the ex t r ace l l u l a r f i e ld po ten t i a l . 67 derived from these laminar profiles provides an indirect measure of the relative current flow along the dendrosomatic axis of pyramidal and dentate cells during the various phases of an evoked response. This information is fundamental to understanding the role of ephaptic interactions in cortical structures. 3.2 SPECIFIC METHODS In order to characterize the spatial distribution of an evoked field potential one ideally needs to simultaneously record the response through a large array of electrodes evenly spaced throughout the neuropil. However the technical problems presented by this approach are considerable. In particular, glass electrodes, necessary for accurate depth recording, cannot be placed close enough together to provide the required spatial resolution, and the sample rates of data acquisition systems available at the time of this study were too slow to simultaneously record the potential from a large number of electrodes. Fortunately, evoked potentials in the hippocampal slice are reasonably consistent for considerable durations of time. Therefore, in the present study, it was possible to use a single electrode to record the evoked potential sequentially from a series of locations. Stimulus frequencies were kept less than 1 per 7 seconds i n CAl and less than 1 per 10 seconds i n the dentate gyrus to minimize any frequency related changes in response amplitude during the long duration of these experiments. A) Recording Laminar Profiles The following technique was used to obtain the spatial distribution of evoked potentials in both dentate and CAl. Recordings were obtained from 68 approx imate ly t h i r t y loca t ions e v e n l y d i s t r i b u t e d at 25 mic ron i n t e r v a l s i n a s t r a i g h t l ine a long the dendro-somat ic axis of the neu rona l popu la t ion . In C A l , the f i r s t r e c o r d i n g loca t ion was pos i t ioned j u s t beyond the t ips of the basa l d e n d r i t e s i n the a lveus . Subsequen t locat ions were a long a l ine p a s s i n g t h r o u g h the c e l l l a y e r and ex t end ing beyond the t i p s of the ap i ca l d e n d r i t e s . I n the dentate g y r u s , the f i r s t r e c o r d i n g loca t ion was j u s t beyond the t i p s of the g r anu l e c e l l d e n d r i t e s i n the outermost l imit of the s t r a tum moleculare , and subsequen t locat ions were pos i t ioned on a l ine ex tend ing beyond the soma we l l in to the h i l u s . I n o r d e r to mainta in accura te c o n t r o l on the r e c o r d i n g locat ions the e lec t rode manipula tor was a lways pos i t ioned w i t h i t s h o r i z o n t a l t r a v e l o r i en ta t ed p e r p e n d i c u l a r to the c e l l l a y e r . I n t h i s way , consecu t ive movements of the e lec t rode r e q u i r e d adjus tment of o n l y one axis of the manipula tor , and the pos i t ion of the r e c o r d i n g loca t ion c o u l d be a c c u r a t e l y de te rmined from the h o r i z o n t a l micrometer scale. I n some exper iments r e c o r d i n g s were ob ta ined at each loca t ion b y i n s e r t i n g the e lec t rode to a spec i f i c d e p t h w i t h i n the s l ice and then a v e r a g i n g f o u r consecu t ive evoked poten t ia l s . T h i s d e p t h was de te rmined before each exper iment b y mov ing the e lec t rode t h r o u g h the s l ice at the l e v e l of the c e l l l a y e r and o b s e r v i n g the ampl i tude of the e v o k e d po ten t ia l . The dep th g i v i n g the h ighes t ampl i tude was then u s e d at a l l r e c o r d i n g loca t ions a long the dendro-somat ic axis of the c e l l popu la t ion . A l t h o u g h t h i s method p r o v e d sa t i s f ac to ry , i t d i d not take in to c o n s i d e r a t i o n v a r i a t i o n s i n the dep th of maximal r e sponse at d i f fe ren t loca t ions a long the c e l l ax is . There fo re , i n most exper iments , s ing le evoked potent ia ls were r e c o r d e d at approx imate ly 15 separate dep ths for each r e c o r d i n g loca t ion . The r e sponses were r e c o r d e d from the top to the 69 bottom surface of the slice in 20 micron intervals and then ten of the middle evoked potentials were averaged to produce a final record. This method avoided the problem of response variations at different depths and gave an average evoked potential for the central portion of the slice. This form of averaged potential was particularly useful in CSD analysis since the CSD was estimated for a "column" of tissue rather than for a single depth. The stability of the evoked response was monitored in one of two ways. In some experiments, a reference recording electrode was placed in the cell layer near the line of main recording locations to continuously monitor the quality of the response. If the reference response changed during data collection the experiment was abandoned. In other experiments, the main recording electrode was used to obtain a series of ten control responses from the cell layer before and after data collection. If the two sets of control responses differed, the experimental data were omitted from further analysis. The evoked potentials were then plotted in the form of "laminar profiles". These profiles were constructed by "stacking" individual traces, above one another, such that each trace shared a common time axis on the abscissa, but was separated vertically from others in the profile. The vertical position of a trace corresponded to its specific recording location along the dendro-somatic axis of the neuronal population. Thus a laminar profile shows the complete spatial distribution of voltage along the line of recording locations for a given evoked response. B) Current-Source Density Calculations Current source density (CSD) refers to the net loss or accumulation of charge in a given volume of extracellular space. The measurement of 70 CSD d u r i n g an evoked po ten t ia l is a power fu l tool for l o c a l i z i n g r eg ions of c u r r e n t flow ac ross neu rona l membranes. F o r example, an o u t w a r d f low, o r " source" , i s detected as an appa ren t accumula t ion of ex t r ace l l u l a r cha rge , whereas an i n w a r d f low, or " s i n k " , i s detected as an appa ren t loss of ex t r ace l lu l a r c h a r g e . These s i n k s and sources p r o v i d e a much more d i sc re t e loca l i za t ion of neu rona l a c t i v i t y than s t a n d a r d f i e ld po ten t ia l a n a l y s i s (Mi tzdor f 1985). C u r r e n t source d e n s i t y i s i n d i r e c t l y est imated from the spa t i a l d i s t r i b u t i o n of ex t r ace l l u l a r po ten t i a l and the c o n d u c t i v i t y of the ex t r ace l lu l a r space. It i s based on the second d i f f e r en t i a l of vo l tage w i th r e spec t to d i s t ance . The fo l lowing formula g ive s the c u r r e n t source d e n s i t y S at time t fo r po in t x , y , z i n 3 d imens iona l space. S(t ,x,y,z)= - ( ox d*V(t)/dx* + oy d * V ( t ) / d y 2 + oz d 2 V ( t ) / d z 2 ) where ox, oy , oz are the c o n d u c t i v i t y va lues for each of the th ree d imens ions , and V i s the ex t r ace l l u l a r vo l tage at time t (Nicholson and Freeman 1975). U n d e r idea l exper imenta l cond i t ions the spa t i a l d i s t r i b u t i o n of vo l tage i s ob ta ined from a th ree d imens iona l a r r a y of equ id i s t an t r e c o r d i n g e lec t rodes (Freeman Nicho l son 1975; Freeman and Stone 1969). However , i n the p re sen t s t u d y , a one d imens iona l CSD a n a l y s i s was per formed a long the dendro-somat ic axis of the neu rona l popu la t ion . T h i s s impl i f i ca t ion i s j u s t i f i e d i n h i g h l y laminar s t r u c t u r e s u n d e r g o i n g s y n c h r o n o u s ac t i va t i on s ince the major i ty of c u r r e n t f lows a long the neu rona l axis (Haber ly and S h e p h e r d 1973). E x t r a c e l l u l a r conduc tance i s v e r y d i f f i c u l t to determine. A t the p resen t time o n l y measurements of r e l a t ive c o n d u c t i v i t y are ava i lab le for . 71 the hippocampus (Jefferys 1984; Holsheimer 1987). These studies report about a two fold difference in conductance between the dendritic and the somatic layers of C A l . Fortunately, Holsheimer (1987) found that ignoring this variation in conductance had only a small influence on the the absolute magnitude of CSD, and did not alter the polarity or location of sinks and sources. Others have also found that ignoring variations in extracellular conductance has only a minor influence on the qualitative results of CSD analysis. As a result, conductivity tensors are often treated as constants (Freeman and Stone 1969; Haberly and Shepherd 1973; Mitzdorf 1980, 1985; Nicholson and Freeman 1975; Swann et al 1986). The working equation for a one dimensional analysis of CSD with a unity conductivity tensor is S(t,x) = - ( ( Vi(t) - V2(t) ) - ( V2(t) - V3(t) ) ) / ( X 3 - Xi)2 where Vi , V 2 , and V 3 are the amplitude of field potentials recorded in three consecutive locations Xi, X2 and X3 at time t. In the present study the distance between recording locations was normalized so that an electrode separation of 25 microns was one unit of distance. This allowed a further simplification to the following equation U(t)= ( Vi(t) - V2(t) ) - ( V2(t) - V3(t) ) = Vi(t) + V3(t) - 2V2(t) This equation is expressed in the arbitrary unit (U) to emphasize the fact that the results are proportional to CSD. It is only the relative magnitude of U that is important in the present context. The data required for this calculation are a series of evoked potentials recorded at equidistant points along the neuronal axis of the cell population. These are precisely the data present in the laminar profiles 72 d e s c r i b e i n the p r e v i o u s sec t ion . The CSD response at a g i v e n loca t ion was ca lcu la ted b y a p p l y i n g the w o r k i n g equa t ion to the vo l tage r e sponse r e c o r d e d at that loca t ion , as we l l as the two adjacent loca t ions . The r e s u l t i n g CSD t races were then d i s p l a y e d above one ano ther i n a manner s imi la r to the vo l tage laminar p ro f i l e s . C) Vol tage G r a d i e n t Ca lcu l a t i pns The major r e s u l t s of th i s chap t e r d e s c r i b e the vo l tage g r a d i e n t p r e s e n t a long a c e l l ax is d u r i n g d i f f e r en t phases of a n e v o k e d r e sponse . A l t h o u g h a l l da ta r e d u c t i o n was a c t u a l l y done b y computer the p r o c e s s i s eas ie r to concep tua l i ze as a se r i es of s teps c a r r i e d out b y h a n d . The f i r s t s tep was to o b t a i n the po ten t i a l p r e sen t at a g i v e n i n s t a n t i n time for each loca t ion a long the c e l l ax i s . Imagine a v e r t i c a l l ine d r a w n t h r o u g h a l l of the r e sponses i n a laminar p ro f i l e at a spec i f i c loca t ion a long the a b s c i s s a . A s t h i s l ine i n t e r s e c t s each e v o k e d response i t i den t i f i e s the vo l t age p r e s e n t at the c o r r e s p o n d i n g r e c o r d i n g loca t ion for one spec i f i c time. If these vo l t ages are t h e n p lo t t ed aga ins t dendro - somat i c loca t ion a "vo l tage p r o f i l e " i s p r o d u c e d (see f i g u r e 3.6 le f t ) . B y c h o o s i n g the a p p r o p r i a t e pos i t i on a long the a b s c i s s a the vo l tage p ro f i l e can be d e r i v e d for a n y phase of the r e sponse . The vo l tage g r a d i e n t ac ross a spec i f i c p o r t i o n of the c e l l axis can t h e n be d e r i v e d b y m e a s u r i n g the slope of the vo l t age p ro f i l e o v e r a spec i f i c dendro - soma t i c r e g i o n of the c e l l popu la t ion . T h i s is accompl i shed b y t a k i n g the vo l t age d i f fe rence between two r e c o r d i n g loca t ions and d i v i d i n g i t b y the d i s t ance s e p a r a t i n g the two loca t ions . The vo l tage g rad ien t , measured i n mi l l i vo l t s pe r mi l l imeter , i nd i ca t e s the d r i v i n g force for c u r r e n t flow t h r o u g h the ex t r ace l l u l a r space. 73 D) Measurement Of The E v o k e d Po t en t i a l Waveform E v o k e d poten t ia l s i n the h ippocampal format ion have complex waveforms of ten c o n s i s t i n g of s e v e r a l d i s t i n c t components . The conven t ions u s e d i n th i s thes i s for naming and measu r ing these v a r i o u s components a re d e s c r i b e d below. F i g u r e 3.5 shows a t y p i c a l po ten t i a l r e c o r d e d i n the c e l l l a y e r of the dentate g y r u s fo l lowing a n o r thodromic s t imulus . The waveform cons i s t s of two pos i t i ve waves , P l and P2, and a s ing le nega t ive wave, N I . The nega t ive wave o n l y o c c u r s i f the s t imulus i n t e n s i t y i s su f f i c i en t to generate ac t ion poten t ia l s i n at least some of the neu rons w i t h i n the popu la t i on . A s the s t imulus i n t e n s i t y i s decreased and the nega t ive wave fa i l s to o c c u r , P l and P2 become a s ing le pos i t i ve wave . T h i s s ing le wave i s a lso r e f e r r e d to as P l . The ampl i tudes of P l and P2 a re measured from the basel ine po ten t i a l to the peak of the c o r r e s p o n d i n g wave . The ampli tude of NI i s measured from the peak of P l to the nega t ive peak of N I . S imi la r waveforms are p re sen t fo l lowing o r thodromic s t imula t ion of C A l and the same naming a n d measurement conven t ions are u s e d . F o l l o w i n g an t id romic s t imula t ion of the dentate g y r u s o r C A l the r e sponse cons i s t s of a s ing le nega t ive wave. T h i s an t id romic popu la t ion s p i k e , also r e f e r r e d to as N I , i s measured from the basel ine po ten t ia l to the minimum poin t on the wave . M _ I E S y j L T S The r e s u l t s d e s c r i b e d i n th i s chap te r are based on 39 complete vol tage p ro f i l e s a l l meet ing the c r i t e r i a for s t a b i l i t y ou t l i ned above. On ly 74 FIGURE 3 . 5 illustrates the method used to measure various components of a population potential recorded in the cell layer of either the hippocampus or the dentate gyrus. A typical orthodromic response recorded from the dentate gyrus is shown. The two positive waves, PI and P 2 , are measured from the pre-response baseline to the peak of the corresponding positive wave. The negative wave, NI, is measured from the peak of PI to the bottom of the sharp negative deflection. With low intensity stimulation the NI potential will not be seen and the response will consist of a single positive potential. This potential is considered to be equivalent to PI. Following antidromic stimulation a single negative potential occurs. Under these circumstances the potential is considered to be equivalent to NI and is measured from the pre-response baseline. 75 NI 76 one p ro f i l e was r e c o r d e d from a g i v e n s l ice and g e n e r a l l y o n l y one o r two s u c c e s s f u l p rof i l es were ob ta ined from a g i v e n exper imenta l animal . Laminar vo l tage p ro f i l e s were r e c o r d e d i n the dentate g y r u s and the C A l r e g i o n of the h ippocampus d u r i n g both an t id romic and o r thodromic s t imula t ion . A l t h o u g h some deta i ls of the laminar p ro f i l e s and CSD a n a l y s i s v a r i e d between exper iments , the gene ra l p a t t e r n of r e sponse was r e m a r k a b l y cons i s t en t . Each g r o u p of r e s u l t s unde rgoes a s imi lar se r ies of da ta a n a l y s i s p re sen ted as fou r separate sec t ions . Each sec t ion emphasizes the r e l a t i onsh ip between CSD, vo l tage g r a d i e n t s , and poss ib le ephapt ic i n t e r a c t i o n . A) Antidromic Dentate Response Reliable da ta were ob ta ined from f ive an t id romic exper iments i n the dentate g y r u s . I n each case the s t imula t ing e lec t rode was p laced i n the u p p e r t h i r d of the h i l u s , w i t h i n the dense mass of g r anu l e c e l l axons, and the r e c o r d i n g pos i t ions were located i n the u p p e r blade a few h u n d r e d microns towards the apex of the dentate g y r u s . Laminar Profiles A laminar p ro f i l e of the ex t r ace l l u l a r f i e ld po ten t ia l s r e c o r d e d i n the dentate g y r u s fo l lowing an t id romic a c t i v a t i o n i s shown on the left of f i g u r e 3.6. The c o r r e s p o n d i n g CSD prof i l e i s shown to the r i g h t . Between these two p ro f i l e s i s a schematic g ranu le c e l l d r a w n approx imate ly to scale. The dot ted l ines a r o u n d the c e l l body ind ica te the pos i t ion and w i d t h of the c e l l l a y e r , and the v e r t i c a l pos i t ion of each waveform ind ica t e s the r e c o r d i n g loca t ion a long the dendro-somat ic axis of the c e l l . 77 The vol tage response i n the c e l l l a y e r (dot) cons i s t ed of a s t imulus a r t i f ac t fol lowed b y a s h a r p NI po ten t i a l . A s the r e c o r d i n g locat ion was moved out of the somal r e g i o n in to the molecular l a y e r the waveform of the vo l tage response g r a d u a l l y changed u n t i l i t had the form of a r o u n d e d pos i t i ve def lec t ion . The CSD t races i nd i ca t ed a s i n k fol lowed b y a source i n the c e l l l a y e r and a source fol lowed b y a s i n k i n the m i d - d e n d r i t i c r e g i o n . A p u r e source was p resen t i n the d i s t a l d e n d r i t e s . The vol tage r e sponse i n the c e l l l a y e r (dot) and the h i l u s (below the dot) had a s imi la r waveform. However , the ampl i tude of the r e sponse g r a d u a l l y d imin i shed as the r e c o r d i n g pos i t i on was moved in to the h i l u s . In con t ra s t to the f i e ld po ten t ia l s , the CSD ampl i tude a b r u p t l y d imin i shed as soon as the r e c o r d i n g loca t ion left the c e l l l a y e r . The loss of CSD def lec t ions i n the h i l u s was a cons i s t en t f i n d i n g i n a l l of the an t id romic r e s u l t s i n the dentate . I t may re f lec t the fact that t h i s r e g i o n has v e r y few somata o r d e n d r i t e s . However , the h i l u s does have a h i g h d e n s i t y of g r anu l e c e l l axons . It i s somewhat s u r p r i s i n g that ac t ion poten t ia l s t r a v e l l i n g i n these axons d i d not generate a CSD def lec t ion . A c lose examinat ion of f i g u r e s i n a paper b y J e f f e r y s (1979) also fa i led to detect a n y c l e a r l y v i s i b l e CSD def lec t ions i n the h i l u s u n d e r s imi lar cond i t i ons . P e r h a p s the CSD def lec t ions are too shor t i n d u r a t i o n to detect o r p e r h a p s the mechanism for ac t ion po ten t ia l t r ansmis s ion i s h i g h l y e f f ic ien t a n d genera tes v e r y small c u r r e n t s that are not seen on the t r aces . Voltage Gradients The k e y o b s e r v a t i o n from the laminar p ro f i l e of f i g u r e 3.6 i s the vo l tage g r a d i e n t a long the dendrosomat ic ax is d u r i n g the NI po ten t ia l . 78 T h i s i s shown more c l e a r l y i n f i g u r e 3.7. The lower t r aces are potent ia ls a n d the top t races a re CSD r e c o r d s t a k e n from the laminar p ro f i l e of the p r e v i o u s f i g u r e . The left t race i n each case was r e c o r d e d i n the c e l l l aye r a n d the r i g h t t race was r e c o r d e d i n the mid d e n d r i t i c r e g i o n . T h i s new o r i en t a t i on i s emphasized b y the h o r i z o n t a l pos i t i on of the schematic g r a n u l e c e l l . The dot on a l l fou r t races c o r r e s p o n d s to the peak of NI i n the c e l l l a y e r (lower lef t ) . The CSD t races show s t r o n g i n w a r d c u r r e n t at the somatic l e v e l and o u t w a r d c u r r e n t i n the d e n d r i t i c r e g i o n d u r i n g N I . The l ine j o i n i n g the dots on the somatic a n d d e n d r i t i c vo l tage t races demonst ra tes the d e n d r o -somatic vo l tage g r a d i e n t ac ross the g r a n u l e popu la t ion . A l t h o u g h somewhat schematic , the dot ted l ine can be imagined as a down h i l l pa th for c u r r e n t flow between the d e n d r i t i c source and the somatic s i n k . Compar ing a response r e c o r d e d i n the c e l l l a y e r w i t h a response r e c o r d e d w i t h i n the d e n d r i t e s i s a q u i c k and simple method of de t ec t i ng the p resence of a vo l tage g r a d i e n t a long the dendro-somat ic axis of a n e u r o n a l popu la t ion . However , i t does not take in to c o n s i d e r a t i o n poss ib le v a r i a t i o n s i n the g r a d i e n t w i t h i n the r e g i o n of t i s sue s epa ra t i ng these two r e l a t i v e l y d i s t an t r e c o r d i n g loca t ions . A more in format ive r ep re sen t a t i on of the vo l tage g r a d i e n t i s shown i n f i g u r e 3.8 (top). The data for th i s g r a p h are d e r i v e d from the laminar p ro f i l e of f i g u r e 3.6. It shows the vo l tage for each r e c o r d i n g loca t ion a long the whole dendro-somat ic axis at the i n s t an t i n time when NI i n the c e l l l a y e r i s at i t s most nega t ive poin t . The loca t ion of each va lue a long the X axis ind ica tes the r e c o r d i n g pos i t ion w i t h i n the dentate g y r u s . The v e r t i c a l l ine marks the c e l l l a y e r and the schematic g ranu le c e l l he lps c l a r i f y the r e l a t i onsh ip of each data poin t to the g ranu le c e l l popu la t ion . The evoked 79 FIGURE 3.6 shows a l aminar p ro f i l e and CSD ana ly s i s of an t id romica l ly e v o k e d potent ia ls i n the dentate g y r u s . The left column of t races are poten t ia l s r e c o r d e d at 25 mic ron i n t e r v a l s a long a l ine pa ra l l e l to the dendro-somat ic axis of the g r anu l e c e l l popu la t ion . The c o r r e s p o n d i n g CSD t races are shown to the r i g h t . Po in t s above the zero l ine on each CSD t race ind ica te i n w a r d c u r r e n t ( s ink) and po in t s below the l ine ind ica te ou tward c u r r e n t ( source) . A schematic g ranu le c e l l between the t races shows the approximate r e l a t i o n s h i p between each t race and the r e c o r d i n g loca t ion a long the c e l l ax is . The h o r i z o n t a l dot ted l ines ind ica te the extent of the c e l l l aye r . T races above the c e l l l aye r , next to the d e n d r i t i c a r b o r i z a t i o n , were r e c o r d e d i n the molecular l aye r and t races below the ce l l l aye r were r e c o r d e d i n the h i l u s . C a l i b r a t i o n : potent ia ls - 5 ms/5 mV, CSD - 5 ms/5 U. 80 Voltage Dentate C S D 81 F I G U R E 3.7 shows the vo l tage g rad i en t p resen t d u r i n g an t id romic ac t iva t ion of the dentate g y r u s . The top pa i r of t races are CSD r eco rds and the bottom pa i r are evoked potent ia ls t aken from the p r e v i o u s f i g u r e . The left t r aces were obta ined from the ce l l l aye r and the r i g h t t races from a mid d e n d r i t i c r e g i o n as i n d i c a t e d by the h o r i z o n t a l o r i en t a t i on of the schematic g r anu l e c e l l . The dots mark the ins t an t of NI as measured on the somatic response . The dashed l ine j o i n i n g the evoked potent ia ls p r o v i d e s a v i s u a l impres s ion of the g r a d i e n t a long the dendro-somat ic axis of the g r anu l e c e l l popu la t ion d u r i n g the an t id romic response . E x t r a c e l l u l a r c u r r e n t w i l l f low down th i s g rad ien t (arrow) from the e x t r a d e n d r i t i c space towards the c e l l l i ne . C a l i b r a t i o n : potent ia ls - 5 ms/5 mV, CSD 5 ms/1 U . 82 5 mv 5 m s e c 83 FIGURE 3.8 Top: spat ial d is t r ibu t ion of extracel lular potential d u r i n g ant idromic act ivat ion of the dentate g y r u s . Bottom: r e g r e s s i o n analys is of voltage grad ient v e r s u s NI ampli tude. The data on the top g r a p h was d e r i v e d from the laminar prof i les of f igure 3.6. Each point shows the potential at a par t icu lar location a long the dendro-somat ic axis of the granule cel l populat ion at the instant the somatic potential reached its most negat ive point (NI). The schematic granule cel l indicates the approximate re la t ionship between cel l s t r u c t u r e and r e c o r d i n g locat ion. A smooth voltage grad ient extends from the posi t ive extracel lu lar potential in the dendr i t ic reg ion towards the negat iv i ty in the cel l layer . The arrow indicates the flow of extracel lular c u r r e n t a long the dendro-somat ic axis of the granule cel l populat ion. The grad ient in this case was about 50 mV/mm. Each point on the bottom g r a p h indicates the magnitude of the dendro-somat ic grad ient v e r s u s the amplitude of NI for each of the ant idromic experiments. Regress ion coeff ic ient: 8.95 mm" 1, S E +/- 0.83 mm- 1 Y in tercept : -1.6 mV/mm S E +/- 3.11 mV/mm 84 D E N T A T E A N T I D R O M I C G R A D I E N T 5-, AMPLITUDE AT CELL LAYER (mv) 85 po ten t i a l inse t on the lower left of the g r a p h shows the an t id romic popu la t ion sp ike r e c o r d e d from the c e l l l a y e r . The marke r u n d e r NI i nd i ca t e s the re fe rence time used to ex t rac t the vo l tage from each e v o k e d po ten t i a l i n f i g u r e 3.6. As shown i n f i g u r e 3.8, the ex t r ace l l u l a r n e g a t i v i t y of the an t id romic popu la t ion s p i k e was w e l l l oca l i zed to the c e l l l a y e r at the i n s t an t of N I . The po ten t ia l t hen v a r i e d smoothly t h r o u g h o u t the d e n d r i t i c r e g i o n u n t i l i t f i n a l l y r e v e r s e d to a pos i t i ve po ten t i a l i n the ou te r molecular l a y e r . A s imi lar loss of n e g a t i v i t y o c c u r r e d o v e r the h i l u s bu t the po ten t i a l d i d not r e v e r s e . A s w i t h the dot ted l ine of f i g u r e 3.7, i t i s h e l p f u l to v i s u a l i z e c u r r e n t f l owing "down h i l l " a long the slope of th i s vo l tage g r a d i e n t p ro f i l e . The g r a p h p r e d i c t s a c u r r e n t flow towards the c e l l l a y e r from both the molecular l a y e r and the h i l u s . A s d i s c u s s e d i n " theore t i ca l concep t s" , a p o r t i o n of a n y ex t r ace l l u l a r c u r r e n t w i l l f low w i t h i n a c e l l u l a r p rocess i n i t s pa th . I n th i s case some of the c u r r e n t f l owing "down h i l l " from the molecular l a y e r c o u l d en te r the d e n d r i t e s and t r a v e l to the soma c a u s i n g a r e l a t ive depo la r i za t ion of ce l l s w i t h i n the popu la t ion . A l t h o u g h the re was also a vo l tage g rad i en t w i t h i n the h i l u s , the ephap t ic i n t e r ac t i ons i n d u c e d b y c u r r e n t f l owing down th i s g r ad i en t shou ld be r e l a t i v e l y smal l . The v e r y fine diameter of the axons i n th i s r e g i o n shou ld p r e sen t a much h i g h e r ax ia l r e s i s t ance to c u r r e n t f low, l im i t i ng the component of ex t r ace l l u l a r c u r r e n t e n t e r i n g the axon and t r a v e l l i n g to the soma ( T r a n c h i n a and Nicho l son 1986). The predominant impor tance of the soma and d e n d r i t e s i n ephapt ic i n t e r ac t i ons i s conf i rmed b y the r e s u l t s of a p p l y i n g a r t i f i c i a l ex t r ace l l u l a r vo l tage g r a d i e n t s to the dentate g y r u s ( Jef fe rys 1981; also see fo l lowing chap te r ) , and to the cerebe l lum 86 ( B r o o k h a r t and B l a c h l y , 1952; Chan and Nicho l son 1986). I n each case the e x c i t a b i l i t y of the neu rona l popu la t ion r e sponded a c c o r d i n g to the d e n d r o -somatic vol tage g r a d i e n t and not the g r ad i en t o v e r the axon. The g r a p h on the bottom of f i g u r e 3.8 shows the r e l a t i o n s h i p of vo l t age g r a d i e n t a long the dendro-somat ic axis of the g r anu l e c e l l popu la t ion (ordinate) to the peak ampl i tude of an an t id romic popu la t ion s p i k e (absc issa) . The g rad i en t (G) was de te rmined u s i n g the fo l lowing ca l cu la t ion . G = ( V 2 - V i ) / ( X 2 - X i ) where V i s vo l tage and X i s the loca t ion of two sequen t i a l r e c o r d i n g pos i t ions . T h i s i s equ iva l en t to c a l c u l a t i n g the slope between two po in t s on the top g r a p h of th i s f i g u r e . The r e c o r d i n g pos i t ions were chosen to span the b o u n d a r y between the p rox imal d e n d r i t i c r e g i o n and the c e l l l a y e r s ince the g r a d i e n t o v e r th i s r e g i o n shou ld be most impor tan t i n g e n e r a t i n g a n ephapt ic in f luence on the somatic membrane po ten t ia l . The r e g r e s s i o n l ine ind ica tes a d i r e c t r e l a t i onsh ip between vol tage g r a d i e n t and the ampl i tude of N I , and p r e d i c t s a pos i t i ve g r a d i e n t for NI poten t ia l s of a l l ampl i tudes . The fo l lowing c o n v e n t i o n i s u sed to s impl i fy the d i s c u s s i o n of ex t r ace l l u l a r vo l tage g r a d i e n t s w i t h r e spec t to t h e i r poss ib le ephap t ic in f luence . Grad i en t s are r e f e r r e d to as " p o s i t i v e " i f the e x t r a d e n d r i t i c po ten t i a l i s more pos i t i ve than that i n the c e l l l a y e r . These g r a d i e n t s are associa ted w i t h c u r r e n t flow towards the c e l l l a y e r and p r e d i c t an ephapt ic depo la r i za t ion of somatic membrane po ten t ia l . Negat ive g r a d i e n t s are associa ted w i t h c u r r e n t flow i n the oppos i te d i r e c t i o n and p r e d i c t ephapt ic dep re s s ion . 87 B) Or thodromic Stable laminar p ro f i l e s were ob ta ined from e igh teen o r thodromic exper iments i n the dentate g y r u s . I n each case the s t imu la t ing e lec t rode was p laced i n the middle of the molecular l a y e r of the u p p e r blade and the r e c o r d i n g loca t ions were pos i t ioned a few h u n d r e d microns away. T h i s a r rangement a l lowed d i r e c t s t imula t ion of pe r fo ran t pa th f i be r s w i t h subsequen t s y n a p t i c a c t i va t i on of the g r anu l e c e l l d e n d r i t e s . Laminar P r o f i l e s The laminar p ro f i l e and CSD a n a l y s i s of an o r t h o d r o m i c a l l y e v o k e d po ten t i a l i n the dentate g y r u s are shown i n f i g u r e 3.9. These p ro f i l e s had a more compl ica ted form than those seen fo l lowing an t id romic ac t i va t i on . T h i s i s not s u r p r i s i n g s ince , i n t h i s case, s y n a p t i c c u r r e n t s are f l owing i n a d d i t i o n to the c u r r e n t s associa ted w i t h ac t ion po ten t ia l genera t ion . The p redominan t ly nega t ive po ten t ia l r e c o r d e d from the c e n t r a l d e n d r i t i c r e g i o n c o r r e s p o n d s to the loca t ion of s y n a p t i c i n p u t . The CSD o v e r t h i s same r e g i o n i nd i ca t ed an i n w a r d movement of c u r r e n t ac ros s the d e n d r i t i c membranes of the g ranu le c e l l popu la t ion . T h i s s i n k was p r e s u m a b l y the r e s u l t of the ac t ive i n w a r d c u r r e n t associa ted w i t h the exc i t a to ry s y n a p t i c d r i v e . The e v o k e d po ten t i a l o b s e r v e d w i t h i n the c e l l l ine had a t r i p h a s i c shape w i t h an i n i t i a l pos i t i ve po ten t i a l (PI) fol lowed b y a nega t ive go ing (NI) and a second pos i t i ve wave (P2). T h i s c h a r a c t e r i s t i c waveform i s more c l e a r l y seen i n f i g u r e 3.10 (lower lef t ) . The peak of P I c o r r e s p o n d e d to a prominent n e g a t i v i t y w i t h i n the d e n d r i t i c a r b o r i z a t i o n ( f igure 3.9). The poten t ia l s r e c o r d e d at locat ions in termedia te to these two extremes showed 88 FIGURE 3,9 shows a laminar p ro f i l e and CSD a n a l y s i s of o r thodromica l ly e v o k e d potent ia ls i n the dentate g y r u s . The left column of t races are poten t ia l s r ecorded at 25 um i n t e r v a l s a long a l ine p a r a l l e l to the d e n d r o -somatic axis of the g ranu le c e l l popu la t ion . The c o r r e s p o n d i n g CSD t races are shown to the r i g h t . Po in t s above the zero l ine on each CSD t race ind ica te i n w a r d c u r r e n t (s ink) and po in t s below the l ine indica te ou tward c u r r e n t (source) . A schematic g ranu le c e l l between the t races shows the approximate r e l a t ionsh ip between each t race and the r e c o r d i n g locat ion a long the ce l l axis . The ho r i zon t a l do t ted l ines ind ica te the extent of the c e l l l a y e r . Traces above the c e l l l a y e r , next to the d e n d r i t i c a r b o r i z a t i o n , were r e c o r d e d i n the molecular l a y e r and t races below the ce l l l a y e r were r e c o r d e d i n the h i l u s . C a l i b r a t i o n : potent ia ls - 5 ms/5 mV, CSD - 5 ms/5 U . 89 Voltage Dentate C S D 90 FIGURE 3.10 shows the voltage gradient present during orthodromic activation of the dentate gyrus. The top pair of traces are CSD records and the bottom traces are evoked potentials taken from the previous figure. Note that the same two evoked potentials are repeated three times. The left traces were obtained from the cell layer and the right traces from the mid dendritic region as indicated by the horizontal orientation of the schematic granule cell. Open circles mark the instant of Pl , filled circles the instant of NI and dotted circles the instant of P2 measured on the somatic response. The dashed line joining the top pair of responses indicates a dendro-somatic gradient along the cell axis during Pl . During this phase extracellular current should flow along the cell axis from the somatic positivity towards the dendritic negativity. The dashed line joining the middle pair of traces shows a minimal gradient during NI. As a result, very little extracellular current flow is expected during this phase of the response. The gradient during P2 is shown by the dashed line joining the lower pair of traces. This gradient is similar to that observed during Pl and should be associated with a similar dendro-somatic flow of extracellular current. Calibration: potentials - 5 ms/5 mV, CSD 5 ms/5 U. 5 in s o i: 92 F I G U R E 3.11 Pl potential Top: spatial distribution of extracellular potential during orthodromic activation of the dentate gyrus. Bottom: regression analysis of voltage gradient versus Pl amplitude. The data on the top graph was derived from the laminar profiles of figure 3.9. Each point shows the potential at a particular location along the dendro-somatic axis of the granule cell population at the instant the somatic potential reached the peak of its initial positivity (Pl). The schematic granule cell indicates the approximate relationship between cell structure and recording location. A smooth voltage gradient extends from the positive extracellular potential in the cell layer towards the negativity in the dendritic region. The arrow indicates the flow of extracellular current along the dendro-somatic axis of the granule cell population. The gradient in this case was about -60 mV/mm. Each point on the bottom graph indicates the magnitude of the dendro-somatic gradient versus the amplitude of Pl for each of the orthodromic experiments. Regression coefficient: -17.0 mm-1, SE +/- 2.4 m r 1 Y intercept: 2.68 mV/mm SE +/- 9.15 mV/mm 93 AMPLITUDE AT CELL LAYER (mv) 94 F I G U R E 3.12 N I po t en t i a l Top : spa t i a l d i s t r i b u t i o n of e x t r a c e l l u l a r p o t e n t i a l d u r i n g o r thod romic a c t i v a t i o n of the denta te g y r u s . Bottom: r e g r e s s i o n a n a l y s i s of vo l tage g r a d i e n t v e r s u s N I ampl i tude . The data on the top g r a p h was d e r i v e d from the laminar p ro f i l e s of f i g u r e 3.9. Each po in t shows the po ten t i a l at a p a r t i c u l a r loca t ion a long the d e n d r o - s o m a t i c ax i s of the g r a n u l e c e l l p o p u l a t i o n at the i n s t an t the somatic po ten t i a l r eached the peak n e g a t i v i t y of the p o p u l a t i o n s p i k e ( N I ) . The schemat ic g r a n u l e c e l l i nd i ca t e s the approximate r e l a t i o n s h i p be tween c e l l s t r u c t u r e and r e c o r d i n g loca t ion . O n l y a minimal vo l t age g r a d i e n t ex i s t s a long the d e n d r o - s o m a t i c ax is i n th i s example. E a c h po in t on the bottom g r a p h i nd i ca t e s the magni tude of the d e n d r o - s o m a t i c g r a d i e n t v e r s u s the ampl i tude of N I f o r each of the o r t hod romic exper iments exper iments . R e g r e s s i o n coeff ic ient : 2.85 m m - 1 , S E + / - 1.1 m m - 1 Y in t e r cep t : -17.0 mV/mm S E +/- 9.81 mV/mm DENTATE N1 VOLTAGE GRADIENT 5-i DENDRO-SOMATIC LOCATION ( microns ) 180 -i 1 6 0 A 1 4 0 A 1 2 0 -1 1 0 0 -> 3 8 0 -AMPLITUDE AT CELL LAYER (mv) 96 FIGURE 3.13 P2 potential Top: B p a t i a l distribution of extracellular potential during orthodromic activation of the dentate gyrus. Bottom: regression analysis of voltage gradient versus P2 amplitude. The data on the top graph was derived from the laminar profiles of figure 3.9. Each point shows the potential at a particular location along the dendro-somatic axis of the granule cell population at the instant the somatic potential reached the peak of its final positivity (P2). The schematic granule cell indicates the approximate relationship between cell structure and recording location. A smooth voltage gradient extends from the positive extracellular potential in the cell layer towards the negativity in the dendritic region. The arrow indicates the flow of extracellular current along the dendro-somatic axis of the granule cell population. The gradient in this case was about -30 mV/MMm. Each point on the bottom graph indicates the magnitude of the dendro-somatic gradient versus the amplitude of Pl for each of the orthodromic experiments experiments. Regression coefficient: -8.6 mm-1, SE +/- 0.9 mm-1 Y intercept: -6.5 mV/mm SE +/- 5.14 mV/ 97 DENTATE P2 VOLTAGE GRADIENT 5n AMPLITUDE AT CELL LAYER (mv) 98 F I G U R E 3«14 compares the vo l tage d i s t r i b u t i o n d u r i n g the three phases of an o r thod romica l l y evoked poten t ia l i n the dentate g y r u s ( P I , NI and P2). The three l ines on the g r a p h are t aken from f i g u r e s 3.11 to 3.13. Each po in t ind ica tes the po ten t i a l at a g i v e n locat ion a long the dendro-somat ic axis of the c e l l popu la t ion at the peak of P I (open c i r c l e s ) , NI (closed c i r c l e s ) and P2 ( t r i ang les ) . The schematic c e l l shows the approximate r e l a t i onsh ip between ce l l s t r u c t u r e and r e c o r d i n g loca t ion . D u r i n g both P I and P2 the ex t r ace l lu l a r po ten t ia l i n the c e l l l a y e r i s pos i t i ve and the potent ia l i n the d e n d r i t i c r e g i o n i s nega t ive g e n e r a t i n g a g rad ien t for ex t r ace l lu l a r c u r r e n t flow away from the c e l l l a y e r a long the dendro-somat ic axis of the g ranu le c e l l popu la t ion . D u r i n g NI the nega t ive shi f t i n the ce l l l a y e r r educes the vo l tage g rad i en t to a minimum l e v e l . DENTATE GRADIENTS o o P1 © e N1 A A P2 1 1 1 1 1 ' f 1 1 1 1 1 300 -200 -100 0 100 200 DENDRO-SOMATIC LOCATION (microns) 100 a g r a d u a l p r o g r e s s i o n from n e g a t i v i t y w i t h i n the d e n d r i t e s to the p o s i t i v i t y o b s e r v e d i n the c e l l l a y e r . A s imi lar r e l a t i onsh ip between the somatic and d e n d r i t i c r e c o r d s was seen d u r i n g the second pos i t i ve peak of the popu la t ion po ten t i a l . The d e n d r i t i c n e g a t i v i t y was s t i l l p r e sen t d u r i n g P2 and the laminar p ro f i l e showed a s imi lar g r a d u a l sh i f t towards the p o s i t i v i t y r e c o r d e d i n the c e l l l a y e r . The CSD d u r i n g P I and P2 bo th i nd i ca t ed a c u r r e n t source at the somatic l e v e l . The nega t ive po ten t ia l , N I , was most prominent i n the c e l l l a y e r . In the p rox imal d e n d r i t i c r e g i o n i t u n d e r w e n t a compl icated r e v e r s a l to f i n a l l y become a shor t d u r a t i o n pos i t i ve def lec t ion super imposed on the o v e r a l l n e g a t i v i t y r e c o r d e d i n the molecular l aye r . In th i s example there was i n w a r d c u r r e n t i n the c e l l l a y e r d u r i n g N I . I n o the r exper iments , p a r t i c u l a r l y w i t h smaller ampl i tude NI poten t ia l s , the CSD con t inued to i nd i ca t e ou tward c u r r e n t d u r i n g t h i s phase . I n the mid d e n d r i t i c r e g i o n the o u t w a r d c u r r e n t seen d u r i n g P I and P2 p e r s i s t e d d u r i n g N I . The poten t ia l s r e c o r d e d i n the h i l u s had bas i ca l l y the same form as those r e c o r d e d i n the c e l l l a y e r . A s the r e c o r d i n g pos i t i on sh i f t ed deeper in to the h i l u s the o v e r a l l ampl i tude of the response s lowly dec l ined and the c o r r e s p o n d i n g CSD r e c o r d s i nd i ca t ed v i r t u a l l y no source o r s i n k a c t i v i t y . Voltage Gradients T h i s complex sequence of potent ia ls genera ted an equa l l y complex se r ies of ex t r ace l l u l a r vo l tage g r a d i e n t s a long the dendro-somat ic axis of the u n d e r l y i n g c e l l popu la t ion . These g r ad i en t s are shown i n f i g u r e 3.10. A l l of the t races are t aken from the laminar p ro f i l e s and CSD r e c o r d s of 101 the p r e v i o u s f i g u r e . The o rgan i za t i on of th i s f i g u r e i s s imi la r to that of f i g u r e 3.7 except that the same e v o k e d poten t ia l s are shown th ree times, once for each major phase of the response . The marke r s ind ica te the i n s t an t i n time at the peak of P l (open c i r c l e s ) , NI ( f i l led c i r c l e s ) and P2 (dot ted c i r c l e s ) r e c o r d e d i n the c e l l l i ne . P l Po ten t i a l A t the time of P l , the CSD on the top of the f i g u r e ind ica tes a c u r r e n t source o v e r the somatic r e g i o n and a s i n k i n the d e n d r i t e s . The dashed l ine j o i n i n g the top p a i r of evoked poten t ia l s immediately u n d e r the schematic g r anu l e c e l l shows the p resence of an ex t r ace l l u l a r vo l tage g r a d i e n t d u r i n g the same phase of the response . The CSD p a t t e r n sugges t s that i n t r a c e l l u l a r c u r r e n t i s f l owing from the d e n d r i t e s towards the c e l l bodies and the vo l tage g r a d i e n t sugges t s that ex t r ace l l u l a r c u r r e n t i s f l owing from the c e l l l a y e r towards the e x t r a d e n d r i t i c space. P r e s u m a b l y c u r r e n t flow i n t h i s loca l c i r c u i t r e s u l t s from s y n a p t i c a c t i v i t y i n the d e n d r i t e s . The vo l t age g r a d i e n t d u r i n g P l i s shown i n more de t a i l i n the top g r a p h of f i g u r e 3.11. The data for t h i s g r a p h was d e r i v e d from the laminar p ro f i l e of f i g u r e 3.9. A t the time of P l the peak pos i t i ve vo l tage was i n the c e l l l a y e r ( v e r t i c a l l ine) and the most nega t ive poin t a long the c e l l axis was i n the mid d e n d r i t i c r e g i o n . Between these two po in t s there was a smooth vo l tage g rad i en t ex t end ing ac ross the en t i r e p rox imal d e n d r i t i c r e g i o n of the u n d e r l y i n g popu la t ion of g ranu le ce l l s . T h i s g r a d i e n t s h o u l d be associa ted w i t h a flow of ex t r ace l lu l a r c u r r e n t from the c e l l l a y e r towards the mid d e n d r i t i c r e g i o n , and p r e d i c t s an ephapt ic d e p r e s s i o n of the somatic membrane po ten t ia l . 102 The r e l a t i o n s h i p of vo l tage g r a d i e n t to the ampl i tude of P l i s shown on the bottom of f i g u r e 3.11. Each poin t r ep r e sen t s the g r ad i en t measured ac ros s the b o u n d a r y between the molecular and c e l l l a y e r . A l t h o u g h some sca t te r i s p resen t , h i g h e r ampl i tude P l potent ia ls were c l e a r l y associa ted w i t h g rea te r vo l tage g r ad i en t s . F u r t h e r m o r e , these g r a d i e n t s were c o n s i s t e n t l y i n the nega t ive d i r e c t i o n , p r e d i c t i n g ephap t ic d e p r e s s i o n t h r o u g h o u t the whole r ange of P l ampl i tudes . N I Po ten t i a l The CSD r e c o r d s on the top of f i g u r e 3.10 ind ica te a r e l a t i ve shi f t towards i n w a r d c u r r e n t i n the c e l l l a y e r and o u t w a r d c u r r e n t i n the d e n d r i t e s d u r i n g the NI po ten t i a l ( f i l led c i r c l e s ) . T h i s was the t r e n d ac ross a l l the exper imenta l r e s u l t s . However a complete r e v e r s a l of CSD i n the c e l l l a y e r was not o b s e r v e d i n a l l cases . In gene ra l , i f NI was of h i g h ampl i tude the sh i f t i n the somatic r e g i o n was adequate to r e v e r s e the CSD (as i n f i g u r e 3.10). D u r i n g lower ampl i tude poten t ia l s the l e v e l of o u t w a r d c u r r e n t s imply decreased . On the o ther hand , the t endency towards o u t w a r d c u r r e n t i n the d e n d r i t e s d u r i n g NI was neve r adequate to r e v e r s e the predominant s i n k i n th i s r e g i o n . The vol tage g r a d i e n t d u r i n g NI i s shown i n the middle pa i r of e v o k e d potent ia ls on f i g u r e 3.10. I n th i s example the g r a d i e n t i s v e r y smal l . Grad ien t s i n o ther exper iments were also small w i t h the major i ty i n d i c a t i n g an ex t r ace l l u l a r flow of c u r r e n t from the c e l l l a y e r towards the e x t r a d e n d r i t i c space. The vo l tage g rad i en t d u r i n g NI i s shown i n more de t a i l i n the top g r a p h of f i g u r e 3.12. These data were also d e r i v e d from the evoked potent ia ls of f i g u r e 3.9. D u r i n g NI the po ten t ia l i n the c e l l l ine was 103 nega t ive . However i t d i d not r each the l e v e l of the p e r s i s t i n g n e g a t i v i t y p r e s e n t i n the mid d e n d r i t i c r e g i o n . The r e s u l t i n t h i s example was a minimal g r a d i e n t w i t h some v a r i a b i l i t y a long the prox imal d e n d r i t i c shaft . The r e l a t i onsh ip of the g r a d i e n t to the ampl i tude of NI i s shown i n the g r a p h on the bottom of the f i g u r e . Fo r a l l NI poten t ia l s of moderate ampl i tude the g r a d i e n t remained i n a nega t ive o r d e p r e s s i n g d i r e c t i o n . On ly the occas iona l h i g h ampl i tude po ten t i a l was able to genera te a pos i t i ve g rad ien t . The loca l c i r c u i t s and c u r r e n t flow are not as easy to w o r k out for NI as t hey were for P I , One way to s impl i fy the s i t ua t ion i s to assume that the even t s of NI are super imposed on a b a c k g r o u n d of o n g o i n g a c t i v i t y i n i t i a t ed d u r i n g P I . Wi th in t h i s f ramework the r e l a t i ve sh i f t towards i n w a r d c u r r e n t i n the somal r e g i o n and ou tward c u r r e n t i n the d e n d r i t e s sugges t s that NI i s assoc ia ted w i th an i n t r a c e l l u l a r movement of c u r r e n t from the soma towards the d e n d r i t e s . The r e l a t i ve shi f t towards ex t r ace l l u l a r n e g a t i v i t y i n the c e l l l ine and p o s i t i v i t y i n the molecular l a y e r sugges t s that NI i s also associa ted w i t h ex t r ace l l u l a r c u r r e n t flow from the d e n d r i t i c r e g i o n towards the c e l l l a y e r . P r e s u m a b l y c u r r e n t flow i n t h i s loca l c i r c u i t r e s u l t s from ac t ion po ten t i a l genera t ion i n the somata of g r a n u l e ce l l s and i s s imi la r i n form to the c u r r e n t flow p r e v i o u s l y d e s c r i b e d d u r i n g the an t id romic popu la t ion s p i k e . However , the net c u r r e n t o b s e r v e d d u r i n g NI i s the summation of the b a c k g r o u n d c u r r e n t s p e r s i s t i n g s ince P I and the c u r r e n t s spec i f ic to th i s phase of the response . E p h a p t i c i n t e rac t ions are o n l y i n f luenced b y the net c u r r e n t flow i n the ex t r ace l l u l a r space. D u r i n g NI ex t r ace l l u l a r c u r r e n t flow a long the dendro-somat ic ax is of the g ranu le c e l l popu la t ion was u s u a l l y towards the dend r i t e s , s u g g e s t i n g a d e p r e s s i o n of somatic e x c i t a b i l i t y . A l t h o u g h the 104 d i r e c t i o n of c u r r e n t flow and poss ib le ephap t ic i n t e r ac t i ons were the same d u r i n g P l and N I , the magni tude d u r i n g NI was g r e a t l y r e d u c e d . P2 Po ten t i a l D u r i n g P2 , the f i n a l phase of the response , the re was a r e t u r n to . cond i t i ons s imi lar to those seen d u r i n g P l . The c u r r e n t source i n the c e l l l a y e r r edeve loped and the d e n d r i t i c s i n k s t r e n g t h e n e d ( f igure 3.10, top t races dot ted c i r c l e ) . The vo l tage g r a d i e n t ac ross the g r a n u l e c e l l popu la t ion a lso s t r e n g t h e n e d (bottom pa i r of potent ia ls ) s u g g e s t i n g a r e t u r n of c u r r e n t flow from the c e l l l a y e r towards the e x t r a d e n d r i t i c space. C u r r e n t f low d u r i n g P2 iB p r e s u m a b l y due to a combina t ion of ac t ion po ten t i a l r e p o l a r i z a t i o n , i n h i b i t o r y s y n a p t i c c u r r e n t s at the somatic l e v e l and a n y r ema in ing exc i t a to ry s y n a p t i c c u r r e n t s i n the d e n d r i t e s . U n f o r t u n a t e l y , i t i s not poss ib le to isolate these v a r i o u s c o n t r i b u t i o n s w i t h the ava i lab le data . The top g r a p h of f i g u r e 3.13 shows the smooth cha rac t e r of the vo l tage g r a d i e n t ac ross the p rox imal d e n d r i t e s and somata of the g r anu l e c e l l popu la t ion d u r i n g P2 . The r e l a t i onsh ip of th i s g r a d i e n t to the ampl i tude of the po ten t i a l i s shown on the bottom of f i g u r e 3.13. I n a l l cases the g r a d i e n t was nega t ive towards the d e n d r i t e s w i t h an i n c r e a s i n g magni tude as P2 ampl i tude i n c r e a s e d . The ephap t ic i n t e rac t ions associa ted w i t h t h i s phase of the response shou ld dep re s s the somata of g ranu le ce l l s as ex t r ace l l u l a r c u r r e n t moves from the c e l l l ine towards the e x t r a d e n d r i t i c space. 105 Summary of P I N I P2 A compar i son of the spa t i a l d i s t r i b u t i o n of vo l tage d u r i n g the three phases of the o r thod romica l l y evoked po ten t ia l i n the dentate g y r u s i s shown i n f i g u r e 3.14. The t races are the same as those shown i n f i g u r e s 3.11, 3.12 and 3.13. D u r i n g each phase the ex t r ace l l u l a r po ten t ia l i n the d e n d r i t i c r e g i o n remained nega t ive whereas the po ten t i a l i n the c e l l l a y e r u n d e r w e n t a l a rge sh i f t f rom pos i t i ve , nega t ive , and back to p o s i t i v e . The vol tage g r a d i e n t ac ross the dendro-somat ic axis was nega t ive d u r i n g P I and P2 . In most cases the g r ad i en t d u r i n g NI was i n the same d i r e c t i o n as d u r i n g P I and P2 bu t at a g r e a t l y r e d u c e d magni tude . The dendro-somat ic g r a d i e n t o n l y r e v e r s e d d i r e c t i o n when the NI po ten t ia l was v e r y l a rge . C) A n t i d r o m i c C A l Response Stable laminar p ro f i l e s of an t id romic evoked potent ia ls were ob ta ined from the h ippocampal C A l r e g i o n i n f i ve exper iments . S t imu la t ing e lec t rodes were p laced i n the a lveus and the r e c o r d i n g pos i t ions were loca ted a few h u n d r e d microns l a t e r a l l y towards CA2. Laminar P r o f i l e s The laminar and CSD pro f i l e of a r ep re sen t a t i ve response are shown i n f i g u r e 3.15. In some re spec t s the an t id romic r e sponse i n C A l was s imi la r to that o b s e r v e d i n the dentate g y r u s . In both cases the h ighes t ampl i tude r e sponse was located i n the c e l l l a y e r i n assoc ia t ion w i t h a we l l loca l i zed c u r r e n t s i n k . D u r i n g th i s same phase, t races r e c o r d e d from d e n d r i t i c loca t ions showed a g r a d u a l r e v e r s a l to a p o s i t i v i t y associa ted w i t h 106 o u t w a r d c u r r e n t . In C A l , bo th d e n d r i t i c r eg ions showed th i s gene ra l p a t t e r n . Voltage Gradients The gene ra l p a t t e r n of CSD and ex t r ace l lu l a r vo l tage g r a d i e n t s p r e sen t d u r i n g the r ep re sen t a t i ve an t id romic response of f i g u r e 3.15 are shown more c l e a r l y i n F i g u r e 3.16. The CSD t races on the top of the f i g u r e show a c u r r e n t s i n k i n the c e l l l ine and c u r r e n t sources i n bo th d e n d r i t i c r eg ions d u r i n g N I . The vo l tage g r a d i e n t s associa ted w i t h t h i s CSD p a t t e r n are i n d i c a t e d b y the dashed l ines j o i n i n g the poten t ia l s on the bottom of the f i g u r e . P r e sumab ly , i n w a r d somatic c u r r e n t d u r i n g ac t ion po ten t ia l gene ra t ion en te r s both d e n d r i t i c a r b o r i z a t i o n s and t h e n r eappea r s as o u t w a r d c u r r e n t w i t h i n the s t r a tum rad ia tum and o r i e n s . The ex t r ace l l u l a r component of th i s p a i r of loca l c i r c u i t s c a r r i e s c u r r e n t from both the a p i c a l and basa l d e n d r i t i c r eg ions towards the c e l l l a y e r . The vo l tage prof i l e d u r i n g NI i s shown on the top g r a p h of f i g u r e 3.17. A s expected , the most nega t ive poin t was i n the c e l l l ine and the po ten t i a l smoothly sh i f t s towards more pos i t i ve vo l tages as the r e c o r d i n g loca t ion was moved ou twards towards the a p i c a l and basa l d e n d r i t i c r eg ions . The g r a d i e n t was c o n s i s t e n t l y s teeper ac ross the basa l than the a p i c a l d e n d r i t e s as seen th i s example. Both of these dendro-somat ic g r a d i e n t s shou ld c a r r y ex t r ace l lu l a r c u r r e n t from the d e n d r i t e s towards the c e l l l a y e r . The ephap t ic in f luence associa ted w i t h each g r a d i e n t w i l l t end to depola r ize the somata, g e n e r a t i n g a to ta l ephap t i c exc i ta t ion g rea te r t han expected for e i the r g r a d i e n t alone. 1 0 7 F I G U R E 3.15 shows a laminar p ro f i l e and CSD ana lys i s of an t id romica l ly e v o k e d potent ia ls i n the h ippocampal C A l r e g i o n . The left column of t races a re potent ia ls r e c o r d e d at 25 mic ron i n t e r v a l s a long a l ine pa ra l l e l to the dendro-somat ic axis of the p y r a m i d a l c e l l popu la t ion . The c o r r e s p o n d i n g CSD t races are shown to the r i g h t . Po in ts above the zero l ine on each CSD t race ind ica te i n w a r d c u r r e n t (s ink) and po in t s below the l ine ind ica te ou tward c u r r e n t (source) . A schematic p y r a m i d a l c e l l between the t races shows the approximate r e l a t ionsh ip between each t race and the r e c o r d i n g loca t ion a long the ce l l ax is . The ho r i zon t a l dot ted l ines ind ica te the extent of the c e l l l aye r . Traces above the ce l l l aye r , next to the basal d e n d r i t i c a r b o r i z a t i o n , were r e c o r d e d i n the s t r a tum o r i e n s and t races below the c e l l -l a y e r , next to the ap i ca l d e n d r i t e s , were r e c o r d e d i n the s t ra tum radia tum. C a l i b r a t i o n : potent ia ls - 5 ms/5 mV, CSD - 5 ms/10 U . 108 109 F I G U R E 3.16 shows the vo l tage g r a d i e n t p re sen t d u r i n g an t id romic ac t i va t i on of the h ippocampal C A l r e g i o n . The t races above the schematic p y r a m i d a l ce l l are CSD r e c o r d s and the t races below the c e l l are e v o k e d potent ia ls t aken from the p r e v i o u s f i g u r e . The left t races were ob ta ined from the mid basal d e n d r i t i c r e g i o n , the middle t races from the c e l l l a y e r and the r i g h t t races from the mid ap i ca l d e n d r i t i c r eg ion . The dots mark the ins tan t of N I as measured on the somatic r esponse . The dashed l ine j o i n i n g the potent ia ls at the time of NI i nd ica te a vol tage g rad i en t ac ross both the ap i ca l and basal d e n d r i t i c r eg ions associa ted w i t h flow of ex t r ace l lu l a r c u r r e n t from the d e n d r i t e s towards the c e l l l a y e r (a r rows) . Ca l i b r a t i on : potent ia ls - 5 ms/5 mV, CSD 5 ms/1 U . 110 BASAL SOMATIC APICAL CSD FIGURE 3.17 Top: spatial d is t r ibu t ion of extracel lular potential d u r i n g ant idromic act ivat ion of the hippocampal C A l area. Bottom: regress ion ana lys is of vol tage grad ient v e r s u s NI amplitude. The data on the top g r a p h was d e r i v e d from the laminar prof i les of f igure 3.15. Each point shows the potential at a par t icu la r location along the dendro-somat ic axis of the pyramida l cel l populat ion at the instant the somatic potential reached its most negat ive point (NI). The schematic cel l indicates the approximate re la t ionship between cel l s t r u c t u r e and r e c o r d i n g location. A smooth voltage grad ient extends from the posi t ive extracel lular potential in the both dendr i t i c reg ions towards the negat iv i ty in the cel l layer . The arrows indicate the flow of extracel lu lar c u r r e n t a long the dendro-somat ic axis of the pyramida l cel l populat ion. The total gradient across both dendr i t ic f ie lds was about 90 mV/mm. Each point on the bottom g r a p h indicates the magnitude of the total dendro -somat ic grad ient v e r s u s the amplitude of NI for each of the ant idromic exper iments. Regress ion coeff ic ient: 14.1 m m - 1 , S E +/- 1.39 m m - 1 Y in tercept : 6.28 mV/mm S E +/- 0.97 mV/mm 112 CA1 ANTIDROMIC GRADIENT 5n ~ i i l I I i 1 1 1 1 1 1 -250-200 -150 -100 - 5 0 0 50 100 150 200 250 300 350 DENDRO-SOMATIC LOCATION ( microns ) 0 2 4 6 8 1 0 AMPLITUDE AT CELL LAYER (mv) 113 The s e n s i t i v i t y of a g i v e n d e n d r i t i c s t r u c t u r e to ephapt ic i n t e r ac t i ons i s dependan t on a number of c h a r a c t e r i s t i c s s u c h as e lec t ro ton ic l e n g t h , membrane r e s i s t ance , b r a n c h i n g p a t t e r n and d i r e c t i o n , as we l l as o ther e v e n less access ib le fac tors ( T r a n c h i n a and Nicho l son 1986). The c h a r a c t e r i s t i c s of ap i ca l and basa l d e n d r i t e s of p y r a m i d a l ce l l s d i f fe r i n many respec t s so i t would not be s u r p r i s i n g i f they also had d i f fe ren t s e n s i t i v i t i e s to ephapt ic i n t e r ac t ions . F o r example the l o n g e lec t ro ton ic l e n g t h of the t h i c k p rox imal shaft of the a p i c a l d e n d r i t e may make i t p a r t i c u l a r l y s ens i t i ve to ex t r ace l lu l a r c u r r e n t s . However the p r e sen t s t u d y does not p r o v i d e a n y data w i t h r e spec t to the r e l a t i ve impor tance of ephap t ic i n t e r ac t i ons i n d i f fe ren t d e n d r i t i c s t r u c t u r e s . There fo re , i n t h i s s t u d y , the a p i c a l and basal d e n d r i t e s of C A l p y r a m i d a l ce l l s are assumed to have equa l s e n s i t i v i t y to the in f luences of ex t r ace l lu l a r c u r r e n t s . The g r a p h on the bottom of f i g u r e 3.17 shows the to ta l g r a d i e n t ac ros s both sets of d e n d r i t e s for a g i v e n ampl i tude of N I . I n each case the two g r a d i e n t s were s imply added toge ther to p roduce a to ta l va lue . The to ta l g r a d i e n t was c o n s i s t e n t l y i n the pos i t i ve d i r e c t i o n and v a r i e d d i r e c t l y w i t h the absolute ampl i tude of N I . A to ta l of 14 s table laminar p ro f i l e s were ob ta ined fo l lowing o r thodromic a c t i v a t i o n of the C A l r e g i o n of the h ippocampus . I n each case the s t imu la t ing e lec t rode was p laced i n the middle of the s t r a tum rad ia tum and the a r r a y of r e c o r d i n g pos i t ions were located s e v e r a l h u n d r e d microns away i n the d i r e c t i o n of CA2. T h i s a r rangement a l lowed d i r e c t s t imula t ion of the Schaf fe r co l l a t e ra l f i be r s as they passed t h r o u g h the s t r a tum 114 rad ia tum before s y n a p s i n g on the ap i ca l d e n d r i t e s of the p y r a m i d a l ce l l s of C A l . Laminar P ro f i l e s The laminar p ro f i l e and CSD a n a l y s i s of a r ep re sen t a t i ve response are shown i n f i g u r e 3.18. M a n y of the c h a r a c t e r i s t i c s o b s e r v e d d u r i n g the o r thodromic C A l response were s imi lar to the equ iva l en t response i n the dentate g y r u s ( f igure 3.9). T h i s s i m i l a r i t y was most o b v i o u s i n the t races r e c o r d e d o v e r the s y n a p t i c a l l y ac t i va t ed d e n d r i t i c r e g i o n and the c e l l l a y e r . The po ten t i a l i n the c e l l l a y e r showed the c h a r a c t e r i s t i c t r i p h a s i c r e sponse w i t h a c lear P l , NI and P2 po ten t ia l (also see f i g u r e 3.19). I n bo th the mid s t r a tum rad ia tum and molecular l a y e r there was a p rominent p e r i o d of n e g a t i v i t y associa ted w i t h an i n w a r d flow of s y n a p t i c c u r r e n t . D u r i n g the e a r l y ( P l ) and late (P2) phase of the r e sponse there was a c o r r e s p o n d i n g pos i t i ve def lec t ion and o u t w a r d movement of c u r r e n t i n the c e l l l a y e r . Between these two phases (NI) there was a s h a r p nega t ive def lec t ion i n the c e l l l a y e r associa ted w i t h a s t r o n g c u r r e n t s i n k . At th i s time the ac t i va t ed d e n d r i t i c r e g i o n u n d e r w e n t a r e l a t ive r e v e r s a l of i t s o v e r a l l n e g a t i v i t y and the CSD showed a r e l a t i ve sh i f t towards ou tward c u r r e n t . The p a t t e r n o v e r the basa l d e n d r i t e s was somewhat d i f fe ren t . D u r i n g P l , the smooth pos i t i ve po ten t ia l seen i n the c e l l l ine s lowly decreased i n ampl i tude as the r e c o r d i n g loca t ion was sh i f t ed ou twards towards the d i s t a l d e n d r i t e s . The small c u r r e n t source seen i n the c e l l l ine also decreased to neg l ig ib l e l eve l s . D u r i n g P2 , the pos i t i ve po ten t i a l dec l ined much more r a p i d l y ac ros s the s t ra tum o r i ens and the somatic source r e v e r s e d to a s ink i n the d e n d r i t e s . A t the b e g i n n i n g of NI a small , bu t sha rp , 115 p o s i t i v i t y appeared o v e r the d i s t a l basa l dend r i t e s . T h i s po ten t ia l t hen r e v e r s e d late i n NI before f i n a l l y s h i f t i n g back towards the base l ine . The CSD d u r i n g th i s wave showed a sha rp source fol lowed b y a s i n k . Vol tage G r a d i e n t s Vol tage g r a d i e n t s ac ros s the dendro-somat ic axis of the p y r a m i d a l c e l l popu la t ion u n d e r w e n t a compl icated sequence of changes d u r i n g an o r thod romica l l y e v o k e d response . These g r a d i e n t s are shown on f i g u r e 3.19 u s i n g potent ia ls and CSD t races from the p r e v i o u s f i g u r e . P I Po t en t i a l D u r i n g P I (open c i r c l e s top row of t races) the vo l tage g r a d i e n t ac ros s the basa l d e n d r i t e s was v e r y small as shown b y the minimal s lope of the dashed l ine j o i n i n g the left two t races . However the g r a d i e n t ac ross the ap i ca l d e n d r i t e s was much s teeper , i n d i c a t i n g a movement of ex t r ace l l u l a r c u r r e n t from the source i n the c e l l l a y e r towards the s i n k i n the s t r a tum rad ia tum. P r e s u m a b l y th i s l oca l c i r c u i t was d r i v e n b y s y n a p t i c a c t i v i t y i n the a p i c a l d e n d r i t e s . The ex t r ace l l u l a r vo l tage g r a d i e n t s d u r i n g P I are seen i n more de ta i l i n the top g r a p h of f i g u r e 3.20. The dendrosomat ic g r a d i e n t ac ros s the a p i c a l d e n d r i t e s formed a smooth downward s lope towards the e x t r a d e n d r i t i c n e g a t i v i t y of s t r a tum rad ia tum. A much sha l lower g r ad i en t was p re sen t ac ross the basa l d e n d r i t e s . Both these g r a d i e n t s are associa ted w i t h movement of c u r r e n t away from the c e l l l a y e r and p r e d i c t ephap t i c d e p r e s s i o n . 116 FIGURE 3.18 shows a laminar profile and CSD analysis of orthodromically evoked potentials in the hippocampal CAl region. The left column of traces are potentials recorded at 25 micron intervals along a line parallel to the dendro-somatic axis of the pyramidal cell population. The corresponding CSD traces are shown to the right. Points above the zero line on each CSD trace indicate inward current (sink) and points below the line indicate outward current (source). A schematic pyramidal cell between the traces shows the approximate relationship between each trace and the recording location along the cell axis. The horizontal dotted lines indicate the extent of the cell layer. Traces above the cell layer, next to the basal dendritic arborization, were recorded in the stratum oriens and traces below the cell layer, next to the apical dendrites, were recorded in the stratum radiatum. Calibration: potentials - 10 ms/10 mV, CSD - 10 ms/20 U. 117 118 FIGURE 3.19 shows the voltage gradient present during orthodromic activation of the hippocampal CAl region. The traces above the schematic pyramidal cell are CSD records and the traces below the cell are evoked potentials taken from the previous figure. Note that the same set of evoked potentials are repeated three times. The left traces were obtained from the mid basal dendritic region, the middle traces from the cell layer and the right traces from the mid apical dendritic region. Open circles mark the instant of PI, filled circles the instant of NI and dotted circles the instant of P2 measured from the somatic response. The dashed line joining the top responses at the time of PI indicates a minimal gradient across the basal dendritic region but a larger gradient carrying extracellular current from the cell layer towards the apical dendrites (arrow). Both dashed lines joining the middle traces show a gradient and associated extracellular current from the dendrites towards the cell line during NI (arrows). The apical and basal dendro-somatic gradients and the associated current flow reverse direction during P2. Calibration: potentials - 5 ms/5 mV, CSD 5 ms/5 U. 119 5 m s e c 120 FIGURB 3.20 PI Potential Top: spatial distribution of extracellular potential during orthodromic activation of the hippocampal CAl area. Bottom: regression analysis of voltage gradient versus PI amplitude. The data on the top graph was derived from the laminar profiles of figure 3.18. Each point shows the potential at a particular location along the dendro-somatic axis of the pyramidal cell population at the instant the somatic potential reached the peak of its initial positivity (PI). The schematic cell indicates the approximate relationship between cell structure and recording location. A smooth voltage gradient extends from the positive extracellular potential in the cell layer towards the negativity of the apical dendritic region and a minimal gradient is present across the basal dendrites. The arrow indicates the major flow of extracellular current along the dendro-somatic axis of the pyramidal cell population. The total gradient across both dendritic fields was about minus 20 mV/mm. Each point on the bottom graph indicates the magnitude of the total dendro-somatic gradient versus the amplitude of PI for each of the orthodromic experiments. Regression coefficient: -11.0 mm-1, SE +/- 2.99 mm-1 Y intercept: -0.3 mV/mm SE +/- 5.97 mV/mm 121 CA1 P1 VOLTAGE GRADIENT 5-1 I 122 F I G U R E 3.21 NI Po t en t i a l Top : spa t i a l d i s t r i b u t i o n of ex t r ace l l u l a r po ten t i a l d u r i n g o r thod romic a c t i v a t i o n of the h ippocampal C A l area . Bottom: r e g r e s s i o n a n a l y s i s of vo l t age g r a d i e n t v e r s u s NI ampl i tude . The da ta on the top g r a p h was d e r i v e d from the laminar p ro f i l e s of f i g u r e 3.18. Each po in t shows the po ten t i a l at a p a r t i c u l a r loca t ion a long the dendro - soma t i c ax is of the p y r a m i d a l c e l l popu la t i on at the i n s t an t the somatic po ten t i a l r e a c h e d i t s most nega t ive va lue (NI) . The schematic c e l l i nd i ca t e s the approximate r e l a t i o n s h i p be tween c e l l s t r u c t u r e and r e c o r d i n g loca t ion . S h a r p vo l tage g r a d i e n t s ex tend from bo th d e n d r i t i c f i e lds towards the e x t r a c e l l u l a r n e g a t i v i t y of the c e l l l a y e r . The a r r o w s ind ica te the flow of e x t r a c e l l u l a r c u r r e n t a long the d e n d r o - s o m a t i c axis of the p y r a m i d a l c e l l popu l a t i on . The to ta l g r a d i e n t a c ro s s bo th d e n d r i t i c f i e lds was about 160 mV/mm. Each po in t on the bottom g r a p h ind ica t e s the magni tude of the to ta l dend ro - soma t i c g r a d i e n t v e r s u s the ampl i tude of NI for each of the o r thod romic exper imen t s . R e g r e s s i o n coef f ic ien t : 15.8 mm" 1 , S E +/- 0.65 m r 1 Y i n t e r c e p t : -30.0 mV/mm SE +/- 9.97 mV/mm 123 CA1 N1 VOLTAGE GRADIENT n 1 1 r -250-200 -150 -100 -50 r 50 100 T 1 r 150 200 250 300 DENDRO-SOMATIC LOCATION ( microns ) 180 4 6 8 AMPLITUDE AT CELL LAYER (mv) 10 124 FIGURE 3.22 P2 Potential Top: B p a t i a l distribution of extracellular potential during orthodromic activation of the hippocampal CAl area. Bottom: regression analysis of voltage gradient versus P2 amplitude. The data on the top graph was derived from the laminar profiles of figure 3.18. Each point shows the potential at a particular location along the dendro-somatic axis of the pyramidal cell population at the instant the somatic potential reached the peak of its final positivity (PI). The schematic cell indicates the approximate relationship between cell structure and recording location. A smooth voltage gradient extends from the positive extracellular potential in the cell layer towards both dendritic regions. The arrows indicate the major flow of extracellular current along the dendro-somatic axis of the pyramidal cell population. The total gradient across both dendritic fields was about minus 90.0 mV/mm. Each point on the bottom graph indicates the magnitude of the total dendro-somatic gradient versus the amplitude of P2 for each of the orthodromic experiments. Regression coefficient: -15.0 mm-1, SE +/- 1.16 mm-1 Y intercept: -1.8 mV/mm SE +/- 6.36 mV/mm 125 CA1 P2 VOLTAGE GRADIENT 1 5 1 ' i i i — i — i — i — i — i — i — i — . -250-200 -150 -100 -50 0 50 100 150 200 250 300 350 DENDRO-SOMATIC LOCATION ( microns ) -20 A s-40 A a - 6 0 < o -80 A -100 2 i 4 6 AMPLITUDE AT CELL LAYER (mv) 126 FIGURE 3.23 compares the voltage distribution during the three phases of an orthodromically evoked potential in the hippocampal CAl region (Pl, NI and P2). The three lines on the graph are taken from figures 3.20 to 3.22. Each point indicates the potential at a given location along the dendro-somatic axis of the cell population at the peak of Pl (open circles), NI (closed circles) and P2 (triangles). The schematic cell shows the approximate relationship between cell structure and recording location. During both Pl and P2 the extracellular potential in the cell layer is positive and the potential in the apical dendritic region is negative, generating a gradient for extracellular current flow away from the cell layer along the apical dendro-somatic axis of the neuronal population. A similar gradient from the cell layer towards the basal dendrites also exists during P2. However, during Pl, the positivity in the basal dendritic region minimizes the voltage gradient across this region. A large negative shift in the cell layer during NI generates a steep gradient for current flow from both the apical and basal dendritic fields towards the somatic region. CM GRADIENTS DENDRO-SOMATIC LOCATION (microns) 128 The g r a p h on the bottom of f i g u r e 3.20 shows the r e l a t i onsh ip between the g r a d i e n t and the ampl i tude of P I . The to ta l g r a d i e n t was c o n s i s t e n t l y i n the nega t ive d i r e c t i o n a n d i n c r e a s e d w i t h the ampl i tude of P I . N I Po t en t i a l The dashed l ines j o i n i n g the middle set of po ten t ia l s on f i g u r e 3.19 ind ica te s t r o n g g r a d i e n t s from both d e n d r i t i c r eg ions towards the c e l l l ine d u r i n g NI (f i l led c i r c l e s ) . P r e sumab ly , c u r r e n t flow down these g r a d i e n t s i s the ex t r ace l l u l a r component of loca l c i r c u i t s genera ted b y somatic ac t ion poten t ia l s w i t h i n the c e l l l a y e r . These g r a d i e n t s are seen i n more de ta i l on the top g r a p h of f i g u r e 3.21. The p a t t e r n of g r a d i e n t s i n the h ippocampus d u r i n g NI was c e r t a i n l y the most complex seen i n th i s s t u d y , and showed the least s i m i l a r i t y to the equ iva l en t da ta r e c o r d e d i n the dentate g y r u s . The most cons i s t en t f i n d i n g was a steep g r a d i e n t ac ros s the basa l d e n d r i t e s towards the c e l l l a y e r . A s imi lar g r a d i e n t was u s u a l l y p re sen t ac ross the a p i c a l dend r i t e s . However , t h i s g r a d i e n t was r e d u c e d o r e v e n r e v e r s e d b y the n e g a t i v i t y associa ted w i t h s y n a p t i c a c t i v i t y i n the s t r a tum rad ia tum. The s h a r p pos i t i ve def lec t ion seen i n the f i g u r e o v e r the prox imal p o r t i o n of the ap i ca l d e n d r i t e s was o b s e r v e d i n s e v e r a l cases , u s u a l l y i n assoc ia t ion w i t h a p a r t i c u l a r l y l a rge NI po ten t ia l . When p resen t , i t t ended to i nc rease the g r a d i e n t o v e r the ap i ca l d e n d r i t e s . The g r a p h on the bottom of f i g u r e 3.21 ind ica tes that the tota l g r ad i en t was i n the nega t ive d i r e c t i o n for small NI ampl i tudes . I n these examples the s y n a p t i c s i n k i n the ap i ca l d e n d r i t e s was the dominant 129 influence on extracellular current. However, in most cases the gradient over the basal dendrites dominated, generating a positive gradient. Current flow down this gradient should ephaptically depolarize the somata of pyramidal cells. P2 Potential During P2, a strong source developed in the cell layer and sinks appeared over the apical and basal dendritic regions (figure 3.19 circle with dot). The extracellular potentials associated with this pattern of CSD generated negative voltage gradients across both dendritic fields. Presumably the somatic source was the result of action potential repolarization, recurrent inhibitory synaptic activity and any remaining excitatory synaptic current entering the apical dendrites. The extracellular voltage gradients present during P2 are shown more clearly in figure 3.22 (top). A well localized positivity within the cell layer diminished rapidly as the recording position was moved outwards along the apical and basal dendritic regions, creating steep voltage gradients across both dendritic fields. These gradients predict an ephaptic depression of the somatic membrane potential. The regression line in the bottom graph indicates that gradient varied indirectly with amplitude of P2 and always remained in the negative direction. Summary P l NI P2 A comparison of the spatial distribution of voltage during the three phases of the orthodromically evoked potential in the hippocampal CAl region is shown in figure 3.23. The traces are the same as those shown in figures 3.20, 3.21 and 3.22. During each phase, the extracellular potential 130 i n the a p i c a l d e n d r i t i c r e g i o n remained nega t ive . The po ten t i a l i n the basa l d e n d r i t e s was pos i t i ve d u r i n g P I and NI and near zero d u r i n g P2 . I n the c e l l l a y e r the po ten t ia l u n d e r w e n t a l a rge shi f t from pos i t i ve to nega t ive and back to pos i t i ve d u r i n g the evoked po ten t ia l . The to ta l ex t r ace l l u l a r g r a d i e n t s were c o n s i s t e n t l y nega t ive d u r i n g P I and P2 and pos i t i ve d u r i n g a l l bu t the smallest NI po ten t ia l . 3.4 DISCUSSION The r e s u l t s c l e a r l y demonstra te the p resence of vo l tage g r a d i e n t s ac ross the dendro-somat ic axis of p r i n c i p a l neurons w i t h i n the dentate g y r u s and h ippocampal C A l r e g i o n d u r i n g evoked po ten t ia l s . T h r o u g h o u t a g i v e n response these g r a d i e n t s u n d e r g o a p red i c t ab l e and h i g h l y s t e r eo typed sequence of changes i n magni tude and p o l a r i t y . T h e y p r o v i d e an i n d i r e c t measure of the flow of ex t r ace l l u l a r c u r r e n t a long the axis of the n e u r o n a l popu la t ion and p r e d i c t the r e l a t i ve p o l a r i t y and magni tude of ephap t ic i n t e r ac t i ons d u r i n g d i f fe ren t phases of an evoked po ten t i a l . Before c o n t i n u i n g w i th a d i s c u s s i o n of these vol tage g r ad i en t s , the fo l lowing sec t ion c l a r i f i e s some impor tan t cons ide ra t ions r e g a r d i n g d e n d r i t i c s p i k e s i n C A l . A) S i te o f A c t i o n Po ten t i a l I n i t i a t i on The ac tua l s i te of ac t ion po ten t i a l i n i t i a t i on w i t h i n a n e u r o n i s a c r i t i c a l fac tor when c o n s i d e r i n g the in f luence of poss ib le ephapt ic i n t e r ac t i ons . A g i v e n ex t r ace l l u l a r c u r r e n t w i l l have an oppos i te in f luence on the somatic and d e n d r i t i c membrane potent ia ls ( T r a n c h i n a and Nicho l son 1986) and w i l l e i the r dep re s s or exci te a n e u r o n d e p e n d i n g on the p r i m a r y si te of ac t ion po ten t i a l genera t ion . A l t h o u g h a complete d i s c u s s i o n of th i s 131 topic is outside the scope of this thesis, the laminar profiles and CSD analysis shown here support the hypothesis that the initial site of action potential generation is in the somata for both granule and pyramidal neurons. In all cases the earliest current sink associated with action potential generation (NI potential) appeared in the vicinity of the cell layer before propagating out into the dendritic fields. This confirms similar findings by others during the orthodromic population response in the dentate (Jefferys 1979, 1981) and during the antidromic response in CAl (Leung 1979b; Taylor and Dudek 1984a). These authors also concluded that the initial site of action potential generation is in or near the somata of the neuronal population. However a major controversy surrounds the sequence of events during the orthodromic response in CAl. The ability to record action potential-like transients in the dendrites of pyramidal neurons has led some investigators to suggest that the initial site of action potential generation during synaptic activation is in the dendrites of these cells (Wong et al 1979). However our laboratory has carried out a critical study of intradendritic and CSD records during orthodromic activation of CAl to specifically address this controversy. We concluded that the initial site of action potential generation is in or near the soma. These action potentials then propagate into both the apical and basal dendrites where they can be detected on intradendritic recordings (Richardson et al 1987; Turner et al 1988a,b). BJ Voltage ..Gradients The sequence of gradients observed in the dentate and CAl during an evoked potential are similar in many respects. In both areas the 132 ex t r ace l l u l a r vo l tage g r a d i e n t i s pos i t i ve d u r i n g the an t id romic NI po ten t ia l a n d nega t ive d u r i n g the o r thodromic P l and P2 poten t ia l s . A sh i f t towards a pos i t i ve g r a d i e n t i s also o b s e r v e d i n bo th the dentate and C A l d u r i n g the o r thodromic NI po ten t i a l . E p h a p t i c i n t e r ac t i ons d u r i n g these g r a d i e n t s s h o u l d depress the o v e r a l l e x c i t a b i l i t y of the neu rona l popu la t ion d u r i n g P l a n d P2 , and inc rease e x c i t a b i l i t y w i t h i n the popu la t ion d u r i n g N I . A n excep t ion to th i s gene ra l i za t ion i s seen for the o r thodromic NI po ten t i a l i n the dentate g y r u s . A l t h o u g h the g r a d i e n t a lways sh i f t s i n the pos i t i ve d i r e c t i o n d u r i n g N I , i t a c tua l l y remains nega t ive d u r i n g a l l bu t the h ighes t ampl i tude e v o k e d poten t ia l s . F o r each phase of the r e sponse the magni tude of the g r a d i e n t has an approx imate ly l i nea r r e l a t i onsh ip to the ampl i tude of the e v o k e d po ten t i a l i n the c e l l l a y e r . The r e g r e s s i o n coeff ic ients for these r e l a t i o n s h i p s are shown aga in i n f i g u r e 3.24. The magni tude of each coeff ic ient i s a r e l a t i ve measure of the t endency to genera te an ex t r ace l l u l a r vo l tage g r a d i e n t d u r i n g a g i v e n phase of the response . The p o l a r i t y of the coeff ic ient i nd ica t e s whe the r the t endency i s towards a nega t ive o r a pos i t i ve g r ad i en t . Compar ing these coeff ic ients r evea l s some impor tant d i f fe rences between the dentate and C A l . Fo r example the t endency to genera te a pos i t i ve g r ad i en t d u r i n g both the an t id romic and o r thodromic NI po ten t ia l i s much g rea te r i n the C A l a rea than the dentate g y r u s . T h i s d i f fe rence i s most s t r i k i n g d u r i n g the o r thodromic response where the coeff ic ient for the dentate i s the smallest of a l l those o b s e r v e d . The r e g r e s s i o n coeff ic ients measured d u r i n g P l and P2 are also d i f f e ren t i n the dentate and C A l . D u r i n g P l the t endency to genera te a negat ive g r a d i e n t i s s t r o n g e r i n the dentate , where as d u r i n g P2 th i s 133 FIGURE 3.24 compares the generat ion of voltage gradients in the dentate g y r u s and hippocampal C A l region. Prev ious f igures showed the re lat ionship between the total voltage gradient along the dendro-somat ic axis of a cell population and the amplitude of a g iven phase of the somatic evoked potential . In the present f igure , all of the regress ion coeff icients are presented in a single bar g raph . Each pair of bars compares the regress ion coeff icient for C A l (filled) and the dentate g y r u s (hatched) d u r i n g antidromical ly (NI left) and orthodromical ly evoked potentials ( P l , NI and P2). E r r o r bars show standard e r ror . There was a s igni f icant d i f ference between the regress ion coeff icients for the orthodromic NI potential in the dentate g y r u s and the hippocampal C A l region (p<.05). 135 r e l a t i onsh ip r e v e r s e s and the s t ronges t t endency is seen i n C A l . However , these d i f fe rences were not s ign i f i can t . A t least some of these d i f fe rences between g r a d i e n t s genera ted i n the the dentate and C A l may r e s u l t from the fact that g r anu l e ce l l s have one and p y r a m i d a l ce l l s have two d e n d r i t i c a r b o r i z a t i o n s . The impor tance of d e n d r i t i c geometry i s most o b v i o u s d u r i n g the NI po ten t i a l of an o r thodromic response . D u r i n g t h i s phase the ex t r ace l l u l a r po ten t i a l i n both the c e l l l a y e r and the ac t iva t ed d e n d r i t i c r e g i o n are nega t ive . T h i s s imi lar sh i f t i n po ten t i a l a long the dendro-somat ic axis tends to cance l a n y vol tage g r a d i e n t s that might o the rwise o c c u r . The r e s u l t of t h i s i n t e r a c t i o n between somatic and d e n d r i t i c even t s i s a minimal t e n d e n c y to deve lop vol tage g r a d i e n t s d u r i n g the o r thodromic NI po ten t ia l i n the dentate g y r u s . The s i tua t ion i n C A l is s imi lar i f one o n l y cons ide r s the g r a d i e n t a long the d e n d r i t e r e c e i v i n g s y n a p t i c i n p u t . However p y r a m i d a l ce l l s are also exposed to vo l tage g r a d i e n t s ac ros s t h e i r uns t imula ted d e n d r i t e s . S ince the ex t r ace l l u l a r po ten t i a l i n the basa l d e n d r i t i c r e g i o n remains pos i t i ve d u r i n g NI a l a rge vo l tage g rad i en t deve lops towards the n e g a t i v i t y of the c e l l l a y e r . There fore the t endency to genera te a net pos i t i ve g r a d i e n t d u r i n g NI shou ld be much s t r o n g e r i n C A l than the dentate g y r u s . T h i s d i f fe rence i s re f lec ted i n the s i g n i f i c a n t l y g rea te r r e g r e s s i o n coeff ic ient for the o r thodromic NI po ten t i a l i n C A l as seen i n f i g u r e 3.24. The p resence of two o p p o s i n g d e n d r i t e s on p y r a m i d a l neurons may also accoun t for the l a r g e r r e g r e s s i o n coeff ic ient measured i n C A l d u r i n g the an t id romic NI po ten t ia l . D u r i n g th i s response the g r a d i e n t a long both d e n d r i t e s has the same p o l a r i t y so the net g r ad i en t w i l l a lways be g rea te r t han tha t c o n t r i b u t e d from e i ther dend r i t e alone. On the o ther hand o n l y 136 a single dendrite contributes to the gradient observed in the dentate gyrus. Relationship To Other Studies The basic characteristics of the laminar profiles obtained in the present study are similar to those reported by other investigators (Andersen 1960; Andersen and Lflmo 1966; Andersen et al 1966; Gloor et al 1963; Jefferys 1981; Leung 1979a,b,c; L/5mo 1971; Fujita and Sakata 1962; Taylor and Dudek 1984a,b; Taylor et al 1984). However, these previous studies did not specifically deal with extracellular voltage gradients. In order to obtain data for comparison with the present findings it was necessary to measure gradients directly from the figures of these publications. The small size and limited resolution of the laminar profiles shown in the publications allowed only rough estimates of the gradients. Further variability was contributed by differences between the experimental preparation, anesthetic agent and recording protocols. A total of 25 extracellular gradients were derived by hand from previous studies. Of these gradients, 22 were obtained from orthodromic responses in CAl and dentate, 3 were obtained from antidromic responses in CAl and none were obtained from antidromic dentate responses. The absolute value of the gradients ranged from 10 to 150 mV/mm with an average of 37 mV/mmM (SE +/- 8 mV/mm). This range is similar to that observed in the present study. It was also possible to measure the somatic amplitude of evoked potentials from the previously published laminar profiles. These additional data provided a more informative way of comparing voltage gradients from other investigations with those from the present study. This comparison 137 was c a r r i e d out b y f i r s t a p p l y i n g the r e g r e s s i o n coeff ic ients d e r i v e d i n the r e s u l t s sec t ion to the somatic e v o k e d potent ia ls measured from the o ther s tud ie s . The ac tua l vo l tage g r a d i e n t o b s e r v e d i n each s t u d y was then exp re s sed as percen tage of the vol tage g r a d i e n t p r e d i c t e d b y the r e g r e s s i o n ca l cu l a t i on . One h u n d r e d pe rcen t impl ied that the hand measured and the p r e d i c t e d g rad i en t were equa l and a percen tage of less t han one h u n d r e d impl ied that the hand measured va lue was smaller t han that p r e d i c t e d b y the p re sen t f i n d i n g s . The percen tages v a r i e d from 50 to 200% w i t h an ave rage of 88% (SE +- 7%). A g a i n these va lues sugges t that the g r a d i e n t s o b s e r v e d i n the p r e sen t s t u d y are s imi lar to those d e r i v e d from p r e v i o u s l y p u b l i s h e d data . 138 CHAJP^ER 4 At this point it is clear that extracellular voltage gradients consistently occur along the axis of principal cells during evoked potentials in the hippocampal formation. But are these gradients of sufficient amplitude to have a significant ephaptic influence on the excitability of the neuronal population? One approach to answering this question is to study the influence of artificially applied voltage gradients on the amplitude of evoked potentials. Brookhart and Blachly (1952) used this approach to examine the importance of standing voltage gradients in the cerebellum. They passed a constant current into the exposed cerebellar cortex and observed the influence of the current on spontaneous activity of Purkinje cells and on the amplitude of stimulus evoked potentials. They observed an increase in spontaneous discharge and evoked potential amplitude when the applied current shifted the extradendritic potential in a positive direction. Similar studies in the cerebral cortex (Bindman et al, 1964; Denny-Brown and Brookhart 1962) indicate that pyramidal cells also become more excitable when the extradendritic space around their primary apical dendrite is shifted in a positive direction. These findings confirm the expected ephaptic excitation associated with extracellular current flow along dendritic structures towards the somata of a cell population. Unfortunately the investigators did not measure the voltage gradient generated by the applied current making it impossible to directly compare their results with the present findings. More recently investigators studying the influence of applied currents have used in vitro techniques. By working with isolated sections of tissue it is much easier to control the uniformity of current flow in the 139 extracellular space and to directly measure the voltage gradients associated with the applied current. Jefferys (1981) studied the influence of applied currents on the excitability of dentate granule cells in the hippocampal slice preparation. He found that the extracellular current associated with a gradient as small as 5 mV/mm was adequate to alter the amplitude of the orthodromic population response. Using the isolated frog cerebellum Chan and Nicholson (1986) found that 15 to 20 mV/mm was required to influence the spontaneous discharge of Purkinje cells. Similarly, gradients as small as 7 mV/mm alter evoked potentials i n the CAl region of the hippocampal slice (Bawin et al, 1986). The voltage gradients observed in the present study were often a fu l l order of magnitude greater than the threshold gradients reported in these earlier studies. This strongly suggests that significant ephaptic interactions occur during evoked potentials within the hippocampal formation. Since this line of evidence is critical to the conclusions derived in this thesis the present chapter further investigates the influence of artificially applied voltage gradients on the excitability of neurons in the hippocampal formation. 4.1 SPECIFIC METHODS Artificial field potentials were applied to the hippocampal slice by placing a pair of current passing electrodes in the ACSF such that a proportion of the current passing between the electrodes travels through the substance of the slice. The voltage gradient generated within the hippocampal formation was then directly assessed by recording the extracellular voltage at a series of points along the current path. This 140 t e chn ique al lowed the app l i ca t i on of a c c u r a t e l y c o n t r o l l e d a n d r e p r o d u c i b l e vo l t age g rad i en t s ac ross the whole h ippocampal s l i ce . However , i t was not poss ib le to genera te loca l ized g r a d i e n t s ac ross spec i f i c s u b r e g i o n s of the neu rona l t i s sue . T h i s l imi ta t ion was not a p rob lem w i t h i n the dentate g y r u s s ince g r anu l e ce l l s have a s ing le d e n d r i t i c a r b o r i z a t i o n l imi ted to one s ide of the c e l l l a y e r . On the o ther hand the p y r a m i d a l ce l l s w i t h i n the h ippocampus have bo th ap i ca l and basa l d e n d r i t i c a r b o r i z a t i o n s ex t end ing from oppos i te s ides of the c e l l l aye r . A n a r t i f i c i a l vo l tage g r a d i e n t app l i ed ac ross the complete dendro-somat ic axis of a p y r a m i d a l c e l l w i l l genera te o p p o s i n g ephapt ic in f luences s ince c u r r e n t f lows towards the c e l l l a y e r i n one d e n d r i t i c r e g i o n and away from the c e l l l a y e r i n the o ther . The net effect w i l l depend on the r e l a t i ve s e n s i t i v i t y of the basa l and a p i c a l d e n d r i t e s . Bawin et a l (1986) f ound that the popu la t ion response i n C A l was enhanced when a n a p p l i e d g rad i en t genera ted an ex t r ace l l u l a r p o s i t i v i t y o v e r the ap i ca l and a n e g a t i v i t y o v e r the basa l d e n d r i t e s s u g g e s t i n g that the ap i ca l d e n d r i t e dominates the s e n s i t i v i t y of C A l p y r a m i d a l ce l l s to ephap t ic i n t e r ac t i ons . I n the p re sen t s t u d y th i s i s sue i s avo ided b y c o n c e n t r a t i n g mainly on the r e s u l t s ob ta ined from the dentate g y r u s where i n t e r p r e t a t i o n of the f i n d i n g s i s more s t r a i g h t f o r w a r d . A) C u r r e n t The c u r r e n t p a s s i n g e lec t rodes were c o n s t r u c t e d from s t a n d a r d g lass microe lec t rodes p u l l e d from c a p i l l a r y t u b i n g as d e s c r i b e d i n the gene ra l methods. T h e y were then " b r o k e n back" to a t ip diameter of approx imate ly 50 to 100 mic rons b y t a p p i n g the e lec t rode t ip on a h a r d sur face . T h i s l a rge diameter lowered the t i p r e s i s t ance of the e lec t rodes to a few 141 h u n d r e d k i lohms and al lowed the passage of much l a r g e r c u r r e n t s t han a s t a n d a r d micro p ipe t te . However , the l a rge t ip also a l lowed the e l ec t ro ly te i n the p ipe t te to f r ee ly exchange w i t h the p e r f u s i o n so lu t ion i n the r e c o r d i n g chamber . S ince the e lec t rode was p laced near o r on the s l i ce i n some exper iments the h i g h l y concen t r a t ed e l ec t ro ly te u s e d i n s t a n d a r d ex t r ace l l u l a r e lec t rodes wou ld have d i f fused in to the s l ice and a l t e r ed the e l ec t ro ly t e concen t r a t i on of the ex t r ace l l u l a r space. To a v o i d t h i s p rob lem, the e lec t rodes were f i l l e d w i t h a r t i f i c i a l C S F t aken from the r e s e r v o i r of p e r f u s i o n so lu t ion feed ing the s l ice chamber . Two of these c u r r e n t p a s s i n g e lec t rodes were connec ted to an i so la ted c u r r e n t source (Grass) and then p laced on oppos i te s ides of the h ippocampal s l ice s u c h that the c u r r e n t f l owing between the e lec t rodes passed t h r o u g h the subs tance of the neu rona l t i s sue . The e lec t rodes were a lways pos i t ioned s u c h that the d i r e c t i o n of c u r r e n t flow was p a r a l l e l w i t h the dendro-somat ic axis of the neu rons u n d e r s t u d y i n o r d e r maximize a n y c u r r e n t i n d u c e d effects . A l t h o u g h c u r r e n t e lec t rodes were n e v e r p laced d i r e c t l y on o r immediately next to the r e g i o n of in t e res t , one e lec t rode was sometimes pos i t ioned at a d i s t an t s i te on the s l i ce . The l e v e l of c u r r e n t c o u l d be a l t e red manual ly o r cou ld be t u r n e d on o r off i n s t an t aneous ly b y a t iming s i g n a l genera ted b y the Dig i t imer . I n gene ra l the f i e lds were app l i ed for shor t pe r iods l a s t i n g from 100 to 500 ms. The f i e ld was " t u r n e d o n " b y the Digi t imer for a p e r i o d of time before the s t imulus evoked po ten t i a l and then t u r n e d off immediately fo l lowing the response . No f i e ld was app l i ed for the 7 or more seconds between s t imulus app l i ca t ions . The vo l tage g r a d i e n t genera ted b y a g i v e n c u r r e n t was d i r e c t l y moni tored b y measu r ing the ex t r ace l l u l a r vo l tage at s e v e r a l po in t s a long 142 the dendro-somat ic axis of the neu rona l popu la t ion , and then p l o t t i n g these vo l tage va lues aga ins t r e c o r d i n g loca t ion . The slope of the g r a p h at a n y po in t gave the vol tage g rad i en t for a g i v e n locat ion a long the d e n d r o -somatic ax i s , and the shape of the c u r v e ind ica t ed how cons i s t en t the g r a d i e n t was t h r o u g h o u t the app l i ed f i e ld po ten t ia l . A n example of the ex t r ace l l u l a r f i e ld po ten t ia l genera ted b y a p p l y i n g c u r r e n t ac ross the dentate g y r u s is shown i n f i g u r e 4.1. Each l ine r e p r e s e n t s the ex t r ace l lu l a r po ten t i a l measured at 25 mic ron i n t e r v a l s a long the d e n d r o -somatic ax is of the g ranu le c e l l popu la t ion at a g i v e n l e v e l of app l i ed c u r r e n t . The numbers ind ica te the approximate vo l tage g r a d i e n t i n mV/mm est imated b y measur ing the s t r a i g h t l ine slope between the ends of each c u r v e . A l t h o u g h the vol tage p rof i l e c u r v e s were neve r p r e c i s e l y l inea r t hey a lways smoothly v a r i e d t h r o u g h o u t the neu rona l t i s sue as seen i n th i s example. I n cases where th i s n o n l i n e a r i t y was more dramat ic the g r a d i e n t was s teepest i n the d i s t a l d e n d r i t e s and g r a d u a l l y decreased towards the c e l l l a y e r . I n these cases the vo l tage g r a d i e n t measurement was t a k e n o n l y o v e r the r e g i o n between the p rox imal d e n d r i t e s and the c e l l l a y e r . .4.2 RESULTS The r e s u l t s r e p o r t e d i n th i s chap te r were ob ta ined from 22 h ippocampal s l ices sub jec ted to v a r i o u s magni tudes of a p p l i e d vol tage g r a d i e n t s and s t imulus i n t ens i t i e s . In most cases the vo l tage g r a d i e n t was v a r i e d whi le the s t imulus i n t e n s i t y was maintained at a cons tan t l e v e l . The in f luence of the app l i ed f i e ld was then assessed b y measur ing the ampl i tudes of the P I , NI and P2 po ten t ia l s . The vol tage g r a d i e n t s u sed i n these exper iments were between p lu s and minus 100 mV/mm. A l t h o u g h 143 FIGURE 4.1 illustrates the extracellular voltage gradients generated during experimentally applied currents. Each line represents the voltage gradient across the dentate gyrus during a given magnitude of applied current. The lines were constructed by recording the extracellular potential at a series of locations along the dendro-somatic axis of the granule cell population (circles). The values to the left of each line indicate its slope in mV/mm. Although the curves are not perfectly straight, they vary smoothly throughout their extent. The point of intersection for all the curves has no particular significance. It simply indicates the balance point between the two current passing electrodes. -20 H -25 --1 1 ! 1 j 1 1 1 I i 1 i I » I ' I 25 -75 -25 25 75 125 175 225 275 DENDRO-SOMATIC LOCATION (microns) 145 these g r a d i e n t s were s u b s t a n t i a l t h e y were w i t h i n the r ange o b s e r v e d d u r i n g the e v o k e d poten t ia l s a n a l y z e d i n the p r e v i o u s chap te r . A) Orthodromic Responses F i g u r e 4.2 shows an example of the in f luence of a p p l i e d vol tage g r a d i e n t s on the o r thod romica l l y e v o k e d po ten t i a l i n the dentate g y r u s . The c o n t r o l r e sponse , e v o k e d w i t h no a p p l i e d f i e l d , has a p rominent P I and NI po ten t ia l and a smaller P2 po ten t i a l . A s l i g h t nega t ive po ten t i a l i s also seen fo l lowing P2 . D u r i n g nega t ive app l i ed f i e lds ( ex t r adendr i t i c po ten t ia l nega t ive w i th r e spec t to extrasomatic potent ia l ) the ampl i tudes of NI and P2 are r e d u c e d and at a nega t ive g r a d i e n t of 34 mV/mm and g r ea t e r o n l y the P I po ten t ia l remains . A s the ampl i tude of these poten t ia l s decreases u n d e r the in f luence of the app l i ed f i e lds , a late nega t ive po ten t i a l becomes more p rominen t and f i n a l l y appea r s to s t a r t immediately af ter P I . On the o the r hand , the ampl i tudes of NI and P2 are m a r k e d l y enhanced u n d e r the in f luence of pos i t i ve vo l tage g r a d i e n t s and the late nega t ive po ten t ia l i s los t i n the p rominent t r a i l i n g edge of P2 . The in f luence of app l i ed vo l tage g r a d i e n t s o b s e r v e d i n f i g u r e 4.2 a re shown g r a p h i c a l l y i n f i g u r e 4.3. Each po in t ind ica tes the ampl i tude of e i the r P I , NI o r P2 d u r i n g a g i v e n magni tude of vo l tage g r a d i e n t . The P I po ten t i a l remains at a f a i r l y cons tan t ampl i tude t h r o u g h o u t the r ange of a p p l i e d f i e ld s , showing o n l y a s l i g h t t r e n d towards a decrease i n ampl i tude d u r i n g nega t ive g r a d i e n t s . The n e a r l y f la t c u r v e shown for P I i s con t r a s t ed b y the s u b s t a n t i a l decrease i n ampl i tude of NI a n d P2 as the vo l tage g r a d i e n t i s i nc r ea sed i n the pos i t i ve d i r e c t i o n . The r e s u l t s shown i n f i g u r e 4.2 and 4.3 were c h a r a c t e r i s t i c of v i r t u a l l y a l l of the t r i a l s i n v o l v i n g o r thodromic a c t i v a t i o n of the dentate 146 FIGURE 4.2 i l l u s t r a t e s the in f luence of a r t i f i c i a l l y genera ted vol tage g r a d i e n t s on the o r thod romica l ly e v o k e d poten t ia l i n the dentate g y r u s . The same s t imulus i n t e n s i t y was u s e d to evoke a l l of the responses shown i n t h i s f i gu re . The o n l y d i f fe rence i n the cond i t ions for each response was the i n t e n s i t y and p o l a r i t y of the app l i ed f i e l d . In th is f i g u r e , and those that fol low, each r e c o r d is the ave rage of 4 evoked potent ia l s , the h o r i z o n t a l l ine ind ica tes zero o r basel ine vol tage and the numbers to the r i g h t of each po ten t ia l ind ica te the magni tude of the g rad ien t (mV/mm) app l i ed a long the dendro-somat ic axis of the ce l l popu la t ion . The u p p e r left potent ia ls were r e c o r d e d d u r i n g a vo l tage g rad i en t wh ich sh i f ted the e x t r a d e n d r i t i c po ten t ia l i n the nega t ive d i r e c t i o n wi th r e spec t to the extrasomatic space (negat ive g r a d i e n t ) . A s th i s g rad ien t was inc reased the NI and P2 potent ia ls p r o g r e s s i v e l y decrease i n ampli tude u n t i l t hey were no longer p resen t . On the o the r hand , the ampli tude of both poten t ia l s was enhanced d u r i n g g r a d i e n t s of the oppos i te po l a r i t y . Note that the app l i ed f ie lds have v e r y l i t t l e in f luence on the ampli tude of the P l po ten t ia l . > -I 5 MSEC Orthodromic Population Spike During Applied Fields M V / M M c o n t r o l 4-92 148 FIGURE 4.3 i s a g r a p h compar ing the change i n ampl i tude of P I , NI and P2 d u r i n g ex te rna l ly app l i ed vo l t age g r ad i en t s . Each point r ep re sen t s the peak ampl i tude of a g i v e n component of the evoked poten t ia l p lot ted aga ins t the magni tude of the e x t e r n a l l y app l i ed f i e ld . The data was d e r i v e d from the responses shown i n f i g u r e 4.2. The ampl i tude of P I decreased o n l y s l i g h t l y d u r i n g i n c r e a s i n g l y negat ive vol tage g rad i en t s and remained v i r t u a l l y cons tan t d u r i n g pos i t i ve g r ad i en t s . On the o ther hand the ampl i tudes of NI and P2 i n c r e a s e d (with opposi te po la r i t i es ) d u r i n g pos i t ive vo l tage g r a d i e n t s and dec reased when the vol tage g rad i en t was i n the nega t ive d i r e c t i o n . No v a l u e s are shown for NI and P2 d u r i n g the most nega t ive g r a d i e n t s s ince these components were complete ly s u p p r e s s e d u n d e r these cond i t i ons . Evoked Potential Ampli tude During Applied Fields 100 - 8 0 - 6 0 - 4 0 - 2 0 0 20 40 60 80 100 APPLIED FIELD (MV/MM) 150 g y r u s . I n each case the ampl i tude of both NI and P2 was i n c r e a s e d b y pos i t i ve and decreased b y nega t ive g r a d i e n t s whi le the ampl i tude of P2 remained nea r ly cons tant . The r e s u l t s were independen t of s t imulus i n t e n s i t y or the p resence of a popu la t ion s p i k e on the c o n t r o l response . T h i s independence is demonst ra ted i n f i g u r e 4.4 where the in f luence of app l i ed f ie lds on o r thodromic r e sponses r e c o r d e d i n the dentate g y r u s i s shown for three d i f fe ren t s t imulus s t r e n g t h s . The responses on each row (hor izonta l ) were exposed to the same vol tage g r a d i e n t and a l l those i n a g i v e n column (ver t ica l ) were e v o k e d u s i n g the same s t imulus i n t e n s i t y . A s expec ted , there was an inc rease i n the ampl i tude of P l , NI and P2 on the c o n t r o l r esponse as the s t imulus s t r e n g t h was i n c r e a s e d . E v e n t hough the c o n t r o l r e sponses d i f f e r ed , the in f luence of the ex te rna l vo l tage g r a d i e n t was s imi lar . . In each case the g r ad i en t had a marked effect on NI and P2 bu t v e r y l i t t l e i f a n y in f luence on P l . The r e l a t i onsh ip between s t imulus i n t e n s i t y and app l i ed f ie lds i s shown g r a p h i c a l l y i n f i g u r e 4.5. The change i n peak ampl i tude of the NI and P2 potent ia ls from the p r e v i o u s f i g u r e are shown for each of the three s t imulus ' i n t ens i t i e s . The c u r v e s for each i n t e n s i t y have the same c h a r a c t e r i s t i c s as those shown i n f i g u r e 4.3 for a s ing le s t imulus s t r e n g t h . I n each case the ampl i tudes of NI and P2 are enhanced b y pos i t ive g r a d i e n t s and dep re s sed b y negat ive g r a d i e n t s i n an approx imate ly l inea r f a sh ion . A l t h o u g h the slope of the NI and P2 c u r v e s for the s t r o n g s t imulus s t r e n g t h is lower than that for the o ther in t ens i t i e s i n th i s p a r t i c u l a r example, the r e l a t i onsh ip between s t imulus s t r e n g t h and slope was not r i g o r o u s l y i n v e s t i g a t e d . The c u r v e s for P l are not shown on the g r a p h s ince they were v i r t u a l l y flat t h roughou t the range of g r ad i en t s . 151 ]O.GJUiyE_JL4 illustrates the characteristics of the orthodromically evoked dentate potential at three different intensities during applied voltage gradients. Each column of responses (vertical) was evoked by the same stimulus intensity and each row (horizontal) was evoked during the same voltage gradient. The relative stimulus intensity is indicated across the top of the responses and the magnitude of the voltage gradient applied to the dentate gyrus is indicated to the left of each row. At all stimulus intensities the amplitudes of NI and P2, but not PI, were influenced by the voltage gradients. Orthodromic Population Spike At Three Intensities During Applied Fields LOW • M E D I U M HIGH 153 FIGURE 4.5 is a graph comparing the change in amplitude of NI and P2 of the orthodromically evoked dentate potential during different applied voltage gradients and different stimulus intensities. The data was derived from the responses shown in figure 4.4. Each point represents the peak amplitude of either NI or P2 plotted against the magnitude of the externally applied field. Different symbols are used to represent responses to stimuli of low (filled circle), medium (open circle) and high (filled diamond) intensity. Increasing the stimulus intensity shifted the initial appearance of the NI and P2 potentials to more negative levels of applied voltage gradient, but had a relatively minor influence on the relationship between voltage gradient and the amplitude of NI and P2. At each stimulus intensity the absolute amplitudes of NI and P2 are enhanced during positive gradients and depressed during negative gradients. The values for P l are not shown since they were virtually flat throughout the whole range of applied gradients. Evoked Potential Ampli tude During Applied Fields APPLIED FIELD (MV/MM) 155 The minimal in f luence of a p p l i e d vo l tage g r a d i e n t s on the ampl i tude of P l was a cons i s t en t and r a t h e r s t r i k i n g r e su l t . T h i s c h a r a c t e r i s t i c of the o r thodromic response i n the dentate g y r u s is c l e a r l y i l l u s t r a t e d i n f i g u r e 4.6. The same s t imulus i n t e n s i t y was used for a l l of the r e sponses . A l t h o u g h NI and P2 a re d ramat ica l ly enhanced b y pos i t i ve g r ad i en t s (bottom) the ampl i tude and shape of P l remains v i r t u a l l y u n c h a n g e d . Negat ive g r a d i e n t s (top) s l i g h t l y dep re s s the peak of P l bu t the r i s i n g edge of the r e sponse remains cons tant . E v o k e d poten t ia l s r e c o r d e d i n the molecular l a y e r of the dentate g y r u s were also i n f l uenced b y the app l i ca t i on of a r t i f i c i a l vol tage g r a d i e n t s . These changes c o r r e s p o n d e d to the p resence o r absence of NI on the somatic r esponse . Whenever the somatic response cons i s t ed of a P l po ten t i a l w i t h no N I , the e x t r a d e n d r i t i c response had the form of a smooth nega t ive po ten t ia l . On the o ther hand , i f NI was p resen t i n the c e l l l a y e r , a na r row pos i t i ve def lec t ion was super imposed on the o v e r a l l n e g a t i v i t y of the d e n d r i t i c r e sponse . As po in ted out i n the las t chap te r , t h i s pos i t i ve def lec t ion i s p r o b a b l y genera ted b y o u t w a r d c u r r e n t from the dend r i t e s of g r anu l e ce l l s d u r i n g ac t ion po ten t i a l genera t ion . It had the same gene ra l form whe the r the popu la t ion sp ike was i n d u c e d b y i n c r e a s i n g the s t imulus i n t e n s i t y or b y a p p l y i n g a pos i t i ve vo l tage g rad ien t . F i g u r e 4.7 shows an example of the changes i n the e x t r a d e n d r i t i c r e sponse d u r i n g app l i ca t i on of vo l tage g r ad i en t s . The extrasomatic r e sponses are i n c l u d e d to emphasize the r e l a t i onsh ip between the pos i t ive e x t r a d e n d r i t i c def lec t ion and the NI po ten t ia l . In th i s p a r t i c u l a r example a small popu la t ion s p i k e was p re sen t on the c o n t r o l r esponse (not shown) . D u r i n g app l i ca t i on of a nega t ive vo l tage g rad i en t (middle t races) NI fa i led to o c c u r and the e x t r a d e n d r i t i c response was a simple nega t ive po ten t ia l . 156 F I G U R E 4.6 f u r t h e r i l l u s t r a t e s the the inf luence of app l i ed vol tage g r a d i e n t s on the c h a r a c t e r i s t i c s of P I , NI and P2 of the o r thodromica l ly e v o k e d dentate response . TOP: supe r impos i t ion of a c o n t r o l response and two responses r eco rded d u r i n g app l i ca t ion of a negat ive vol tage g rad ien t . The rate of r i se of the po ten t ia l remains e s sen t i a l ly u n c h a n g e d and on ly a s l i g h t decrease i n the ampli tude of P I is o b s e r v e d . Note that the negat ive po ten t ia l fo l lowing P I is also enhanced . Bottom: supe r impos i t ion of the same c o n t r o l response and three responses r eco rded d u r i n g app l i ca t ion of a pos i t ive vo l tage g rad ien t . There i s a v e r y s t r i k i n g enhancement of NI and P2 but the r i se time and ampli tude of P I remain v i r t u a l l y u n c h a n g e d . 157 Orthodromic Population Spike During Applied Fields CONTROL > 3 MSEC 158 F I G U R E 4.7 compares the extradendritic to the extrasomatic potential orthodromically evoked in the dentate gyrus during applied voltage gradients. Three groups of evoked potentials are shown. In the left and middle group the potential recorded in the cell layer is superimposed on the response recorded under identical conditions in the middle of the molecular layer. The numbers above, indicate the magnitude of the applied voltage gradient (mV/mm). All of the potentials shown in the left two groups are superimposed in the group on the right to allow a more detailed comparison. Visual separation of the extrasomatic and extradendritic potentials is easiest in the middle group of records where the somatic response is predominantly positive and the dendritic response is negative. In the left and right groups, the initial rising edge of a given potential identifies its recording location as somatic (positive going) or dendritic (negative going). Orthodromic Population Spike During Applied Fields + 40 - 4 0 + 4 0 / -i 5 MSEC 160 D u r i n g app l i ca t i on of a pos i t i ve g r ad i en t (left t races) a l a rge NI po ten t ia l was p re sen t and the e x t r a d e n d r i t i c response deve loped a p rominent pos i t ive def lec t ion . When both e x t r a d e n d r i t i c r e sponses were super imposed ( r i gh t t races) i t became c lear that the pos i t i ve def lec t ion was ac tua l l y a p o s i t i v e -nega t ive complex. D u r i n g NI the complex was pos i t ive w i t h r e spec t to the smooth nega t ive po ten t ia l o b s e r v e d d u r i n g d e p r e s s i o n of the popu la t ion s p i k e , bu t t hen sh i f t ed to a r e l a t i v e l y nega t ive pos i t ion d u r i n g P2. A l so note the smooth supe r impos i t i on of the l e ad ing edges of both e x t r a d e n d r i t i c r e sponses even t h o u g h they were r e c o r d e d u n d e r the in f luence of oppos i te vo l tage g r a d i e n t s . I n gene ra l , the simple nega t ive po ten t i a l seen i n the absence of a popu la t ion s p i k e was o n l y minimal ly i n f luenced b y the app l i ed vo l tage g r a d i e n t s . I n th i s r e spec t the e x t r a d e n d r i t i c response was s imi la r to the P l po ten t ia l r e c o r d e d i n the c e l l l a y e r . B) A n t i d r o m S u r p r i s i n g l y , an t i d romica l l y evoked potent ia ls i n the dentate g y r u s were also modif ied b y the p resence of ex t e rna l l y app l i ed vo l tage g r ad i en t s . The in f luence was s imi lar to that o b s e r v e d on the o r thodromic popu la t ion po ten t ia l . Pos i t i ve g r a d i e n t s enhanced and nega t ive g r a d i e n t s dep re s sed the ampl i tude of N I . Th i s r e s u l t i s i l l u s t r a t e d i n f i g u r e 4.8 u s i n g two d i f fe ren t s t imulus in t ens i t i e s (medium and low). In add i t i on to i n c r e a s i n g the ampl i tude , pos i t i ve g r a d i e n t s also decreased the peak l a t ency of the response . T h i s in f luence on l a t ency was oppos i te to that o b s e r v e d as s t imulus i n t e n s i t y was i n c r e a s e d . As seen i n th i s f i g u r e , the peak l a t ency is g rea te r fo l lowing a medium i n t e n s i t y s t imulus even t hough the ampli tude of the r e sponse i s s u b s t a n t i a l l y i n c r e a s e d . 161 F I G U R E _ 4.8 i l l u s t r a t e s the in f luence of app l i ed vol tage g rad ien t s on the an t i d romica l l y evoked dentate response . The six super imposed sweeps shown in the f i gu re were a l l r e c o r d e d from the same locat ion i n the g ranu le ce l l l aye r . Responses were e v o k e d u s i n g e i ther a low o r medium s t imulus i n t e n s i t y u n d e r one of th ree magnitudes of app l i ed vol tage g rad ien t (mV/mm). The ampl i tude of the response was enhanced by a pos i t ive g r ad i en t and dep re s sed b y a negat ive g rad ien t at both s t imulus in tens i t i e s . 162 Antidromic Populat ion Spike During Applied Fields 163 F I G U R E 4.9 f u r t h e r cha rac t e r i ze s the in f luence of app l i ed vol tage g rad i en t s on the an t i d romica l l y evoked dentate response . The i n s e r t shows f ive super imposed an t idromic responses r e c o r d e d i n the g r anu l e c e l l l aye r u s i n g the same s t imulus i n t e n s i t y , bu t d u r i n g d i f fe ren t magni tudes of vol tage g rad ien t . Each point on the g r a p h shows the peak n e g a t i v i t y of the an t idromic potent ia l (mV) p lo t ted aga ins t the c o r r e s p o n d i n g l eve l of app l i ed g rad ien t (mV/mm). Antidromic Potential Amplitude During Applied Fields •60 -40 -20 0 20 40 APPLIED FIELD (MV/MM) 80 100 165 The r e l a t i onsh ip between the magni tude of app l i ed vol tage g rad i en t a n d the ampl i tude of the an t id romic popu la t ion response is shown i n f i g u r e 4.9. The re is an approximate ly l inea r inc rease i n response ampl i tude as the g r a d i e n t i s sh i f ted from negat ive towards more pos i t i ve l eve l s . The appa ren t f l a t t en ing of the c u r v e ove r the extreme nega t ive r ange of g r a d i e n t s was also o b s e r v e d i n o ther examples bu t was not sys temat ica l ly i n v e s t i g a t e d . C) M u l t i - s p i k e a n d B u r s t D i s c h a r g e U n d e r normal cond i t ions i n the dentate g y r u s e v e n a v e r y in tense s t imulus w i l l evoke o n l y a s ing le popu la t ion s p i k e pe r r e sponse . However , on s e v e r a l occas ions i n the p re sen t s t u d y , a s t imulus app l i ed d u r i n g a pos i t i ve vol tage g r a d i e n t evoked a second popu la t ion s p i k e on the f a l l i n g edge of the P2 po ten t ia l . When th i s o c c u r r e d , the app l i ed g rad i en t was t u r n e d off and at tempts were made to generate a " secondary popu la t ion s p i k e " b y g r e a t l y i n c r e a s i n g the s t imulus s t r e n g t h . I n a l l cases the h i g h s t imulus i n t e n s i t y alone was not adequate to i nduce mul t ip le sp ike d i s c h a r g e . U n f o r t u n a t e l y the i n f r equen t o c c u r r e n c e of s econda ry popu la t ion s p i k e s i n dentate g y r u s d i d not allow f u r t h e r c h a r a c t e r i z a t i o n of th i s phenomenon. Most of the s tud ies i n v o l v i n g app l i ed vo l tage g rad i en t s were c a r r i e d out i n the dentate g y r u s to avo id the compl ica t ions of the o p p o s i n g d e n d r i t i c t rees on C A l p y r a m i d a l ce l l s (see i n t r o d u c t i o n of th i s chap te r ) . In the small number of t r i a l s c a r r i e d out i n C A l the app l i ed vo l tage g r a d i e n t s had a marked effect on the prominence of the NI and P2 po ten t i a l i n a s imi lar f a sh ion to that o b s e r v e d i n the dentate g y r u s . A r e l a t i v e l y pos i t i ve ex t r ace l lu l a r po ten t ia l i n the r e g i o n of the ap i ca l 166 d e n d r i t e s enhanced NI and P2 and r e v e r s i n g the g r a d i e n t had the opposi te effect . T h u s i t appea red that the ap i ca l d e n d r i t e was more s ens i t i ve to the a p p l i e d g r a d i e n t s s ince the expected in f luence of the g r a d i e n t s on the basa l d e n d r i t e s i s opposi te to that a c t u a l l y o b s e r v e d . U n l i k e the dentate g y r u s , mul t ip le popu la t ion s p i k e s were f r e q u e n t l y o b s e r v e d i n C A l d u r i n g the app l i ca t i on of pos i t i ve vo l tage g r ad i en t s . F u r t h e r m o r e , h i g h i n t e n s i t y s t imula t ion alone cou ld i nduce secondar ies i n a few of these cases . A p a r t i c u l a r l y s t r i k i n g m u l t i - s p i k e d i s c h a r g e was o b s e r v e d s e v e r a l times whi le a p p l y i n g vol tage g r a d i e n t s to C A l . T h i s a c t i v i t y wou ld b e g i n spon taneous ly as the app l i ed vol tage g r a d i e n t was i nc rea sed i n the pos i t ive d i r e c t i o n and con t inued u n t i l the g r a d i e n t was removed . There was no need to s t imulate C A l to s t a r t o r to mainta in these con t inuous b u r s t s of a c t i v i t y . A n example i s shown i n f i g u r e 4.10. The top two t races show the a c t i v i t y r e c o r d e d i n the p y r a m i d a l l a y e r d u r i n g a two second p e r i o d . In each case the app l i ed vo l tage g r a d i e n t was s lowly i n c r e a s e d to minus 60 mV/mm (top) o r p l u s 60 mV/mm (middle) , he ld cons tan t for about 1 second and then s lowly r e t u r n e d to ze ro . The basel ine shi f t seen on each t race i s s imply the change i n extrasomatic po ten t ia l i n d u c e d b y the app l i ed vo l tage g rad i en t . A l t h o u g h the magni tude and p o l a r i t y of th i s sh i f t a re dependent on the placement of the c u r r e n t p a s s i n g e lec t rodes i n a n y p a r t i c u l a r exper iment , i t i s s t i l l u se fu l as an i nd i ca to r of the r e l a t i ve vo l tage g r a d i e n t a p p l i e d to the neu rona l t i s sue . No spontaneous a c t i v i t y o c c u r r e d d u r i n g app l i ca t i on of a negat ive vo l tage g r a d i e n t (top) bu t a b u r s t of popu la t ion potent ia ls o c c u r r e d as the g r a d i e n t was sh i f t ed i n the pos i t i ve d i r e c t i o n (middle) . The a c t i v i t y con t inued u n t i l the g r ad i en t was r e t u r n e d towards zero . In o the r exper iments the b u r s t was maintained for more than t h i r t y seconds w i th no 167 FIGURE 4.10 shows a b u r s t of popula t ion responses r e c o r d e d i n the ce l l l a y e r of C A l d u r i n g an ex te rna l ly app l i ed vol tage g rad ien t . The top two t races were r e c o r d e d on a v e r y slow time base d u r i n g the g r a d u a l onset and offset of a vo l tage g rad ien t (2 seconds pe r sweep). D u r i n g the i n i t i a l f lat p o r t i o n of each t race the vol tage g rad ien t was zero . The g rad i en t was then s lowly i nc r ea sed by hand to a l eve l of about 60 mV/mm, held cons tan t for a b r i e f pe r iod and then s lowly r e t u r n e d to a zero l e v e l . A p p l i c a t i o n of a pos i t i ve g r ad i en t i n d u c e d a b u r s t of popula t ion potent ia ls (middle trace) whereas a nega t ive g r a d i e n t fa i led to generate a n y spontaneous a c t i v i t y (upper t race) . The bottom pa i r of t races are expanded por t ions of the b u r s t of popu la t ion re sponses seen on the middle t race . Note that the p o l a r i t y of these g r a d i e n t s were named wi th r e spec t to the ap ica l d e n d r i t i c a r b o r i z a t i o n . For example, a pos i t ive g r a d i e n t p r o d u c e d a re la t ive p o s i t i v i t y i n the ex t r ace l l u l a r space s u r r o u n d i n g the ap i ca l d e n d r i t e s and a r e l a t ive n e g a t i v i t y i n the r e g i o n of the somata and basal dend r i t e s . 168 Synchronous Discharge During Applied Fields 169 appa ren t damage to the neu rona l t i s sue . When the b u r s t was d i s p l a y e d on an expanded time scale (bottom) each event appeared to have a form s imilar to an an t id romic popu la t ion s p i k e . The p r e v i o u s chap te r e s t ab l i shed the p resence of l a rge vol tage g r a d i e n t s ac ros s the axis of both C A l and the dentate g y r u s d u r i n g evoked potent ia ls i n the h ippocampal format ion. The r e s u l t s i n the p resen t chap te r ind ica te that ex t e rna l l y app l i ed vo l tage g r ad i en t s i n the same range of magni tudes can p r o f o u n d l y in f luence the s e n s i t i v i t y of h ippocampal neurons to both a n t i and o r thodromic s t imu l i . A) Orthqdro The p re sen t r e s u l t s s u p p o r t the f i n d i n g s b y o the r s that an ex t r ace l l u l a r vo l tage g r a d i e n t app l i ed a long the dendro-somat ic axis of neurons w i t h i n a c o r t i c a l r e g i o n can lead to an a l t e red l e v e l of e x c i t a b i l i t y i n the u n d e r l y i n g neu rona l popu la t ion (Bawin et a l , 1986; Bindman et a l , 1964; Chan and Nicho l son , 1986; Denny and B r o o k h a r t , 1962; J e f f e ry s 1981). The major in f luence of ex t e rna l l y a p p l i e d vo l tage g r a d i e n t s was on the ampl i tude of the NI and P2 poten t ia l s . In con t ras t , the P I po ten t ia l was o n l y s l i g h t l y a l t e red e v e n d u r i n g the s t ronges t g r ad i en t s . A l t h o u g h not s p e c i f i c a l l y s ta ted i n the f i n d i n g s of J e f f e ry s (1981) and Bawin et a l (1986), c lose examinat ion of f i g u r e s i n these pub l i ca t i ons also sugges t s that app l i ed g r a d i e n t s p redominan t ly in f luence NI and P2. T h i s p a t t e r n of s e n s i t i v i t y sugges t s that app l i ed f ie lds in f luence the number of a c t i v e l y d i s c h a r g i n g ce l l s w i t h i n a popu la t ion wi thout a l t e r i n g 170 s y n a p t i c d r i v e . The f a i lu re of app l i ed g rad i en t s to in f luence the r i s i n g edge of the e x t r a d e n d r i t i c potent ia l f u r t h e r s u p p o r t s th i s c o n c l u s i o n . It may seem that the add i t i ona l i n t r a d e n d r i t i c c u r r e n t i n d u c e d b y a vo l tage g rad i en t shou ld add to s y n a p t i c c u r r e n t f lowing from the d e n d r i t e s to the soma, and as a r e su l t , in f luence the extrasomatic po ten t ia l . The appa ren t i n s e n s i t i v i t y of the s y n a p t i c component of the response i s based on the s teady state na tu re of the app l i ed f i e l d . S ince the flow of c u r r e n t associa ted w i t h the f i e ld i s con t inuous t h r o u g h o u t the r e sponse , a n y d i r e c t in f luence i t may have on the ex t r ace l lu l a r po ten t ia l w i l l a lso be con t inuous . A s a r e su l t , o n l y the c u r r e n t s d i r e c t l y associa ted w i t h t r ans i en t even t s , s u c h as s y n a p t i c a c t i v i t y and ac t ion po ten t ia l genera t ion , shou ld in f luence the shape of the evoked response . In o ther words the c h a r a c t e r i s t i c s of P l , NI and P2 i nd ica te the r e l a t i ye c o n t r i b u t i o n of s y n a p t i c and ac t ion po ten t i a l c u r r e n t s whereas c u r r e n t s d i r e c t l y re la ted to the app l i ed f ie ld are los t w i t h i n the s teady state shi f t of the ex t r ace l lu l a r po ten t ia l . B) A n U The s e n s i t i v i t y of the an t id romic popu la t ion s p i k e to ex te rna l ly app l i ed f i e ld po ten t ia l s i s an i n t r i g u i n g r e su l t . It impl ies that at least a p o r t i o n of the neu rona l d i s c h a r g e c o n t r i b u t i n g to the popu la t ion s p i k e may not be an t id romic i n the c l a s s i ca l sense. A t least some of the ce l l s may be d i s c h a r g e d as a d i r e c t r e s u l t of t r ans i en t ephapt ic c u r r e n t s genera ted b y the s t r o n g vol tage g r a d i e n t s p resen t d u r i n g the evoked po ten t ia l . If th i s i s so, t hen a change i n the r e s t i n g membrane po ten t ia l i n d u c e d b y an ex t e rna l l y app l i ed f i e ld wou ld a l te r the p o r t i o n of ce l l s ephap t i ca l l y d i s c h a r g e d l e a d i n g to a change i n ampl i tude of the response . Those 171 neu rons d i r e c t l y i n v a d e d b y an te rog rade ac t ion potent ia ls wou ld cont inue to d i s c h a r g e i r r e s p e c t i v e of the app l i ed g rad ien t . C e r t a i n l y , one must also cons ide r the p o s s i b i l i t y that c h a n g i n g the membrane po ten t i a l may ac tua l ly b lock a t r ue an t id romic ac t ion po ten t i a l i n the axon before i t i n v a d e s the soma. T h i s p o s s i b i l i t y seems p a r t i c u l a r l y reasonable when c o n s i d e r i n g v e r y l a rge sh i f t s i n membrane poten t ia l . However i n the next chap te r i t becomes c lear that e p h a p t i c a l l y i n d u c e d changes i n membrane po ten t i a l a re o n l y i n the o r d e r of a few mi l l i vo l t s . S e v e r a l o ther o b s e r v a t i o n s also sugges t that a component of the an t id romic popu la t ion s p i k e is a d i r e c t r e s u l t of ephapt ic i n t e r ac t ions . 1) E v e n s t r o n g nega t ive vo l tage g r a d i e n t s fa i led to complete ly s u p p r e s s an t id romic potent ia l s . T h i s sugges t s that at least a p o r t i o n of the neu rona l d i s c h a r g e s were i n s e n s i t i v e to sh i f t s i n membrane po ten t i a l . I n con t r a s t , o r thodromic popu la t ion s p i k e s were ea s i ly s u p p r e s s e d b y l a rge g r a d i e n t s . 2) I n t r a c e l l u l a r r e c o r d i n g s d u r i n g an t id romic s t imula t ion of p y r a m i d a l neu rons ind ica te that s h i f t i n g the membrane po ten t ia l as l i t t l e as one mi l l ivo l t w i th c u r r e n t i n j ec t ion can sometimes b lock an an t id romic ac t ion po ten t ia l . I n o the r ce l l s even s t r o n g h y p e r p o l a r i z i n g c u r r e n t s f a i l to b lock the d i s c h a r g e 3) I n the fo l lowing chap te r i n t r a c e l l u l a r r e co rds from p y r a m i d a l ce l l s show c lea r examples of ac t ion potent ia ls d i r e c t l y ac t iva ted b y the ephap t ic in f luence of an an t id romic popu la t ion s p i k e . (also see T a y l o r and Dudek 1984a,b; Yim et a l 1986). CJMulti-spike The con t inuous b u r s t s of popula t ion potent ia ls i n d u c e d b y ex t e rna l l y app l i ed vo l tage g r a d i e n t s i n C A l were s t r i k i n g l y s imi lar to low calc ium b u r s t s . However , the ep i lep t i fo rm a c t i v i t y o c c u r r e d i n normal s l ices wi th 172 no r equ i r emen t for e l ec t ro ly te o r pharmacologic manipula t ions of the p e r f u s i o n media. F u r t h e r m o r e the d i s c h a r g e cou ld be t u r n e d on and off almost i n s t a n t l y b y a d j u s t i n g the l e v e l of app l i ed vo l tage g rad i en t . The b a c k g r o u n d l e v e l of spontaneous s ing le u n i t a c t i v i t y i n C A l of ten i nc rea sed d u r i n g app l i ca t i on of a pos i t i ve g r ad i en t and was p a r t i c u l a r l y prominent immediately before onset of the b u r s t a c t i v i t y . The next chap te r i nves t i ga t e s a poss ib le mechanism l ead ing to s y n c h r o n i z a t i o n of these ac t ion po ten t ia l s . DJ CqnclusM The r e s u l t s p r e sen t ed i n th i s chap te r sugges t that vo l tage g r a d i e n t s app l i ed d u r i n g o r thodromic s t imula t ion of the dentate g y r u s can a l te r the p r o p o r t i o n of g r anu l e ce l l s r e s p o n d i n g w i t h an ac t ion po ten t i a l wi thout a l t e r i n g the s y n a p t i c d r i v e associa ted w i th the s t imulus . T h i s i n f luence on n e u r o n a l e x c i t a b i l i t y i s far from sub t le . B y a p p l y i n g vo l tage g r a d i e n t s i n the r ange o b s e r v e d d u r i n g evoked potent ia ls i t was poss ib le to a l t e r the c h a r a c t e r i s t i c s of the response from that equ iva l en t to a s u b t h r e s h o l d s t imulus to a s t imulus p r o d u c i n g a near maximal popu la t ion s p i k e . Therefore i t seems l i k e l y that the ex t r ace l lu l a r f i e lds genera ted d u r i n g e v o k e d poten t ia l s w i l l a lso have a s u b s t a n t i a l in f luence on the behav io r of the v e r y neurons r e spons ib l e for g e n e r a t i n g the f i e ld i n the f i r s t p lace . A l t h o u g h s u c h r e c u r s i v e i n t e r ac t i ons are bound to be complex and d i f f i c u l t to d i s sec t from more conven t iona l phenomena, t hey cou ld p r o f o u n d l y in f luence the dynamic behav io r of a neu rona l popu la t ion . F o r th i s reason the fo l lowing chap te r at tempts to d i r e c t l y measure ephapt ic in f luences on the membrane potent ia l of i n d i v i d u a l neu rons u s i n g i n t r a c e l l u l a r r e c o r d i n g t echn iques . 173 CHAPTER 5:_TO This chapter investigates the role of ephaptic interactions within the hippocampal formation at the single cell level. The objective is to measure as precisely as possible the specific potentials influencing voltage dependent processes of an individual neuron during an evoked response. These measurements should help characterize the ephaptic influence of an evoked potential, and show to what degree this influence can modify the electrical behavior of a neuron. A resurgence of interest in ephaptic interactions in the hippocampal formation followed the discovery of low calcium bursting (Jefferys and Haas, 1982; Taylor and Dudek 1982). These studies focused on ephaptic interactions under conditions of synaptic blockade (Taylor and Dudek 1984ab). By avoiding situations where synaptic activity participates in a response these investigators simplified interpretation of their results. However i t made it more difficult to relate their findings to physiological conditions where synaptic transmission is often important. The present study extends the intracellular study of ephaptic interactions to include experimental protocols where synaptic function is intact. It provides evidence for both inhibitory and excitatory ephaptic interactions during a single orthodromically evoked response and shows how these interactions can modify the discharge characteristics of a neuronal population. The findings support the hypothesis that ephaptic interactions play a crucial role in recruitment and synchronization of epileptiform activity. 174 The t ransmembrane po ten t i a l (TMP) i s the dominant fac tor c o n t r o l l i n g the e l e c t r i c a l behav io r of a n e u r o n and modif icat ion of the T M P b y the p resence of ex t r ace l l u l a r f i e ld po ten t ia l s i s the bas is for ephap t ic i n t e r a c t i o n s . There fo re i t i s impor tan t to c l a r i f y what i s meant b y the term T M P and to show how i t can be measured . S imp ly s ta ted , the TMP i s the vo l tage expres sed ac ros s a neu rona l membrane. A t r a d i t i o n a l i n t r a c e l l u l a r r e c o r d i n g p r o v i d e s a good approx imat ion of the TMP d u r i n g r e s t i n g cond i t ions bu t i s inadequate i n the p resence of an ex t r ace l l u l a r f i e l d po ten t i a l . T h i s p rob lem i s i l l u s t r a t e d i n f i g u r e 5.1. A s t a n d a r d i n t r a c e l l u l a r e lec t rode i s shown p e n e t r a t i n g the soma of a p y r a m i d a l n e u r o n and an E P S P r e c o r d e d b y t h i s e lec t rode i s shown above . It i s impor tan t to rea l i ze that the vo l tage r e c o r d e d b y the e lec t rode i s " r e f e r r e d " to a d i s t an t g r o u n d , u s u a l l y a s i l v e r w i r e i n the medium of a s l ice chamber or the s k i n on an in tac t animal . T h i s d i s t an t po in t i s assumed to have the same po ten t i a l as the ex t r ace l lu l a r space i n the immediate v i c i n i t y of the c e l l . Often t h i s i s t rue . I n the example shown, i f no f i e ld po ten t ia l s are p re sen t i n the ex t r ace l l u l a r space, the " g r o u n d r e f e r e n c e d " i n t r a c e l l u l a r po ten t i a l w i l l be v i r t u a l l y the same as the T M P . On the o ther hand , i f t h i s c e l l i s o n l y one n e u r o n w i t h i n a popu la t i on u n d e r g o i n g o r thodromic ac t i va t i on , a l a rge ex t r ace l l u l a r po ten t ia l i s p r o b a b l y p r e s e n t i n the ex t r ace l lu l a r env i ronment . T h i s vo l tage w i l l modify the TMP but w i l l not be de tec ted b y the r e c o r d i n g e lec t rode . F i g u r e 5.2 shows the same i n t r a c e l l u l a r e lec t rode and E P S P (top) bu t t h i s time a second e lec t rode is i n the immediate ex t r ace l lu l a r space of the ce l l (middle) . T h i s e lec t rode i s also g r o u n d re fe renced and o n l y detects the d i f fe rence between the vo l tage at i t s t i p and that at the d i s t an t 175 F l | G J J j R E _ 5 J L i s a d iagram showing the normal a r rangement for r e c o r d i n g i n t r a c e l l u l a r potent ia ls i n e l e c t r o p h y s i o l o g i c a l exper iments . A n e lect rode is r e c o r d i n g the po ten t ia l from one of three neu rons w i t h i n the confines of a c e l l l aye r , s u c h as that found i n C A l . The po ten t ia l i s measured w i th r e spec t to a d i s t an t g r o u n d , i nd i ca t ed b y the symbol to the r i g h t . Th i s reference po in t is u s u a l l y a wi re p laced i n the f l u i d of the r e c o r d i n g chamber. A n example of an E P S P r e c o r d e d from w i t h i n a p y r a m i d a l neu ron , u s i n g th is c o n f i g u r a t i o n , is shown at the top of the f i g u r e . S u c h r e c o r d i n g s o n l y ind ica te how the i n t r a c e l l u l a r po ten t ia l d i f f e r s from that i n the bath whereas the c e l l membrane of the soma i s exposed to the local ex t r ace l lu l a r po ten t ia l . G R O U N D R E F E R E N C E D  P O T E N T I A L S 177 FIGURE 5.2 shows a two e lec t rode a r rangement for r e c o r d i n g the t ransmembrane po ten t ia l . On the left , a g r o u n d reference electrode has pene t ra t ed a schematic C A l p y r a m i d a l ce l l and a second g r o u n d re fe renced e lec t rode i s i n i ts immediate ex t r ace l lu l a r env i ronment . To the r i g h t are samples of r ea l potent ia ls r e co rded b y two s i m i l a r l y p laced e lec t rodes d u r i n g an o r t h o d r o m i c a l l y evoked response . The t ransmembrane po ten t ia l (lowest trace) is then ca lcu la ted b y s u b t r a c t i n g the ex t race l lu la r from the i n t r a c e l l u l a r r e c o r d . Note that a l l three r e c o r d s are d r a w n to the same scale . 179 ground. The difference in potential between the two electrodes is the TMP, the actual voltage drop across the neuronal membrane. Note that the TMP (bottom) is markedly different from either the ground referenced intracellular or extracellular potential. It may not be obvious to the casual observer that the TMP is the dominant factor controlling the electrical behavior of a neuron. Figure 5.3 attempts to put this concept into perspective. A highly schematic neuronal membrane is shown dividing the left and right halves of the figure. The membrane has an inner surface in contact with the intracellular space and an outer surface in contact with the extracellular space. Ionic channels within the substance of the membrane are drawn as dot filled blobs. The voltage dependent properties of these channels ultimately control ionic conductances, action potential threshold and most of the other functional properties of the neuron. The voltage controlling these channels is not the absolute voltage on either surface of the membrane but rather the voltage drop from one surface to the other. In this example, the intracellular potential is minus 60 mV and the extracellular potential is minus 10 mV. This pattern could represent an inactive cell exposed to an antidromic population spike. In this case the TMP, the difference between the intra and extracellular potential, is minus 50 mV. Note that a negative extracellular potential actually represents a depolarizing influence on the cell. 5.2 SPECIFIC METHODS During the course of the experiments two different methods were employed to measure the TMP. One method used a single electrode but required two separate phases of data collection. First, a stable 180 F I G U R E 5 . 3 i l l u s t r a t e s the concept of t ransmembrane po ten t ia l (TMP) A h i g h l y diagrammatic r eg ion of ce l l membrane i s seen i n the center of the f i g u r e . The two v e r t i c a l l ines form the i n n e r (left) and outer ( r igh t ) sur face of the membrane. The la rge o v a l s t r u c t u r e s w i t h i n the membrane r ep re sen t v a r i o u s vol tage dependan t ion ic channe ls r e spons ib l e for the e l ec t r i ca l c h a r a c t e r i s t i c s of the neu ron . A s t anda rd g r o u n d re fe renced electrode is measur ing an i n t r a c e l l u l a r po ten t ia l of -60 mV (Vi) , and at the same ins tan t a second g r o u n d r e fe renced e lec t rode is measu r ing an ex t race l lu la r po ten t ia l of -10 raV (Ve). The po ten t ia l genera ted ac ross the vol tage sens i t ive channe l s i n the membrane is the vo l tage d i f fe rence between the ins ide and ou t s ide of the c e l l ( V i - V e ) , i n th i s case 50 mV. Th i s " t ransmembrane po ten t i a l " u l t imate ly in f luences the e l e c t r i c a l behav io r of the neuron . 181 T R A N S M E M B R A N E POTENTIAL (TMP) VOLTAGE DEPENDENT CHANNELS TMP=Vi-Ve=-50 mv 182 in t rasomat ic r e c o r d i n g was ob ta ined u s i n g a s t a n d a r d g r o u n d re fe renced i n t r a c e l l u l a r e lec t rode . A number of i n t r a c e l l u l a r r e sponses were then e v o k e d u s i n g a p rede f ined sequence of s t imulus parameters . Each s t imulus was repea ted at least fou r times to al low for s i g n a l a v e r a g i n g and the r e sponses were s t o r e d on l ine for la te r use . Fo l lowing co l l ec t ion of the i n t r a c e l l u l a r da ta the e lec t rode was s lowly w i t h d r a w n from the c e l l and then r ep laced to the o r i g i n a l dep th . At t h i s po in t the e lec t rode was i n the ex t r ace l lu l a r space v e r y near i t s o r i g i n a l pos i t i on . A ser ies of ex t r ace l l u l a r r e c o r d i n g s was made from th i s loca t ion u s i n g the i d e n t i c a l sequence of s t imulus parameters used to evoke the i n t r a c e l l u l a r r e sponses . The data a n a l y s i s p r o g r a m on the computer t hen s u b t r a c t e d the ex t r a from the i n t r a c e l l u l a r po ten t ia l s to genera te the c o r r e s p o n d i n g TMP r e c o r d s . The second method u s e d a pa i r of e lec t rodes to r e c o r d the e x t r a -and i n t r a c e l l u l a r potent ia ls s imul taneous ly . T h i s a r rangement had the advantage that d i f f e r en t i a l ampl i f ica t ion of the two potent ia ls p r o v i d e d a r ea l time r e c o r d of T M P . In p rac t i ce , a s table i n t r a c e l l u l a r r e c o r d i n g was f i r s t a c h i e v e d and then the ex t r ace l l u l a r e lec t rode was pos i t ioned i n the immediate v i c i n i t y of the n e u r o n b y use of a manipula tor w i th a p a i r of pos i t i on con t ro l s . F o l l o w i n g data co l l ec t ion the i n t r a c e l l u l a r e lec t rode was w i t h d r a w n and the ex t r ace l l u l a r potent ia ls r e c o r d e d w i t h th i s e lec t rode were compared to those of the second e lec t rode . The p r e v i o u s l y co l lec ted TMPs were accepted o n l y i f d i f f e r en t i a l ampl i f ica t ion of the two e lec t rodes p r o d u c e d vol tage def lec t ions approx ima t ing b io log ica l noise . U n f o r t u n a t e l y , d i f f i cu l t i e s associa ted w i th the p r a c t i c a l app l i c a t i on of the two e lec t rode t echn ique were often p r o h i b i t i v e . F o r ins tance , movement of the second e lec t rode t h r o u g h the s l ice of ten d i s r u p t e d the i n t r a c e l l u l a r r e c o r d i n g , l e a d i n g to loss of the c e l l . F u r t h e r m o r e , a t t a in ing close 183 approx imat ion between the two e lec t rodes was d i f f i c u l t and even small v a r i a t i o n s i n pos i t i on cou ld a l te r the c h a r a c t e r i s t i c s of the response . A more sub t le bu t equa l l y se r ious source of e r r o r was i n t r o d u c e d b y poss ib le d i f fe rences i n the phase and g a i n c h a r a c t e r i s t i c s of the i n t r a and ex t r ace l l u l a r r e c o r d i n g sys tems. T h i s p rob lem was minimized b y u s i n g a matched pa i r of ampl i f ie r s (WPI M707) and choos ing a p a i r of e lec t rodes w i t h s imi lar t ip impedances . I n con t ra s t , the s ing le e lec t rode t echn ique p r o v i d e d re l i ab le i n t r a c e l l u l a r r e c o r d i n g s and al lowed p r ec i s e placement of the ex t r ace l lu l a r e lec t rode . F u r t h e r m o r e i t complete ly avo ided the p rob lem of poor match ing between e lec t rodes and ampl i f i e r s . There fo re , the s ing le e lec t rode t echn ique was u s e d to ob ta in the major i ty of r e c o r d i n g s i n t h i s chap te r . 5 i 3 . . . . . R E S U L T S The f i e ld po ten t ia l ana ly s i s p re sen ted i n chap te r three d e s c r i b e d spec i f ic sequences of ex t r ace l l u l a r vo l tage g r a d i e n t s d u r i n g e v o k e d popu la t ion responses i n the h ippocampal format ion. That chap te r went on to p r e d i c t that ephapt ic i n t e rac t ions associa ted w i th these vol tage g r a d i e n t s shou ld dep re s s neu rona l d i s c h a r g e d u r i n g P l and P2 and enhance d i s c h a r g e d u r i n g N I . If t h i s hypo the s i s i s c o r r e c t , the TMP of p y r a m i d a l and g r anu l e ce l l s shou ld be modified b y the p resence of ex t r ace l l u l a r f i e ld s , and ac t ion po ten t i a l d i s c h a r g e shou ld be d i r e c t l y l i n k e d to depo la r i za t i on of the TMP r a the r than the g r o u n d re fe renced membrane po ten t ia l . The p r e sen t chap te r examines these pos s ib i l i t i e s b y s t u d y i n g the r e l a t i onsh ip between g r o u n d re fe renced i n t r a c e l l u l a r po ten t ia l , T M P , ex t r ace l lu l a r po ten t ia l , and ac t ion po ten t i a l d i s c h a r g e . 184 The r e s u l t s were ob ta ined from 63 C A l p y r a m i d a l and 15 dentate g r a n u l e ce l l s a l l meet ing the acceptance c r i t e r i a ou t l i ned i n chap te r 2. The smal ler number of r e c o r d i n g s from g ranu l e ce l l s re f lec t s the r e l a t ive d i f f i c u l t y i n o b t a i n i n g stable pene t ra t ions i n the dentate g y r u s . E v e n so, a n adequate number of r e c o r d s from g ranu le ce l l s are i n c l u d e d i n the s t u d y for compar i son w i t h the C A l r e s u l t s and for compar i son w i t h the r e s u l t s of the p r e v i o u s chap te r s . A) C A l - j D r ^ Low i n t e n s i t y o r thodromic s t imula t ion of p y r a m i d a l neu rons genera ted a g r a d e d E P S P o r E P S P / I P S P sequence and h i g h e r i n t ens i t i e s also evoked a n ac t ion po ten t ia l . The ex t r ace l lu l a r po ten t i a l assoc ia ted w i t h a j u s t t h r e s h o l d s t imulus v a r i e d w i d e l y from c e l l to c e l l . A P I po ten t ia l was a lways p re sen t bu t the NI and P2 poten t ia l s r a n g e d from neg l ig ib l e to near maximal. Excep t for a few extreme cases , the major i ty of neu rons reached t h r e s h o l d when the popu la t ion s p i k e was between 2 and 7 mV i n ampl i tude . T h i s v a r i a t i o n i n t h r e s h o l d p r e s u m a b l y accounts for the g r a d e d na tu re of the popu la t ion s p i k e . A more de ta i led a n a l y s i s of the r e l a t i onsh ip between i n t r a - and ex t r ace l l u l a r r e sponses to t h r e s h o l d s t imula t ion was p a r t i c u l a r l y i l l u m i n a t i n g . F i r s t , cons ide r the case where an impaled c e l l has a low t h r e s h o l d r e l a t i ve to the res t of the popu la t ion . S ince v e r y few o the r ce l l s are d i s c h a r g e d b y the s t imulus , the ex t r ace l lu l a r f i e ld w i l l cons i s t of o n l y a P I po ten t i a l . The i n t r a c e l l u l a r r e c o r d s from a c e l l of th i s t y p e are shown on the top t race of f i g u r e 5.4. J u s t s u b t h r e s h o l d s t imula t ion e v o k e d a smooth E P S P on the g r o u n d re fe renced r e c o r d and a low ampl i tude P I i n the ex t r ace l l u l a r space. On occas ions when response j i t t e r b r o u g h t the 185 I3GURE_ _5A compares the i n t r a c e l l u l a r , ex t r ace l lu l a r and t ransmembrane po ten t ia l of an o r thod romica l l y evoked response i n C A l . A: supe r imposed , g r o u n d re fe renced r e c o r d s of a sub and j u s t t h r e s h o l d i n t r a c e l l u l a r response wi th an ac t ion po ten t ia l a r i s i n g from the peak of the E P S P . In this ce l l the t h r e sho ld for ac t ion po ten t ia l genera t ion was below t h r e s h o l d for an NI po ten t ia l i n the s u r r o u n d i n g ex t r ace l lu l a r space. B: g r o u n d re fe renced r eco rds from another c e l l w i t h a t h r e s h o l d above that for genera t ion of an NI po ten t ia l . The sub and j u s t t h r e s h o l d i n t r a c e l l u l a r responses are super imposed a long wi th the ex t r ace l l u l a r po ten t ia l r e c o r d e d immediately af ter w i t h d r a w a l from the c e l l . The s u b t h r e s h o l d and ex t r ace l lu l a r response were evoked u s i n g the same s t imulus i n t e n s i t y . C: s u b t h r e s h o l d t ransmembrane po ten t i a l ob ta ined by s u b t r a c t i n g the ex t r a from the s u b t h r e s h o l d i n t r a c e l l u l a r r e sponse . The a r r o w ind ica tes a b reak i n the r i s i n g phase of the t ransmembrane po ten t ia l equ iva l en t i n l a t ency to the f a l l i n g phase of the popu la t ion s p i k e . D: s u b t h r e s h o l d t ransmembrane potent ia l a n d g r o u n d re fe renced r e c o r d are super imposed for compar i son . 186 Orthodromic Ca1 - T r a n s m e m b r a n e Potent ia l A. Intra 187 E P S P to t h r e s h o l d , the ac t ion po ten t ia l a lways arose from the h ighes t po in t on the i n t r a c e l l u l a r r e c o r d . T h i s r e l a t i onsh ip between membrane po ten t ia l and s p i k e genera t ion i s reasonable for a n e u r o n w i th an absolu te t h r e s h o l d vo l t age . The in f luence of the small P I po ten t ia l on the TMP was minimal i n t h i s case. When the TMP and i n t r a c e l l u l a r response were compared , the TMP had a s l i g h t l y lower ampl i tude bu t a v e r y s imi lar waveform. T h u s the normal i n t r a c e l l u l a r method p r o v i d e d a reasonable measure of vo l tages i n f l u e n c i n g the a c t i v i t y th i s c e l l . S imi la r r e s u l t s were ob ta ined for o the r low t h r e s h o l d neu rons . D u r i n g the cour se of the s t u d y o n l y a small number of v e r y low t h r e s h o l d neu rons were impaled. The major i ty o n l y reached t h r e s h o l d af ter the s t imulus i n t e n s i t y was suf f i c ien t to genera te a popu la t ion s p i k e of 2 mV or more. The r e c o r d s shown i n B of f i g u r e 5.4 a re t y p i c a l of th i s g r o u p of ce l l s . A t j u s t s u b t h r e s h o l d s t imulus i n t e n s i t y the g r o u n d re fe renced i n t r a c e l l u l a r r e sponse cons i s t ed of an E P S P w i t h a d i s t i n c t "no t ch" on i t s r i s i n g edge. On occas ions when response j i t t e r d i s c h a r g e d the c e l l , an ac t ion po ten t ia l a rose from the no tch , not from the peak of the E P S P as expec ted . T h i s no tch had a po ten t i a l c l e a r l y below the peak of the s u b t h r e s h o l d E P S P and there fore below the apparen t t h r e s h o l d for the c e l l . The same s t imulus i n t e n s i t y e v o k e d a f u l l y deve loped popu la t ion po ten t ia l i n the ex t r ace l l u l a r space immediately s u r r o u n d i n g the c e l l . There was a s t r i k i n g co r r e spondence between the l a t ency of the i n t r a c e l l u l a r no tch and the f a l l i n g phase of the popu la t ion s p i k e . F u r t h e r m o r e , the no tch was o n l y p r e sen t at i n t ens i t i e s su f f i c ien t to evoke an NI po ten t ia l . I n a l l of the neurons s t u d i e d , the p resence of f u l l y deve loped popu la t ion po ten t i a l had a p r o f o u n d in f luence on the t ransmembrane po ten t ia l . I n f i g u r e 5.4 s u b t r a c t i o n of the e x t r a - from the i n t r a c e l l u l a r 188 poten t ia l s generate the TMP r e c o r d shown alone i n C, and super imposed o n the g r o u n d r e fe renced po ten t ia l i n D. The TMP had a s h o r t e r peak l a t e n c y , g rea te r peak vo l tage , and a fas te r i n i t i a l decay than the g r o u n d r e fe renced po ten t ia l . The peak depo la r i za t i on of t h i s TMP c o r r e s p o n d e d w i t h i n i t i a t i o n of the ac t ion po ten t i a l shown i n C. A more de ta i led examinat ion of the TMP shows that i t s r i s i n g edge had a n i n i t i a l slow component fol lowed b y a more r a p i d ra te of r i s e . The i n f l e c t i o n between the two components (arrow) o c c u r r e d at the same l a t ency a n d vol tage as the no tch o b s e r v e d on the g r o u n d r e fe renced response , a n d at a l a t ency c o r r e s p o n d i n g to the nega t ive go ing phase of the ex t r ace l l u l a r popu la t ion s p i k e . F i g u r e 5.5 f u r t h e r i l l u s t r a t e s the close r e l a t i o n s h i p between the i n t r a c e l l u l a r (A), ex t r ace l l u l a r (B) and t ransmembrane po ten t ia l s (C) u s i n g data from ano the r p y r a m i d a l n e u r o n . The super imposed r e c o r d s show the in f luence of i n c r e a s i n g s t imulus i n t ens i t i e s . A t the lowest i n t ens i t i e s no popu la t ion s p i k e was p r e sen t and the r i s i n g edge of the E P S P was smooth. A t h i g h e r i n t ens i t i e s the NI po ten t ia l o n the ex t r ace l l u l a r r esponse and the no tch on the r i s i n g edge of E P S P became v i s i b l e . A s the i n t e n s i t y was i n c r e a s e d , both the NI po ten t i a l and the no tch d i s p l a y e d a p a r a l l e l sh i f t i n l a t ency a n d a n inc rease i n ampl i tude . F i n a l l y at t h r e s h o l d an ac t ion po ten t i a l arose from the nega t ive no tch on the E P S P . The TMP for t h i s c e l l i s shown i n C. Fo l lowing the lowest in t ens i t i e s of s t imula t ion the smooth depo la r i za t ion of the TMP had a lower ampl i tude bu t o the rwise s imi la r waveform to the i n t r a c e l l u l a r r e sponses shown i n A . A s the s t imulus i n t e n s i t y was i nc rea sed an i n f l ec t i on appeared on the r i s i n g edge of the TMP and i t s peak l a t ency decreased . Th i s i n t e n s i t y c o r r e s p o n d e d to the appearance of the NI po ten t ia l i n the ex t r ace l lu l a r 189 F I G U R E 5.5 shows the in f luence of i n c r e a s i n g the o r thodromic s t imulus i n t e n s i t y on the t ransmembrane po ten t ia l r e co rded from C A l p y r a m i d a l neu rons . A : super imposed g r o u n d re fe renced i n t r a c e l l u l a r responses e v o k e d at i n c r e a s i n g s t imulus i n t ens i t i e s . A negat ive go ing no tch appear s on the r i s i n g edge of the E P S P on the t h i r d i n t e n s i t y and becomes more prominent as the s t imulus i n t e n s i t y is i nc r ea sed . The ac t ion po ten t i a l a r i se s from th i s no tch at the h ighes t i n t e n s i t y . B: ex t r ace l lu l a r popu la t ion poten t ia l s r e c o r d e d at the same s t imulus in tens i t i e s as i n A , immediately af ter w i t h d r a w from the c e l l . C: Transmembrane potent ia ls ob ta ined b y s u b t r a c t i n g the ex t r a from the i n t r a c e l l u l a r response . Note the appearance of a s h a r p t ransmembrane depo la r i za t ion w i th a l a t ency and ampl i tude that v a r i e s d i r e c t l y w i th the f a l l i n g phase of the ex t r ace l lu l a r popu la t ion s p i k e . The ac t ion po ten t i a l a r i s e s from the r i s i n g edge of one of these depo la r i za t ions . D: an example of super imposed t ransmembrane potent ia ls evoked at i n c r e a s i n g i n t ens i t i e s u s i n g the two e lec t rode t echn ique . The ac t ion po ten t ia l a r i se s from the t ip of a sha rp depo la r i za t ion . 190 O r t h o d r o m i c CA1 - T r a n s m e m b r a n e Po ten t i a l (at i n c r e a s i n g s t i m u l u s in tens i t i es ) 20 mV 10 msec D. I 5 msec 191 space. As the intensity was further increased, the amplitude and peak latency of the TMP displayed a parallel shift to that of the population spike. At these higher intensities it appeared as if the TMP had a depolarizing wave superimposed on a more gently contoured wave form. At the highest stimulus intensity an action potential arose from this depolarizing wave. The close relationship between the population spike, TMP and spike generation is illustrated by another pyramidal cell on the bottom of figure 5.5. Both the depolarizing wave of the TMP and the population spike displayed a parallel increase in amplitude as stimulus was increased, and at threshold, the action potential clearly arose from the peak depolarization of the TMP. Virtually all of the pyramidal cells with a moderate or high threshold showed a similar association between the depolarizing wave of the TMP and spike generation. .?)_.PA1.™ In order to investigate the possible dependence of the TMP depolarizing wave on synaptic transmission, the TMP, population potential and intracellular response were studied following antidromic activation. These experiments were performed in a medium modified to block all synaptic transmission in order to guarantee the quality of the antidromic responses. Two types of intracellular response were recorded from pyramidal neurons following antidromic stimulation. In a small number of cells the action potential arose directly from the baseline membrane potential. Hyperpolarization of these cells, by current injection, usually failed to block the antidromic response. In the second type of cell, the action 192 po ten t i a l a rose from w i t h i n a nega t ive "no t ch" on the membrane po ten t ia l . These ac t ion potent ia ls were ea s i ly b l o c k e d b y h y p e r p o l a r i z i n g the membrane po ten t i a l b y as l i t t l e as one o r two mi l l i vo l t s . Records from a c e l l s howing these c h a r a c t e r i s t i c s i s shown i n f i g u r e 5.6. The i n t r a c e l l u l a r r e sponses to s e v e r a l s t imulus i n t ens i t i e s are super imposed i n B . A nega t ive no tch deve loped as the s t imulus i n t e n s i t y was i nc rea sed and an an ac t ion po ten t i a l arose from w i t h i n t h i s no tch . The c o r r e s p o n d i n g ex t r ace l l u l a r r e c o r d s showed an an t id romic popu la t ion s p i k e w i t h a n i n c r e a s i n g l y nega t ive NI po ten t i a l (C). The no tch and the NI po ten t i a l had the same l a t ency and d i s p l a y e d a p a r a l l e l sh i f t i n ampl i tude i n a s imi lar f a sh ion to that of the o r thodromic r e sponse . The TMP potent ia l s genera ted d u r i n g the an t id romic r e sponse are shown i n D. A d e p o l a r i z i n g wave was seen a r i s i n g from the base l ine , and at t h r e s h o l d i n t e n s i t y an ac t ion po ten t i a l was in i t i a t ed from the peak of the wave . The l a t ency , ampl i tude , a n d d u r a t i o n of t h i s d e p o l a r i z i n g wave v a r i e d w i t h the c h a r a c t e r i s t i c s of the an t id romic popu la t ion s p i k e . C) The data p r e sen t ed so fa r s u p p o r t s e v e r a l impor tan t conc lus ions . A d i s c u s s i o n of these f i n d i n g s shou ld he lp ease the r e ade r ' s b u r d e n d u r i n g the r ema in ing sec t ions . TMP Versus Cell Discharge The close r e l a t i onsh ip between peak depo la r i za t ion of the TMP and ac t ion po ten t i a l d i s c h a r g e i n p y r a m i d a l ce l l s s u p p o r t s the hypo the s i s that e l e c t r i c a l behav io r of a n e u r o n i s d i r e c t l y i n f l uenced by the T M P . T h i s 193 FIGURE 5.6 shows the relationship between the transmembrane potential and action potential discharge during antidromic stimulation of C A l . A: orthodromically evoked intracellular response of a pyramidal cell before and 30 minutes after synaptic blockade in a medium containing 4 mM MnSO« and 0.8 mM CaCh. All further records in this figure where obtained during this synaptic blockade. B: ground referenced intracellular responses to antidromic stimulation at increasing intensities. Note the presence of the negative going potential immediately following the stimulus artifact. With increasing stimulus intensities this negative potential increases in size and finally, an action potential arises from its peak negativity. C: extracellular antidromic population spikes recorded using the same stimulus intensities as in B, immediately following withdrawal from the cell. D: transmembrane potentials obtained by subtracting the extra from intracellular responses. Note the prominent depolarizing deflection following the stimulus artifact and the action potential arising from its peak. The amplitude and latency of this depolarization varied directly with that of the population spike. 194 Antidromic CA1 — Transmembrane Potential (at increasing stimulus intensities) CONTROL POST 4mM M n + + INTRA B. r _JT0 mV 5 msec INTRA EXTRA D. 10 mV 5 msec 195 influence is not dependent on chemically mediated synaptic transmission and can discharge a cell in the absence of an underlying EPSP. Although the TMP may be the measurement of choice for intracellular studies, ground referenced potentials are usually recorded on the implicit assumption that they provide an adequate approximation of the TMP. This assumption is reasonable only in the absence of extracellular fields since the relationship between standard intracellular recordings and the TMP is distorted by evoked potentials in the immediate vicinity of a cell. In the present study this distortion caused action potentials to appear as if they arose from a potential below threshold, a paradoxical behavior clarified only after calculating the TMP. Depolarizing Wave Versus Ephaptic Interactions The depolarizing wave of the TMP observed in pyramidal cells during evoked potentials was predicted by the field potential study in chapter three. Following either anti- or orthodromic stimulation a strongly positive voltage gradient was found in the extracellular space of CAl during the NI phase of the response. The ephaptic currents associated with this gradient should, on theoretical grounds, depolarize the TMP of pyramidal cells and increase their level of excitability. The close relationship between the amplitude of the NI potential and that of the depolarizing wave of the TMP supports this hypothesis. Before concluding that ephaptic interactions induced the depolarizing wave of the TMP observed in pyramidal cells, other factors must be considered. For instance, an efflux of potassium ions during discharge of a large number of cells within the population may alter the extracellular 196 po tass ium i o n concen t r a t i on s u f f i c i e n t l y to depola r ize the membrane po ten t i a l i n n e i g h b o r i n g neurons . I f t h i s was the p r i m a r y mechanism, a d e p o l a r i z i n g shi f t wou ld appear on the g r o u n d re fe renced po ten t i a l d u r i n g N I . However , i n the p re sen t s t u d y , NI was a lways associa ted w i t h a nega t ive g o i n g no tch on i n t r a c e l l u l a r r e c o r d s . F u r t h e r m o r e , measurements of ex t r ace l l u l a r po tass ium concen t ra t ions u s i n g i o n - s e l e c t i v e e lec t rodes r e p o r t a maximal va lue of o n l y 0.2 mM for the change i n ex t r ace l lu l a r po tass ium associa ted w i t h a s ing le evoked C A l popu la t ion s p i k e (Beninger et a l | 1980). A l t h o u g h th i s va lue may underes t imate the ac tua l change i n potass ium ion concen t r a t i on , i t i s we l l below the change r e q u i r e d to account for the magni tudes of depo la r i za t ions o b s e r v e d i n th i s s t u d y (Lothman et a l , 1975). A l t e r n a t i v e l y , the d e p o l a r i z i n g wave may o c c u r as c u r r e n t f lows between e l ec t ro ton i ca l l y coup led ce l l s w i t h i n the popu la t ion . A l t h o u g h th i s may seem a reasonable explana t ion , s e v e r a l l ines of ev idence a r g u e aga ins t i t . F i r s t , not e v e r y p y r a m i d a l n e u r o n i s coup led to o the r s (Taylor and Dudek , 1982), whereas the TMP depo la r i za t ion was found i n e v e r y c e l l examined i n the p resence of a popu la t ion s p i k e of 2 mV or g rea te r . Second , an e l ec t ro ton ica l ly mediated depo la r i za t ion cou ld o c c u r d u r i n g a n y phase of a n o r thodromic response s ince the i n t r a c e l l u l a r po ten t i a l w i t h i n the popu la t ion i s pos i t i ve d u r i n g P I , NI and P2 . However , i n the p re sen t s t u d y the d e p o l a r i z i n g wave was o n l y seen when the popu la t ion po ten t i a l had a c l e a r l y v i s i b l e NI phase. F i n a l l y , an e l ec t ro ton ica l ly mediated depo la r i za t i on shou ld p r o d u c e a pos i t i ve def lec t ion on s t a n d a r d i n t r a c e l l u l a r r e c o r d s , not a nega t ive go ing no tch as o b s e r v e d here . On the o the r hand , the fact that the d e p o l a r i z i n g wave i n py ramida l ce l l s (1) was p r e sen t o n l y on t ransmembrane poten t ia l r e c o r d i n g s and o n l y 197 when the s t imulus i n t e n s i t y was su f f i c i en t to evoke an NI component on the popu la t ion po ten t i a l , (2) d i s p l a y e d a p a r a l l e l sh i f t i n ampl i tude and l a tency to NI, and (3) was p r e d i c t e d b y the vo l tage g r a d i e n t s s t u d y of chap te r th ree , s u p p o r t s the c o n c l u s i o n that the d e p o l a r i z i n g wave was genera ted b y ephap t i c c u r r e n t s associa ted w i th the popu la t ion r e sponse . E p h a p t i c In t e r ac t i ons V e r s u s P a t t e r n of D i s c h a r g e The p resence of ephapt ic i n t e r ac t i ons may have an impor tan t in f luence on the c h a r a c t e r i s t i c s of neu rona l d i s c h a r g e i n C A l . F o r in s t ance , ex t r ace l l u l a r f i e lds genera ted b y ac t ion poten t ia l s i n a small number of neu rons c o u l d lead to r ec ru i tmen t of o the rwise s u b t h r e s h o l d neu rons t h r o u g h an ephap t ic depo la r i za t ion of t h e i r T M P . T h i s in f luence would tend to ampl i fy the number of ce l l s r e s p o n d i n g to a g i v e n s t imulus and inc rease the ampl i tude of the popu la t ion s p i k e beyond that r e s u l t i n g from s y n a p t i c a c t i va t i on alone. F i e l d i n d u c e d in t e r ac t i ons may also modify the average d i s c h a r g e l a t ency of a popu la t ion of ce l l s r e s p o n d i n g to an o r thodromic s t imulus . The d e p o l a r i z i n g wave associa ted w i th NI s h o u l d add to the s y n a p t i c component of the TMP a n d , as a r e su l t , b r i n g the c e l l to t h r e s h o l d at a s h o r t e r l a t ency than expected for s y n a p t i c c u r r e n t s alone. T h i s mechanism may c o n t r i b u t e to the t endency for the NI po ten t ia l to o c c u r v e r y e a r l y i n the r e sponse , u s u a l l y on what appears to be the r i s i n g edge of the u n d e r l y i n g po ten t ia l . F i n a l l y , the ephap t ic depo la r i za t ion associa ted w i th NI may c o n t r i b u t e to s y n c h r o n i z a t i o n of neu rona l d i s c h a r g e fo l lowing both a n t i - and 198 orthodromic stimulation. This synchronizing influence is based on the fact that many neurons in the population are simultaneously exposed to the same field potential. As a result, the depolarizing wave should also occur simultaneously within many cells bringing them to threshold at approximately the same time. This synchronizing influence was most dramatic during the antidromic response where many cells discharged in synchrony with their neighbors even though they were not directly activated by the stimulus. The results of chapter three also predicted a relatively strong ephaptic depression during PI and P2. These depressions may contribute to the pattern of discharge in at least two ways. For example, ephaptic depression associated with P2 may contribute to other inhibitory influences active in the period immediately following cell discharge. Ephaptic depression during PI and P2 may also enhance the slope on either side of the depolarizing wave of the TMP. A sharper peak on the depolarizing wave would, in turn, sharpen the synchronization of discharge within the population. D) C A l -mJ^te^vM<m Ephaptic interactions in CAl may contribute to the various forms of potentiation characteristic of the hippocampus. For example, if we assume that some primary potentiating mechanism increases the amplitude of the NI potential, then recruitment of additional cell discharge by ephaptic interactions could contribute to the overall level of potentiation. The role of ephaptic interactions was investigated in two types of potentiation. In the first type, referred to as paired pulse potentiation, the amplitude of a population response is greater on the second pulse of a 199 pair of stimuli (Creager et al 1980; Steward et al 1976). In the second type , re fe r red to as f r e q u e n c y potentiat ion, each success ive pulse in a t ra in of stimuli evokes a populat ion response of increas ing amplitude (Andersen and L^mo 1967; Creager et al 1980). A n example of the potentials recorded d u r i n g pa i red pulse potentiat ion is shown in f igure 5.7. The g r o u n d re ferenced intracel lu lar (A), extracel lular (B) and TMP (C) t races are shown for three di f ferent st imulus intensit ies. At each in tens i ty , the amplitude of the i n t r a - and extracel lular responses were substant ia l ly greater fol lowing the second st imulus. In par t icu lar , the NI extracel lular potential , the notch on the r i s i n g edge of the E P S P and the depolar iz ing wave of the TMP were all potentiated on the test response . A similar potentiation of the depolar iz ing wave of the TMP was observed d u r i n g t ra ins of stimuli . F igure 5.8 shows the f i rs t , second, f o u r t h , and sixth response to a 10 Hz stimulus t ra in . As the NI potential of the extracel lular response potent iated, the notch on the r i s i n g edge of the E P S P and the depolar iz ing wave of the TMP also increased in amplitude unt i l f ina l ly , on the sixth response , an action potential arose from the depolar iz ing wave. The g r a p h on the r igh t of the f igure indicates the paral le l potentiation of the NI potential (filled circles) and the depolar iz ing wave of the TMP (open c i rc les ) . E ) C A l -After a pro longed per iod of incubat ion the evoked potentials recorded in most sl ices decreased in amplitude and eventual ly faded away. However, on occasion, a deter iorat ing slice developed epileptic act iv i ty , often in the form of a t ra in of populat ion spikes following each st imulus. These sl ices 200 FIGURE 5.7 compares the i n t r a c e l l u l a r and t ransmembrane potent ia ls of a p y r a m i d a l c e l l d u r i n g i n c r e a s i n g in t ens i t i e s of pa i r ed pu lse s t imula t ion. A : super imposed g r o u n d re fe renced r e c o r d s at three in tens i t i e s . A negat ive g o i n g no tch is v i s i b l e on the r i s i n g edge of the E P S P wi th a peak la tency equ iva l en t to that of the popu la t ion sp ike seen i n B . Both the E P S P and no tch are potent ia ted on the second pu l se , and an ac t ion potent ia l a r i ses from the notch fo l lowing s t imula t ion at the h ighes t i n t ens i t y , B: ex t r ace l lu l a r popula t ion responses r e c o r d e d at the same s t imulus in tens i t i e s immediately after w i t h d r a w a l from the c e l l . C: t ransmembrane potent ia ls ob ta ined by s u b t r a c t i n g the ex t r a from the i n t r a c e l l u l a r r esponses . The s h a r p d e p o l a r i z i n g waves p re sen t on these r e c o r d s v a r i e d i n ampl i tude and l a t ency wi th the popu la t ion s p i k e s shown i n B . The ac t ion po ten t ia l i s now seen a r i s i n g from a l a t ency c o r r e s p o n d i n g to the r i s i n g edge of a d e p o l a r i z i n g wave. Both the popu la t ion s p i k e and the d e p o l a r i z i n g wave were potent ia ted fo l lowing the second s t imulus . 201 Orthodromic CA1 - Transmembrane Potential (paired pulse st imulation at increasing intensit ies) A. B . C. 10 mV i 10 msec 202 FIGURE 5.8 compares the intracellular and transmembrane potentials of pyramidal cell during orthodromic stimulation at 10 Hz. A: ground referenced intracellular potentials. Pulse numbers 1, 2, 4 and 6 are shown sequentially on the left, and superimposed on the right for comparison. The stimulus intensity was chosen such that an action potential occasionally occurred on the sixth pulse of a trial. A response with an action potential is shown superimposed on response number six in the figure. Both the EPSP amplitude and the notch on its rising edge increase in amplitude during the train. On the sixth response the action potential arises from the notch. B: extracellular responses recorded using the same stimulus parameters as in A, immediately after withdrawal from the cell. C: transmembrane potential calculated by subtracting the extra from the intracellular potential. Note the development of a sharp depolarizing wave in the presence of an extracellular population spike. Both the depolarizing wave and population spike increase in amplitude during the stimulus train and the action potential arises from the peak of the depolarizing wave. D: Plot of population spike amplitude (filled circles) and depolarizing wave (open circles) for the responses shown in A-C. Insert indicates the method used to measure the amplitude of the depolarizing wave on the transmembrane potential. Note the parallel variation in the amplitude of the population spike and depolarizing wave during the stimulus train. Orthodromic CA1 - Transmembrane Potential (during frequency potentiation) 1 2 4 6 10 msec 2 4 6 8 PULSE NUMBER PULSE NUMBER 204 p r o v i d e d an idea l o p p o r t u n i t y to s t u d y the r e l a t i onsh ip between ex t r ace l l u l a r f i e ld po ten t ia l s and the TMP d u r i n g ep i lep t i fo rm d i s c h a r g e . A n example of th i s ep i l ep t i c a c t i v i t y is shown i n f i g u r e 5.9. P a i r e d pu l se s t imula t ion was used to p r o l o n g the m u l t i - s p i k e d i s c h a r g e on the second response ( S c h w a r t z k r o i n 1975). N e g a t i v e - g o i n g notches were c l e a r l y v i s i b l e on the g r o u n d re fe renced r e c o r d (A) and each one c o r r e s p o n d e d to an NI po ten t i a l i n the ex t r ace l lu l a r space (B). The f i r s t no t ch fo l lowing each s t imulus (arrow) ac tua l l y had a nega t ive peak below the p r e s t imu lus membrane po ten t i a l and an ac t ion po ten t i a l appeared to a r i se from w i t h i n the no tch of the second response . T h i s pa radox ica l p a t t e r n of d i s c h a r g e was c l a r i f i e d on the TMP r e c o r d where the ac t ion po ten t i a l arose from the top of a d e p o l a r i z i n g wave (arrow C) . The re was a close c o r r e l a t i o n between the l a t ency , ampl i tude and number of NI poten t ia l s (B) and the d e p o l a r i z i n g waves of the TMP (C). A l t h o u g h the si te of ac t ion po ten t ia l i n i t i a t i o n often v a r i e d from s t imulus to s t imulus , s p i k e s a lways arose from a no tch on the g r o u n d re fe renced po ten t ia l or a d e p o l a r i z i n g wave on the TMP . Fo r example, an ac t ion po ten t i a l i s seen a r i s i n g from e i the r the f i r s t o r second no tch on the g r o u n d r e fe renced r e c o r d shown i n C. The mechanisms u n d e r l y i n g v a r i o u s forms of po ten t ia t ion o b s e r v e d i n the h ippocampus remain c o n t r o v e r s i a l . Some i n v e s t i g a t o r s sugges t that t h i s phenomenon i n v o l v e s an inc rease i n the p r e s y n a p t i c release of t r ansmi t t e r (Creager et a l 1980; T u r n e r and M i l l e r 1982; White et a l 1979), whi le o the r s have found an a l t e ra t ion i n s e n s i t i v i t y of the p o s t s y n a p t i c c e l l ( B u z a k i and Czeh 1981; B u z a k i and E i d e l b e r g 1982). However , i r r e s p e c t i v e of the 205 FIGURE 5.9 compares the i n t r a c e l l u l a r and t ransmembrane potent ia ls of a p y r a m i d a l c e l l i n a s l ice demons t r a t ing mult iple popu la t ion s p i k e s fo l lowing o r thod romic s t imula t ion . P a i r e d pu lse s t imula t ion was u s e d to increase the b u r s t d u r a t i o n on the second pu l se . A: g r o u n d r e fe renced i n t r a c e l l u l a r r e sponse showing mult iple nega t ive go ing notches on the E P S P fo l lowing each s t imulus . In both cases the f i r s t notch peaked below the p re s t imu lus membrane po ten t ia l (a r rows) . A n ac t ion po ten t ia l is seen a r i s i n g from a l e v e l below basel ine on the second response . B: ex t r ace l l u l a r popula t ion potent ia ls r e c o r d e d at the same dep th and i n t e n s i t y immediately fo l lowing w i t h d r a w a l from the c e l l . Note that both the ampl i tude and number of popu la t ion poten t ia l s are enhanced on the second pu lse and each popu la t ion s p i k e c o r r e s p o n d s to a negat ive notch on the g r o u n d r e fe renced response i n A . C: t ransmembrane po ten t ia l ob ta ined by s u b t r a c t i n g the ex t r a from the i n t r a c e l l u l a r response . The s h a r p depo la r iza t ions of the t ransmembrane po ten t i a l v a r y d i r e c t l y w i th the l a t ency , ampl i tude and number of popu la t ion s p i k e s seen i n B. Note that the ac t ion po ten t ia l a r i se s from an in f l ec t i on on the r i s i n g edge of the second response (a r row) . D: g r o u n d r e fe renced responses from the same c e l l showing the ac t ion po ten t i a l a r i s i n g from e i the r the f i r s t o r second negat ive go ing no tch . 206 Orthodromic CA1 - Transmembrane Potential (during burst discharge) 20 mV 20 mV 10 msec 207 " p r i m a r y " mechanisms i n v o l v e d , a potent ia ted popu la t ion s p i k e shou ld inc rease the ephapt ic depo la r i za t ion associa ted w i t h the NI po ten t ia l . T h i s depo la r i za t i on c o u l d d i s c h a r g e add i t i ona l neu rons f o r c i n g the l e v e l of po ten t ia t ion beyond that d i r e c t l y genera ted b y the " p r i m a r y " mechanism. The p r e sen t s t u d y s u p p o r t s t h i s hypo the s i s b y demons t ra t ing a s u b s t a n t i a l inc rease i n the d e p o l a r i z i n g wave of the TMP d u r i n g p a i r e d pu l se and f r e q u e n c y po ten t ia t ion . On the o the r hand , the r e s u l t s do not imp ly that ephap t i c i n t e r ac t i ons pa r t i c ipa t e i n the " p r i m a r y " po ten t i a t ing mechanism. In fact , the p r o p o r t i o n a l sh i f t i n ampl i tude of the popu la t ion s p i k e and the d e p o l a r i z i n g wave of the TMP ( f igure 5.8 inse t ) sugges t s that the s e n s i t i v i t y to ephap t ic i n t e r ac t i ons remains cons tan t d u r i n g a s t imulus t r a i n . The p re sen t r e s u l t s also sugges t that ephapt ic i n t e r ac t i ons c o n t r i b u t e to the c h a r a c t e r i s t i c s of neu rona l d i s c h a r g e d u r i n g ep i lep t i fo rm a c t i v i t y i n C A l . D e p o l a r i z i n g waves were o b s e r v e d on the TMP r e c o r d for each NI po ten t i a l d u r i n g t r a i n s of popu la t ion s p i k e s seen i n d e t e r i o r a t i n g s l i ces . The p resence of these depo la r i za t ions s h o u l d inc rease the number of c e l l s d i s c h a r g i n g d u r i n g each NI po ten t i a l and s y n c h r o n i z e the d i s c h a r g e to the peak of the TMP depo la r i za t i on . The end r e s u l t shou ld be a s h a r p e r and h i g h e r ampl i tude se r ies of NI po ten t ia l s t han would have o c c u r r e d i n the absence of s i g n i f i c a n t ex t r ace l l u l a r f i e l d s . S imi la r t r a i n s of popu la t ion r e sponses were r e p o r t e d b y T a y l o r and Dudek (1982) fo l lowing an t id romic s t imula t ion of C A l i n s l ices exposed to low ca lc ium l eve l s . T h e y a lso o b s e r v e d d e p o l a r i z i n g waves of the TMP d u r i n g nega t ive components of the ex t r ace l lu l a r response and conc luded that ephap t ic i n t e r ac t i ons c o n t r i b u t e to s y n c h r o n i z a t i o n of neu rona l a c t i v i t y d u r i n g low ca lc ium b u r s t s . I n fact , i t seems l i k e l y that s imi lar ephapt ic 208 in t e r ac t i ons are a p r i m a r y s y n c h r o n i z i n g mechanism d u r i n g o the r forms of ep i l ep t i fo rm a c t i v i t y i n C A l . G) Dente^ AppkedFields Orthodromic s t imula t ion of g r anu l e ce l l s genera ted g r a d e d E P S P s on g r o u n d r e fe renced i n t r a c e l l u l a r r e c o r d s . A t j u s t t h r e s h o l d s t imulus i n t ens i t i e s two d i f fe ren t E P S P waveforms were r e c o r d e d . I n some ce l l s the E P S P had a smooth waveform as shown i n the lower p h o t o g r a p h of f i g u r e 5.10. The ex t r ace l l u l a r r esponse i n the v i c i n i t y of these ce l l s cons i s t ed of o n l y a P I po ten t ia l . I n o the r ce l l s the top of the E P S P had a wide no tch as shown i n the u p p e r p h o t o g r a p h . I n these cases the ex t r ace l l u l a r f i e ld was made u p of a f u l l P I , NI and P2 sequence . The ampl i tude a n d l a t ency of the no tch v a r i e d d i r e c t l y w i t h the NI po ten t i a l i n a manner v e r y s imi lar to that o b s e r v e d i n p y r a m i d a l neu rons . Fo l lowing j u s t t h r e s h o l d s t imula t ion , s p i k e s gene ra l l y o r i g i n a t e d af ter the peak of the g r o u n d r e fe renced E P S P at a po ten t i a l a p p a r e n t l y below t h r e s h o l d . Both the ce l l s i n f i g u r e 5.10 i l l u s t r a t e th i s c h a r a c t e r i s t i c . A compar i son of the TMP and the g r o u n d re fe renced po ten t i a l i n g r anu l e ce l l s i n d i c a t e d that the peak depo la r i za t ion of the TMP was u s u a l l y lower i n ampl i tude and had a l o n g e r l a t ency than that of the s t a n d a r d i n t r a c e l l u l a r r e c o r d . A n example i s shown i n f i g u r e 5.11. In th i s case the r i s i n g edge of the TMP (dotted l ine) f la t tens out to a shou lde r before c o n t i n u i n g on to the peak. T h i s f lat shou lde r o c c u r r e d d u r i n g P I and a s imi la r d e p r e s s i o n o c c u r r e d d u r i n g P2 . A l t h o u g h the peak depo la r i za t ion of the TMP i s associa ted w i t h N I , i t i s o n l y s l i g h t l y g rea te r t han the c o r r e s p o n d i n g g r o u n d re fe renced po ten t ia l . 209 F I G U R E 5.10 shows g r o u n d re fe renced i n t r a c e l l u l a r responses of dentate g r a n u l e ce l l s to o r thodromic s t imula t ion . TOP: four super imposed i n t r a c e l l u l a r r e sponses to a s t imulus i n t e n s i t y v e r y near t h r e s h o l d . A prominen t negat ive no tch is p r e sen t immediately fo l lowing the peak of the E P S P and an ac t ion po ten t ia l on one of the responses i s seen a r i s i n g from w i t h i n the no tch . The ex t r ace l lu l a r po ten t ia l at th i s s t imulus i n t e n s i t y had a l a rge NI po ten t ia l . BOTTOM: four super imposed i n t r a c e l l u l a r r e sponses at i n c r e a s i n g s t imulus in t ens i t i e s . Note that the ac t ion po ten t ia l a r i se s after , and at a lower po ten t ia l , than the peak of the E P S P . In th i s example, the ex t r ace l l u l a r r e sponse cons i s t ed of o n l y a P l po ten t i a l . 210 Dentate Granule Cell Intracellular Records Threshold Intensity Series 20 MSEC 211 FIGURE 5.11 shows the TMP d u r i n g an o r thodromic popula t ion potent ia l i n the dentate g y r u s . A n ex t r ace l lu l a r (extra) , g r o u n d re fe renced i n t r a c e l l u l a r (dotted i n t r a ) , and t ransmembrane po ten t ia l (sol id in t ra ) are super imposed i n the f i g u r e . The s t imulus i n t e n s i t y was j u s t s u b t h r e s h o l d for ac t ion po ten t ia l genera t ion . A Prominent P l , NI and P2 potent ia l are p resen t on the ex t r ace l lu l a r r e c o r d and a l a rge negat ive notch i s seen on the top of the E P S P . The t ransmembrane po ten t i a l remains dep res sed u n t i l i t peaks at a l a t ency after the peak depo la r i za t ion seen on the g r o u n d re fe renced r e c o r d . C a l i b r a t i o n : 3 mV/5 ms 213 FIGURE 5.12 shows the influence of applied voltage gradients on the intracellular and transmembrane potentials of a dentate granule cell. All of the traces in this figure were evoked by the identical orthodromic stimulus. A: superimposition of two intracellular responses recorded during application of a positive and of a negative voltage gradient (mV/mm). The rising edge of the EPSP is unchanged by the gradient but the remaining part of the potential is highly altered. To the right are the equivalent extracellular traces. The large NI and P2 potentials present during the positive gradient are completely suppressed when the gradient is reversed. B: the transmembrane potential is shown for two levels of positive and two levels of negative applied gradient. The traces are drawn with all of the baselines superimposed to clarify changes in the transmembrane potential. The positive gradients induced a long lasting relative depolarization and negative gradients induced a long lasting relative hyperpolarization of the transmembrane potential. C: the same transmembrane records are shown again but with their true resting membrane potentials. The resting potential shifts approximately 5 mV when the applied voltage gradient is changed from its most negative to most positive value. 214 Orthodromic Dentate - Transmembrane Potential (influence of applied fields) 215 The dentate g y r u s also a f forded the o p p o r t u n i t y to r e c o r d the TMP whi le a p p l y i n g ex t r ace l l u l a r vo l tage g r a d i e n t s of d i f fe ren t magni tudes . The r e s u l t s of one of these exper iments i s shown i n f i g u r e 5.12. I n th i s example the vo l tage g r a d i e n t s r a n g e d from minus to p l u s 100 mV/mm. The TMP t races i n C are shown w i t h t h e i r t r ue basel ine poten t ia l s bu t basel ines of a l l o ther t races were normal ized to ze ro . U n d e r the in f luence of l a rge vo l tage g r a d i e n t s the ex t r ace l lu l a r evoked po ten t ia l (A r i g h t ) changed from a s imple P I po ten t i a l (negat ive g rad ien t ) to a f u l l popu la t ion s p i k e w i t h a P I , NI a n d P2 po ten t ia l (pos i t ive g r a d i e n t ) . The g r o u n d r e fe renced i n t r a c e l l u l a r po ten t i a l was also h i g h l y modified b y ex t r ace l l u l a r f i e ld s . D u r i n g nega t ive g r a d i e n t s the E P S P was smooth and q u i c k l y r e t u r n e d to a po ten t i a l below basel ine where i t remained for a p ro longed p e r i o d . D u r i n g pos i t i ve g r a d i e n t s the E P S P deve loped a no tch on i t s r i s i n g edge, r eached a much g rea te r peak po ten t ia l , and then remained i n a r e l a t i v e l y depo la r i zed s tate . The no tch c o r r e s p o n d e d i n time to NI and the l a rge peak c o r r e s p o n d e d to P2. The TMP r e c o r d e d i n the same c e l l d u r i n g d i f f e ren t magni tudes of a p p l i e d g r a d i e n t s are shown i n C w i t h each t race pos i t ioned a c c o r d i n g to i t s t r u e base l ine po ten t i a l . P o s i t i v e vo l tage g r a d i e n t s depo la r i zed the r e s t i n g membrane po ten t i a l and i nc rea sed the peak ampl i tude of the r e sponse whereas nega t ive g r a d i e n t s had the oppos i te effect . The same t r aces are shown i n B w i t h t he i r basel ines supe r imposed . The peak po ten t i a l sh i f ted i n ampl i tude w i t h the a p p l i e d f i e ld bu t to a l e s se r degree than o b s e r v e d i n C. Therefore a sh i f t i n basel ine as we l l as a change i n the r e l a t i ve ampl i tude of the d e p o l a r i z i n g po ten t ia l c o n t r i b u t e to the o v e r a l l sh i f t i n peak ampl i tude of the T M P . 216 A compar i son of the i n t r a c e l l u l a r , TMP and ex t r ace l l u l a r r e c o r d s d u r i n g d i f fe ren t l eve l s of a p p l i e d vo l tage g rad i en t was also very-in fo rmat ive . T races from the same exper iment are shown i n a new a r rangement i n f i g u r e 5.13. Each g r o u p of t races cons i s t s of the i n t r a , ex t r a and TMP t races r e c o r d e d u n d e r the in f luence of a g i v e n app l i ed f i e l d . A l l the t races are shown on a normal ized basel ine . U n d e r the in f luence of c o n t r o l o r nega t ive g r a d i e n t s the ampl i tude of the TMP was lower and i t peaked la te r t han the g r o u n d r e fe renced E P S P . A t p l u s 15 mV/mm the E P S P deve loped a no tch on top, bu t the TMP s t i l l peaked la te r and at a lower po ten t i a l t h a n the g r o u n d r e fe renced r e c o r d . A t p l u s 80 mV/mm, the peak of the TMP extended beyond tha t of the E P S P , and f i n a l l y at p l u s 100 mV/mm the no tch sh i f t ed to the r i s i n g edge of the E P S P . U n d e r the in f luence of t h i s g r a d i e n t the TMP deve loped a s h a r p d e p o l a r i z i n g wave w i t h a s h o r t e r peak l a t ency than the g r o u n d re fe renced r e c o r d . The peak depo la r i za t i on of the TMP d u r i n g o r thodromic s t imula t ion of g r a n u l e ce l l s c o n s i s t e n t l y had a l a t ency equa l to or g rea te r t han that of the g r o u n d r e f e r enced E P S P . T h i s o b s e r v a t i o n was t r ue whe the r o r not an NI po ten t i a l was p r e sen t i n the ex t r ace l l u l a r space. The TMP was dep re s sed d u r i n g P l and P2, and o n l y minimal ly enhanced d u r i n g N I , as p r e d i c t e d b y the f i e ld po ten t i a l s t u d y i n chap te r th ree . The r e s u l t was a b l u n t i n g of the r i s i n g edge of the TMP w h i c h tended to de lay the peak depo la r i za t i on . These f i n d i n g s s u p p o r t the p r e d i c t i o n i n chap te r three that P l and P2 shou ld be associa ted w i th ephapt ic dep re s s ion . 217 FIGURE 5.13 shows how app l i ed vo l tage g rad ien t s a l te r the r e l a t ionsh ip between g r o u n d re fe renced and t ransmembrane potent ia ls d u r i n g o r thod romica l l y evoked responses i n the dentate g y r u s . A d i f fe ren t magni tude of vol tage g r a d i e n t (mV/mm) was app l i ed d u r i n g each g r o u p of super imposed responses but a l l the r e sponses i n the f i gu re were evoked u s i n g the i den t i ca l s t imulus . To help v i s u a l l y i d e n t i f y the separate t races note that , i n each g r o u p , the r i s i n g edge of the g r o u n d re fe renced i n t r a c e l l u l a r po ten t ia l ( in t ra) has a s h o r t e r l a t ency than the c o r r e s p o n d i n g t ransmembrane po ten t i a l (TMP), and the ex t r ace l lu l a r response (extra) has the lowest ampl i tude . There i s a p r o g r e s s i v e shi f t i n the r e l a t i onsh ip between the peak l a t ency of the i n t r a c e l l u l a r and the t ransmembrane response as the app l i ed vo l tage g r a d i e n t is i nc r ea sed . U n d e r c o n t r o l cond i t i ons , and d u r i n g app l i ca t i on of nega t ive g rad i en t s , the t ransmembrane po ten t ia l peaks la te r t han the i n t r a c e l l u l a r t race . However , th i s r e l a t i onsh ip r e v e r s e s as the popu la t ion sp ike increases i n ampl i tude u n d e r the in f luence of more pos i t i ve vo l tage g r ad i en t s . U n d e r the in f luence of +100 mV/mm the sha rp no tch on the r i s i n g edge of the i n t r a c e l l u l a r r e sponse , and the e a r l y d e p o l a r i z i n g wave on the t ransmembrane potent ia l ,are t y p i c a l of those o b s e r v e d i n C A l r a the r than the dentate g y r u s . 218 Orthodromic Dentate - Transmembrane Potential (influence of applied fields) -100 control +15 +80 + 100 219 Dentate Versus CAl The most s t r i k i n g d i f fe rence between the dentate g y r u s and C A l was i n the r e l a t i onsh ip of s p i k e d i s c h a r g e to the u n d e r l y i n g E P S P waveform. In C A l , the s t r o n g ephapt ic depo la r i za t ion d u r i n g NI tended to sh i f t the ac t ion po ten t i a l o r i g i n towards s h o r t e r l a tenc ies . I n the dentate g y r u s , the p rominent ephap t ic d e p r e s s i o n d u r i n g P l and r e l a t i ve l ack of ephapt ic depo la r i za t ion d u r i n g NI t ended to sh i f t the ac t ion po ten t i a l o r i g i n to l onge r la tenc ies . It wou ld appear that the vol tage g r a d i e n t s genera ted d u r i n g an e v o k e d po ten t i a l a re impor tan t de te rminants of the p a t t e r n of c e l l d i s c h a r g e . I f t h i s i s t rue , t h e n mod i fy ing the vo l tage g r a d i e n t s i n the ex t r ace l l u l a r env i ronment shou ld in f luence the p a t t e r n of ac t ion po ten t ia l d i s c h a r g e w i t h i n a popu la t ion of neu rons . A pos i t i ve g r a d i e n t app l i ed to the dentate g y r u s shou ld enhance the u s u a l l y minimal ephap t i c depo la r i za t ion associa ted w i t h N I , and minimize the s t r o n g d e p r e s s i o n associa ted w i t h P l and P2 . T h i s o v e r a l l sh i f t towards depo la r i za t ion s h o u l d r e c r u i t add i t i ona l c e l l d i s c h a r g e and also sh i f t the l a t ency to peak depo la r i za t ion of the TMP to an ea r l i e r l a t ency . T h i s i s exac t ly what i s o b s e r v e d i n f i g u r e 5.13. A s the app l i ed g rad i en t is i nc r ea sed the TMP i s enhanced and i t s peak sh i f t s to an ea r l i e r pos i t ion on the response . In th i s sense, as the app l i ed f i e ld enhances exc i t a to ry and depresses i n h i b i t o r y ephap t ic i n t e r ac t ions , the response beg ins to look as i f i t was r e c o r d e d i n C A l , r a the r t han the dentate g y r u s . T h i s o b s e r v a t i o n impl ies that at least some of the d i f fe rences between the c h a r a c t e r i s t i c s of evoked po ten t ia l s i n C A l and the dentate g y r u s are based on d i f fe rences i n the ephapt ic i n t e r ac t i ons i n d u c e d d u r i n g an evoked response . 220 Steady State Shifts In The TMP Application of extracellular voltage gradients to the dentate gyrus should, on theoretical grounds, shift the baseline TMP. Since this shift is the basis for ephaptic interactions it was reassuring to observe a graded shift in the baseline TMP in the appropriate direction during application of various magnitudes of voltage gradients. The TMP shifted approximately plus to minus 5 mV during application of the full range of gradients observed during evoked potentials in the hippocampal formation. A rather unexpected finding was the prolonged shift in TMP following synaptic activation of granule cells exposure to applied voltage gradients. The shift was depolarizing during positive gradients and hyperpolarizing during negative gradients. This finding implies that granule cells are more sensitive to ephaptic interactions during the post synaptic period. This enhanced sensitivity could result from 1) a decrease in somatic membrane conductance, 2) an increase in extracellular resistance, or 3) an increase in dendritic membrane conductance. On intuitive grounds, a prolonged increase in dendritic conductance initiated by synaptic activity seems a likely possibility. Unfortunately resolution of this problem must await further investigation. 5.4 CONCLUSION The intracellular studies in this chapter demonstrate a close relationship between extracellular field potentials and the TMP of hippocampal neurons. Since the TMP was found to be an important determinant of action potential discharge it seems reasonable to conclude that extracellular fields present during an evoked potential can, indeed, modify the pattern of discharge within a population of neurons 221 C H A P T E R ^ 6^  G E N E R A L p i S T h i s s t u d y es tab l i shes the impor tance of ephapt ic i n t e rac t ions i n h ippocampal p h y s i o l o g y . It demonstra tes the potent in f luence of ex t r ace l l u l a r f i e ld po ten t ia l s on n e u r o n a l e x c i t a b i l i t y and shows how these f i e lds can a l te r the o v e r a l l p a t t e r n of d i s c h a r g e w i t h i n a neu rona l r e g i o n . The r e c r u i t i n g and s y n c h r o n i z i n g in f luence of d e p o l a r i z i n g ephapt ic i n t e r ac t i ons sugges t that s imi lar i n t e r ac t i ons may also c o n t r i b u t e to ep i l ep t i c d i s c h a r g e i n the h ippocampus as we l l as o ther c o r t i c a l r eg ions . The concep t s and da ta pe r t i nen t to these conc lus ions are summarized i n the fo l lowing sec t ion . 6.1 S U M J ^ y The p r ec i s e o r g a n i z a t i o n of the h ippocampal format ion in to d i s t i n c t somatic and d e n d r i t i c l a y e r s makes the s t r u c t u r e idea l for ephap t i c i n t e r ac t i ons (Green and Maxwel l 1961). T h i s anatomical a r rangement causes ex t r ace l l u l a r c u r r e n t s genera ted d u r i n g s y n c h r o n o u s neu rona l a c t i v i t y to accumulate ac ros s many ce l l s , l e ad ing to l a rge ex t r ace l lu l a r f i e ld po ten t ia l s . If a p r o p o r t i o n of t h i s c u r r e n t f lows t h r o u g h the i n t r a c e l l u l a r space, i t shou ld a l t e r the l e v e l of e x c i t a b i l i t y w i t h i n the whole neu rona l popu la t ion . A l t h o u g h s u c h ephap t ic i n t e rac t ions s h o u l d o c c u r on theore t i ca l g r o u n d s , t h e i r magni tude and s ign i f i cance were u n k n o w n u n t i l r e c e n t l y . The f i r s t phase of th i s p ro j ec t e s t ab l i shed the p resence of s u b s t a n t i a l ex t r ace l l u l a r vol tage g r a d i e n t s a long the dendro-somat ic axis of h ippocampal neu rons d u r i n g evoked poten t ia l s . The c h a r a c t e r i s t i c s of these g r a d i e n t s were used to p r e d i c t e d the p o l a r i t y and r e l a t i ve magni tude of ephap t ic i n t e r ac t i ons t a k i n g place d u r i n g a popu la t ion response . The 222 de ta i l ed c h a r a c t e r i s t i c s of the vo l tage g r a d i e n t s v a r i e d between C A l and the dentate g y r u s and also between a n t i - and o r thodromic responses . I n gene ra l , P I and P2 were associa ted w i t h h y p e r p o l a r i z i n g and NI w i t h d e p o l a r i z i n g g r ad i en t s . These g r a d i e n t s were of ten more than an o r d e r of magni tude g rea t e r t han the smallest g r a d i e n t k n o w n to in f luence g ranu le c e l l a c t i v i t y ( Je f fe rys 1982). A n except ion to th i s r u l e was the minimal g r a d i e n t o b s e r v e d d u r i n g NI of the o r thodromic dentate response . The second phase of t h i s s t u d y demonst ra ted the remarkab le s e n s i t i v i t y of dentate g r anu l e ce l l s to exper imenta l ly a p p l i e d vo l tage g r a d i e n t s i n the r ange o b s e r v e d d u r i n g evoked poten t ia l s . These vo l tage g r a d i e n t s cou ld a l t e r the N1 /P2 component of an e v o k e d po ten t i a l from near minimal to near maximal. S u r p r i s i n g l y , even an t id romic poten t ia l s were i n f l uenced b y ex t r ace l l u l a r g r a d i e n t s . On the o ther hand , vo l tage g rad i en t s d i d not in f luence the P I component of the somatic response o r the i n i t i a l component of the e x t r a d e n d r i t i c po ten t i a l . These f i n d i n g s e s t ab l i sh that ex t r ace l l u l a r c u r r e n t s can in f luence the l e v e l of e x c i t a b i l i t y w i t h i n a neu rona l popu la t ion wi thou t a l t e r i n g s y n a p t i c d r i v e . The t h i r d phase of s t u d y d i r e c t l y measured ephap t ic i n t e rac t ions at the s ing le c e l l l e v e l . A case was made for the impor tance of measu r ing the T M P , r a t h e r t han the g r o u n d r e fe renced i n t r a c e l l u l a r response when an impaled n e u r o n i s exposed to ex t r ace l l u l a r f i e ld po ten t ia l s . The a rgument was based on the fact that the e l e c t r i c a l behav io r of a c e l l s h o u l d o n l y depend on the loca l vo l tage d r o p ac ros s the neu rona l membrane. The close r e l a t i o n s h i p between the TMP and ac t ion po ten t ia l genera t ion conf i rmed th i s t heo re t i ca l a rgument . I n o r d e r to measure the i n t r a c e l l u l a r in f luence of f i e ld po ten t ia l s , the TMP was moni tored whi le the impaled c e l l was exposed to ex t r ace l l u l a r 223 vol tage g r a d i e n t s s p a n n i n g the r ange o b s e r v e d d u r i n g e v o k e d potent ia ls . The TMP sh i f ted b y as much as p lu s o r minus 5 mV, d e p e n d i n g on the ampl i tude and p o l a r i t y of the g rad ien t . S u c h a l a rge sh i f t i n TMP ac ross a popu la t ion of neu rons cou ld c e r t a i n l y account for the a l t e red popu la t ion s p i k e ampl i tudes o b s e r v e d d u r i n g app l i ed f i e lds . S imi la r sh i f t s i n TMP were o b s e r v e d d u r i n g evoked poten t ia l s . Fo r example, a d e p o l a r i z i n g wave of the TMP o c c u r r e d d u r i n g the NI component of a n t i and o r thodromic C A l r e sponses . T h i s d e p o l a r i z i n g wave was capable of i n i t i a t i n g ac t ion potent ia ls and tended to decrease the l a t ency to d i s c h a r g e d u r i n g o r thodromic r e sponses . 6.2 I J ^ L i CATIONS A) Experimented C l e a r l y , ephap t i c i n t e r ac t i ons must be c o n s i d e r e d d u r i n g a n a l y s i s of a n y exper imenta l da ta r e c o r d e d d u r i n g l a rge ex t r ace l lu l a r f i e ld po ten t ia l s . F a i l u r e to do so cou ld lead to se r ious d i f f i cu l t i e s i n i n t e r p r e t a t i o n of i n t r a c e l l u l a r waveforms, p a r t i c u l a r l y i f the i n v e s t i g a t o r fa i l s to r ecogn ize the l imi ta t ions of g r o u n d re fe renced po ten t ia l s . Fo r example, Fla tman and Lamber t (1979) found that g r o u n d r e fe renced i n t r a c e l l u l a r r e sponses c o u l d underes t imate the ac tua l TMP b y as much as 50 mV when r e c o r d e d d u r i n g ion tophore t i c app l i c a t i on of exc i t a to ry amino ac ids . A s a r e su l t , t hey recommended the use of an ex t r ace l lu l a r e lec t rode to monitor the often s u b s t a n t i a l sh i f t s i n ex t r ace l l u l a r po ten t ia l assoc ia ted w i t h ion tophore t i c app l i ca t i on of many subs tances . Ephap t i c i n t e r ac t i ons also make ex t r ace l lu l a r da ta more d i f f i cu l t to i n t e r p r e t . F o r example, a component of the poten t ia t ion o b s e r v e d d u r i n g 224 p a i r e d pu l se and f r e q u e n c y s t imula t ion is d i r e c t l y re la ted to ephapt ic i n t e r a c t i o n s . I t seems l i k e l y that s imi lar i n t e rac t ions may in f luence the ampl i tude of evoked potent ia ls i n long term and o ther forms of po ten t ia t ion . The a d d i t i o n of th i s n o n - s y n a p t i c component may complicate at tempts to i d e n t i f y the p r i m a r y mechanisms i n v o l v e d i n these phenomenon. B) Phy8iqlogi Since ephap t ic i n t e rac t ions in f luence the p a t t e r n of neu rona l d i s c h a r g e d u r i n g evoked poten t ia l s , i t i s n a t u r a l to wonder whe the r t hey also in f luence normal , p h y s i o l o g i c a l forms of a c t i v i t y . F o r example, do ephap t ic i n t e r ac t i ons c o n t r i b u t e to the mechanisms u n d e r l y i n g the ta r h y t h m i n the h ippocampal formation? D u r i n g the ta the vo l tage g r a d i e n t a long the dendro-somat ic ax is of g r anu l e and p y r a m i d a l ce l l s osc i l la tes between p l u s and minus about 2 to 4 mV/mm (Fox - pe r sona l communicat ion) . The p o l a r i t y of these g r a d i e n t s p r e d i c t s that t h e y shou ld enhance d i s c h a r g e d u r i n g the ac t ive phase and dep re s s d i s c h a r g e d u r i n g the s i l en t phase of the ta . T h i s pos i t i ve feed back b y ephapt ic i n t e rac t ions may c o n t r i b u t e to the o s c i l l a t o r y na tu re of theta a c t i v i t y . However , the g r a d i e n t s are v e r y small compared to those s tud ied i n th i s thes i s . I n fact , t hey are s l i g h t l y smaller t han the minimal g r a d i e n t capable of i n f l u e n c i n g evoked potent ia ls i n the dentate g y r u s ( Je f fe rys 1982). I f ephapt ic i n t e r ac t i ons are impor tan t d u r i n g the ta a c t i v i t y the effect must be v e r y sub t le . Other p h y s i o l o g i c a l forms of s y n c h r o n o u s or r h y t h m i c a l neu rona l a c t i v i t y are also associa ted w i t h ex t r ace l l u l a r f i e ld po ten t ia l s (slow wave s leep, s leep s p i n d l e s , PG0 waves etc . ) . The ex t r ace l lu l a r vol tage g r a d i e n t s and ephap t ic i n t e r ac t i ons genera ted d u r i n g these ac t i v i t i e s are not k n o w n but t hey are u n l i k e l y to generate the v e r y la rge g rad i en t s o b s e r v e d 225 d u r i n g evoked po ten t ia l s . On th i s bas i s , i t seems that ephap t ic i n t e rac t ions are not fundamenta l to normal b r a i n f u n c t i o n . On the o the r hand , ephapt ic i n t e r ac t i ons do r ep re sen t a form of " c r o s s t a lk" between adjacent r eg ions of n e u r o n a l t i s sue . T h i s c r o s s t a lk c o u l d a l t e r the spec i f ic in format ion c a r r i e d b y i n d i v i d u a l neu rons and degrade the o v e r a l l performance of the b r a i n . Katz and Schmi t t (1940) demonst ra ted that ephap t ic i n t e rac t ions can a l te r the v e l o c i t y of ac t ion poten t ia l s t r a v e l l i n g a long p e r i p h e r a l n e r v e s . S imi la r i n t e r ac t i ons cou ld a l t e r the t iming of s p i k e s w i t h i n c lose ly apposed c e n t r a l neu rons and d i s t o r t the in format ion r e p r e s e n t e d b y the i r p a t t e r n of d i s c h a r g e . How de t r imenta l t h i s form of in t e r f e rence i s to normal b r a i n f u n c t i o n remains u n k n o w n . C) Pathologi Se izu re d i s c h a r g e i s one form of a c t i v i t y were ephapt ic i n t e r ac t i ons are c l e a r l y impl ica ted . The work p re sen ted i n th i s thes i s , as we l l as b y o the r s ( Je f fe rys and Haas 1982; R i c h a r d s o n et a l , 1984; T a y l o r and Dudek , 1982, 1984a,b; T a y l o r et a l , 1984), demonstra tes the r e c r u i t i n g and s y n c h r o n i z i n g in f luence of ex t r ace l lu l a r f ie lds d u r i n g b u r s t a c t i v i t y i n the h ippocampal format ion. S imi la r i n t e r ac t i ons may c o n t r i b u t e to i n t e r i c t a l d i s c h a r g e and o the r forms of focal ep i l ep t i c a c t i v i t y associa ted w i t h h i g h ampl i tude f i e ld po ten t ia l s . E p h a p t i c i n t e r ac t i ons may also p l a y an impor tan t role i n the sp read of s e i zu re a c t i v i t y ac ross normal b r a i n t i s sue . Fo r example, d i s c h a r g e w i t h i n an ac t ive ep i l ep t i c focus may occas iona l ly generate vo l tage g rad i en t s of su f f i c ien t magni tude to r e c r u i t a c t i v i t y i n n e a r b y neurons . D i scha rge of these add i t i ona l ce l l s cou ld , enhance f i e ld potent ia ls i n the v i c i n i t y of the 226 focus , l e ad ing to ephapt ic r ec ru i tmen t of e v e n more d i s t an t neu rons . Con t inua t ion of t h i s pos i t ive feed back p rocess cou ld e f f ec t ive ly sp read s e i zu re a c t i v i t y ac ross the cor tex . 6.3 C p N C L U S I O N The major i ty of s tud ies dea l ing w i t h c o r t i c a l neu rob io logy have i g n o r e d the poss ib le impor tance of ephapt ic i n t e r ac t ions , i m p l i c i t l y a s suming that ex t r ace l l u l a r c u r r e n t s associa ted w i t h f i e ld potent ia ls are i n s i g n i f i c a n t to b r a i n func t i on . The work p re sen ted here has concen t r a t ed on th i s l o n g i g n o r e d e l e c t r i c a l p r o p e r t y of the n e u r o p i l , and has demonst ra ted an impor tan t ro le for ephap t ic i n t e r ac t i ons i n the r ec ru i tmen t and s y n c h r o n i z a t i o n of neu rona l a c t i v i t y . I hope that t h i s effor t has c o n t r i b u t e d to o u r u n d e r s t a n d i n g of the n e r v o u s sys tem and w i l l p r o v i d e a bas is for f u r t h e r s t u d y of both p h y s i o l o g i c a l and pa tho log ica l c o r t i c a l f u n c t i o n . 227 R E F E R E N C E S A d r i a n , E .D. (1930) The effects of i n j u r y on mammalian n e r v e f i b e r s . P r o c . Roya l Soc. B106:596-618. A i r d , R . B . , M a s l a n d , R . L . and Woodbury , D.M (1984) The ep i leps ies : A c r i t i c a l r ev i ew . Raven P r e s s , New Y o r k , p p . 1-41. Ajmone M a r s a n , C. (1969) Acu te effects of t op ica l ep i l ep togen ic agents . In : Bas ic Mechanisms of the E p i l e p s i e s , ed i ted b y H.H. J a spe r , A . A . Ward , J r . and A . Pope, p p . 299-319, L i t t l e , B r o w n and Co. , Bos ton . A l g e r , B . E . and N i c o l l , R .A . (1980) Ep i l ep t i fo rm b u r s t h y p e r p o l a r i z a t i o n : Ca lc ium-dependen t potass ium po ten t i a l i n h ippocampal C A l p y r a m i d a l ce l l s . Sc ience , 210:1122-1124. A l g e r , B . E . a n d N i c o l l , R .A . (1982) Feed fo rward d e n d r i t i c i n h i b i t i o n i n ra t h ippocampal p y r a m i d a l ce l l s s t ud i ed i n v i t r o . J . P h y s i o l . , 328:105-123. A l g e r , B . E . , M c C a r r e n , M . and F i s h e r , R . S . (1983) On the p o s s i b i l i t y of s imul taneous ly r e c o r d i n g from two ce l l s w i t h a s ing le microe lec t rode i n the h ippocampal s l i ce . B r a i n Res. , 270:137-141. A l l e n , G.I . , E c c l e s , J . , N i c o l l , R . A . , Oshima, T. and R u b i a , F . H . (1977) The ion ic mechanisms conce rned i n g e n e r a t i n g the I P S P s of the h ippocampal p y r a m i d a l ce l l s . P r o c . R. Soc. L o n d . B 198:363-384.99 A n d e r s e n , P . (1959) In t e rh ippocampa l Impulses I O r i g i n , cou r se and d i s t r i b u t i o n i n cat , r a b b i t and ra t . A c t a p h y s i o l . s cand . 47:63-90. A n d e r s e n , P . (1960) In t e rh ippocampa l Impulses II A p i c a l d e n d r i t i c a c t i va t i on of C A l neu rons . A c t a p h y s i o l . s cand . 48:178-208. A n d e r s e n , P . (1983) Bas ic mechanisms of p e n i c i l l i n - i n d u c e d ep i l ep t i fo rm d i s c h a r g e s . In : E p i l e p s y : A n Upda te on Resea rch and T h e r a p y . A . R . L i s s , Inc . , New Y o r k p g 3-13. A n d e r s e n , P . , B l i s s , T . V . P . , and S k r e d e , K . K . (1971a) U n i t ana ly s i s of h ippocampal popu la t ion s p i k e s . E x p . B r a i n Res. 13:208-221. A n d e r s e n , P . , B l i s s , T . V . P . , and S k r e d e , K . K . (1971b) Lamel lar Organ iza t ion of Hippocampal E x c i t a t o r y Pa thways . E x p . B r a i n Res. 13: 222-238. A n d e r s e n , P . , Ecc l e s , J . C . and L ^ y n i n g , Y . (1964) Pa thway of p o s t s y n a p t i c i n h i b i t i o n i n the h ippocampus . J . N e u r o p h y s i o l . 27:608-619. A n d e r s e n , P . , G je r s t ad , L . and Langmoen, I .A.I (1978) A c o r t i c a l ep i l epsy model i n v i t r o . In : N . Cha lazon i t i s and M . Boi s son (eds.) , Abnorma l Neurona l D i scha rges . Raven P r e s s . New Y o r k : 29-36. 228 A n d e r s e n P . , Gros s , G .N. , L^raio, T. and S v e e n , 0 . (1969) P a r t i c i p a t i o n of i n h i b i t o r y and exc i t a to ry i n t e r n e u r o n s i n the c o n t r o l of h ippocampal c o r t i c a l ou tpu t . I n : The i n t e r n e u r o n . e d . M . A . B . B r a z i e r . B e r k e l y U n i v . Cal i f . P r e s s , p p 415-465. A n d e r s e n , P . , Holmqvis t , B . and Voorhoeve , P . E . (1966) E n t o r h i n a l a c t i va t i on of dentate g ranu le ce l l s . A c t a p h y s i o l . s cand . V o l . 66:448-460. A n d e r s e n , P . , a n d Lfixno, T. (1967) C o n t r o l of h ippocampal ou tpu t b y af ferent v o l l e y f r e q u e n c y . In : P r o g r e s s i n b r a i n r e s e a r c h (ed.) W.R. A d e l y and T. Tok i zane . V o l 27:400-412. A n d e r s e n , T . E . and Rut ledge , L . T . (1979) I n h i b i t i o n i n p e n i c i l l i n - i n d u c e d foc i . E l e c t r o e n c e p h . c l i n . N e u r o p h y s i o l . 46:498-509 A n d r e w , R.D. , T a y l o r , C P . , Snow, R.W. a n d Dudek , F . E . (1982) C o u p l i n g i n r a t h ippocampal s l i ces : d y e t r a n s f e r between C A l p y r a m i d a l ce l l s . B r a i n Res. B u l l . , 8:211-222. A r v a n i t a k i , A . (1942) Effec ts evoked i n an axon b y the a c t i v i t y of a con t iguous one. J . N e u r o p h y s i o l . , 5:89-108. A v o l i , M . (1983) Is e p i l e p s y a d i s o r d e r of i n h i b i t i o n o r exci ta t ion? I n : E p i l e p s y : A n Update on Resea rch and T h e r a p y . A . R . L i s s , Inc . , New Y o r k p g . 23-37. A y a l a , G .F . (1983) The pa roxysma l d e p o l a r i z i n g sh i f t . In : E p i l e p s y : A n Update on Resea rch and T h e r a p y . A . R . L i s s , Inc . New Y o r k , p g 15-21. A y a l a , G .F . , D ich te r , M , Gumnit , R . J . Matsumoto, H . , and Spence r , W.A. (1973) Genesis of ep i l ep t i c i n t e r i c t a l s p i k e s . New knowledge of c o r t i c a l feedback sys tems sugges t s a n e u r o p h y s i o l o g i c a l explana t ion of b r i e f pa roxysms . B r a i n Res. , 52:1-18. A y a l a , G .F . , Matsumoto, H. and Gumnit , R . J . (1970) E x c i t a b i l i t y changes a n d i n h i b i t o r y mechanism i n neocor t i ca l neu rons d u r i n g s e i z u r e s . J . N e u r o p h y s i o l . , 33:73-85. y B a k e r , R. and L l i n a s , R. (1971) E lec t ro ton ic c o u p l i n g between neu rons i n the r a t mesencephal ic nuc l eus . J . P h y s i o l . (Lond . ) , 212:45-63. Bawin S . M . , A b u - A s s a l , M . L . , S h e p p a r d A . R . , Mahoney, M.D. and A d e y W.R. (1986) L o n g - t e r m effects of s i n u s i o d a l ex t r ace l l u l a r e l ec t r i c f i e lds i n p e n i c i l l i n - t r e a t e d r a t h ippocampal s l i ces . B r a i n Res. 399:194-199 B a w i n , S . M . , S h e p p a r d , A . R . , Mahoney, M.D. and A d e y , W.R. (1984) Inf luences of s i n u s o i d a l e l ec t r i c f ie lds on e x c i t a b i l i t y i n the ra t h ippocampal s l i ce . B r a i n Res. 323:227-237. Bawin , S . M . , S h e p p a r d , A . R . , Mahoney, M.D. , A b u - A s s a l , M and A d e y , W.R. (1986) Compar i son between the effects of ex t r ace l lu l a r d i r e c t and s i n u s o i d a l c u r r e n t s on e x c i t a b i l i t y i n h ippocampal s l i ces . B r a i n Res. 362:350-354. 229 Bennet t , M . V . L . (1977) E l e c t r i c a l t r ansmis s ion : a f unc t i ona l ana ly s i s and compar i son to chemica l t r ansmis s ion . In : Handbook of P h y s i o l o g y . The N e r v o u s Sys tem. Be thesda , MD:Am. P h y s i o l . Soc. sect . 1, v o l . 1, p a r t 1, chap te r 11, p . 357-416. Benne t t , M . V . L . , Nakaj ima, Y . and Pappas , G.D. (1967) P h y s i o l o g y and u l t r a s t r u c t u r e of e lec t ro ton ic j u n c t i o n s . III . Giant e lectromotor neurons of M a l a p t e r u r u s e l e c t r i c u s . J . N e u r o p h y s i o l . , 30:209-235. Bennet t , M . V . L . , Pappas , G.D., A l j u r e , E . and Nakajima, Y . (1967) P h y s i o l o g y a n d u l t r a s t r u c t u r e of e lec t ro ton ic j u n c t i o n s . II S p i n a l and medu l l a ry e lect romotor n u c l e i i n mormyr id f i s h . J . N e u r o p h y s i o l . , 30:180-208. Bennet t , M . V . L . , Pappas , G.D. Gimenez, M . and Nakajima, Y . (1967) P h y s i o l o g y and u l t r a s t u r c t u r e of e lec t ro ton ic j u n c t i o n s . IV. M e d u l l a r y e lect romotor n u c l e i i n gymno t id f i s h . J . N e u r o p h y s i o l . , 30:236-300. B e n n i n g e r , C , K a d i s , J . , and P r i n c e , D.A. (1980) E x t r a c e l l u l a r ca lc ium and potass ium changes i n h ippocampal s l i ces . B r a i n Res. 187:165-182. Bindman, L . J . , L i p p o l d , O .C . J , and Redfea rn , J .W.T. (1964) The ac t ion of b r i e f p o l a r i z i n g c u r r e n t s on the c e r e b r a l cor tex of the r a t (1) d u r i n g c u r r e n t flow and (2) i n the p r o d u c t i o n of l o n g - l a s t i n g a f te r -e f fec t s . J . P h y s i o l . (London) , 172:369-382. B l a i r , E . A . a n d E r l a n g e r , J . (1940) In t e r ac t i on of medul la ted f i b e r s of a n e r v e tes ted w i t h e l ec t r i c shock . Amer . J . P h y s i o l . 131:483-493. B l i s s , T . V . P . (1979) S y n a p t i c p l a s t i c i t y i n the h ippocampus . T r e n d s N e u r o s c i . 4:42-45. B r o c a , P . (1878) Anatomie comparee des c i r c o n v o l u t i o n s cerebrale^s. Le g r a n d lobe l imbique et l a s c i s s u r e dans l a ser ie des mammiferes. Review A n t h r o p . 1:385-498. B r o o k h a r t , J . M . and B l a c h l y , P . H . (1952) Cerebe l l a r u n i t r e sponses to DC p o l a r i z a t i o n . Amer . J . P h y s i o l . 171: 711. B u l l o c k , A . G . M . a n d Ka te r , S .B. (1981) Se lec t ion of a nove l connec t ion b y adu l t mol luscan neurons . Sc ience , 212:79-81. B u z a k i , G. and C z e h , G. (1981) Commissura l and pe r fo ran t pa th i n t e r connec t ions i n the ra t h ippocampus . E x p . B r a i n Res. 43:429-438. B u z a k i , G. and E i d e l b e r g , E . (1982) Conve rgence of assoc ia t iona l and commissura l pa thways on C A l p y r a m i d a l ce l l s of the r a t h ippocampus . B r a i n Res. 237:283-295. / s. Ca ja l , S. Ramon y (1911) His tologie d u systeme n e r v e u x de l'homme et des v e r t e b r e s . A . Maloine . P a r i s . V o l . I I . C h a n , C . Y . and Nicho l son , C. (1986) Modula t ion b y a p p l i e d e l e c t r i c a l f i e lds of P u r k i n j e and s tel la te c e l l a c t i v i t y i n the i so la ted t u r t l e ce rebe l lum. J . P h y s i o l . (Lond.) 371:89-114. 230 C l a r k , J . and P lonsey , R. (1966) A mathematical eva lua t ion of the core conduc to r model B i o p h y s . J . 6:95-112. C o l l i n s , R .C . , Olney , J.W. and Lothman, E.W. (1983) Metabol ic and pa tho log ica l consequences of focal s e i zu re s . In : E p i l e p s y , ed i t ed b y A . Ward , K. P e n r y , and D.P. P u r p u r a , New Y o r k : Raven , p g 87-107. C o n n o r s , B.W. (1984) In i t i a t ion of s y n c h r o n i z e d n e u r o n a l b u r s t i n g i n neocortex. Nature V o l . 310:685-687. Creage r , R., Dunwidd ie , T. and L y n c h G. (1980) P a i r e d pu l se and f r e q u e n c y f a sc i l i t a t i on i n the C A l r e g i o n of the i n v i t r o r a t h ippocampus . J . P h y s i o l , (london) 299:409-424. C reu t z f e ld t , O.D., G e r h a r d , H .F . a n d Kapp , H. (1962) Inf luence of t r a n s c o r t i c a l DC c u r r e n t s on c o r t i c a l neu rona l a c t i v i t y . E x p . Neuro l . 5:436-452. C r i l l , W.E. (1980) Neurona l mechanisms of se iqu ; re i n i t i a t i o n . In : A n t i e p i l e p t i c D r u g s : Mechanisms of ac t ion , ed . b y G.H. Glaser , J . K . P e n r y , and D.M. Woodbury , New Y o r k : Raven , p . 169-183. C u r t i s , D.R., Fe l i x , D. and M c L e n n a n , H. (1970) GABA and h ippocampal i n h i b i t i o n . B r i t . J . Pharmacol . 40:881-883. C u t l e r , W.D. and Y o u n g , J . (1979) The effect of p e n i c i l l i n o n the release of gamma-aminobutyr ic ac id from c e r e b r a l cor tex s l i ces . B r a i n Res. 170:157-163. Da lka ra , K . , K r n j e v i c , K . , Roper t , N . and Y i m , C . Y . (1986) Chemical modula t ion of ephap t i c a c t i v a t i o n of CA3 h ippocampal p y r a m i d s . Neurosc ience 17:361-370. D e n n e y - B r o w n , D. and B r o o k h a r t , J . M . (1962) The effects of a p p l i e d po l a r i z a t i on on evoked e l e c t r o - c o r t i c a l waves i n the cat . E l e c t r o e n c e p h . c l i n . N e u r o p h y s i o l . 14:885-897. D ich t e r , M . , and Spence r , W.A. (1969a) P e n i c i l l i n - i n d u c e d i n t e r i c t a l d i s c h a r g e s from the cat h ippocampus . I. C h a r a c t e r i s t i c s and t o p o g r a p h i c a l fea tures . J . N e u r o p h y s i o l . , 32:649-662. D ich te r , M . , a n d Spence r , W.A. (1969b) P e n i c i l l i n - i n d u c e d i n t e r i c t a l d i s c h a r g e s from the cat h ippocampus . I I . Mechanisms u n d e r l y i n g o r i g i n and r e s t r i c t i o n . J . N e u r o p h y s i o l . , 32:663-687. D ich t e r , M . , Hermann, C. and Se lze r , M . (1973) P e n i c i l l i n e p i l e p s y i n isola ted i s l a n d s of h ippocampus . E l ec t roenceph . C l i n . N e u r o p h y s i o l . , 34:631-638. Die tze l , I . , Heinemann, U . , Hofmeier, G. and L u x , H.D. (1980) T r a n s i e n t changes i n the s ize of the ex t r ace l l u l a r space i n sensor imotor cor tex of ca ts i n re la t ion to s t imulus i n d u c e d changes i n potass ium concen t r a t i on . E x p . B r a i n Res. , 40:432-439. 231 Die tze l , I . , Heinemann, U . , Hofmeier, G. and L u x , H.D. (1982a) Changes i n ex t r ace l l u l a r volume i n the c e r e b r a l cor tex of cats i n r e l a t i on to s t imulus i n d u c e d ' ep i lep t i fo rm a f t e rd i s cha rges . In : P h y s i o l o g y and Pharmaco logy of Ep i l ep togen ic Phenomena, ed . b y M.R. Kee, H.D. L u x a n d E . J . Speckmann , p p . 5-12. Raven , New Y o r k . Die tze l , I., Heinemann, U . , Hofmeier, G. and L u x , H.D. (1982b) S t imulus i n d u c e d changes i n ex t r ace l lu l a r Na+ and C l - concen t r a t i on i n r e l a t ion to changes i n the s ize of the ex t r ace l lu l a r space. E x p . B r a i n Res. , 46:73-84. D ing led ine , R. a n d Gje r s t ad , L . (1979) P e n i c i l l i n b l o c k s h ippocampal I P S P s u n m a s k i n g p ro longed E P S P s . B r a i n Res. 168:205-209. D ing led ine , R. and G j e r s t a d , L . (1980) Reduced i n h i b i t i o n d u r i n g ep i l ep t i fo rm a c t i v i t y i n the i n v i t r o h ippocampal s l i ce . J . P h y s i o l . (Lond.) 305:297-313. D ing led ine , R. and Langmoen, I .A . (1980) Conduc tance changes and i n h i b i t o r y ac t ions of h ippocampal r e c u r r e n t I P S P s . B r a i n Res. 185:277-287. Dudek , R . E . , A n d r e w , R.D. , Mac V i c a r , B . A . , Snow, R.W. and T a y l o r , D.P . (1983) Recent ev idence for a n d poss ib le s ign i f i cance of gap j u n c t i o n s and e lec t ron ic synapses i n the mammalian b r a i n . In : Bas ic mechanisms of neu rona l h y p e r e x c i t a b i l i t y . ed . b y H.H. J a s p e r and N . M . v a n Gelder , New Y o r k : L i s s p . 31-73. Dudek , F . E . , Snow, R.W. and T a y l o r , D.P. (1986) Role of e l e c t r i c a l i n t e r ac t i ons i n s y n c h r o n i z a t i o n of ep i lep t i fo rm b u r s t s . In : A d v a n c e s i n Neuro logy , V o l . 44, ed . b y A . V . De lgado-Escue ta , A . A . Ward , D .M. Woodbury and R . J . P o r t e r Raven P r e s s , New Y o r k , pp 593-617. Dunwidd ie , T . V . (1981): A g e - r e l a t e d d i f fe rences i n the i n v i t r o r a t h ippocampus . Dev. N e u r o s c i . , 4:165-175. E l u l , R. (1962) Dipoles of spontaneous a c t i v i t y i n the c e r e b r a l cor tex . E x p . Neuro l , 6:285-299. F e d e r , R. a n d Ranck J r . , J . B . (1973) S tud i e s on s ing le neu rons i n d o r s a l h ippocampal format ion and septum i n u n r e s t r a i n e d r a t s . I I . Hippocampal slow waves and the ta c e l l f i r i n g d u r i n g bar p r e s s i n g and o ther b e h a v i o r s . E x p . N e u r o l . 41:53-555. F la tman, J . A . and Lamber t , J .D .C . (1979) Sus t a ined ex t r ace l lu l a r po ten t ia l s i n the cat s p i n a l c o r d d u r i n g the micro ion tophore t i c app l i ca t i on of exc i t a to ry amino ac ids . J . N e u r o s c i . Meth . 1:205-218. Freeman, J . A . a n d Nicho l son , C. (1975) Exper imen ta l op t imiza t ion of c u r r e n t s o u r c e - d e n s i t y t echn ique for A n u r a n ce rebe l lum. J . N e u r o p h y s i o l . 38: 369-382. Freeman, J . A . and Stone, J . (1969) A t echn ique for c u r r e n t d e n s i t y a n a l y s i s of f i e ld po ten t ia l s and i t s app l i ca t i on to the f r o g cerebe l lum. In : Neurob io logy of Ce rebe l l a r E v o l u t i o n and Development, ed i ted b y R. L l i n a s . Amer i can Med ica l Assoc i a t i on , p.421-430. 232 F r i c k e , R .A. and P r i n c e , D.A. (1984) Elec t ro-phys io logy of Dentate C y r u s Granu le Ce l l s . J . of N e u r o p h y s . V o l . 51 No. 2:195-209. F u j i t a , Y and Saka ta H. (1962) E l e c t r o p h y s i o l o g i c a l p r o p e r t i e s of C A l and CA2 a p i c a l d e n d r i t e s of r a b b i t h ippocampus . J . N e u r o p h y s . 25:209-222. F u r s h p a n , E . J . and Po t te r , D.D. (1959) T r a n s m i s s i o n at the g ian t motor synapse of the c r a y f i s h . J . P h y s i o l . (Lond.) 145:289-325. F u r u k a w a , T. and F u r s h p a n , E . J . (1963) Two i n h i b i t o r y mechanisms i n the mauthner neu rons of g o l d f i s h . J . N e u r o p h y s i o l o g y 26:140-176. Fu tamachi , K . S . and P r i n c e , D.A. (1975) Effec t of p e n i c i l l i n on an exc i t a to ry synapse . B r a i n Res., 100:589-597. G a r d n e r - M e d w i n , A . R . (1983) A s t u d y of the mechanisms b y w h i c h potass ium moves t h r o u g h b r a i n t i s sue i n the ra t . J . P h y s i o l . (Lond) , 335:353-374. G a r d n e r - M e d w i n , A . R . , Coles , J . A . and Tsacopoulos , M . (1981) Clearance of ex t r ace l l u l a r potass ium: ev idence for spa t i a l b u f f e r i n g b y g l i a l ce l l s i n the r e t i n a of the d rone . B r a i n Res. , 200:452-457. Gasser , H.S. (1938) Recru i tment of n e r v e f i b e r s . Am. J . P h y s i o l . , 121:193-202. Gaustaut , H . and B r o u g h t o n , R. (1973) E p i l e p t i c s e i zu re s : T h e i r c l i n i c a l na ture , pa thophys io log i c and d i f f e r en t i a l d i agnos i s . In : A n t i c o n v u l s a n t D r u g s , ed : J . M e r c i e r , Pwergamon P r e s s , New Y o r k . V o l . 1 p g 3-44. Ge t t i ng , P . A . and Willows, A.O.D. (1974) Modi f i ca t ion of n e u r o n p r o p e r t i e s by-e lec t ron ic synapses . I I . B u r s t format ion b y e lec t ron ic synapses . J . N e u r o p h y s i o l . 37:858-868. G je r s t ad , L . , Langmoen, I .A. and A n d e r s e n , P . (1978) Fac to r s a f f ec t ing ep i l ep t i fo rm p y r a m i d a l c e l l d i s c h a r g e s i n v i t r o . I n : A d v a n c e s i n E p i l e p t o l o g y . ed : M e i n a r d i H. Rowan A . J . Amsterdam, L i s s e : Swets and Ze i t l i nge r . p g 443-449. Gloor , P . , V e r a , C L . and S p e r t i , L . (1963) E l e c t r o p h y s i o l o g i c a l s tud ie s of h ippocampal neu rons . I. C o n f i g u r a t i o n and laminar ana ly s i s of the " r e s t i n g " po ten t i a l g rad ien t , of the ma in - t r ans i en t r esponse to pe r fo ran t pa th , f i b r i a l and mossy f ibe r v o l l e y s a n d of "spontaneous" a c t i v i t y . E l e c t r o e n c e p h . c l i n . N e u r o p h y s i o l . 15:353-378. G r a y ' s Anatomy (1973) 35th E d i t i o n , ed . b y R.Warwick and P . L . Williams. Longman Group L t d . Green , J .D. and Maxwel l , D.S. (1961) Hippocampal e l e c t r i c a l a c t i v i t y . I. M o r p h o l o g i c a l aspec ts . E l ec t roenceph . C l i n . N e u r o p h y s i o l . 13:837-846. Grund fe s t , H. (1967) S y n a p t i c and ephapt ic t r ansmis s ion . In : The Neurosc iences b y Q. Quar ton , T. Melnechuk and F . Schmit t . (eds . ) Rockefe l l e r U n i v . P r e s s , N . Y . p 353-372. Grund fe s t , H . and Magnes , J . (1951) E x c i t a b i l i t y changes i n d o r s a l roots p r o d u c e d b y e lec t ro ton ic effects from adjacent af ferent a c t i v i t y . Am. J . P h y s i o l . , 164:502-508. 233 G u t n i c k , M . J . and P r i n c e , D.A. (1981) Dye c o u p l i n g and poss ib le e lec t ron ic c o u p l i n g i n the gu inea p i g neocor t i ca l s l i ce . Sc ience , 211:67-70. Haas, H . L . and J e f f e r y s , J .G.R. (1984) Low ca lc ium f ie ld b u r s t d i s c h a r g e s of C A l p y r a m i d a l neurones i n ra t h ippocampal s l i ces . J . P h y s i o l . 354:185-201. H a b e r l y , L . B . and S h e p h e r d , G.M. (1973) C u r r e n t - s o u r c e a n a l y s i s of summed evoked po ten t ia l s i n Opossum P r e p y r i f o r m cor tex . J . N e u r o p h y s i o l . 36: 789-802. Hab l i t z , J . J . (1981) Effec ts of i n t r a c e l l u l a r in jec t ions of c h l o r i d e and EGTA on p o s t e p i l e p t i f o r m - b u r s t h y p e r p o l a r i z a t i o n s i n h ippocampal neurons . N e u r o s c i . Le t t . , 22:159-163. Hab l i t z , J . J . a n d J o h n s t o n , D. (1981) The endogenous na tu re of spontaneous b u r s t s i n h ippocampal p y r a m i d a l neu rons . C e l l Mol . Neurob io l . , 1:325-334. Hansen , A . J . , Hounsgaa rd , J . and J ahnsen , H. (1982) Anox ia inc reases po tass ium conduc tance i n h ippocampal n e r v e ce l l s . A c t a P h y s i o l . Scand . , 115-301-310. Heinemann, U . and G u t n i c k , M . J . (1979) Rela t ion between ex t r ace l lu l a r potass ium and neu rona l a c t i v i t i e s i n cat thalamus (VPL) d u r i n g p r o j e c t i o n of c o r t i c a l ep i l ep t i fo rm d i s c h a r g e . E l ec t roencepha logy C l i n . N e u r o p h y s i o l . , 47: 345-357. Heinemann, U . and L u x , H.D. (1983) Ionic changes d u r i n g exper imenta l ly i n d u c e d ep i l eps ies . In : P r o g r e s s i n E p i l e p s y Resea rch , ed . b y F . C . Rose, p p . 87-102. P i tman Med ica l , L o n d o n . Heinemann, U . , L u x , H.D. and G u t n i c k , M . J . (1977) E x t r a c e l l u l a r free ca lc ium and po tass ium d u r i n g pa roxysma l a c t i v i t y i n c e r e b r a l cor tex of the cat . E x p . B r a i n Res. , 27:237-243. Heinemann, U . , L u x , H.D. and G u t n i c k , M . J . (1978) Changes i n ex t r ace l lu l a r free ca lc ium and potass ium a c t i v i t y i n the somatosensory cor tex of ca ts . In : A b n o r m a l Neurona l D i scha rges , ed . b y M . Cha lazon i t i s , and M . Bo i s son , p p . 329-345. Raven , New Y o r k . H jo r th -S imonsen , A . and Jeune , B . (1972) O r i g i n and te rmina t ion of the h ippocampal pe r fo ran t pa th i n the ra t s t ud i ed b y s i l v e r impregna t ion . J . Comp. Neuro l . , 144:215-232. Holsheimer , J . (1987) E l e c t r i c a l c o n d u c t i v i t y of the h ippocampal C A l l a y e r s a n d app l i ca t i on of c u r r e n t source d e n s i t y a n a l y s i s . E x p . B r a i n Res. 67:402-410. Hotson, J .R . , S y p e r t , G.W. and Ward , A . A . (1973) E x t r a c e l l u l a r potass ium concen t r a t i on changes d u r i n g p ropaga ted se i zu res i n neocortex. E x p . Neuro l . , 38:20-26. Ito, M . and Oshima, T. (1964) The e x t r u s i o n of sodium from cat s p i n a l motoneurones. P r o c . Roy. Soc. B . (Lond . ) , 161:109-131. 234 J a c k s o n , J . H . (1870) A s t u d y of c o n v u l s i o n s . T r a n s . St . A n d r e w s Med . G r a d . A s s n . , 3:1-45. J a spe r , H.H. (1969) Mechanisms of p ropaga t ion : E x t r a c e l l u l a r s tud ies . I n : Bas ic Mechanisms of the Ep i l eps i e s , ed . b y H.H. J a spe r , A . A . Ward , J r . and A. Pope, pp.421-438. L i t t l e , B r o w n & Co. , Bos ton . J a spe r , H.H. and Monn ie r , A . M . (1938) T r a n s m i s s i o n of exc i ta t ion between exc i sed non-mye l ina ted n e r v e s . A n a r t i f i c i a l synapse . J . C e l l . Comp. P h y s i o l . , 11:259-277. J e f f e r y s , J .G.R. (1979) In i t i a t i on and s p r e a d of ac t ion potent ia ls i n g r anu l e ce l l s mainta ined i n v i t r o i n s l ices of g u i n e a - p i g h ippocampus . J . P h y s i o l . 289:375-388. J e f f e r y s , J .G.R. (1981) Inf luence of e l ec t r i c f i e lds on the e x c i t a b i l i t y of g r anu l e ce l l s i n g u i n e a - p i g h ippocampal s l i ces . J . P h y s i o l . (London) 319:143-152. J e f f e r y s , J .G.R. (1984) C u r r e n t flow t h r o u g h h ippocampal s l i ces . Soc. N e u r o s c i . A b s t r . 14:1074. J e f f e r y s , J .G.R. and Haas, H . L . (1982) S y n c h r o n i z e d b u r s t i n g of C A l , h ippocampal p y r a m i d a l ce l l s i n the absence of s y n a p t i c t r ansmis s ion . Nature (London) , 300:448-450. J o h n s t o n , D. and B r o w n , T .H . (1981) Giant s y n a p t i c po ten t ia l h y p o t h e s i s for ep i lep t i fo rm a c t i v i t y . Sc ience 211:294-297. J o h n s t o n , D. and B r o w n T .H . (1984) The s y n a p t i c na tu re of the pa roxysma l d e p o l a r i z i n g sh i f t i n h ippocampal neurons . A n n Neuro l . , 16:S65-S71. Johns ton ,D. , Hab l i t z , J . J . a n d Wilson, W.A. (1980) Voltage clamp d i sc loses slow i n w a r d c u r r e n t i n h ippocampal b u r s t - f i r i n g neurones . Na tu re , 285:391-393. J u n g R., and K o r n u m u l l e r , A . E . (1938) Eine methodik de r A b l e i l u n g l o k a l i s i e r t e r p o t e n t i a l s c h w a n k u n g e n aus s u b c o r t i k a l e n H i rngeb i e t en . A r c h . P s y c h i a t r . N e r v e n k r 109:1-30 K a n d e l , E.R. a n d Spence r , W.A. (1961a) E l e c t r o p h y s i o l o g y of h ippocampal neu rons . I I . A f t e r potent ia ls and r e p e t i t i v e f i r i n g . J . N e u r o p h y s i o l . , 24:243-259. K a n d e l , E .R . , and Spence r , W.A. (1961b) Exc i t a t i on and i n h i b i t i o n of s ing le p y r a m i d a l ce l l s d u r i n g h ippocampal s e i zu re . E x p . Neuro l . , 4:162-179. Kass , I .S. and L i p t o n , P . (1982) Mechanisms i n v o l v e d i n i r r e v e r s i b l e anoxic damage to the i n v i t r o ra t h ippocampal s l i ce . J . P h y s i o l . , 332:459-472. Ka tz , B, (1969) The release of n e u r a l t r ansmi t t e r subs tances . The S h e r r i n g t o n l e c tu r e . Thomas, S p r i n g f i e l d , 111. Ka tz , B . and Scmni t t , O.H. (1940) E l e c t r i c a l i n t e r ac t i ons between two adjacent n e r v e f i b e r s . J . P h y s i o l . 97:471-488. 235 K e l l y , D.D. (1981) P h y s i o l o g y of s leep and d reaming . In : P r i n c i p l e s of N e u r a l Sc ience , ed . b y E .R .Kande l and J . H . S c h w a r t z . E l s e v i e r Nor th Hol land , Inc . New Y o r k . p g . 473-485. K l u v e r , H. and B u c y , P . C . (1939) P r e l i m i n a r y ana ly s i s of func t ions of the tempora l lobes i n monkeys . A r c h . Neuro l . P s y c h i a t r y 42:979-1000. Knowles , W.D. and S c h w a r t z k r o i n , P . A . (1981) L o c a l c i r c u i t s y n a p t i c i n t e r ac t i ons i n h ippocampal b r a i n s l i ces . J . N e u r o s c i . , 1:318-322. Knowles , W.D., P u n c h , P .G . and S c h w a r t z k r o i n , P .A . (1982) E lec t ro ton ic and dye c o u p l i n g i n the h ippocampal s l i ce . Neurosc ience , 7:1713-1722. Ko ike , H . , Mano, N . , Okada , Y . and Oshima, T. (1972) A c t i v i t i e s of the sodium pump i n cat p y r a m i d a l t r a c t ce l l s i n v e s t i g a t e d w i t h i n t r a c e l l u l a r i n j ec t i on of sodium ions . E x p . B r a i n Res. , 14:489-503. K o n n e r t h , A . , Heinemann, U . a n d Y a a r i , Y . (1984) Slow t r ansmis s ion of n e u r a l a c t i v i t y i n h ippocampal a rea C A l i n absence of ac t i ve chemical synapses . Nature (Lond . ) , 307:69-71. K o n n e r t h , A . , Heinemann, U . , and Y a a r i , Y . (1986) Nonsynap t i c ep i lep togenes i s i n the mammalian h ippocampus i n v i t r o 1. Development of s e i z u r e l i k e a c t i v i t y i n low ex t r ace l l u l a r ca lc ium. J . N e u r o p h y s i o l . 56:409-423. K o r n , H. and A x e l r a d , H. (1980) E l e c t r i c a l i n h i b i t i o n of P u r k i n j e ce l l s i n the ce rebe l lum of the ra t . P r o c . Na t l . A c a d . S c i . U . S . A . , 77:6244-6247. K o r n , H. and Fabe r , D.S. (1975) A n e l e c t r i c a l l y mediated i n h i b i t i o n i n go ld f i sh medul la . J . N e u r o p h y s . 38:452-471. K o r n , H . , Sotelo, C. and C r e p e l , F . (1973) E lec t ro ton ic c o u p l i n g between neu rons i n the r a t l a t e r a l v e s t i b u l a r nuc leus . E x p . B r a i n Res. , 16:255-275. L a n d a u , W.M., B i s h o p , G.H. and Cla re , M . H . (1965) Si te of exc i ta t ion i n s t imula t ion of the motor cor tex . J . N e u r o p h y s i o l 28:1206-1222. Lee , K . S . , O l ive r M.O. , Scho t t l e r F . and L y n c h G.S. (1981) E l e c t r o n microscope s tud ies of b r a i n s l i ces : the effects of h i g h - f r e q u e n c y s t imula t ion on d e n d r i t i c u l t r a s t r u c t u r e . In : E l e c t r o p h y s i o l o g y of i so la ted mammalian CNS p repa ra t i ons , ed . G.A. K e r k u t and H.W. Wheal, Academic P r e s s Inc . , L o n d o n , p p 189-211. Le rma , J . , H e r r e r a s , O., Munoz , D. and So l i s , J . M . (1984) In te rac t ions between h ippocampal p e n i c i l l i n s p i k e s and the ta r h y t h m . E l e c t r o e n c e p h a l o g r a p h y and C l i n i c a l N e u r o p h y s i o l o g y , E l s e v i e r Sc ien t i f i c P u b l i s h e r s I r e l a n d , L t d . 57:532-540. L e u n g , L . S . (1979a) Potent ia l s e v o k e d b y A l v e a r t r a c t i n h ippocampal C A l r e g i o n of r a t s . I. T o p o g r a p h i c a l p ro j ec t i on , component a n a l y s i s , and c o r r e l a t i o n w i t h u n i t a c t i v i t i e s . J . of N e u r o p h y s . V o l . 42, No. 6:1557-1570. 236 L e u n g , L . S . (1979b) Potent ia l s E v o k e d b y A l v e a r t r ac t i n h ippocampal C A l r e g i o n of r a t s . II Spa t i a l f i e ld a n a l y s i s . J . of N e u r o p h y s . V o . l 42, No. 6:1571-1589. L e u n g , L . S . (1979c) Or thodromic ac t i va t i on of h ippocampal C A l r e g i o n of the ra t . B r a i n Res. 176:49-63. L e w i s , D.V. and Schue t t e , W.H. (1975) NADH f luorescence and [K+]o changes d u r i n g h ippocampal e l e c t r i c a l s t imula t ion . J . N e u r o p h y s i o l . , 38:405-417. Loewens te in , W.R. (1981) J u n c t i o n a l I n t e r c e l l u l a r Communicat ion: The c e l l - t o -c e l l membrane channe l . P h y s i o l o g i c a l Rev iews . Vo. 61, No. 4, p .829-913. L^mo, T. (1971a) Pa t t e rn s of a c t i va t i on i n a monosynapt ic c o r t i c a l pa thway: The pe r fo ran t pa th i n p u t to the dentate a rea of the h ippocampal format ion. E x p . B r a i n Res . 12:18-45. L^mo. T. (1971b) Po ten t ia t ion of monosynapt ic E P S P s i n the pe r fo ran t pa th -den ta te g r anu l e c e l l synapse . E x p . B r a i n Res. 12:46-63. Lowens t e in , W.R. (1981) J u n c t i o n a l I n t e r c e l l u l a r communicat ion: The c e l l to c e l l membrane channe l . P h y s i o l o g i c a l Rev iews , V o l 61, No. 4:829-9013. Loren te de N O T R . (1934) S tud ie s on the s t r u c t u r e of the c e r e b r a l cor tex II . Con t inua t ion of the s t u d y of the ammonic sys tem. J . P s y c h o l . Neu ro l . , 46: 113-177. Lo ren t e de No', R. (1947) A c t i o n po ten t i a l of the motorneurons of the h y p o g l o s s u s nuc leus . J . C e l l . Comp. P h y s i o l . 29:207-228 Lothman, E.W., and Somjen, G.G. (1975) E x t r a c e l l u l a r po tass ium a c t i v i t y , i n t r a c e l l u l a r and ex t r ace l l u l a r po ten t i a l r e sponses i n the s p i n a l c o r d . J . P h y s i o l . (Lond.) 252:115-136. Mac V i c a r , B . A . and Dudek, F . E . (1980) Dye c o u p l i n g between CA3 p y r a m i d a l ce l l s i n s l ices of r a t h ippocampus . B r a i n Res. , 196:494-497. M a c V i c a r , B . A . and Dudek, F . E . (1980) Loca l s y n a p t i c c i r c u i t s i n ra t h ippocampus : i n t e rac t ions between p y r a m i d a l ce l l s . B r a i n Res. 184:220-223. M a c V i c a r , B . A . and Dudek , F . E . (1982) E lec t ro ton ic c o u p l i n g between g ranu le ce l l s of ra t dentate g y r u s : P h y s i o l o g i c a l and anatomical ev idence . J . N e u r o p h y s i o l . , 47:579-592. M a c V i c a r , B . A . and Dudek, F.E.(1981) E l e c t r o n i c - c o u p l i n g between CA3 p y r a m i d a l ce l l s : a d i r e c t demons t ra t ion i n ra t h ippocampal s l i ces . Sc ience 213:782-785 M a c V i c a r , B . A . , Roper t , N . and K r n j e v i c , K. (1982) D y e - c o u p l i n g between p y r a m i d a l ce l l s of ra t h ippocampus i n v i v o . B r a i n Res. 238:239-244. M a d r y g a , F . J . , G o d d a r d , G.V. and Rasmusson, D.D. (1975) The k i n d l i n g of motor s e i zu re s from hippocampal commissure of ra t . P h y s i o l . P s y c h o l . 3(4):369-373. 237 M a r r a z z i , A . S . and L o r e n t e de No, R. (1944) In t e r ac t i on of n e i g h b o r i n g f i b r e s i n myel ina ted n e r v e . J . P h y s i o l . V o l . 7:83-101. Matsumoto, H. and Ajmone M a r s a n , C. (1964a) C o r t i c a l c e l l u l a r phenomena i n exper imenta l ep i l epsy : I n t e r i c t a l manifes ta t ions . E x p . Neuro l . , 9:286-304 Matsumoto, H. and Ajmone M a r s a n , C. (1964b) C o r t i c a l c e l l u l a r phenomena i n exper imenta l e p i l e p s y : I c t a l manifes ta t ions . E x p . Neuro l . , 9:305-326. Matsumoto, H . , A y a l a , G .F . and Gumnit , R . J . (1969) Neurona l behav io r and t r i g g e r i n g mechanism i n c o r t i c a l ep i l ep t i c focus . J . N e u r o p h y s i o l . , 32:688-703. McDonald , R. and B a r k e r , J . L (1977) Pen ty l ene t e t r azo l and p e n i c i l l i n are se lec t ive an tagon i s t s of GABA mediated p o s t s y n a p t i c i n h i b i t i o n i n c u l t u r e d mammalian neurons . Nature (Lond.) 267:720-721. McNamara, J .P . , Pepe r A . M . and Pa t rone , V . (1980) Repeated s e i zu re s i nduce l o n g - t e r m inc rease i n h ippocampal benzodiazep ine r ecep to r s . P r o c . Na t l . A c a d . S c i . U S A , 77:3029-3032. Mi les , R. and Wong, R . K . S . (1983) S ing le neurones can in i t i a t e s y n c h r o n i z e d popu la t ion d i s c h a r g e i n the h ippocampus . Nature (Lond.) 306:371-373. Mi l e s , R., Wong, R . K . S . and T r a u b , R.D. (1984) S y n c h r o n i z e d a f t e rd i s cha rges i n the h ippocampus : C o n t r i b u t i o n of loca l s y n a p t i c i n t e r a c t i o n . Neurosc ience , 12:1179-1189. M i l n e r , B . (1966) Amnesia fo l lowing opera t ion on the temporal lobes . In : Amnesia , ed . b y C.W.M. Whi t ty and O.L . Zangwi l l . pp 109-133. Mi t zdo r f , U . (1980) J u s t i f i c a t i o n of the assumpt ion of cons tan t r e s i s t i v i t y u s e d i n c u r r e n t source d e n s i t y ca lcu la t ions . J . P h y s i o l . L o n d o n 304:216-220. M i t z d o r f , U . (1985) C u r r e n t - s o u r c e d e n s i t y method and app l i ca t i on i n cat c e r e b r a l cor tex : i n v e s t i g a t i o n of evoked potent ia ls and E E G phenomenon. P h y s i o l . Rev . 65: 37-100. Nelson , P .G . and F r a n k , K. (1964) E x t r a c e l l u l a r po ten t ia l f i e lds of s ing le s p i n a l motoneurons . J . N e u r o p h y s i o l . , 27:913-927. Nelson , P .G . (1966) In t e rac t ion between s p i n a l motoneurons of the cat . J . N e u r o p h y s i o l . , 29:275-287. N icho l son , C. (1973) Theore t i ca l a n a l y s i s of f i e ld potent ia ls i n an i so t rop ic ensembles of neu rona l elements. I E E E T r a n s . Biomed. E n g . B M E -20:278-288. N icho l son , C. and Freeman, J . A . (1975) T h e o r y of c u r r e n t s o u r c e - d e n s i t y a n a l y s i s and de te rmina t ion of c o n d u c t i v i t y t ensor for A n u r a n ce rebe l lum. J . N e u r o p h y s i o l . 38: 356-368. Nicho l son , C. and L l i n a s , R. (1971) F i e l d potent ia ls i n the a l l iga tor ce rebe l lum and t h e o r y of t he i r r e l a t i onsh ip to p u r k i n j e c e l l d e n d r i t i c s p i k e s . J . N e u r o p h y s i o l . V o l . 34:509-531 238 N i z n i k , H .B . , Burnham, . W.M., and K i s h , S . J . (1984) Benzodiazep ine r ecep to r b i n d i n g fo l lowing a m y g d a l a - k i n d l e d c o n v u l s i o n s : d i f f e r i n g r e s u l t s i n washed and unwashed b r a i n membranes. J . Neurochem. , 43:1732-1736. Noebels , J . L . and P r i n c e , D.A. (1978) Development of focal s e i zu re s i n c e r e b r a l cor tex: role of axon t e rmina l b u r s t i n g . J . N e u r o p h y s i o l . 41:1267-1281. O l i v e r , M.W. (1986) E l e c t r o p h y s i o l o g i c a l p r o p e r t i e s of the h ippocampal format ion i n ra t : an i n v i t r o s t u d y . Thes i s submi t ted for P h . D. i n Dept. of P h y s i o l o g y , U . B . C . V a n c o u v e r , B . C . , Canada O l i v e r , M.W. and M i l l e r J . J . (1985) A l t e r a t i ons of i n h i b i t o r y p rocesses i n the dentate g y r u s fo l lowing k i n d l i n g - i n d u c e d e p i l e p s y . E x p . B r a i n Res. , 57:443-447. Pa lay , S . L . and C h a n - P a l a y , V . (1974) Ce rebe l l a r cor tex , c y t o l o g y and o r g a n i z a t i o n . ( S p r i n g e r , B e r l i n , H e r d e l b e r g , New Y o r k ) . Papez , J.W. (1937) A p r p o s e d mechanism of emotion. A r c h . N e u r o l . P s y c h i a t r y 38:725-743. Pen f i e ld , W. (1958) F u n c t i o n a l loca l i za t ion i n tempora l and deep S y l v i a n areas . Res. P u b l . A s s o c . Res. N e r v . Ment . Dis . 36:210-226. Pen f i e ld , W. and E r i c k s o n , T .C . (1941) The h i s t o r y of e p i l e p s y . I n : E p i l e p s y a n d c e r e b r a l l oca l i za t ion . C C . Thomas. I l l i no i s . 4-10. P i t t s , W. (1952) Inves t i ga t i ons on s y n a p t i c t r ansmis s ion . In : C y b e r n e t i c s , T r a n s . N i n t h Conf . , ed i t ed b y H. v o n F o e r s t e r . New Y o r k : Jo s i ah M a c y , p . 159-162 P r i n c e , D.A. (1968) The depo la r i za t ion sh i f t i n "ep i l ep t i c " neu rons . E x p . N e u r o l . 21:467-485. P r i n c e , D.A. (1966) Modi f i ca t ion of focal c o r t i c a l ep i lep togenic d i s c h a r g e b y afferent i n f luences . E p i l e p s i a , 7:181-201. P r i n c e , D.A. (1967) E l e c t r o p h y s i o l o g y of ep i l ep t i c neu rons . E lec t roencepha lage C l i n . N e u r o p h y s i o l . 23:83-84. P r i n c e , D.A. (1978) N e u r o p h y s i o l o g y of E p i l e p s y . A n n u . Rev . N e u r o s c i . 1:395-415. P r i n c e , D.A. (1982) Neurona l even ts u n d e r l y i n g ep i lep togenes i s . In : Top ics i n C h i l d Neuro logy , V o l . 2, ed . b y R.R. O u v r i e r and P .G. P r o c o p i d , p p . 35-53. Spec t rum, New Y o r k . P r i n c e , D.A. and Connor s , B.W. (1986) Mechanisms of I n t e r i c t a l Ep i l ep togenes i s . In : A d v a n c e s i n Neuro logy , V o l . 44, ed . b y A . V . De lgado-Escue ta , A . A . Ward , D.M. Woodbury and R . J . P o r t e r Raven P r e s s , New Y o r k , pp 275-299. P r i n c e , D.A. , L u x , H.D., and Neher , E . (1973) Measurements of ex t r ace l lu l a r potass ium a c t i v i t y i n cat cor tex . B r a i n Res. , 50:489-495. 239 P u r p u r a , D.P. (1969) Mechanisms of p ropaga t ion : I n t r a c e l l u l a r s tud ies . In : Bas ic Mechanisms of the Ep i l eps i e s , ed . b y H.H. J a spe r , A . A . Ward . J r . and A . Pope, p p . 441-451. L i t t l e B r o w n & Co. Bos ton . P u r p u r a , D.P . , M c M u r t r y , J . G . , L e o n a r d , C F . and Ma l l i an i , A . (1966) E v i d e n c e for d e n d r i t i c o r i g i n of s p i k e s wi thou t d e p o l a r i z i n g p repo ten t i a l s i n h ippocampal neu rons d u r i n g and af ter s e i zu re . J . N e u r o p h y s i o l . 29:954-977,1966. P u r p u r a , D.P. and M a l l i a n i , A . (1966) S h o r t Communicat ion S p i k e Genera t ion and p ropaga t ion in i t i a t ed i n d e n d r i t e s b y t r ansh ippocampa l po l a r i za t i on . B r a i n Resea rch , 1 p . 403-406. P u r p u r a , D.P. and M c M u r t r y , J .G . (1965) I n t r ace l l u l a r a c t i v i t i e s and evoked po ten t ia l changes d u r i n g po l a r i za t i on of motor cor tex . J . N e u r o p h y s i o l . 28:166-185. Racine , R . J . , Mi lg ram, N.W. and Hafner S. (1983) L o n g - t e r m poten t ia t ion phenomena i n the ra t l imbic f o r e b r a i n . B r a i n Res. , 260-:217-231. Raisman, G. , Cowan, W.M. and Powel l , T . P . S . (1965) The e x t r i n s i c afferent , commissura l and assoc ia t ion f i b r e s of the h ippocampus . B r a i n Res. 88:963-998. Raisman, G . , Cowan, W.M. and Powel l , T . P . S . (1965) The e x t r i n s i c af ferent , commissura l and asoc ia t ion f i be r s of the h ippocampus . B r a i n Res. 88:963-998. Ra ls ton , B . L . (1958) The mechanism of t r a n s i t i o n of i n t e r i c t a l s p i k i n g foc i in to i c t a l s e i zu re d i s c h a r g e . E l ec t roenceph . c l i n . N e u r o p h y s i o l . , 10:217-232. Ramon, F . and Moore , J.W. (1978) Ephap t i c t r ansmis s ion i n s q u i d g ian t axons . A m . J . P h y s i o l . 234:162-169. Ranck , J . B . J r . (1975) Which elements are exc i ted i n e l ec t r i c a l s t imula t ion of mammalian c e n t r a l n e r v o u s sys tem: a r e v i e w . B r a i n Resea rch , 98:417-440. Renshaw, B . and Therman , P.O. (1941) Exc i t a t i on of i n t r a s p i n a l mammalian axons b y n e r v e impulses i n adjacent axons. 133:96-105. R ibak , C , V a u g h n , J . and Sai to , K. (1978) Immunbocytochemical loca l i za t ion of g lutamic ac id deca rboxy lase i n neu rona l somata fo i l lowing co lch ic ine i n h i b i t i o n of axonal t r a n s p o r t . B r a i n Res. 140:315-332. R ibak , C . E . , B r a d b u r n e , R . M . and H a r r i s , A . B . (1982) A p r e f e r e n t i a l loss of G A B A n e r g i c symmetr ic synapses i n ep i l ep t i c foc i : A quan t i t a t ive u l t r a s t r u c t u r a l ana ly s i s of monkey neocortex. J . Neu rosc i . , 2:1725-1735 R i c h a r d s o n , T . L . , T u r n e r , R.W. and M i l l e r , J . J . (1984) E x t r a c e l l u l a r f ie lds in f luence t ransmembrane potent ia ls and s y n c h r o n i z a t i o n of h ippocampal neu rona l a c t i v i t y . B r a i n Res. 294:255-262. 240 Richardson, T . L . , Turner, R.W. and Miller, J .J . (1984b) Extracellular voltage gradients and ephaptic interactions in the hippocampal formation. Soc. Neurosci. Abstr. 14, 204. Richardson, T . L . , Turner, R.W. and Miller, J .J . (1987) Action potential discharge in hippocampal CAl pyramidal neurons: current source density analysis. J.Neurophys. 58:981-996. Rosenblueth, A. (1941) The stimulation of myelinated axons by nerve impulses in adjacent myelinated axons. Amer. J . Physiol. 132:119-128. Rudell, A.P., Fox, S.E., and Ranck Jr . , J.B. (1980) Hippocampal excitability phase-locked to the theta rhythm in walking rats. Exp. Neurol. 68:87-96. Schaffer, K. (1892) Beitrag zum Histologie der Ammonshornformation. Archiv. fur Mikroskopische Anatomie 39:611-632. Schmalbruch, H. and Jahnsen, H. (1981) Gap junctions on CA3 pyramidal cells of guinea pig hippocampus shown by freeze-fracture. Brain Res. 217:175-178. Schwartzkroin, P.A. (1975) Characteristics of C A l neurons recorded intracellularly in the hippocampal in vitro slice preparation. Brain Research, 85:423-436. Schwartzkroin, P.A. (1977) Further characteristics of hippocampal CAl cells in vitro. Brain Res. 128:53-68. Schwartzkroin, P.A. (1986) Hippocampal slices in experimental and human epilepsy. Adv. in Neurol. Vol. 44, Raven Press. Schwartzkroin, P.A. and Prince, D.A. (1977) Penicillin-induced epileptiform activity in the hippocampal in vitro preparation. Ann. Neurol., 1:463-469. Schwartzkroin, P.A. and Prince, D.A. (1978) Cellular and field potential properties of epileptogenic hippocampal slices. Brain Research, 147:117-130. Schwartzkroin, P.A. and Stafstrom, E.E. (1980) Effects of EGTA on the calcium-activated afterhyperpolarization in hippocampal CA3 pyramidal cells. Science, 210:1125-1126. Schwartzkroin, P.A. and Wyler, A.R. (1980) Mechanisms underlying epileptiform burst discharge. Ann. Neurol. 7:95-107. Seress, L. and Pokorny, J . (1981) Structure of the granular layer of the rat dentate gyrus. A light microscopic and Golgi study. J . Anat., 133:181-195. Seress, L. and Ribak, C E . (1983) GABAergic cells in the dentate gyrus appear to be local curcuit and projection neurons. Exp. Brain Res. 50:173-182. 241 Sheppard, A.R., Bawin, S.M. Mahoney, M.D. and Adey, W.R. (1983) The effects of orientation of dc and ac extraceilular fields on excitability in the hippocacmpal slice. Soc. Neurosci. Abstr. 9:678. Skrede, K.K. and Westgaard, R.H. (1971) The transverse hippocampal slice: a well-defined cortical structure maintained in vitro. Brain Res. 35:589-593. Snow, R.W. and Dudek, F .E . (1984a) Electrical fields directly contribute to action potential synchronization during convulsant-induced epileptiform bursts. Brain Research, 323:114-118. Snow, R.W. and Dudek, F .E . (1984b) Synchronous epileptiform bursts without chemical transmission in CA2, CA3 and dentate areas of the hippocampus. Brain Research, 298:382-385. Snow, R.W., and Dudek, F .E . (1986) Evidence for neuronal interactions by electrical field effects in the CA3 and dentate regions of rat hippocampal slices. Brain Res. 367:292-295. Sotelo, C , Llinas, R. and Baker, R. (1974) Structural study of inferior olivary nucleus of the cat: morphological correlates of electrotonic coupling. J . Neurophysiol., 27:541-559. Spencer, W.A. and Kandel, E.R. (1961) Electrophysiology of hippocampal neurons. IV. Fast prepotentials. J . Neurophysiol., 24:272-285. Steward, 0. (1976) Topographical organization of the projections from the entorhinal area to the hippocampal formation of the rat. J . Comp. Neurol. 17:296-314. Stewart, W.W. (1978) Functional connections between cells as revealed by dye-coupling with a highly fluorescent napthalimide tracer. Cell, 14:741-759. Steward, 0., White F.W., Cotman CW. and Lynch, G. (1976) Potentiation of exctitatory synaptic transmission in the normal and in the reinnervated dentate gyrus. Exp. Brain Res. 26:423-441. Strumwasser, F. and Rosenthal, S. (1960) Prolonged and patterned direct extracellular stimulation of single neurons. Am. J . of Physiol. Vol. 198:405-413. Swann, J.W., Brady, R.J. , Friedman, R.J. and Smith, E .J . (1986) The dendritic origins of penicillin-induced epileptogenesis in CA3 hippocampal pyramidal cells. J . Neurophys. 56:1718-1736 Swanson, L.W. and Cowan, W.M. (1977) An autoradiographical study of the organization of the efferent connections of the hippocampal formation in the rat. J . Comp. Neurol. 112:49-84. Taylor, C P . and Dudek, F .E . (1982a) A physiological test for electronic coupling between CAl pyrimadal cells in rat hippocampal slices. Brain Res. 235:351-357. 242 Taylor, C P . and Dudek, F .E. (1982b) Synchronous Neural Afterdishcharges in rat hippocampal slices without active chemical synapses. Science, 218:810-812. Taylor, C P . and Dudek, F .E . (1984a) Excitation of hippocampal pyramidal cells by an electrical field effect. J . of Neurophysiol. Vol. 52, No. 1:126-142. Taylor, C P . and Dudek, F .E . (1984b) Synchronization without active chemical synapses during hippocampal afterdischarges. J . of Neurophysiol. Vol 52, No. 1:143-155. Taylor, C P . , Krnjevic, K. and Ropert, N. (1984) Facilitation of hippocampal CA3 pyramidal cell firing by electrical fields generated antidromically. Neuroscience 11:101-109. Terzuolo, C A . and Bullock, T.H. (1956) Measurement of imposed voltage gradient adequate to modulate neuronal firing. Proc. N.A.S. Vol. 42:687-694. Tombol, T., Babosa, M., Hajdu, F. and Somogi, G. (1979) Interneurons: an electron microscope study of the cat's hippocampal formation, II. Acta morphologica Acad. Sci. Hung. 27:297-313. Tranchina, D. and Nicholson, C. (1986) A model for the polarization of neurones by extrinsically applied electric fields. Biophys. J . 50:1139-1156. Traub, R.D., Miles, R. and Wong, R.K. (1987) Models of synchronized hippocampal bursts in the presence of inhibition. 1. Single population events. J . Neurohpys. 58:739-751. Traub, R.D. and Wong, R.K.S. (1983) Synaptic mechanisms underlying interictal spike initiation in a hippocampal network. Neurology 33: 257-266. Traub, R.D. and Wong, R.K.S. (1981) Penicillin-induced epileptiform activity in the hippocampal slice: a model of synchronization of CA3 pyramidal cell bursting. Neuroscience : 223-230. Traub, R.D. and Wong, R.K.S. (1982) Cellular mechanism of neuronal synchronization in epilepsy. Science 216:745-747. Traub, R.D. and Wong, R.K.S. (1983) Synchronized burst discharge in disinhibited hippocampal slice. II model of cellular mechanism. J . Neurophysiol., 49:442-458. Traub, R.D., Dudek, F . E . , Snow, R.W. and Knowles, W.D. (1985) Computer simulations indicate that electrical field effects contribute to the shape of the epileptiform field potential. Neuroscience Vol. 15, No. 4:947-958 Traub, R.D., Dudek, F . E . , Taylor, C P . and Knowles, W.D. (1985) Simulation of hippocampal afterdischarges synchronized by electrical interactions. Neuroscience Vol. 14, No. 4:1033-1038 243 Tuff, L.P. , Racine, R.J. and Adamec, R. (1983a) The effects of kindling on Gaba-mediated inhibition in the dentate gyrus of the rat: paired-pulse depression. Brain Res., 277:79-90. Tuff, L.P. , Racine, R.J. and Mishra, R.K. (1983b) The effects of kindling on GABA-mediated inhibition in the dentate gyrus of the rat. II. Receptor binding. Brain Res., 277:91-98. Turner, R.W. (1985) Action potential discharge in somata and dendrites of CAl pyramidal neurons of mammalian hippocampus: an electrophysiological analysis. Thesis submitted for Ph. D. in Dept. of Physiology, U.B.C., Vancouver, B.C., Canada. Turner, R.W. and Miller, J .J . (1982) Effects of extracellular calcium on low frequency induced potentiation and habituation in the in vitro hippocampal slice preparation. Can J . Pharmacol. 60:266-272. Turner, R.W., Richardson, T .L . and Miller, J .J . (1982) Intracellular correlates of paired pulse potentiation in hippocampal pyramidal cells: Relationship to hyperpolarization. Soc. Neurosci. Abstr. Turner, R.W., Richardson, T .L . and Miller, J .J . (1983) Role of ephaptic interactions in paired pulse and frequency potentiation of hippocampal field potentials. Soc. Neurosci. Abstr. Turner, R.W., Richardson, T . L . and Miller, J .J . (1984) Ephaptic interactions contribute to paired pulse and frequency potentiation of hippocampal field potentials. Exp. Brain Res. 54:567-570. Turner, R.W., Richardson, T .L . and Miller J .J . (1988) Comparative analysis of intracellular, somatic and dendritic spike discharge in CAl hippocampal pyramidal neurons. J . Neurophys. (accepted). Turner, R.W., Richardson, T . L . and Miller J .J . (1988) Intracellular analysis of the site for spike initiation along the dendrosomatic axis of CAl hippocampal pyramidal neurons. J . Neurophys. (accepted). Vanderwolf, C H . (1969) Hippocampal electrical activity and voluntary movement in the rat. Electroenceph. Clin. Neurophysiol. 26:407-418. Westrum, L . E . and Blackstad, T.W. (1962) An electron microscopic study of the stratum rdiatum of the rat hippocampus (regio superior, CAl) with particular emphasis on synaptology. J . Comp. Neurol. 119:281-309. White, W.F., Nadler J.V., Cotman CW. (1979) Analysis of short-term plasticity at the perforant path-granule cell synapse. Brain Res. 178:41-53. Wong, R.K.S. and Prince, D.A. (1978) Participation of calcium spikes during intrinsic burst firing in hippocampal neurons. Brain Res. 159:385-390. Wong, R.K.S. and Prince, D.A. (1979) Dendritic mechanisms underlying penicillin-induced epileptiform activity. Science 204:1228-1231. 244 Wong, R.K.S. and Traub, R.D. (1983) Synchronized burst discharge in the disinhibited hippocampal slice. I Initiation in the CA2-Ca3 region. J. Neurophysiol., 49:459-471. Wong, R.K.S., Prince, D.A. and Basbaum, A.EI. (1979) Intradendritic recordings from hippocampal neurons. Proc. Natl. Acad. Sci. U.S.A., 76:986-990. Wong, R.K.S., Traub, R.D. and Miles, R. (1986) Cellular Basis of Neuronal Synchrony in Epilepsy. In: Advances in Neurology, Vol. 44, ed. by A.V. Delgado-Escueta, A.A. Ward, Jr . , D.M. Woodbury and R.J. Porter. Raven Press, New York, pp 583-592. Woodbury J.W. (1960) Potentials in a volume conductor. In Medical Physiology and Biophysics ed: T.C. Ruch and J .F. Fulton; W.B. Saunders Co. Philadelphia and London pp. 83-91. Wyss, J.M. (1981) An autographic study of the efferent connections of the entorhinal cortex in the rat. J . Comp. Neurol., 199:495-512. Yaari, Y, Konnerth, A. and Heinemann, U. (1983) Spontaneous epileptiform activity of CAl hippocampal neurons in low extracellular calcium solutions. Exp. Brain Res., 51:153-156. Yamamoto, C. (1972) Intracellular study of seizure-like after discharge elicited in thin hippocampal sections in vitro. Exp. Neurol., 35:154-164. Yim, C.C., Krnjevic, K. and Dalkara, T. (1986) Ephaptically generated potentials in CAl neurons of rat's hippocampus in situ. J . Neurophys. 56:99-122 PUBLICATIONS Turner R.W., Richardson. T.L. and M i l l e r J.J. Comparative analysis of i n t r a c e l l u l a r somatic and dendritic spike discharge in CAl hippocampal pyramidal neurons. J. Neurophys. Accepted 1988 Turner R.W., Richardson. T.L. and M i l l e r J.J. I n t r a c e l l u l a r analysis of the s i t e for spike i n i t i a t i o n along the dendrosomatic axis of CAl hippocampal pyramidal neurons. J. Neurophys. Accepted 1988 Richardson T.L., Turner R.W. and M i l l e r J.J. Action potential discharge in hippocampal CAl pyramidal neurons: current source density analysis. J. Neurophys. 58:981-996 1987 Turner R.W., Richardson. T.L. and M i l l e r J.J. Ephaptic interactions contribute to paired pulse and frequency potentiation of hippocampal f i e l d potentials. Exp. Brain Res. 54:567-570 1984 Richardson T.L., Turner R.W. and M i l l e r J.J. E x t r a c e l l u l a r f i e l d s influence transmembrane potentials and synchron-i z a t i o n of hippocampal neuronal a c t i v i t y . Brain Res. 294:255-262 1984 Oliver M.W., Richardson T.L. and M i l l e r J.J. Electrophysio-l o g i c a l properties of hippocampal pyramidal and dentate granule c e l l s following kindling-induced epilepsy. Can. J. Physiol. Pharmacol. Vol. 61 1983 Hall J.G., Hicks T.P., McLennan H., Richardson T.L., and Wheal H.V. The exci t a t i o n of mammalian central neurons by amino acids. J. Physiol. Lond. 286:29-39 1979 Hall J.G., Hicks T.P., McLennan H. , Richardson T.L., and Wheal H.V. The excitation of mammalian central neurons by amino acids. J. Physiol. Lond. 286:29-39 1979 Richardson T.L., M i l l e r J.J. and McLennan H. Mechanisms of ex c i t a t i o n and i n h i b i t i o n in the n i g r o - s t r i a t a l system. Brain Res. 127:219-234 1977 Mi l l e r J . J . , Richardson T.L., Fibiger H. C. and McLennan H. Anatomical and electrophysiological i d e n t i f i c a t i o n of a projection from the mesencephalic raphe to the caudate-putamen in the rat. Brain Res. 97:133-138 1975 Pearson J.A. and Richardson T.L. The influence of stimulus intensity on s e n s i t i z a t i o n of the flexor r e f l e x . Exp. Neurol. 47:194-197 1975 

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