UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Electrophysiological properties of the hippocampal formation in rat : an in vitro study Oliver, Michael W. 1986

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1987_A1 O54.pdf [ 9.18MB ]
Metadata
JSON: 831-1.0097484.json
JSON-LD: 831-1.0097484-ld.json
RDF/XML (Pretty): 831-1.0097484-rdf.xml
RDF/JSON: 831-1.0097484-rdf.json
Turtle: 831-1.0097484-turtle.txt
N-Triples: 831-1.0097484-rdf-ntriples.txt
Original Record: 831-1.0097484-source.json
Full Text
831-1.0097484-fulltext.txt
Citation
831-1.0097484.ris

Full Text

ELECTROPHYSIOLOGICAL PROPERTIES OF THE HIPPOCAMPAL FORMATION IN RAT: AN IN VITRO STUDY by Michael W. O l i v e r B . S c , Univers i ty of C a l i f o r n i a , I rv ine , 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Physiology) We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1986 copyright (c) Michael W. O l i v e r , 1986 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. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) A b s t r a c t The e l ec t rophys io log i ca l propert ies of dentate granule c e l l s and hippocampal pyramidal neurons were examined with e x t r a c e l l u l a r and i n t r a c e l l u l a r recording techniques in the hippocampal s l i c e . I n t r a c e l l u l a r analys i s revealed that there may exist two populations of granule c e l l s d i s t inguishable by the presence or absence of non- l inear current-vol tage (I-V) membrane propert ies (anomalous r e c t i f i c a t i o n , AR). The granule c e l l s exh ib i t ing AR also maintained greater rest ing membrane potent ia l s and act ion potent ia l (AP) amplitude values . The membrane input res is tance (R n) and time constant (T c ) measurements were s imi lar between the populations in response to hyperpolar iz ing current i n j e c t i o n , but granule c e l l s d i sp lay ing AR had s i g n i f i c a n t l y higher R n and T c values in response to depolar iz ing pulses . Both groups also responded to maintained depo lar iz ing current i n j e c t i o n with r e p e t i t i v e AP discharges; however, t h i s response accommodated. Upon termination of the depolar iz ing current i n j e c t i o n , an a f terhyperpo lar iza t ion (AHP) resu l ted , the amplitude of which appeared to depend on the duration of the depo lar iz ing pulse and not on the number of APs generated during the pulse . Stimulat ion of e i ther the l a t e r a l (LPP) or medial (MPP) perforant paths evoked a monosynaptic EPSP followed by a depo lar iz ing a f t e r p o t e n t i a l (DAP) and a long a f terhyperpolar izat ion (LHP). In contras t , antidromic st imulat ion e l i c i t e d a depolariz ing-IPSP (D-IPSP) and a LHP. Both the DAP and D-IPSP were reversed by membrane d e p o l a r i z a t i o n , whereas, the LHP was inverted by membrane hyperpo lar i za t ion . In a l l cases, however, the EPSP could not be inverted . Af terpotent ia l s were associated with an increase in conductance, but the change accompanying the LHP was less than the DAP and D-IPSP. In a d d i t i o n , by reducing the [ C a ] c and increasing the [Mg] D , the DAP was attenuated and the LHP e l iminated. S imi lar resul t s were also obtained with the GABAB agonist , baclofen. Paired pulse s t imulat ion of e i ther the LPP or MPP resul ted in the potent iat ion of the i n t r a c e l l u l a r EPSP at cond i t ion- te s t (C-T) in t erva l s less than 100 ms; however, simultaneous e x t r a c e l l u l a r records from the granule c e l l layer (GCL) i l l u s t r a t e d depression of the EPSP. The discrepancy between the ex tra - and i n t r a c e l l u l a r recordings was shown to be re lated to the presence of the DAP. In a d d i t i o n , the MPP evoked test EPSP at C-T in terva l s greater than 150 ms exhibited i n h i b i t i o n regardless of whether i t was recorded inside or outside the granule c e l l and t h i s EPSP depression was p a r t i a l l y due to the granule c e l l LHP. The LPP evoked test EPSP potentiated at a l l C-T in terva l s less than 1s when recorded from the outer molecular layer iv (OML) but was i n h i b i t e d at both the GCL and i n t r a c e l l u l a r recording s i t e s . These data confirmed that postsynaptic processes contribute to the short-term a l t e r a t i o n s observed with paired pulse s t imulat ion . The t y p i c a l i n h i b i t i o n - p o t e n t i a t i o n - i n h i b i t i o n sequence of the perforant path (PP) evoked population spike (PS) was noted at C-T in terva l s of 20, 80 and 400 ms, re spec t ive ly . The i n h i b i t i o n of the PS at 20 ms was abol ished with perfusion of the GABA antagonist , b i c u c u l l i n e . In contrast , the PS i n h i b i t i o n at 400ms was unaffected by th i s treatment but was s l i g h t l y attenuated by the gKca antagonist TEA. A number of factors appeared to contr ibute to the potent iat ion of the PS: 1) reduction in AP threshold; 2) the presence of the DAP; and 3) extrasynaptic events. In addi t ion to the PS data from normal t i s sue , hippocampal s l i c e s from c h r o n i c a l l y kindled rats exhibited depression of the PS at a l l C-T in terva l s tes ted. This augmentation of i n h i b i t i o n was dependent on the presence of hippocampal afterdischarges but not on motor se izures . Perfusing the kindled s l i c e s with e i ther b i c u c u l l i n e or lowered [ C l ] 0 d id not markedly reverse the enhanced i n h i b i t i o n at C-T in terva l s which displayed dramatic f a c i l i t a t i o n in normal s l i c e s . I n t r a c e l l u l a r recordings of granule c e l l s obtained from kindled s l i c e s also exhibi ted an increase in the R n and T c . Both the a l t e r a t i o n s in i n h i b i t i o n and membrane c h a r a c t e r i s t i c s appear to be V l o c a l i z e d to . the granule c e l l s , since these changes were not observed in CA1 pyramidal neurons. These data indicate that short-term and long-term a l t e r a t i o n s in granule c e l l neuronal e x c i t a b i l i t y are p a r t i a l l y due to changes in the postsynaptic membrane. v i TABLE OF CONTENTS C e r t i f i c a t e of Examination i Abstract i i Table of Contents v i L i s t of Abbreviations x L i s t of Figures x i i i L i s t of Tables x v i i L i s t of Reversed Pages x v i i i Acknowledgements x x i i CHAPTER 1; INTRODUCTION 1 1.1 Hippocampal formation: dentate gyrus and hippocampus proper 2 1.2 Neurons of the hippocampal formation 3 1.3 I n t r i n s i c connections of the hippocampal formation 6 1.4 E l e c t r o p h y s i o l o g i c a l c h a r a c t e r i s t i c s of synaptic responses in the hippocampal formation 8 1.5 St imulation-induced neurop las t i c i ty . 14 1.6 Topic of thesis 22 CHAPTER 2: GENERAL METHODS 24 2.1 Introduction 24 2.2 The in v i t r o hippocampus 25 2.3 Composition of the a r t i f i c i a l cerebrospinal f l u i d (ACSF) 27 2.4 St imulation parameters 30 2.5 E x t r a c e l l u l a r recording of the population potent ia l s evoked in the dentate gyrus 31 v i i 2.6 Analys is of e x t r a c e l l u l a r f i e l d potent ia l s 32 2.7 I n t r a c e l l u l a r recording of dentate gyrus granule c e l l s 38 2.8 Analys is of i n t r a c e l l u l a r membrane c h a r a c t e r i s t i c s and evoked potent ia l s 38 CHAPTER 3: AN INTRACELLULAR CHARACTERIZATION OF THE DENTATE GYRUS GRANULE CELLS FROM THE IN VITRO RAT HIPPOCAMPUS 47 3.1 Introduction 47 3.2 Methods 53 3.3 Results . . . r . . . . . . . . . . . 55 3.3.1 Membrane c h a r a c t e r i s t i c s of granule c e l l s 55 3.3.2 Antidromic evoked responses in granule c e l l s 63 3.3.3 Perforant path evoked responses in granule c e l l s 71 3.3.4 The ef fect of ionic subst i tu t ion on the membrane c h a r a c t e r i s t i c s and perforant path evoked responses of granule c e l l s 86 Potassium 86 Barium 90 Calcium 105 Baclofen 108 3.4 Discussion 109 CHAPTER 4: THE ROLE OF POSTSYNAPTIC PROCESSES IN MEDIATING PAIRED PULSE PHENOMENA IN THE DENTATE GYRUS 124 4.1 Introduction 124 4.2 Methods 128 4.3 Results 133 v i i i 4.3.1 I n t r a c e l l u l a r corre la tes of paired pulse s t imulat ion in the l a t e r a l and medial perforant paths . . . . 133 4.3.2 The poss ible mechanisms underlying the potent iat ion of the population spike and enhanced p r o b a b i l i t y of granule c e l l discharge 154 4.3.3 The ionic mechanisms underlying paired pulse i n h i b i t i o n of granule c e l l a c t i v i t y 161 4.4 Discussion 169 CHAPTER 5: AN EXTRA- AND INTRACELLULAR ANALYSIS OF .THE DENTATE GYRUS DURING AND FOLLOWING KINDLING-INDUCED EPILEPSY 182 5.1 Introduction 182 5.2 Methods 188 5.3 Results 192 5.3.1 E x 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 the a l t e r a t i o n s produced during the development of k ind l ing 192 Afterdischarges 192 In v i t r o experiments 195 5.3.2 I n t r a c e l l u l a r comparison of the membrane propert ies and synaptic potent ia l s between contro l and kindled granule c e l l s 209 5.4 Discussion 213 CHAPTER 6: INHIBITORY PROCESSES OF HIPPOCAMPAL CA1 PYRAMIDAL NEURONS FOLLOWING KINDLING-INDUCED EPILEPSY IN THE RAT 219 6.1 Introduction 219 6.2 Methods 223 6.3 Results 225 6.4 Discussion 238 CHAPTER 7: CONCLUSIONS 245 REFERENCES 256 X L I S T O F A B B R E V I A T I O N S A C S F A r t i f i c i a l C e r e b r o s p i n a l F l u i d A D A f t e r d i s c h a r g e A l v A l v e u s A P A c t i o n P o t e n t i a l AR A n o m a l o u s R e c t i f i c a t i o n A S A n t i d r o m i c S p i k e B a c l o f e n ( + / - ) B - p - c h l o r o p h e n y l G A B A C A C o r n u A m m o n i s ( h i p p o c a m p a l s u b f i e l d s 1 - 3 ) C a B P C a l c i u m - B i n d i n g P r o t e i n C N S C e n t r a l N e r v o u s S y s t e m C - T C o n d i t i o n - T e s t D A P D e p o l a r i z i n g A f t e r p o t e n t i a l DG D e n t a t e G y r u s D - I P S P D e p o l a r i z i n g - I n h i b i t o r y P o t e n t i a l E a h p E q u i l i b r i u m P o t e n t i a l o f A H P E C 1 E q u i l i b r i u m P o t e n t i a l o f C l ~ E d a p E q u i l i b r i u m P o t e n t i a l o f D A P E d - i p s p E q u i l i b r i u m P o t e n t i a l o f D - I P S P E e p s p E q u i l i b r i u m P o t e n t i a l o f E P S P E g a b a E q u i l i b r i u m P o t e n t i a l o f G A B A E l h p E q u i l i b r i u m P o t e n t i a l o f L H P E P S P E x c i t a t o r y P o s t s y n a p t i c P o t e n t i a l F P P F a s t P r e - P o t e n t i a l G A B A g a m m a - A m i n o b u t y r i c A c i d g K P o t a s s i u m C o n d u c t a n c e xi g k C a Calcium-act ivated Potassium Conductance HF Hippocampal Formation Ir; a Calcium Current 1^  Inward Current I]_ e a l c Leak Current at RMP IML Inner Molecular Layer of DG 1-0 Input-Output I PI Inh ib i t ion-Potent ia t i o n - I n h i b i t i o n IPSP Inhib i tory Postsynaptic Potent ia l I Q Q -Current I S I Inter-st imulus Interval LHP Late Hyperpolar izat ion LPP L a t e r a l Perforant Path MF Mossy Fibers MML Middle Molecular Layer of DG MPP Medial Perforant Path OML Outer Molecular Layer of DG PC Pyramidal C e l l PP Perforant Path PS Population Spike R<3ap Membrane Resistance Associated with DAP R d - i p s p Membrane Resistance Associated with D-IPSP R^p Sp Membrane Resistance Associated with IPSP R^hp Membrane Resistance Associated with LHP RMP Resting Membrane Potent ia l R n Resting Membrane Input Resistance SA Action Potent ia l Amplitude x i i Small Amplitude Spike Standard Error of Mean Stratum Lucidum Stratum Lacumnosum-Molecular Stratum Oriens Stratum Pyramidale Stratum Radiatum Membrane Time Constant Membrane Time Constant Measured 0-4 ms Tetraethylammonium 4 ,5 ,6 ,7 , - t e t rahydr i soxazo lo [5 ,4 , -c ] p y r i d i n e - 3 - o l Membrane Time Constant Measured greater than 4 ms Voltage of Condit ioning Response Voltage of Test Response Concentration of I n t r a c e l l u l a r Ion (X) Concentration of E x t r a c e l l u l a r Ion (X) x i i i LIST OF FIGURES F i g . 2.1 Schematic diagram of the hippocampal s l i c e 28 F i g . 2.2 E x t r a c e l l u l a r potent ia l s of the dentate gyrus 33 F i g . 2.3 T y p i c a l e x t r a c e l l u l a r responses in the paired pulse paradigm 36 F i g . 2.4 Typ ica l i n t r a c e l l u l a r records of a perforant path-evoked granule c e l l . 39 F i g . 2.5 Determination of membrane input resistance and time constant 43 F i g . 3.1 Current-voltage p r o f i l e s for two t y p i c a l granule c e l l s 51 F i g . 3.2 Accommodation of r epe t i t i ve f i r i n g to prolonged depolar izat ion 56 F i g . 3.3 Af terhyperpolar izat ion of granule c e l l s . 58 F i g . 3.4 Ant idromical ly evoked potent ia l s in the dentate gyrus 61 F i g . 3.5 Reversal po ten t ia l for the h i l a r -evoked D-IPSP 64 F i g . 3.6 Reversal po ten t ia l for the h i l a r -evoked LHP 66 F i g . 3.7 T y p i c a l ex tra - and i n t r a c e l l u l a r records of perforant path-evoked EPSPs 69 F i g . 3.8 Orthodromically evoked small amplitude spikes in granule c e l l s . 72 F i g . 3.9 Reversal po ten t ia l for perforant path-evoked EPSP 74 F i g . 3.10 Perforant path-evoked DAP and LHP . 77 F i g . 3.11 Equi l ibr ium potent ia l s for the DAP and LHP 79 xiv F i g . 3.12 Al tera t ions in membrane res i s tance ' associated with the DAP and LHP . . . 82 F i g . 3.13 Effect of e x t r a c e l l u l a r [K + ] on MPP-evoked EPSP and membrane proper t i e s . 84 F i g . 3.14 Ef fec t of subst i tut ing B a 2 + for C a 2 + on membrane propert ies of granule c e l l s 87 F i g . 3.15 Ef fec t of 1 mM B a 2 + on MPP-evoked responses 91 F i g . 3.16 Ef fec t of 2 mM B a 2 + on MPP-evoked responses 93 F i g . 3.17 Spontaneous rhythmic burst discharges in granule c e l l s induced by prolonged exposure to B a 2 + 96 F i g . 3.18 A l t e r a t i o n in membrane resistance associated with the post-burst AHP . 98 F i g . 3.19 Augmented spontaneous burst discharges induced by further exposure to B a 2 + . 101-F i g . 3.20 Ef fec t of lowering [ C a 2 + ] D and r a i s i n g [ M g 2 + ] 0 on MPP-evoked potent ia l s . . . 103 F i g . 3.21 Ef fec t of 10~5 M baclofen on MPP-evoked potent ia l s 106 F i g . 4.1 Ef fec t of paired MPP st imulat ion on EPSP and PS 129 F i g . 4.2 Histograms of test EPSP and PS in response to MPP st imulat ion 131 F i g . 4.3 Ef fec t of paired LPP st imulat ion on EPSP 134 F i g . 4.4 Histograms of test EPSP in response to LPP st imulat ion 136 F i g . 4.5 I n t r a c e l l u l a r potent ia l s evoked by LPP and MPP st imulat ion 139 F i g . 4.6 Ef fec t of PP st imulat ion on the i n t r a c e l l u l a r EPSP 141 F i g . 4.7 Histograms comparing the e f f e c t . o f LPP and MPP on EPSPs and PSs 143 X V F i g . 4.8 Ef fect of measuring the change in voltage of the i n t r a c e l l u l a r test EPSP 145 F i g . 4.9 Effect of paired PP st imulat ion in the absence of a DAP 148 F i g . 4.10 Ef fec t of LHP on EPSP i n h i b i t i o n . . . 150 F i g . 4.11 Effect of AHP on EPSP i n h i b i t i o n . . . 152 F i g . 4.12 Comparison of the e f fect of LHP and AHP on EPSP i n h i b i t i o n 155 F i g . 4.13 Effect of paired PP st imulat ion on granule c e l l discharge 157 F i g . 4.14 Histograms comparing effect of PP s t imulat ion on EPSPs and PS 159 F i g . 4.15 Effect of paired pulse st imulat ion on AP threshold 162 F i g . 4.16 Effect of paired s t imulat ion on MF evoked response 164 F i g . 4.17 Effect of b i c u c u l l i n e on recurrent i n h i b i t i o n 167 F i g . 4.18 Effect of ouabain on PS i n h i b i t i o n . 170 F i g . 4.19 Effect of Cs and TEA on PS i n h i b i t i o n 172 F i g . 5.1 Ef fect of k ind l ing on hippocampal ADs 190 F i g . 5.2 Ef fec t of k ind l ing on paired pulse PS response 193 F i g . 5.3 Ef fec t of stimulus in tens i ty on paired pulse i n h i b i t i o n of PS 196 F i g . 5.4 Ef fec t of ADs on paired pulse PS responses 199 F i g . 5.5 Graph showing ef fect of ADs on paired pulse potent ia t ion of PS . . . . 201 F i g . 5.6 Ef fec t of low [ C l ] 0 on paired pulse responses in contro l and kindled preparations 204 xvi F i g . 5.7 Ef fec t of 6 weeks without a k ind l ing stimulus on paired pulse responses . 207 F i g . 5.8 I n t r a c e l l u l a r records from contro l and kindled granule c e l l s 211 F i g . 6.1 Schematic diagram of hippocampal s l i c e 221 F i g . 6.2 I n t r a c e l l u l a r records from contro l and kindled pyramidal neurons 226 F i g . 6.3 Ef fect of synaptic a c t i v a t i o n on pyramidal c e l l i n h i b i t i o n 229 F i g . 6.4 Ef fec t of r e p e t i t i v e s t imulat ion on pyramidal c e l l i n h i b i t i o n 232 F i g . 6.5 Ef fec t of r e p e t i t i v e s t imulat ion on recurrent i n h i b i t i o n 235 F i g . 6.6 Ef fect of r e p e t i t i v e s t imulat ion on R i p s p 239 xvi i LIST OF TABLES Table 3.1 E l e c t r o p h y s i o l o g i c a l propert ies of granule c e l l s 54 Table 3.2 Ef fec t s of B a 2 + on granule c e l l membrane propert ies 89 Table 5.1 E l e c t r o p h y s i o l o g i c a l propert ies of granule c e l l s from contro l and kindled preparations 210 Table 6.1 E l e c t r o p h y s i o l o g i c a l propert ies of pyramidal c e l l s from control and kindled preparations 224 Table 6.2 Ef fect of r epe t i t i ve st imulat ion on recurrent i n h i b i t i o n 237 xvi i i LIST OF REVERSED PAGES F i g . 2.1 Schematic diagram of the hippocampal s l i c e 28 F i g . 2.2 E x t r a c e l l u l a r potent ia l s of the dentate gyrus 33 F i g . 2.3 Typ ica l e x t r a c e l l u l a r responses in the paired pulse paradigm 36 F i g . 2.4 Typ ica l i n t r a c e l l u l a r records of a perforant path-evoked granule c e l l . 39 F i g . 2.5 Determination of membrane input resistance and time constant 43 F i g . 3.1 Current-voltage p r o f i l e s for two t y p i c a l granule c e l l s 51 F i g . 3.2 Accommodation of r e p e t i t i v e f i r i n g to prolonged depolar izat ion 56 F i g . 3.3 Af terhyperpolar izat ion of granule c e l l s 58 F i g . 3.4 Ant idromica l ly evoked potent ia l s in the dentate gyrus 61 F i g . 3.5 Reversal po tent ia l for the h i l a r -evoked D-IPSP 64 F i g . 3.6 Reversal po ten t ia l for the h i l a r -evoked LHP 66 F i g . 3.7 T y p i c a l ex tra - and i n t r a c e l l u l a r records of perforant path-evoked EPSPs 69 F i g . 3.8 Orthodromically evoked small amplitude spikes in granule c e l l s . 72 F i g . 3.9 Reversal po ten t ia l for perforant path-evoked EPSP 74 F i g . 3.10 Perforant path-evoked DAP and LHP . 77 F i g . 3.11 Equi l ibr ium potent ia l s for the DAP and LHP 79 xix F i g . 3.12 A l t e r a t i o n s in membrane resistance associated with the DAP and LHP . . . 82 F i g . 3.13 Ef fec t of e x t r a c e l l u l a r [K + ] on MPP-evoked EPSP and membrane proper t i e s . 84 F i g . 3.14 Effect of subs t i tu t ing B a 2 + for C a 2 + on membrane propert ies of granule c e l l s 87 F i g . 3.15 Effect of 1 mM B a 2 + on MPP-evoked responses ; 91 Fig'. 3.16 Effect of 2 mM B a 2 + on MPP-evoked responses 93 F i g . 3.17 Spontaneous rhythmic burst discharges in granule c e l l s induced by prolonged exposure to B a 2 + 96 F i g . 3.18 A l t e r a t i o n in membrane res is tance associated with the post-burst AHP . 98 F i g . 3.19 Augmented spontaneous burst discharges induced by further exposure to B a 2 + . 101 F i g . 3.20 Ef fec t of lowering [ C a 2 + ] D and r a i s i n g [ M g 2 + ] D on MPP-evoked potent ia l s . . . 103 F i g . 3.21 Effect of 10~5 M baclofen on MPP-evoked potent ia l s 106 F i g . 4.1 Ef fec t of paired MPP st imulat ion on EPSP and PS 129 F i g . 4.2 Histograms of test EPSP and PS in response to MPP st imulat ion 131 F i g . 4.3 Effect of paired LPP st imulat ion on EPSP 134 F i g . 4.4 Histograms of test EPSP in response to LPP st imulat ion 136 F i g . 4.5 I n t r a c e l l u l a r potent ia l s evoked by LPP and MPP st imulat ion 139 F i g . 4.6 Ef fec t of PP st imulat ion on the i n t r a c e l l u l a r EPSP 141 F i g . 4.7 Histograms comparing the ef fect of LPP and MPP on EPSPs and PSs 143 XX F i g . 4.8 Ef fect of measuring the change in voltage of the i n t r a c e l l u l a r test EPSP . . 145 F i g . 4.9 Ef fec t of paired PP st imulat ion in the absence of a DAP 148 F i g . 4.10 Ef fec t of LHP on EPSP i n h i b i t i o n . . . 150 F i g . 4.11 Ef fec t of AHP on EPSP i n h i b i t i o n . . . 152 F i g . 4.12 Comparison of the effect of LHP and AHP on EPSP i n h i b i t i o n 155 F i g . 4.13 Ef fec t of paired PP st imulat ion on granule c e l l discharge 157 F i g . 4.14 Histograms comparing effect of PP st imulat ion on EPSPs and PS 159 F i g . 4.15 Ef fec t of paired pulse st imulat ion on AP threshold 162 F i g . 4.16 Ef fec t of paired st imulat ion on MF evoked response 164 F i g . 4.17 Effect of b i c u c u l l i n e on recurrent i n h i b i t i o n 167 F i g . 4.18 Ef fec t of ouabain on PS i n h i b i t i o n . 170 F i g . 4 .19 Ef fec t of Cs and TEA on PS i n h i b i t i o n 172 F i g . 5.1 Ef fec t of k ind l ing on hippocampal ADs 190 F i g . 5.2 Ef fec t of k ind l ing on paired pulse PS response 193 F i g . 5.3 Ef fec t of stimulus in tens i ty on paired pulse i n h i b i t i o n of PS 196 F i g . 5.4 Ef fec t of ADs on paired pulse PS responses 199 F i g . 5.5 Graph showing ef fect of ADs on paired pulse potent iat ion of PS . . . . 201 F i g . 5.6 Ef fec t of low [ C l ] 0 on paired pulse responses in contro l and kindled preparations 204 xx i F i g . 5.7 Effect of 6 weeks without a k ind l ing stimulus on paired pulse responses . 207 F i g . 5.8 I n t r a c e l l u l a r records from contro l and kindled granule c e l l s 211 F i g . 6.1 Schematic diagram of hippocampal s l i c e 221 F i g . 6.2 I n t r a c e l l u l a r records from contro l and kindled pyramidal neurons 226 F i g . 6.3 Ef fect of synaptic a c t i v a t i o n on pyramidal c e l l i n h i b i t i o n 229 F i g . 6.4 Ef fect of r e p e t i t i v e s t imulat ion on pyramidal c e l l i n h i b i t i o n 232 F i g . 6.5 Ef fect of r e p e t i t i v e s t imulat ion on recurrent i n h i b i t i o n 235 F i g . 6.6 Effect of r e p e t i t i v e s t imulat ion on R i p s p 2 3 9 xxi i Acknowledgements I would l i k e to express my appreciat ion to V i c k i e Asbra for her support and to Drs . Kenneth Baimbridge, Steven Kehl , Istvan Mody, Raymond Pederson and Tom Richardson and Patr ick Leung for the ir f r i endsh ip . I would a lso l i k e to acknowledge Dr. J . J . M i l l e r for h i s support and the opportunity to f u l f i l l my academic ambitions. I am grate fu l to my Supervisory Commitee and Dr. Bradley E . Alger for taking the time to read th i s thes i s . F i n a l l y , I wish to dedicate t h i s thesis to my brother Sean, and to Nancy and V i c k i T o o t e l l who could not share th i s with me. 1 Chapter 1 Introduction Comprehension of the processes whereby neuronal a c t i v i t y and interact ions are trans la ted into a funct ioning memory has long been the goal of neurobio log i s t s . Fol lowing the discovery that r e p e t i t i v e s t imulat ion a l tered synaptic e f f icacy at the neuromuscular junc t ion , i t was proposed that s imi lar changes in the centra l nervous system (CNS) may subserve the formation of memories (for a review see E c c l e s , 1964). In th i s regard, a number of pathways associated with the hippocampal formation (HF) have demonstrated a richness in neuronal p l a s t i c i t y without equal . This i s p a r t i c u l a r l y exc i t ing because the HF is bel ieved to be c r u c i a l for the conso l idat ion of new memories (Swanson et a l . , 1982). Of importance in the present study are the a l t e r a t i o n s in synaptic e f f i cacy which resul t from r e p e t i t i v e e l e c t r i c a l s t imulat ion of HF a f ferents . S p e c i f i c a l l y , those processes subserving short-term change in synaptic responses and c e l l u l a r e x c i t a b i l i t y of the dentate gyrus (DG). Although the much of the evidence indicates that short-term ef fects on synaptic e f f i cacy are re lated to changes in the quanti ty of neurotransmitter released ( i . e . , presynaptic) 2 (Lomo,1971b, McNaughton, 1980), other studies have i l l u s t r a t e d that postsynaptic mechanisms may contr ibute to the a l t e r a t i o n s exhibi ted by the DG (Assaf and M i l l e r , 1981; Douglas et a l . , 1983; Lomo, 1971b). Thus, given the importance attatched to a l t e r a t i o n s in synaptic e f f i cacy as a mechanism for memory storage, i t i s e s s en t ia l to understand a l l components underlying th i s process. With th is in mind, the present study invest igated the i n t r a c e l l u l a r propert ies and the synapt ica l ly evoked potent ia l s in the DG granule c e l l s and the poss ible contr ibut ion that these postsynaptic events play in short-term a l t e r a t i o n s of synaptic e f f i c a c y . In order to appreciate how f indings from th i s study are re lated to neuronal processing throughout the HF, i t is e s sent ia l to have an understanding of the connections and the var ie ty of synaptic responses which occur in the HF. Hence i t i s appropriate to begin with a review of the known anatomical and neurophysiological features of the HF. 1.1. Hippocampal formation; dentate gyrus and hippocampus  proper The hippocampal formation is an elongated structure which includes the dentate gyrus and hippocampus proper (or Ammon's horn) . These s tructures are joined by i n f o l d i n g and considered to be part of the l imbic system. The DG and hippocampus proper (HP) are highly organized and laminated s t ruc tures . The DG cons is t s of a C-shaped granule c e l l layer 3 (stratum granulosum) which surrounds the h i l a r zone (CA4 o f Lorente de No/ 1934). The h i l u s i s comprised of polymorphic c e l l s and granule c e l l axons. The granule c e l l s are bordered s u p e r f i c i a l l y by the molecular layer (stratum moleculare) which contains the dendr i t i c processes of the granule c e l l s and is the primary target for afferent project ions (see F i g . 2 .1) . The hippocampus proper (HP) is organized around a re-shaped layer of pyramidal neurons which has been d iv ided into subf ie lds cornu ammonis (CA) 1-3 (Lorente de No; 1934). The CA regions are d i s t inguished from one another on the basis of anatomical connections and morphological di f ferences in pyramidal neurons. Each CA subf ie ld i s further separated into six s trata along the dendro-somatic axis of the pyramidal c e l l : 1) stratum lacunosum-moleculare (SLM) occupies the d i s t a l area of the a p i c a l dendrites; 2) stratum radiatum (SR) defines the middle a p i c a l dendr i t i c region; 3) stratum lucidum (SL) contains the proximal a p i c a l dendrites; 4) stratum pyramidale (SP) comprises the somas; 5) stratum oriens (SO) the basalar dendrites; and 6) alveus (Alv) contains the pyramidal c e l l axons ( F i g . 2 .1) . 1.2 Neurons of the hippocampal formation As stated prev ious ly , the dentate gyrus and the hippocampus proper contain granule and pyramidal neurons, re spec t ive ly . These two c e l l groups account for 4 approximately 95% of a l l neurons found in the HF (Seress and Pokorny, 1981). The granule c e l l s (GC) are approximately 10-15 microns in diameter and form a dense band 4-7 c e l l s in width. These neurons maintain elaborate dendr i t i c arbor iza t ions in the molecular layer and send fine c o l l a t e r a l axonal branches to the h i l a r polymorph c e l l s , as well as a major axon to the CA3 pyramidal neurons. The l a t t e r project ion is the mossy f iber system (MF) which maintains some of the largest terminals in the CNS ( i . e . , 0.2 microns) and makes en passage synaptic contacts with CA3 pyramidal c e l l s (Gaarskjaer, 1981; Laatsch and Cowan, 1966; Lorente de No, 1 934). The pyramidal c e l l s of the hippocampal subf ie lds vary in morphology. The CA3 pyramidal neurons are the largest (approximately 60 microns in diameter) and form a loose band several layers in width. The primary di f ferences between the CA3 and the CA1 neurons are: 1) the former maintain thick spines on the a p i c a l dendr i t i c shaft , whereas the l a t t e r i s r e l a t i v e l y free of spines in th i s area; 2) the a p i c a l dendrites of CA3 pyramids arborize in stratum lacunosum-moleculare — CA1 neurons s tart to branch in the stratum radiatum; and 3) the CA3 pyramids have larger diameter axons (Lorente de No, 1934). Besides the pyramidal and granule c e l l s , a var i e ty of other l o c a l c i r c u i t neurons (interneurons) are dispersed throughout the HF. Based on locat ion and morphology, f ive types of interneurons (basket c e l l s ) have been described in 5 the DG. The majority of these c e l l s occupy the h i l a r region (60%) or the inner molecular layer (35%) with the rest being located in the granule c e l l layer (Seress and Pokorny, 1981). These neurons maintain various dendr i t i c branching conformations which terminate in the molecular layer and h i l u s of DG (Lorente de No, 1934; Seress and Pokorny, 1981). One in teres t ing feature of these neurons i s the i r axonal d i s t r i b u t i o n . Axons from these basket c e l l s sometimes extended for 0.5 mm along the granule c e l l layer and e i ther form a plexus around GC somas or branch into the molecular layer (Seress and Pokorny, 1981). These neurons exhibi t immunoreactivity to glutamate decarboxylase ant i sera (Ribak et a l . , 1978), ind ica t ing the presence of gamma-aminobutyric ac id (GABA). Although the term interneuron has been used to describe a l l basket c e l l s in the DG, there is growing evidence that some of these neurons contribute a commissural project ion which innervates the c o n t r a l a t e r a l DG (Seress and Ribak, 1983; Seroogy et a l . , 1983; and Swanson et a l . , 1981). In addi t ion to the c l a s s i c a l basket c e l l , Lorente de No (1934) described th ir teen d i f f erent interneurons. These interneurons, l i k e those of the DG, appear to be GABAergic (Storm-Mathisen, 1977). 6 1.3. I n t r i n s i c connections of the hippocampal formation  Dentate gyrus The primary afferent connection to the HF, the perforant path (PP), or ig inates from the l a t e r a l and the medial components of the entorhinal cortex (EC) and makes en  passage synaptic contacts with the outer and middle one-t h i r d of the dentate granule c e l l dendrites ( F i g . 2.1; Hjorth-Simonsen and Jeune, 1972; Steward, 1976; Wyss, 1981). The granule c e l l s receive another major project ion onto the inner one- th ird of the i r dendrites from i p s i l a t e r a l and c o n t r a l a t e r a l neurons occupying the h i l a r area of the DG (see section 1.2; Seress and Ribak, 1983; Seroogy et a l . , 1983; Swanson et a l . , 1981; Voneida et a l . , 1981). CA3 The p r i n c i p a l afferent to CA3 pyramidal neurons i s the mossy f iber project ion from DG granule c e l l s which terminates onto the proximal a p i c a l dendri tes . The d i s t a l -most a p i c a l dendrites of CA3 pyramidal c e l l s (SLM) are innervated by the PP (Lorente de No, 1934). Assoc ia t iona l and commissural f ibers o r i g i n a t i n g from other CA3 PCs make synaptic contact with the stratum radiatum and stratum oriens (Laurberg, 1979; Laurberg and Sorensen, 1981; Swanson et a l . , 1978; Voneida et a l . , 1981). 7 CA1 CA1 pyramidal neurons receive an i p s i l a t e r a l (Schaffer c o l l a t e r a l s ) and c o n t r a l a t e r a l projec t ion from CA3 neurons which terminates in s t ra ta radiatum and oriens (Laurberg and Sorensen, 1981; Swanson et a l . , 1978; Voneida et a l . , 1981). CA1 neurons appear to receive other inputs into s trata oriens and radiatum from c o n t r a l a t e r a l CA1 pyramidal c e l l s (Voneida et a l . , 1981) and a PP input onto the d i s t a l a p i c a l dendrites (SLM) (Lorente de No, 1934). By v i r t u e of the successive connections ( i . e . , PP, MF, Schaffer c o l l a t e r a l s ) the i n t r i n s i c c i r c u i t r y of the hippocampus forms a t r i - s y n a p t i c loop which l i n k s the entorhinal cortex to the CA1. A l l of the primary project ion systems within the hippocampus are exc i tatory with the poss ible exception of the h i l a r commissural f ibers which may form both exc i tatory and i n h i b i t o r y synapses with granule c e l l s (Buzsaki and Czeh, 1981; Douglas et a l . , 1983; Seroogy et a l . , 1983). Furthermore the majority of the afferents and ef ferents associated with the hippocampus are laminated in a homotopic manner and thus, as pointed out by Lorente de No (1934), a transverse section of the hippocampus w i l l maintain the synaptic i n t e g r i t y of the t r i - s y n a p t i c c i r c u i t r y . It is th i s anatomical feature of the hippocampus which has been exploi ted with tremendous success both in vivo and in v i t r o in determining the e l e c t r o p h y s i o l o g i c a l 8 propert ies of both the synaptic connections and the p r i n c i p a l neurons. 1.4. E l e c t r o p h y s i o l o g i c a l c h a r a c t e r i s t i c s of synaptic  responses in the hippocampal formation Pyramidal c e l l s While the general anatomical and morphological c h a r a c t e r i s t i c s of the hippocampal formation (HF) were f i r s t described at the turn of th i s century (see Lorente de No, 1934), the e l ec t rophys io log i ca l events associated with the primary afferent and efferent systems of the hippocampus are s t i l l under inves t iga t ion . One of the techniques which has aided the invest igat ion of hippocampal processes i s the in v i t r o preparation introduced by Yamamoto and Mcllwain ( 1966) . The PC of the hippocampus i s probably the most studied CNS neuron with i n t r a c e l l u l a r recordings having been made since the ear ly 1960's ( e . g . , Kandel et a l . , 1961; Andersen et a l . , 1964). As previous ly stated, the i n t r i n s i c connections of the hippocampus are exc i tatory and when st imulated e l e c t r i c a l l y produce an i n i t i a l monosynaptic exc i ta tory postsynaptic po ten t ia l (EPSP) (Andersen et a l . , 1966a; Kandel et a l . , 1961; Schwartzkroin, 1975). The putat ive transmitter mediating exc i ta t ion i s glutamic ac id (Co l l ingr idge et a l , 1983; Storm-Mathisen, 1977). Following the EPSP there is a depo lar iz ing a f t e r p o t e n t i a l (Kandel and 9 Spencer, 1961a; Schwartzkroin, 1975) and at least two temporally d i s t i n c t i n h i b i t o r y a f t e r p o t e n t i a l s , the i n h i b i t o r y postsynaptic po tent ia l (IPSP) (Kandel et a l . , 1961; Andersen et a l . , 1964; Dingledine and Langmoen, 1980; Knowles et a l . , 1984; Schwartzkroin, 1975) and the la te hyperpolar izat ion (LHP) (Alger, 1984; Alger and N i c o l l , 1982a; Knowles et a l . , 1984; Lancaster and Wheal, 1984; Newberry and N i c o l l , 1984a). The depolar iz ing a f t erpoten t ia l (DAP) has a duration of approximately 10 ms and seems to be a mixed potent ia l that p a r t i a l l y resu l t s from the ac t iva t ion of a C a 2 + conductance (Wong and Pr ince , 1981). The IPSP has been associated with an interneuron ( i . e . the basket c e l l ) which releases gamma-aminobutyric ac id (GABA) onto PCs. GABA produces the i n h i b i t o r y po tent ia l by increasing membrane permeabil i ty to ch lor ide ions (Alger and N i c o l l , 1982b; A l l en et a l . , 1977; Andersen et a l . , 1964; C o l l i n g r i d g e et a l . , 1984; C u r t i s et a l . , 1970; Dingledine and Langmoen, 1980; Eccles et a l . , 1977; Storm-Mathisen, 1977). Recent studies have shown that r e p e t i t i v e alvear s t imulat ion resu l t s in a reduction of the IPSP amplitude (Al len et a l . , 1977; McCarren and Alger , 1985; O l i v e r and M i l l e r , 1985), whereas s imi lar stimulus parameters de l ivered to the stratum radiatum reverse the IPSP to a depo lar iz ing po tent ia l (Wong and Watkins, 1982). Iontophoretic a p p l i c a t i o n of GABA to the somatic or dendr i t i c areas of the PC also resu l t s in the hyperpolar izat ion and depolar izat ion 10 of PCs, respect ive ly (Alger and N i c o l l , 1979; 1982a; Andersen et a l . , 1980; Thalmann et a l . , 1981). The GABA-induced dendr i t i c depolar izat ion may be a t t r i b u t e d to GABA act ing on extrasynaptic receptors (Alger and N i c o l l , 1982b). Inhib i tory PSPs were bel ieved to be mediated exc lus ive ly by recurrent ac t iva t ion of GABAergic interneurons (Kandel and Spencer, 1961b; Andersen et a l . , 1964; Schwartzkroin, 1975). However, biochemical , anatomical and e l ec t rophys io log i ca l evidence suggests that the IPSP may be evoked in a feed-forward as well as in a feed-back manner (for a review see Buzsaki, 1984). It has been known for some time that glutamate decarboxylase (GAD), an enzmye required for the production of GABA, i s l o c a l i z e d to the c e l l layers and dendr i t i c zones of the HP (Storm-Mathisen-> 1977) supporting the view that GABAergic neurons make synaptic contact with the dendrites of pyramidal c e l l s . A combined degeneration-GAD immunohistochemical study showed that fol lowing f imbr ia l t ransec t ion , degenerating terminals made synaptic contact with GAD-immunoreactive non-pyramidal neurons (Frotscher et a l . , 1984), implying that commissural f ibers d i r e c t l y innervate GABAergic interneurons which may i n i t i a t e i n h i b i t i o n of PCs in a feed-forward manner. In a d d i t i o n , e x t r a c e l l u l a r and i n t r a c e l l u l a r recordings obtained from i d e n t i f i e d non-pyramidal c e l l s have shown that these neurons respond to the s t imulat ion of a number of orthodromic pathways ( e . g . , commissural) at stimulus i n t e n s i t i e s which do not ac t ivate recurrent c i r c u i t r y 11 (Ashwood et a l . , 1984; Buzsaki and E ide lberg , 1982; Knowles and Schwartzkroin, 1981; Schwartzkroin and Mathers, 1978). In tradendr i t i c (Wong and Pr ince , 1979) and i n t r a c e l l u l a r (Alger and N i c o l l , 1982a) recordings from PCs have demonstrated the presence of orthodromical ly evoked IPSPs. Furthermore, pyramidal c e l l s may receive a d i r e c t GABAergic projec t ion from the medial septal nucleus (Kohler et a l . , 1984). Thus, there i s evidence ind ica t ing that feed-forward i n h i b i t i o n occurs through monosynaptic (septal) and disynapt ic (commissural v ia interneurons) pathways. The LHP is d i f f erent from the IPSP in that i t is chloride-independent and unaffected by t y p i c a l GABA antagonists , e .g . b i c u c u l l i n e (Alger, 1984; Knowles et a l . , 1984; Newberry and N i c o l l , 1984a; Thalmann, 1984). Several studies suggest that the LHP is a potassium-dependent po tent ia l which is not C a 2 + - a c t i v a t e d (Alger, 1984; Knowles et a l . , 1984; Lancaster and Wheal, 1984; Newberry and N i c o l l , 1984b; Thalmann, 1984). Although the mechanism subserving the LHP is not understood, i t does not appear to be l inked to the sodium/potassium pump since ouabain was ine f f ec t ive in reducing th i s po tent ia l (Alger, 1984). It has been suggested that the LHP i s an interneuron-mediated synaptic event (Alger, 1984; Knowles et a l . , 1984; N i c o l l and A lger , 1981; Thalmann, 1984). In support of t h i s , GABA and i t s analog beta-p-chlorophenyl-GABA (baclofen) have been shown to produce a potassium-dependent hyperpolar izat ion in PCs presumably by in terac t ing with the b i c u c u l l i n e -12 insens i t ive GABAg receptor (Ault and Nadler, 1983a; Gahwiler and Brown, 1985; Newberry and N i c o l l , 1984b). Thus i t i s poss ible that both the IPSP and LHP are mediated by GABAergic interneurons, with the d i s s i m i l a r i t i e s between potent ia l s being re lated to the a c t i v a t i o n of d i f f erent populations of GABA receptors . Granule c e l l s St imulation of the PP resu l t s in a monosynaptic exc i tatory postsynaptic po tent ia l (EPSP) in granule c e l l s ( C r u n e l l i et a l . , 1983; Deadwyler et a l . , 1975; Fournier and Crepe l , 1984; Fr icke and Pr ince , 1984; Lomo, 1971a; McNaughton et a l . , 1981; Thalmann and Ayala , 1982). Although there is evidence to suggest that the medial (MPP) and l a t e r a l (LPP) perforant paths act on d i f f erent receptors to produce the EPSP (Col l ingr idge et a l . , 1982; Koerner and Cotman, 1981; C r u n e l l i et a l . , 1983), i t i s general ly bel ieved that e i ther aspartate or glutamate i s the neurotransmitter mediating the EPSP (Storm-Mathisen, 1977; Wheal and M i l l e r , 1980; White et a l . , 1977). Following the EPSP, granule c e l l s exhib i t e i ther an i n h i b i t o r y postsynaptic po ten t ia l (IPSP; Andersen et a l . , 1966a; F r i c k e and Pr ince , 1984; Lomo, 1971b) or a depo lar iz ing a f t e r p o t e n t i a l (DAP; Assaf et a l . , 1981; Deadwyler et a l . , 1975; McNaughton et a l . , 1981; Thalmann and Ayala , 1982). These responses are p a r t i a l l y mediated by 13 GABA and appear to be s i m i l a r , since both are sens i t ive to changes in external C l ~ concentration ( [ C l ] 0 ) and GABA antagonists (Fricke and Pr ince , 1984; Thalmann and Ayala , 1982. Thus the IPSP and DAP are probably generated by the same mechanism and the discrepancy between studies in the p o l a r i t y of these potent ia l s has yet to be resolved. It has been suggested that the hippocampal proper IPSP and the granule c e l l IPSP/DAP are mediated v ia recurrent a c t i v a t i o n of interneurons (Andersen et a l . , 1966a). However, s t imulat ion of the c o n t r a l a t e r a l h i lu s (Buzsaki and Czeh, 1981) or medial septal nucleus (McNaughton and M i l l e r , 1984) resul t s in the d i r e c t a c t i v a t i o n of neurons that have the f i r i n g c h a r a c t e r i s t i c s of putat ive interneurons. Furthermore st imulat ion of the c o n t r a l a t e r a l h i l u s reduces the PS induced by a paired perforant path stimulus (Douglas et a l . , 1983). These data, together with the anatomical f inding of GAD immunoreactivity in h i l a r c e l l s which make-up the commissural /associat ional input to granule c e l l s (Seress and Ribak; 1983; Seroogy et a l . , 1983), provide convincing evidence that GABA-mediated events may be evoked in a feed-forward manner in the DG. Synaptic a c t i v a t i o n of the granule c e l l s e l i c i t s a prolonged hyperpolar izat ion (LHP) which, l i k e the pyramidal c e l l LHP, i s not sens i t ive to a l t e r a t i o n s in [ C l ] c or the GABA antagonist p i cro tox in (Thalmann and Ayala , 1982). The LHP appears to be a K +-dependent po tent ia l (Thalmann and 14 Ayala , 1982), but the mechanism subserving the event ( e . g . , synaptic) i s not presently known. 1.5. St imulat ion-induced neurop las t i c i ty It i s beyond the scope of t h i s thesis to review the l i t e r a t u r e perta in ing to a l l the events and the a l t e r a t i o n s induced by e l e c t r i c a l s t imulat ion of hippocampal connections. However, th i s section w i l l describe only some of these changes in synaptic e f f i cacy associated with repe t i t i ve s t imulat ion and the poss ible mechanisms by which they occur. Paired-pulse f a c i l i t a t i o n A p p l i c a t i o n of a s ingle conditoning pulse to the Schaffer c o l l a t e r a l s (SC) can f a c i l i t a t e the EPSP evoked by subsequent test st imulat ions in pyramidal c e l l s (Creager et a l . , 1980). The degree of paired-pulse potent iat ion depends on the i n t e r v a l between the condi t ioning and test (C-T) pulses . Concommitant with the increase in EPSP amplitude i s an enhancement in the number of discharging pyramidal c e l l s (as measured by the amplitude of the e x t r a c e l l u l a r population spike; Creager et a l . 1980). The augmentation of the EPSP and population spike (PS) amplitude resu l t s from homosynaptic a l t e r a t i o n s , since comparable changes are not observed i f the test stimulus i s de l ivered to another input 15 (heterosynaptic) . Thus postsynaptic processes evident ly do not contribute to the paired pulse phenomena in CA1. Furthermore, increasing or decreasing the external C a 2 + concentration reduces and enhances the amount of f a c i l i t a t i o n , respect ive ly (Creager et a l . , 1980). These data are s imi lar to the ef fects of a l tered [ C a ] Q on the neuromuscular end-plate p o t e n t i a l , where i t has been hypothesized that f a c i l i t a t i o n resu l t s from a res idua l C a 2 + -complex (CaX) which enhances transmitter release (Katz and M i l e d i , 1968; Rahamimoff, 1968). The perforant path-granule c e l l synapse a lso exhib i t s synaptic p l a s t i c i t y in response to paired-pulse s t imulat ion , presynaptic a l t e r a t i o n s in transmitter release do not completely explain • the observed changes. Paired s t imulat ion of the medial perforant path (MPP) or l a t e r a l perforant path (LPP) resu l t s in depression and f a c i l i t a t i o n of the EPSP, respect ive ly (McNaughton, 1980; McNaughton and Barnes, 1977). The depression of the MPP-evoked test EPSP has been hypothesized to occur because a greater amount of neurotransmitter i s released in response to the i n i t i a l stimulus (McNaughton, 1980). In agreement with data obtained from the neuromuscular junct ion , as the condi t ioning pulse releases more of the ava i lab le transmitter ( e . g . , by increasing the external C a 2 + concentration) the depression of the test EPSP i s enhanced (McNaughton, 1980). Thus, a l t e r a t i o n s in the perforant path EPSP induced by pa i red -pulse s t imulat ion may be explained on the basis of changes 16 in the presynaptic release of neurotransmitter (Lomo, 1971b; McNaughton, 1980; Racine and Milgram, 1983). It should be noted that the changes in the dentate population spike response do not p a r a l l e l those of the EPSP. For example, paired st imulat ion of the MPP enhances the PS during periods when the test EPSP is i n h i b i t e d (McNaughton and Barnes, 1977). In a d d i t i o n , the augmentation of the PS response occurs fol lowing heterosynaptic or antidromic st imulat ion (Assaf and M i l l e r , 1981; Douglas et a l . , 1983; Lomo, 1971b; McNaughton and M i l l e r , 1984). Hence, the potent iat ion of the PS is p a r t i a l l y dependent on postsynaptic a l t era t ions in the granule c e l l s (Lomo, 1971b). The process subserving th i s heterosynaptic f a c i l i t a t i o n is undetermined; however, the mechanism underlying the augmentation of disparate inputs in the DG may be the key to understanding the funct ional d i f ferences between the hippocampus proper and dentate gyrus. Frequency potent iat ion Repet i t ive s t imulat ion of the primary HP afferents resul t s in potent iat ion of the EPSP and PS during and fol lowing termination of the te tani (Alger and T e y l e r , 1976; Andersen and Lomo, 1967; Creager et a l . , 1980; Racine and Milgram, 1983; Turner and M i l l e r , 1982). The f a c i l i t a t i o n of the EPSP during the te tani apparently resu l t s from summation of the mechanism underlying paired-pulse f a c i l i t a t i o n 17 (Creager et a l . , 1980; Racine and Milgram, 1983). If the r e p e t i t i v e s t imulat ion is maintained for a prolonged per iod , then the EPSP and PS amplitudes are decreased and increased, respect ive ly (Alger and Tey ler , 1978; Creager et a l . , 1980). This las t e f fect i s observed when e i ther homo- or heterosynaptic pathways are ac t ivated and has been shown to p a r a l l e l an increase in the e x t r a c e l l u l a r K + concentration (Alger and Tey ler , 1978). Following the termination of a frequency t r a i n ( i . e . , greater than 1 Hz) , subsequent homosynaptic s t imulat ion evoked a potent iat ion of the EPSP and PS (post- tetanic potent ia t ion; Alger and Tey ler , 1976; Racine and Milgram, 1983). Racine and Milgram (1983) found that the post - te tanic potent iat ion (PTP) induced with f i m b r i a l s t imulat ion decayed as a double exponential with time constants of 61s and 554s and labeled the two processes potent iat ion 1 and 2, r e spec t ive ly . Presynaptic Ca accumulation and augmentation of transmitter release i s suggested as one pos ible mechanism subserving both potent iat ion 1 and 2 (Racine and Milgram, 1983). During r e p e t i t i v e s t imulat ion the perforant path-granule c e l l complex i n i t i a l l y exh ib i t s depression of the EPSP and PS amplitudes (Alger and Tey ler , 1976; Andersen et a l . , 1966a; Racine and Milgram, 1983; White et a l . , 1979). Maintained s t imulat ion of the perforant path may enhance the PS (Andersen et a l . , 1966a; Gloor et a l . , 1964), but th i s augmentation probably i s in response to an increase in [ K ] 0 18 ( F r i t z and Gardner-Medwin, 1976). Regardless of whether the EPSP and PS are depressed d u r i n g r e p e t i t i v e a c t i v a t i o n , p o s t - t e t a n i c p o t e n t i a t i o n of these p o t e n t i a l s i s observed (Dudek et a l . , 1976; Gloor et a l . , 1964; McNaughton, 1982; Racine and Milgram, 1983). F a c i l i t a t i o n of t r a n s m i t t e r r e l e a s e probably u n d e r l i e s the PTP e x h i b i t e d i n the DG (McNaughton, 1982; Racine and Milgram, 1983). Long-term p o t e n t i a t i o n In a d d i t i o n to the short-term a l t e r a t i o n s i n s y n a p t i c e f f i c a c y , high frequency t e t a n i z a t i o n (e.g., 1 s, 100 Hz) of hippocampal pathways produces a long-term p o t e n t i a t i o n of the EPSP (Alger and T e y l e r , 1976; B l i s s and Gardner-Medwin., 1973; B l i s s and Lomo, 1973; Douglas and Goddard, 1975; Dunwiddie and Lynch, 1978). Long-term p o t e n t i a t i o n (LTP) appears to represent a permanent change i n s y n a p t i c f u n c t i o n s i n c e the e f f e c t has been observed to l a s t at l e a s t two months (Douglas and Goddard, 1975). Although there are r e p o r t s that an i n c r e a s e i n n e u r o t r a n s m i t t e r r e l e a s e i s c o r r e l a t e d with LTP i n the hippocampus (Dolphin et a l . , 1982; Skrede and Malthe-Sorrenssen, 1981), the m a j o r i t y of evidence suggests a p o s t s y n a p t i c l o c u s ( f o r reviews see E c c l e s , 1983; Swanson et a l . , 1982; V o r o n i n , 1983). Evidence f o r the l a t t e r i n c l u d e : 1) the u l t r a s t r u c t u r a l changes in d e n d r i t i c spines ( F i f k o v a and Van H a r r e v e l d , 1977; Lee et a l . , 1981); 2) an i n c r e a s e i n the number of glutamate 19 receptors (Baudry et a l . , 1980); 3) an enhancement of i n t r a d e n d r i t i c C a 2 + accumulation (Kuhnt et a l . , 1985); 4) the necessity for the postsynaptic neuron to discharge for the induction of LTP (Scharfman and Sarvey, 1985); 5) the magnitude of LTP exhibited by a s ingle pathway increased when co-act ivated with another input (heterosynaptic cooperat iv i ty ; Barrionuevo and Brown, 1983; Lee, 1983; McNaughton et a l . , 1978; Wigstrom and Gustafsson, 1983a); 6) the e l iminat ion of LTP with the N-methyl-D-aspartate receptor antagonist , amino-phosphonovalerate (APV) (Col l ingr idge et a l . , 1983; Harr i s et a l . , 1984); and 7) the abolishment of LTP with postsynaptic in j ec t ion of the C a 2 + -che la tor , EGTA (Lynch et a l . , 1983). Although these data r e f l e c t apparently disparate f indings , a mechanism that incorporates the events l i s t e d above, has been put forward (Eccles , 1983; Lynch and Baudry, 1984; Wigstrom and Gustafsson, 1985). P r i n c i p a l l y , s t imulat ion of presynaptic f ibers resul t s in neurotransmitter release ( i . e . , glutamate) and the a c t i v a t i o n of two types of postsynaptic receptors: 1) the c l a s s i c a l transmit ter-receptor complex which p a r t i c i p a t e s in EPSP generation; and 2) a voltage-dependent NMDA receptor which is coupled to a C a 2 + - i o n o p h o r e . A c t i v a t i o n of the second receptor type w i l l only occur during r e p e t i t i v e s t imulat ion and resu l t s in a s i g n i f i c a n t inf lux of C a 2 + which t r iggers events necessary for the induction of LTP. Tetanic s t imulat ion of heterosynaptic inputs should enhance 20 C a 2 + in f lux by increasing membrane d e p o l a r i z a t i o n . I n t r a c e l l u l a r Ca^ induces a number of biochemical events, one of which is the a c t i v a t i o n of a calcium-dependent protease, c a l p a i n , which has been suggested to degrade a cytoskeleton-bound p r o t e i n , brain spectr in or f o d r i n . This l a t t e r prote in i s responsible for the capping of c e l l surface receptors such that in the presence of ca lpa in and C a 2 + fodr in is broken down and glutamate receptors are unmasked. It was further hypothesized that ca lpa in may degrade other cytoskeleton proteins involved in the regulat ion of c e l l shape (or more s p e c i f i c a l l y dendr i t i c spine shape; Lynch and Baudry, 1984). The increase in glutamate receptors and change in spine conformation are poss ible mechanisms for the synaptic enhancement observed with LTP. Augmenting the number of glutamate receptors would increase the p r o b a b i l i t y of forming transmit ter-receptor complexes and i n i t i a t i n g larger synaptic currents , whereas, an a l t e r a t i o n in spine morphology may enhance the coupling between synaptic current and d e n d r i t i c depolar izat ion (Lee et a l . , 1980). Kind l ing K i n d l i n g i s the process whereby repeated presentation of an i n i t i a l l y subconvulsive s t imula t ion , de l ivered to any one of a number of l imbic s tructures ( e . g . , hippocampus), leads to the development of seizures (Goddard et a l . , 1969). 21 In order to induce k ind l ing the stimulus must evoke a high frequency burst response ( i . e . , afterdischarge) in the primary target of the act ivated pathway (Racine, 1972b). The progression of the phenomenon is character ized by a reduction in the threshold in tens i ty required to evoke the afterdischarge (AD), as well as an increase in AD duration (Goddard et a l . , 1969; P ine l et a l . , 1976; Racine, 1972a). Following k i n d l i n g , there i s an increase in the synaptic e f f i cacy i s present which appears s imi lar to LTP (Douglas and Goddard, 1975; Racine, 1975). Moreover the f inding that LTP could not be produced in kindled animals led Racine et a l . (1983) to postulate that k ind l ing and LTP may share common mechanisms. In support of th i s hypothesis , LTP and k ind l ing are associated with an increase in the number of glutamate receptors (Baudry et a l . , 1980; Savage et a l . , 1982); these phenomena are f a c i l i t a t e d by d i srupt ion of GABA-mediated i n h i b i t i o n (Kalichman et a l . , 1981; Wigstrom and Gustafsson, 1983b, c ) ; and the NMDA-receptor antagonist APV blocks the i r development (Col l ingr idge et a l . , 1983; Harr i s et a l . , 1984; Peterson et a l . , 1983). Although th i s evidence indicates that some of the mechanisms underlying LTP and k ind l ing may be shared, i t does not address the p o s s i b i l i t y that k ind l ing resu l t s from mult ip le processes: one re lated to LTP and another, as yet , undetermined. The fact that more than one mechanism may be required for k ind l ing was suggested by Racine and co-workers (1981) when they observed the 22 development of new synaptic components which tr iggered burst responses in kindled preparat ions . A reduction in i n h i b i t o r y processes was postulated as the substrate for the burst response and k indl ing (Racine et a l . , 1981). 1.6. Topic of the thes is The a l t e r a t i o n s in synaptic e f f i cacy described above are bel ieved to contribute to both memory formation ( e . g . , LTP) and epileptogenesis ( i . e . , k i n d l i n g ) . However, before one can hope to comprehend the complex in teract ions in c e l l u l a r events that subserve in neuronal p l a s t i c i t y , i t is imperative to understand those processes responsible for normal c e l l function and communication. The fact that we db not know whether an IPSP or depolar iz ing a f t e r p o t e n t i a l succeeds the perforant path evoked EPSP in DG granule c e l l s is c l ear evidence that we have an incomplete understanding of the mechanisms supporting neuronal p l a s t i c i t y in th i s area . The primary reason for the slow progress in speci fy ing the e lectrophysiology of the granule c e l l s i s the i r r e l a t i v e l y small s ize ( i . e . , less than 15 microns in diameter) . This has hampered the successful i n t r a c e l l u l a r impalement for long durations e s sent ia l to the charac ter i za t ion of any neuronal populat ion. The recent advances in both i n t r a c e l l u l a r microelectrodes and techniques ( e . g . , the use of in v i t r o preparations) have 23 enhanced the p r o b a b i l i t y ' o f recording from granule c e l l s and i t i s now poss ible to character ize these neurons. Accordingly , th i s thes is w i l l concern i t s e l f with the charac ter iza t ion of the i n t r i n s i c and the synaptic propert ies of granule c e l l s . Following th i s assessment, the i n t r a c e l l u l a r corre la tes of the paired-pulse a l t e r a t i o n s in synaptic e f f i cacy and heterosynaptic potent iat ion of the PS w i l l be examined. S p e c i f i c a l l y , the influence of granule c e l l synaptic potent ia l s on the mechanisms subserving paired-pulse phenomena w i l l be determined. In addi t ion to these s tudies , a l t e r a t i o n s in DG and granule c e l l function w i l l be assessed in rats predisposed to seizure a c t i v i t y through k ind l ing (see Chapter 5). The s p e c i f i c i t y of these changes w i l l be determined by comparing the changes in CAT pyramidal c e l l s induced by k i n d l i n g . Because changes in i n h i b i t o r y processes have been proposed as the basis of k indl ing (Racine et a l . , 1981), th i s study w i l l focus on a l t e r a t i o n s in GABA-mediated i n h i b i t i o n . To f a c i l i t a t e the i n t r a c e l l u l a r recording of neurons a l l experiments were c a r r i e d out on hippocampal s l i c e preparat ions . 24 CHAPTER 2 General Methods 2.1. Introduction The c o r t i c a l s l i c e preparation was introduced by Yamamoto and Mcllwain (1966) with the hope that i t would s impl i fy the recording of evoked potent ia l s without compromising the i n t e g r i t y of the neuronal c i r c u i t r y and physiology. Due to the advantages offered by th i s preparation (for a review of the technique and advantages of the hippocampal s l i c e , see Schwartzkroin ,1981), i t has been extensively u t i l i z e d in the determination of the e l e c t r o p h y s i o l o g i c a l substrates of several d i f f eren t forms of neuronal p l a s t i c i t y (Swanson et a l . , 1982), i d e n t i f i c a t i o n of intrahippocampal pathways ( e . g . , feed-forward i n h i b i t i o n , Alger and N i c o l l , 1982a), and the charac ter i za t ion of poss ible neurotransmitter candidates ( e . g . , Co l l ingr idge et a l . , 1983). Furthermore, i t appears almost without exception that the resu l t s obtained from the hippocampal s l i c e preparation are observed in the intact animal (Schwartzkroin, 1975). The present chapter gives a descr ip t ion of the hippocampal s l i c e preparation and the techniques common in the subsequent chapters. 25 2.2 The in v i t r o hippocampus Male Wistar rats (150-350 g) were decapitated and the c r a n i a l bones were removed. Once the brain was exposed i t was superfused with co ld ( 4 - 8 ° C ) oxygenated a r t i f i c i a l cerebrospinal f l u i d (ACSF). The dura was c a r e f u l l y cut and the whole brain was removed from the cranium and placed into cooled oxygenated ACSF. This was done to slow metabolism and to "firm" the neural t i ssue so that less damage occurred during the s l i c i n g procedure. In order to dissect the hippocampus, the cerebellum was removed and the hemispheres were separated by a s a g i t t a l cut . A hemisphere was set on i t s l a t e r a l surface and the hippocampus was removed by inser t ing a non-metalic blunt d i s sec t ing instrument between the fornix and the diencephalon, and gently r o l l i n g the hippocampus d o r s a l l y while freeing i t from the brain by making cuts at the septal and temporal poles . After the hippocampus was f u l l y exposed, the remaining neocor t i ca l t i s sue as well as the majority of the f imbria were excised. The hippocampus was then pos i t ioned on a S o r v a l l t i s sue chopper with i t s l ong i tud ina l axis perpendicular to the blade in order to maintain the i n t r i n s i c hippocampal c i r c u i t r y . Six dorsal hippocampal s l i c e s , approximately 425 microns in thickness , were cut and removed from the chopping blade with a fine brush and placed into a transfer d i sh 26 f i l l e d with cooled, oxygenated ACSF. The s l i c e s were placed on a nylon mesh net within the recording chamber, by e i ther a f ine brush or a large-mouthed p ipet te , where they were maintained at approximately 3 4 ° C . The time required to complete the preparation of the s l i c e s ranged from 7-10 minutes. S l i c e s in the recording chamber were maintained in a f lu id /gas in ter face , with the sides and lower port ions of the s l i c e being continuously perfused (1-1.5 ml/min, gravi ty fed, flow meter contro l l ed) with prewarmed and oxygenated ACSF while the top of the s l i c e was saturated by a warmed and humidified gas mixture (95% C^: 5% CO2). The l eve l of the ACSF was kept constant by suct ioning off excess medium. The hippocampal s l i c e s maintained in th i s fashion adhere to the nylon net great ly enhancing the a b i l i t y to sustain i n t r a c e l l u l a r penetrat ions . This method, however, increased the e q u i l i b r a t i o n time required for examining the ef fects of drugs or changes in the ionic composition of the medium on neuronal a c t i v i t y . Depending on the manipulation, e q u i l i b r a t i o n times ranged between 5 and 30 minutes. S l i c e s e q u i l i b r a t e d for a minimum of 1.5 hours p r i o r to the recording of evoked p o t e n t i a l s . A l l s l i c e s used in th i s study met cer ta in e l e c t r o p h y s i o l o g i c a l c r i t e r i a : 1) the absence of secondary or mult ip le population spikes in response to r e l a t i v e l y high s t imulat ion i n t e n s i t i e s de l ivered to e i ther the perforant path or mossy f i b e r s ; and 2) the presence of both an early ( i . e . , approximately 20 ms) 27 and a late ( i . e . , approximately 0.2-8 s) i n h i b i t i o n when assessed by paired perforant path s t imulat ion . These c r i t e r i a were chosen because they re f l ec t the v i a b i l i t y of the i n h i b i t o r y processes, which have been shown to be most susceptible to damage re la ted to the in v i t r o technique (Dunwiddie, 1981). When these i n h i b i t o r y mechanisms were present, the exci tatory potent ia l s were e a s i l y evoked and very robust . Thus, the tes t ing of the i n h i b i t o r y processes allows one to standardize the resu l t s and provide a basis for comparisons between s l i c e s , as well as to extrapolate the f indings in the s l i c e to the intact animal with some degree of c e r t a i n t y . 2.3 Composition of the a r t i f i c i a l cerebrospinal f l u i d  (ACSF) ' The ACSF is a minimal medium which mimics only the ionic environment ( i . e . , amino acids and/or proteins are not added to the ACSF) of mammalian cerebrospinal f l u i d (CSF). The ACSF used in these experiments represents a modified CSF solut ion which has been shown to maintain v iable s l i c e s (Lee et a l . , 1981). The normal ACSF u t i l i z e d in these experiments contained in mM: 124 NaCl , 3 KC1, 1.25 KHP0 4 , 2 C a C l 2 , 2 MgS0 4 , 24 NaHC0 3 , and 10 D-glucose. In the presence of the 95% 0 2 : 5% C 0 2 gas mixture the bicarbonate acts as a buffer to bring the pH of the ACSF to 7.4. In s p e c i f i c experiments the e f fects of a l t e r i n g the ionic composition of the ACSF were observed on c e l l u l a r 28 Figure 2.1. (A) Schematic of the hippocampal s l i c e and (B) the placements of the recording and st imulat ing electrodes in the dentate gyrus. St imulat ing electrodes and S2 act ivate the LPP and MPP, re spec t ive ly . The population EPSPs evoked from these pathways were recorded in the OML (R 1 ) and MML (R2K The reversed population EPSP and population spike were recorded in the GCL (R3). St imulat ion in the h i l u s (S3) ant idromica l ly evoked the granule c e l l s v ia the MF which was recorded in the GCL (R3). 29 30 funct ion . The changes used in these studies included: 1) the subst i tu t ion of barium ch lor ide for calcium c h l o r i d e ; a l so , because barium prec ip i ta t e s with su l fa te , magnesium sulfate had to be replaced by magnesium c h l o r i d e ; 2) the reduction of ch lor ide to 40 mM by replac ing the equivalent amount of NaCl by sodium proprionate; and 3) a decrease in calcium which had to be compensated with an increase in magnesium (14 mM) to i n h i b i t the development of spontaneous c e l l d i scharge. 2.4 Stimulation parameters Bipolar s t imulat ing electrodes (twisted, insulated 62 micron, nichrome wire) were pos i t ioned into e i ther the outer (OML; S ^ or middle (MML; S2) molecular layer of the dentate gyrus to evoke a c t i v i t y in the l a t e r a l (LPP) and medial (MPP) perforant paths, re spec t ive ly . In some experiments an electrode (S3) was also placed into the h i l u s to act ivate the mossy f ibers (MF) which ant idromica l ly evoked granule c e l l discharges and orthodromical ly act ivated the recurrent i n h i b i t o r y interneurons ( F i g . 2.1; Lomo, 1971b). An e l e c t r i c a l l y i so la ted constant voltage unit de l ivered s t imul i (0.1 ms in duration) at i n t e n s i t i e s of 5-80 V (approximately 10-200 microamperes) to the perforant path or MF once every 30 s (0.033 Hz) to obtain the contro l responses. This long interst imulus in terva l (ISI) was necessary to insure complete independence between contro l 31 responses, since several groups have found that the granule c e l l s habituate at stimulus frequencies as low as 0.05 Hz (Alger and Tey ler , 1976). Frequently the experimental protocol c a l l e d for a second stimulus (test) to be de l ivered fol lowing the contro l (condition) response by a short i n t e r v a l . This paired pulse paradigm was used to invest igate the mechanisms that subserve the short-term a l t e r a t i o n s in neuronal e x c i t a b i l i t y found in the dentate gyrus (Alger and T e y l e r , 1976; Lomo,1971b; McNaughton and Barnes, 1977). Furthermore, because the condi t ion- tes t (C-T) i n t e r v a l could be as long as 8 s, the ISI was changed such that a 30 s i n t e r v a l always existed between the test response and the next C-T p a i r . Thus, the independence of each C - T . p a i r was maintained. 2.5 E x t r a c e l l u l a r recording of the population potent ia l s  evoked in the dentate gyrus E x t r a c e l l u l a r potent ia l s were recorded with glass micropipettes (Frederic Haer, Omega Dot 1.5 mm O.D.) pul led on a Narishige PE-2 p u l l e r to a t i p diameter of 1-2 microns. The e lectrodes had res istances of 2-8 Mohms when f i l l e d with a 2M NaCl s o l u t i o n . The output of these e lectrodes was connected to a high-input impedance ampl i f i er and referenced to an ind i f f erent e lectrode ( i . e . , Ag/AgCl wire) located in the recording chamber. The DC s igna l was then f i l t e r e d (3 kHz) and displayed on a storage osc i l loscope for photography or led to a PDP 11/23 computer for on- l ine 32 s igna l averaging or stored on floppy disk for la ter r e t r i e v a l and a n a l y s i s . F i n a l data output was p lo t ted on a Tektronix 4662 X-Y p l o t t e r . Recording electrodes were pos i t ioned , with the a id of a d i s sec t ing microscope, into e i ther the OML (R 1 ) or the MML (R 2 ) in order to record the population exc i tatory postsynaptic potent ia l s (EPSP) evoked by LPP and MPP s t imulat ion , respec t ive ly . An electrode was a lso posi t ioned in the granule c e l l layer (GCL; R 3 ) in order to record the reversed population EPSP at the soma or the population spike (PS) generated by the synchronous discharge of a number of granule c e l l s ( F i g . 2 .1, 2 .2) . The amplitude of the population potent ia l s was optimized by lowering the electrodes into the s l i c e u n t i l the maximal potent ia l generated by a perforant path stimulus was observed. 2.6 Analys is of the e x t r a c e l l u l a r f i e l d potent ia l s Data c o l l e c t i o n was f a c i l i t a t e d by simultaneous c o l l e c t i o n of both dendr i t i c and somatic evoked potent ia l s on separate channels of a PDP 11/23 computer. The analog to d i g i t a l (A/D) conversion rate per channel was approximately 60 microseconds, thus an evoked potent ia l that was 6 ms in duration would have 100 sampling points from which the EPSP could be reconstructed. This sampling rate was fast enough to detect accurately the peak of EPSPs, PSs, and act ion p o t e n t i a l s . Moreover, the s e n s i t i v i t y of the measurement 33 F i g u r e 2 . 2 . E x t r a c e l l u l a r records of the d i f f e r e n t p o t e n t i a l s observed i n the dentate gyrus. (Top t r a c e ) The EPSP recorded i n e i t h e r the OML or MML i n response to LPP and MPP s t i m u l a t i o n , r e s p e c t i v e l y . (Middle t r a c e ) The reversed p o p u l a t i o n EPSP ( P ^ was recorded from the GCL i n response to p e r f o r a n t path s t i m u l a t i o n and i f the stimulus i n t e n s i t y was in c r e a s e d to di s c h a r g e the granule c e l l s , the EPSP was followed by a p o p u l a t i o n spike (PS). A l l EPSP (P 1) values i n t h i s study were obtained i n the absence of a PS. (Lower t r a c e ) H i l a r s t i m u l a t i o n a n t i d r o m i c a l l y evoked a granule c e l l p o p u l a t i o n spike (AS). The method f o r o b t a i n i n g the amplitudes of each p o t e n t i a l i s i l l u s t r a t e d by the dotted l i n e s and a l l v a l u e s represent an average of four responses. 34 35 was increased because a minimum of 4 evoked potent ia l s were averaged to obtain the amplitudes of the EPSP and PS. The amplitude of the population EPSP responses was ca lcu la ted from the baseline to e i ther the peak negat iv i ty for dendr i t i c EPSPs or the peak p o s i t i v i t y for somatic EPSPs ( F i g . 2.2 A, B) . Since the ionic fluxes during a PS can a l t e r the peak potent ia l of an EPSP, the EPSP values used in a l l studies were determined from potent ia l s which were subthreshold for PS discharge. The PS amplitude was ca lcu la ted from the peak of the somatic EPSP (P-^ ) to the negative peak of the PS potent ia l ( F i g . 2.2 B) . Since no EPSP was associated with the ant idromica l ly evoked population spike (AS), i t was measured from basel ine to the peak negat iv i ty ( F i g . 2.2 C ) . Analys is of data derived from the paired pulse experiments was handled in a s imi lar manner to that of the s ingle pulse experiments. The paired pulse experiments, however, provide an ind ica t ion of the a l t e r a t i o n s in c e l l u l a r e x c i t a b i l i t y which occur in response to previous a c t i v a t i o n . Therefore a l l paired pulse data were expressed in the following way: amplitude of the test (V t ) response . X100 (Formula 1) amplitude of the condi t ioning (V c ) response where the amplitude of the test response was the average of 4 evoked potent ia l s that were i n i t i a l l y paired with the ir condi t ion ing responses ( F i g . 2 .3) . 36 Figure 2.3. Typ ica l e x t r a c e l l u l a r responses i l l u s t r a t i n g the paired pulse paradigm and how the values for the condi t ioning (V c ) and test (V t ) responses were c a l c u l a t e d . The a l t e r a t i o n s in the potent ia l s at varying C-T in terva l s were defined by the changes in the amplitude of V t with respect to the amplitude of V c (see Formula 1). The top trace i l l u s t r a t e s potent iat ion of a dendr i t i c EPSP at the C-T shown. The middle trace represents EPSPs recorded from the granule c e l l layer in which the test EPSP is reduced. Bottom trace shows the potent iat ion of the population spike recorded from the granule c e l l l ayer . 37 38 2.7 I n t r a c e l l u l a r recording of dentate granule c e l l s I n t r a c e l l u l a r recordings from granule c e l l s were made using 35-60 Mohm microelectrodes (Frederic Haer, Omega Dot 1.5 mm O.D.) f i l l e d with 1 M potassium acetate . The microelectrodes were connected to a WPI M707 electrometer, bridge balanced and capacity compensated. A constant current device (designed by Dr. Thomas Richardson) was used to contro l the current i n j e c t i o n from the electrometer to the microelectrode. A l l i n t r a c e l l u l a r recording electrodes were checked for the ir a b i l i t y to pass injected current without causing voltage def lec t ions pr ior to and fol lowing the withdrawal from granule c e l l s , since s i g n i f i c a n t errors could ar i se in the determination of cer ta in i n t r a c e l l u l a r propert ies i f non-l inear electrodes were u t i l i z e d . Moreover, the capacity compensation contro l was equipped with a switch that caused th i s c i r c u i t to offset and produce large (15V) voltage t rans ient s . This 'buzzer' was u t i l i z e d to a id in the penetration of c e l l s . The output from the electrometer was f i l t e r e d (3 kHz) and led into a storage osc i l lo scope or. to the computer for on- l ine data c o l l e c t i o n . 2.8 Analys i s of i n t r a c e l l u l a r membrane c h a r a c t e r i s t i c s and  evoked potent ia l s Granule c e l l s were i d e n t i f i e d by the fol lowing e l e c t r o p h y s i o l o g i c a l c r i t e r i a : 1) perforant path s t imulat ion 39 Figure 2.4. Typ ica l i n t r a c e l l u l a r records of a perforant path evoked EPSP (A) and granule c e l l act ion p o t e n t i a l (B). The amplitudes of the condi t ioning EPSP ( V c ) , test EPSP (V f c) and act ion potent ia l ( V a p ) were determined from baseline (RMP) to peak p o s i t i v i t y . Voltage and time bases are d i f f erent between (A) and (B). 40 A. B. 41 evoked a monosynaptic EPSP, as determined by i t s latency to on-set fol lowing the stimulus; 2) perforant path st imulat ion could evoke an act ion po tent ia l (AP); 3) the neuron responded with a s ingle AP when evoked e i ther by perforant path or MF s t imulat ion; 4) res t ing membrane potent ia l (RMP) had a negative value greater than -60 mV; 5) c e l l input resistance (R n) was greater than 20 Mohms; and 6) that AP amplitudes (SA) were greater than 80 mV. Although the preceding c r i t e r i a had to be s a t i s f i e d before any c e l l was included in th i s study, one c r i t e r i o n that was only used for the experiments u t i l i z i n g paired pulse st imulat ion was that the changes in the amplitude of the PS had to be appropriate ly mimicked by the impaled c e l l ' s a b i l i t y to generate an AP. That i s , i f the PS exhibited i n h i b i t i o n at a cer ta in C-T i n t e r v a l , then the p r o b a b i l i t y of discharge had to decl ine in the impaled granule c e l l . I n t r a c e l l u l a r EPSP amplitudes were measured from the RMP to the peak p o s i t i v i t y and only those EPSPs which did not discharge the granule c e l l were included in the analys is ( F i g . 2.4 A, V"c) . When paired pulse s t imulat ion was u t i l i z e d , the amplitude of the test EPSP was measured from the o r i g i n a l RMP, even though the i n t r a c e l l u l a r po tent ia l was rare ly equal to the RMP during the majority of the C-T i n t e r v a l s ( F i g . 2.4 A, V t ) . A l s o , the assessment of the a l t e r a t i o n s in neuronal e x c i t a b i l i t y , as measured by paired pulse s t imulat ion , was done exactly as stated in the 42 e x t r a c e l l u l a r a n a l y s i s (see Formula 1). The a c t i o n p o t e n t i a l amplitude (SA) was measured from RMP to the peak p o s i t i v i t y of the AP i n response to p e r f o r a n t path s t i m u l a t i o n ( F i g . 2.4 C). The RMP was monitored c o n t i n u o u s l y d u r i n g an experiment by a d i g i t a l voltmeter which was zeroed p r i o r to the impalement of the granule c e l l . The v a l i d i t y of these v a l u e s was checked at the end of an experiment by measuring the volta g e d i f f e r e n c e on an o s c i l l o s c o p e between the RMP and the e x t r a c e l l u l a r p o t e n t i a l a f t e r the e l e c t r o d e was withdrawn from the c e l l . If there was a discrepancy between the two methods, the p o t e n t i a l measured from the o s c i l l o s c o p e was co n s i d e r e d the true v a l u e . The f i r i n g and s y n a p t i c i n t e g r a t i o n c h a r a c t e r i s t i c s of neurons c h i e f l y depends on the e l e c t r i c a l p r o p e r t i e s of the c e l l membrane. Th e r e f o r e , two of the more important p r o p e r t i e s , the input r e s i s t a n c e (R n) and the time constant ( T Q ) were determined f o r granule c e l l s . The values were c a l c u l a t e d from the averaged (n=4) recorded v o l t a g e d e f l e c t i o n s produced by a s e r i e s of c u r r e n t pulses (50-100 ms, -0.8 to +0.6 nA) i n j e c t e d through the r e c o r d i n g e l e c t r o d e ( F i g . 2.5 A). To o b t a i n the Rn the amplitude of each v o l t a g e d e f l e c t i o n was p l o t t e d with respect to c u r r e n t ( F i g . 2.5 B). The slope of the b e s t - f i t t i n g l i n e from the c u r r e n t - v o l t a g e (I-V) p l o t , as c a l c u l a t e d by a computer using l i n e a r - r e g r e s s i o n a n a l y s i s , was taken as the Rn. Because some granule c e l l s d i s p l a y e d n o n - l i n e a r I-V p r o f i l e s ( i . e . , r e c t i f i c a t i o n ) , the R n w a s ' c a l c u l a t e d using only the l i n e a r p o r t i o n of the I-V p l o t . 43 Figure 2.5. The determination of the membrane input resistance (R n) and time constant ( T c ) . (A) Current inje c t i o n (upper trace) causes voltage displacements (lower trace) in the membrane potential of granule c e l l s . This current-voltage (I-v) relat i o n s h i p (voltage values were determined at the time demarcated by the open arrow) is shown in an I-V plot which represents the average of four responses per point (B). The R n was calculated by l i n e a r -regression analysis as the slope of the best f i t t i n g l i n e through the data points. Because of the non-linearity of some of the I-V p r o f i l e s (note that the open c i r c l e s do not f a l l on the given l i n e ) , the R n values were only taken from the linear portion (e.g., the f i l l e d c i r c l e s ) of a part i c u l a r I-V plot. In a l l I-V pl o t s , the diameter of the c i r c l e was chosen to represent the greatest SEM exhibited by a voltage response to the same current i n j e c t i o n . (C) A voltage p r o f i l e resulting from hyperpolarizing current in j e c t i o n is shown. In order to calculate the T c, l i n e a r -regression was performed over a 1 ms period ( s o l i d lines) to get an average change in voltage (dV/dt). The 1 ms period was then incremented at 10~^S (dashed lines) along the voltage p r o f i l e to obtain numerous values of dV/dt (D). The natural logarithm of dV/dt was plotted against time and the T c became the slope of the best f i t t i n g l i n e through the data as determined by linear-regression analysis. Since the 44 time constant d id not always appear to be the same along the extent of the voltage p r o f i l e , T c ' s were determined for the f i r s t 4 ms (T e ) and for 4 ms and on (T-^). 45 I-V Analysis mV Time Constant Analysis c 46 The T c is defined as the time required for a change in voltage to reach 1/e ( i . e . , approximately 62%) of i t s maximal value . Since previous studies have determined the T c associated with the res t ing membrane (Brown et a l . , 1981; Durand et a l . , 1983), th i s study determined the T c at hyperpolarized and depolarized membrane p o t e n t i a l s . To obtain the T c , the computer averaged a minimum of four voltage p r o f i l e s generated at a s ingle current in tens i ty and d i f f e r e n t i a t e d the voltage p r o f i l e with respect to time (dV/dt ) . Due to noise f luctuat ions in the voltage p r o f i l e s , however, dV/dt values were ca l cu la ted from the average change in voltage over a 1 ms p e r i o d , as ca lcu la ted by l inear -regres s ion analys i s (F ig . 2.5 C, s o l i d l i n e s ) . To ensure a good approximation of the T c , the 1 ms period def in ing dV/dt was incremented at 10~^S in terva l s along the voltage p r o f i l e u n t i l dV/dt equaled zero (F ig . 2.5 D). Due to the exponential nature of the dV/dt values, the T c was taken as the negative slope value obtained by l i n e a r -regression analys i s from the plot of the natural logarithm of dV/dt versus rea l time (F ig . 2.5 E ) . Occas ional ly granule c e l l s exhibi ted two T c ' s , a r e l a t i v e l y fast one which occurred ear ly ( less than 4 ms) in the voltage p r o f i l e and another which happened at a longer duration ( g r e a t e r than 4 ms). The voltage p r o f i l e , therefore , was separated into these two ranges with time constants T e and T^ being i n d i v i d u a l l y determined for the 0-4 ms and the greater than 4 ms periods , r e spec t ive ly . 47 Chapter 3 An i n t r a c e l l u l a r charac ter i za t ion of the dentate gyrus granule c e l l s from the in v i t r o rat hippocampus 3.1. Introduction In order to comprehend f u l l y the function of any CNS s tructure , i t is necessary to character ize the membrane propert ies and synaptic responses of the p r i n c i p a l neurons. In th i s respect , the pyramidal neurons of the hippocampus proper have been extensively studied with i n t r a c e l l u l a r recording techniques (Alger, 1984; Alger and N i c o l l , 1979; Andersen et a l . , 1964; Brown and G r i f f i t h , 1983a; Brown and Johnston, 1983; Hotson et a l . , 1979; Kandel et a l . , 1961; Knowles et a l . , 1984; Madison and N i c o l l , 1984; Schwartzkroin, 1975; Schwartzkroin, 1977; Wong and Pr ince , 1981; Yamamoto, 1982). Although much is known about the pyramidal neurons, the function of the hippocampus remains e lus ive , in part , due to the lack of information concerning the other p r i n c i p a l c e l l type within the HF, the DG granule c e l l . Granule c e l l s receive the primary c o r t i c a l input , the perforant path, to the HF (Lorente de No, 1934). Thus, determining the role of the hippocampus w i l l require a 48 descr ip t ion of both pyramidal and granule c e l l s . With few exceptions e l ec t rophys io log i ca l studies of the dentate gyrus have been r e s t r i c t e d to the ana lys i s of population responses (Andersen et a l . , 1966a; Douglas et a l . , 1983; Lomo, 1971a; McNaughton and Barnes, 1977; Racine and Milgram, 1983). These e x t r a c e l l u l a r studies have provided valuable information concerning the physiology of synaptic responses and neuronal in tegrat ion; however, analys is of population potent ia l s remains speculative without i n t r a c e l l u l a r corroborat ion . This i s p a r t i c u l a r l y true for i d e n t i f y i n g the presence of smaller e l e c t r i c a l events which may go undetected by e x t r a c e l l u l a r recording . The paucity of i n t r a c e l l u l a r recordings from granule c e l l s i s presumably due to the d i f f i c u l t y in penetrating these small neurons which are less than 15 microns in diameter. From the few i n t r a c e l l u l a r studies which have been completed, some important questions have ar isen regarding the v a l i d i t y of e x t r a c e l l u l a r recordings obtained from the DG. For example, i t was postulated that i n h i b i t i o n in the DG resul ted from a hyperpolar izat ion mediated by the recurrent a c t i v a t i o n of GABAergic interneurons (Lomo, 1971a,b). I n t r a c e l l u l a r recordings , however, have been unable to e s t a b l i s h c l e a r l y the presence of a GABA-mediated hyper-p o l a r i z a t i o n in granule c e l l s . St imulation of the perforant path (PP) has reportedly evoked an inh ib i tory postsynaptic po ten t ia l (IPSP) (Fricke and Pr ince , 1984; Lomo, 1971a), a depo lar iz ing a f t e r p o t e n t i a l (DAP) (Deadwyler et a l . , 1975; 49 Thalmann and Ayala , 1982), or both (Fournier and Crepe l , 1984; Misgeld et a l . , 1982). S i g n i f i c a n t l y , the IPSP and DAP in granule c e l l s have s imi lar time courses and are sens i t ive to a l t e r a t i o n s in external ch lor ide concentrations and GABA antagonists (Fricke and Pr ince , 1984; Misgeld et a l . , 1982; Thalmann and Ayala , 1982), suggesting that they are the same p o t e n t i a l . This issue is further confused by the fact that b i c u c u l l i n e - s e n s i t i v e IPSPs, evoked by antidromic s t imula t ion , and DAPs can occur in the same granule c e l l (Thalmann and Ayala , 1982). Although i t is not c lear what response i s generated by the synaptic release of GABA in the dentate gyrus, iontophoresis of GABA on the soma or dendrites of granule c e l l s produces a depolar izat ion (Assaf et a l . , 1981). This i s in contrast with the iontophoresis of GABA on pyramidal c e l l s which e l i c i t s a hyperpolar izat ion or a depolar izat ion when appl ied to the soma and dendrites , respect ive ly (Alger and N i c o l l , 1982a; Andersen et a l . , 1980; Thalmann et a l . , 1981). This d i f f e r e n t i a l s e n s i t i v i t y to iontophore t i ca l ly appl ied GABA between the pyramidal and the granule c e l l s implies that the processes mediated by GABA may be func t iona l ly d i f f erent between these neurons. This p o s s i b i l i t y i s supported by the effect iveness of GABA antagonists in e l i c i t i n g ep i l ept i form discharges from pyramidal but not granule c e l l s (Fricke and Pr ince , 1984; Misgeld et a l . , 1982). Furthermore when ep i l ept i form a c t i v i t y i s evoked in the hippocampal formation by prolonged 50 r e p e t i t i v e st imulat ion of the PP, the pyramidal neurons exhib i t marked degeneration even though the granule c e l l s are large ly spared (Olney et a l . , 1983; S l o v i t e r , 1983). It i s apparent then that fundamental di f ferences in the GABA-e l i c i t e d response exis t between the two p r i n c i p a l c e l l types within the hippocampal formation. Synaptic e x c i t a t i o n , evoked from the PP, a lso act ivates a la te hyperpolar izat ion (LHP) in granule c e l l s (Thalmann and Ayala , 1982). The LHP i s most l i k e l y potassium-dependent, but i t i s not known whether th i s p o t e n t i a l is calc ium- or t ransmi t ter - sens i t ive (Thalmann and Ayala , 1982). Recent data from the pyramidal c e l l indicates i t s LHP is a potassium-dependent po tent ia l that may be s y n a p t i c a l l y -mediated v ia interneurons (Alger, 1984; Knowles et a l . , 1984; Thalmann, 1984). Given the importance of neuronal integrat ion in CNS funct ion , and the opposing consequences of membrane hyperpolar izat ion and depolar izat ion on th i s c e l l u l a r property, i t is c r i t i c a l to determine whether granule c e l l s exhib i t e i ther an IPSP or a DAP fol lowing the EPSP. Therefore, the present study was undertaken to further character ize the synapt ical ly-evoked potent ia l s in granule c e l l s . S p e c i f i c a l l y , i n t r a c e l l u l a r recordings from granule c e l l s examined act ive ( i . e . , voltage-dependent) membrane processes and synaptic responses e l i c i t e d by both PP and h i l a r ( i . e . antidromic) s t imula t ion . This paradigm should determine i f the di f ference in p o l a r i t y of these potent ia l s 51 Figure 3.1 . The current-voltage (I-V) p r o f i l e s and p lots for two t y p i c a l granule c e l l s are shown. The f i r s t neuron (A) exhibi ted a non-l inear I-V r e l a t i o n s h i p which was considered evidence for anomalous r e c t i f i c a t i o n . From the I-V plot (B) one can observe a decrease in the membrane slope resistance to hyperpolar iz ing pulses ( s o l i d l i n e ; 20 Mohms) when compared to depolar iz ing pulses (dashed l i n e ; 50 Mohms). The other neuron displayed a l inear I-V r e l a t i o n s h i p (D) as indicated by the membrane slope resistance being defined by a s ingle l i n e (E; 36 Mohms). Although the I-V p r o f i l e s for these neurons were d i f f e r e n t , the ir membrane time constants (T c ) ca lcu la ted for hyperpolar iz ing pulses ( f i l l e d arrows) were s imi lar (C, 3 ms; F , 3 ms). However, the T c ' s in the depolar iz ing range were longer in the non-ohmic c e l l (12 ms) than in the ohmic neuron (3 ms). Open arrows on voltage p r o f i l e s (A,B) indicate where voltage measurements were determined. 52 53 resu l t s from the a c t i v a t i o n of feed-forward versus recurrent interneurons. F i n a l l y , the p o s s i b i l i t y of the LHP being synaptical ly-mediated was examined. 3.2. Methods The majority of the methods used in th i s chapter are described in Chapter 2. In add i t ion , the changes in membrane res is tance associated with the DAP (Rdap) and the LHP (Rihp) were determined by in jec t ing a series of current pulses (-1.0 to 1.0 nA), approximately 50 ms in durat ion, during these d i f f erent p o t e n t i a l s . The R^ap a n c ^ R l h p v a l u e s were then ca lcu la ted from these I-V re la t ionsh ips by computer using l inear -regres s ion a n a l y s i s . Equ i l ibr ium potent ia l s for the DAP (E<jap) and LHP (E^^p) a l so were determined by a l t e r i n g the res t ing membrane potent ia l (RMP) with constant d . c . current injected through the i n t r a c e l l u l a r electrode and then st imulat ing the PP to evoke the a f t e r p o t e n t i a l s . By p l o t t i n g the change in membrane potent ia l associated with the peak amplitude of the a f t erpotent ia l s versus the current- induced sh i f t in membrane p o t e n t i a l , E dap a n c ^ E l h p could be defined by the ir respect ive x- intercept values obtained with l inear regression a n a l y s i s . In several experiments the e f fects of: 1) a l t e r i n g the e x t r a c e l l u l a r potassium concentration ( [ K ] Q ; 4.25 to 8 . 0 54 Table 3 .1 . The mean +/- SEM are given for granule c e l l s exh ib i t ing non-l inear and l inear I-V p r o f i l e s . S ign i f i cant di f ferences in res t ing membrane potent ia l (RMP; p < 0.01) and spike amplitude (SA; p < 0.005) were found between the two groups and the other membrane propert ies (input resistance (R n ) and membrane time constant ( T c ) ) showed no d i f f erence . A l s o , within a group the ear ly ( T e ) and late (T^) time constants were not s i g n i f i c a n t l y d i f f e r e n t . A l l T c values were obtained from hyperpolar iz ing voltages . S t a t i s t i c a l measurements were ca lcu la ted using the two-t a i l e d Student t - t e s t . GRANULE CELLS Non-Linear (n) Linear (n) RMP (-mV) 76 +/- 1.3 (68-85) (20) 70 +/- 1. (60-85) 7 (22) SA (mV) 105 +/- 1.6 (93-116) (15) 96 +/- 2. (85-110) 1 (15) R n (Mohms) 36 +/- 2.7 (20-56) (20) 32 +/- 2. (20-50) 3 (22) T e (ms) 4 +/- 0.3 (2-6) (9) 4 +/- 0. (2-6) 6 (6) Tj_ (ms) 4 +/- 0.5 (2-7) (9) 5 +/- 0. (3-7) 6 (6) 55 mM); 2) subst i tut ing barium for calcium; 3) lowering e x t r a c e l l u l a r calcium ( [Ca] Q ; 2 mM to 1 mM) and r a i s i n g e x t r a c e l l u l a r magnesium ([Mg] 0 ; 2 mM to 14 mM); and 4) perfusing 10"^ M (+/-) B-p-chlorophenyl GABA (baclofen) were invest igated on the PP evoked synaptic potent ia l s and membrane propert ies of granule c e l l s . 3.3. Results 3 .3 .1 . Membrane c h a r a c t e r i s t i c s of granule c e l l s The resul ts of the present study are based on the i n t r a c e l l u l a r recordings from for ty -e ight DG granule c e l l s , a l l of which met the minimal e l e c t r o p h y s i o l o g i c a l c r i t e r i a described in Chapter 2. Analys i s of the I-V re la t ionsh ips suggested the p o s s i b i l i t y that two populations of granule c e l l s occupy the DG. T y p i c a l examples of the d i f f erent I-V p r o f i l e s are shown in F i g . 3.1. The f i r s t response consisted of a non-l inear I-V re la t i onsh ip in response to depo lar iz ing and hyperpolar iz ing current in j ec t ion ( F i g . 3.1 A ) . This non-ohmic response to current i n j e c t i o n was exhibi ted by 20/42 granule c e l l s and indicates the presence of anomalous r e c t i f i c a t i o n (AR) (Brown and G r i f f i t h , 1983a; Hotson et a l . , 1979; Johnston et a l . , 1980). The second I-V r e l a t i o n s h i p , exhibi ted by 22/42 granule c e l l s , was character ized by a r e l a t i v e l y l inear change in voltage over the ser ies of injected currents ( F i g . 3.1 D) . 56 Figure 3.2. The effect of increasing the duration of a depolar iz ing current pulse on the repe t i t i ve f i r i n g of a granule c e l l is shown. As the current pulse becomes longer (A-D; 10, 50, 100, and 150 ms) the number of act ion potent ia l s i n i t i a l l y increases; however, granule c e l l s accommodate ( i . e . , decrease discharge rate) with prolonged depolar izat ion (D). A l l APs were c l ipped during photography. 58 Figure 3.3. Granule c e l l s are character ized by the ir a b i l i t y to produce a graded a f terhyperpo lar iza t ion (AHP) in response to longer depolar iz ing current pulses . This granule c e l l exhibited accommodation (see F i g . 3.2) such that more APs were evoked in (B) than in e i ther (C) or (D). A l l spikes were c l ipped during photography. 59 A. B. C. D. -I InA J 10 mV 0.5 s 60 The dif ferences in the I-V r e l a t i o n s h i p between these neurons was further i l l u s t r a t e d by the I-V p l o t s . In the examples shown, the I-V plot of the granule c e l l exh ib i t ing AR was defined by two l ines g iv ing membrane input resistances (R n) of 20 Mohms ( s o l i d l ine ) and 50 Mohms (dashed l ine ) for hyperpolar iz ing and depolar iz ing current pulses , respect ive ly ( F i g . 3.1 B) . Because R n values included in the present study were only determined in the l inear range of the I-V r e l a t i o n s h i p , th i s p a r t i c u l a r granule c e l l measured 24 Mohms. The I-V plot of the other granule c e l l was l inear g iv ing a s ingle R n of 36 Mohms ( F i g . 3.1 E) . Granule c e l l s exh ib i t ing AR maintained s i g n i f i c a n t l y greater res t ing membrane potent ia l s (RMP; p< 0.01) and act ion p o t e n t i a l amplitudes (SA; p< 0.005) when compared to • the other granule c e l l s (Table 3 .1) . R n values and membrane time constants (T c ) associated with hyperpolar iz ing current pulses were s imi lar between the groups. The d e p o l a r i z a t i o n -induced AR prolonged the T c (12 ms as compared to 3 ms). Despite the aforementioned d i f ferences , depo lar iz ing current in jec t ions that discharged granule c e l l s revealed two i n t r i n s i c propert ies held in common. F i r s t , increasing the duration of depolar iz ing current pulses resul ted in the r e p e t i t i v e f i r i n g of APs. Granule c e l l s displayed accommodation of AP generation based on the observation that the i n t e r v a l between successive APs increased ( F i g . 3.2 D) . Second, termination of the depo lar iz ing pulses 61 Figure 3.4. St imulation of the h i lu s evoked a graded, short - la tency , negative-going spike (antidromic spike, AS) when recorded e x t r a c e l l u l a r l y (A, B; top traces) which represents the synchronous discharge of a population of granule c e l l s . When the stimulus intens i ty i s subthresold for a c t i v a t i o n of the granule c e l l , a graded depo lar iz ing potent ia l (D-IPSP) i s observed with i n t r a c e l l u l a r recording (A, lower t r a c e ) . Increasing the stimulus intens i ty discharged the granule c e l l (B; lower trace) and evoked a s l i g h t l y larger D-IPSP which required approximately 50 ms to return to RMP. r T 2 m V 5ms [20 mV 63 c h a r a c t e r i s t i c a l l y resulted in an a f terhyperpolar izat ion (AHP) ( F i g . 3.3, arrow). The amplitude of t h i s AHP was dependent on the duration of the current i n j e c t i o n but not on the number of APs, since the number of APs generated did not necessar i ly increase with longer pulses ( e . g . , accommodation). This resu l t d i f f e r s from CA1 pyramidal c e l l s , where a c o r r e l a t i o n between the number of spikes and the amplitude and duration of the AHP ex is t s (Hotson and Pr ince , 1980; Madison and N i c o l l , 1983). 3 .3 .2 . Antidromic evoked responses in granule c e l l s E x t r a c e l l u l a r recordings from the granule c e l l layer (GCL) revealed that low intens i ty h i l a r s t imulat ion evoked a short latency ( less than 2.5 ms) negative-going population spike (AS) ( F i g . 3.4 A, B; upper t r a c e ) , which has been a t t r i b u t e d to the antidromic a c t i v a t i o n of granule c e l l s v ia the MFs (Andersen et a l . , 1966a; Lomo, 1971a). Simultaneous i n t r a c e l l u l a r recordings (n=6) showed, that at low stimulus i n t e n s i t i e s , granule c e l l s exhibi ted a monosynaptic depo lar iza t ion which had a duration between 15-50 ms (F ig . 3.4 A; lower t r a c e ) . Increasing the stimulus intens i ty de l ivered to the h i l u s demonstrated that the e x t r a c e l l u l a r AS corre la ted in time to the discharge of granule c e l l s , thereby supporting the in terpre ta t ion that the AS is the e x t r a c e l l u l a r r e f l e c t i o n of granule c e l l APs ( F i g . 3.4 B) . The depolar iz ing po tent ia l associated with h i l a r st imulat ion probably represented the a c t i v a t i o n of GABAergic 64 Figure 3.5. I n t r a c e l l u l a r recordings i l l u s t r a t e that s t imulat ion of the h i lu s evoked a D-IPSP from th i s granule c e l l . A l t e r i n g the membrane potent ia l with current in j ec t ion inverted the D-IPSP at depolarized potent ia l s (A, arrow). The current-vol tage re la t i onsh ip for the membrane (open c i r c l e ) and D-IPSP ( f i l l e d c i r c l e ) are p lo t ted (B). L i n e a r -regression analys is gave membrane slope res istances of 39 Mohms and 14 Mohms for R n and R d - i p S p , respec t ive ly . The dashed l ines in (A) were added to c l a r i f y the experimental paradigm. 65 10ms (nA) oRn-35MXi 66 Figure 3.6. H i l a r s t imulat ion evoked a D-IPSP which was reversed by current i n j e c t i o n at -55 mV ( f i l l e d arrow) and became more depolar iz ing as the membrane p o t e n t i a l was hyperpolarized (-75 mV). At a membrane po tent ia l of -55 mV a second la te hyperpolar izat ion (LHP) becomes obvious (open arrow). The change in resistance associated with the LHP evoked with h i l a r s t imulat ion is shown in (B). The R n of the granule c e l l was ca lcu la ted from the membrane potent ia l p r i o r to the h i l a r s t imulat ion (47 Mohms) and R]_hp values were determined 0.2 s (41 Mohms) and 1.0 s (44 Mohms) fol lowing h i l a r s t imulat ion . The upper traces in (B) are the current pulses; the lower traces are the voltage responses. C a l i b r a t i o n of current pulses in (B) i s 10 mV = 1 nA. 67 68 interneurons which are orthodromical ly evoked by st imulat ion of the MFs (Andersen et a l . , 1966a; Lomo, 1971b); and since the function of th i s depolar izat ion is most l i k e l y i n h i b i t o r y , i t w i l l be d is t inguished from an EPSP and be referred to as a depolar iz ing inh ib i tory postsynaptic po ten t ia l (D-IPSP). In order to determine the . equ i l ibr ium p o t e n t i a l of the D-IPSP ( E d- ipsp^' antidromic st imulat ion was presented during a ser ies of i n t r a c e l l u l a r current pulses . The D-IPSP reversed at about 6 mV pos i t ive to the RMP giv ing an E<a-ip Sp of -61 mV (F ig . 3.5 A, arrow). The D-IPSP also induced a s i g n i f i c a n t a l t e r a t i o n in membrane resistance ( F i g . 3.5 B; R n = 35 Mohms; R d - i p S p = 1 ^ Mohms). The i n h i b i t o r y function of- th i s potent ia l was shown by i t s maintaining an equi l ibr ium potent ia l only s l i g h t l y pos i t ive to the RMP and below AP threshold. Antidromic s t imulat ion also t y p i c a l l y evoked a long a f terhyperpo lar iza t ion (LHP, open arrow) fol lowing the D-IPSP ( f i l l e d arrow; F i g . 3.6 A ) . As shown in F i g . 3.6 (A), the LHP was more e a s i l y observed by depolar iz ing the granule c e l l and in most cases a reversa l potent ia l could not be determined. The LHP was associated with a var iab le reduction in membrane res istance which ranged from 6 Mohms at the peak of the LHP (200 ms) to 3 Mohms at 1 s ( F i g . 3.6 B; c f . , Thalmann and Ayala , 1982). 69 Figure 3.7. Increasing the in tens i ty of the stimulus de l ivered to the perforant path i l l u s t r a t e d the graded nature (input-output re la t ionsh ip) of the e x t r a c e l l u l a r and i n t r a c e l l u l a r EPSPs recorded in the DG. The e x t r a c e l l u l a r potent ia l s (A, C) were recorded from the granule c e l l layer in close proximity to the granule c e l l being i n t r a c e l l u l a r l y recorded. (D) Note that when the EPSP reached threshold that a large amplitude (greater than 90 mV) spike was observed. 70 71 3.3 .3 . Perforant path evoked responses in granule c e l l s C h a r a c t e r i s t i c e x t r a c e l l u l a r records obtained from the GCL showed that increasing the stimulus in tens i ty de l ivered to the PP (input) resul ted in a graded EPSP (output) ( F i g . 3.7 A ) . This input-output (I-O) r e l a t i o n s h i p was simultaneously recorded inside a granule c e l l ( F i g . 3.7 B) . With an increase in the stimulus i n t e n s i t y , the EPSP t y p i c a l l y exhibi ted: 1) a reduction in the latency to peak amplitude; 2) an enhanced rate of r i s e ; and 3) an increase in the rate of decay of the EPSP (arrow). The las t observation may be re lated to the a c t i v a t i o n of i n h i b i t o r y processes. It i s noteworthy that the f i e l d potent ia l s d id not r e f l e c t the i n t r a c e l l u l a r events which occurred af ter the decay of the EPSP. Suprathreshold s t imulat ion de l ivered to the PP t y p i c a l l y evoked a negative-going spike (PS) at the peak of the population EPSP which has been interpreted as the synchronous discharge of many granule c e l l s ( F i g . 3.7 C; Andersen et a l . , 1966a; Lomo, 1971a). This in terpre ta t ion was substantiated by i n t r a c e l l u l a r recordings which showed that granule c e l l s f i r e d from the peak of the EPSP and that the PS was corre la ted in time to the presence of the granule c e l l AP ( F i g . 3.7 D) . The mean (+/- SEM) EPSP amplitude that i n i t i a t e d an AP was 29 +/- 1.3 mV (range: 19-39 mV; n=28) and, under normal condi t ions , PP st imulat ion evoked only a s ingle AP. 72 Figure 3.8. Occasional ly (n = 4) s t imulat ion of the perforant path evoked a small amplitude spike (SAS) -on the peak of the EPSP. (A) shows the s e n s i t i v i t y of the SAS (arrow) to synaptic a c t i v a t i o n . The subthreshold EPSP was evoked with a stimulus in tens i ty of 15.5 V; the SAS became evident when the in tens i ty was increased by 1 V (A). S u r p r i s i n g l y , the granule c e l l d id not discharge off the SAS (arrow), even though i t at ta ined the greatest voltage (B). Granule c e l l spike was c l ipped during photography. 73 ims 74 Figure 3.9. The ef fects of a l t e r i n g the granule c e l l membrane potent ia l on the perforant path evoked EPSP. Hyperpolar iz ing the membrane po tent ia l increased the amplitude and decreased the r i s e time of the EPSP, whereas, depolar izat ion reduced the amplitude and lengthened the r i s e time of the EPSP. Membrane Potential (mV) - 5 5 - 6 5 - 7 5 , l5mV 5 ms 76 In 'four granule c e l l s , a second type of a l l -or -none spike was evoked by PP st imulat ion that was considerably smaller in amplitude than the AP and w i l l be referred to as a small amplitude spike (SAS; F i g . 3 . 8 A, arrow). The SAS was s imi lar to the fast prepotent ia l (FPP) reported by Spencer and Kandel (1961) in the hippocampal pyramidal neurons and may represent e l ec tro ton ic coupling between granule c e l l s (MacVicar and Dudek, 1982). It was in teres t ing to note that in one granule c e l l the neuron discharged on the f a l l i n g phase of the SAS, suggesting that AP generation is not l inked to the presence of SASs (F ig . 3.8 B, arrow). The ef fect of a l t e r i n g the membrane po tent ia l on the i n t r a c e l l u l a r EPSP amplitude was examined in an attempt to determine i t s equi l ibr ium potent ia l (Eepsp^* A s shown in F i g . 3.8, hyperpolar iz ing and depolar iz ing the membrane potent ia l increased and decreased the amplitude of the EPSP, re spec t ive ly . Unfortunately, the E e psp could not be accurately ca lcu la ted from any neuron (n=6) because the EPSP was contaminated by another postsynaptic po tent ia l (note the hyperpolar izat ion fol lowing the EPSP evoked from a membrane potent ia l of -55 mV). Although we were unable to obtain the E e p S p , others have estimated the E e p S p in granule c e l l s to be approximately -5 mV ( C r u n e l l i et a l . , 1984). In addit ion to the EPSP evoked by PP s t imulat ion , granule c e l l s also exhibited a depo lar iz ing a f t erpoten t ia l (DAP) and a late hyperpolar iz ing potent ia l (LHP) (Thalmann and Ayala , 1982). Immediately fol lowing the EPSP, a DAP was 77 Figure 3.10. Perforant path st imulat ion evoked a depo lar iz ing a f t e r p o t e n t i a l (DAP) and a l ong - la s t ing hyperpolar izat ion (LHP) fol lowing the EPSP. Depolar iz ing the membrane potent ia l of the granule c e l l by 10 mV resu l t s in the reversa l of the DAP ( f i l l e d arrow). Conversely, when the membrane potent ia l was hyperpolarized the DAP amplitude increased. In a d d i t i o n , the LHP (open arrow) that followed the DAP increased in amplitude upon membrane depo lar izat ion and reversed to a depolar iz ing potent ia l when the membrane was hyperpolar ized. Note the longer time scale required to observe the LHP. 78 DAP LHP 79 Figure 3.11. Determination of the equi l ibr ium potent ia l s for the DAP (Eaap) and the LHP ( E l n p ) . A l t e r i n g the membrane po tent ia l (abscissa) changed the amplitude (ordinate) and/or p o l a r i t y of the DAP and the LHP. The DAP amplitude ( f i l l e d c i r c l e s ) increased with membrane hyperpolar izat ion and was reversed by membrane depo lar i za t ion . The E^gp was ca lcu la ted for s ix granule c e l l s to be -61 mV. In contras t , LHP amplitudes (open c i r c l e s ) were enhanced by membrane depolar izat ion and became p o s i t i v e . fol lowing membrane hyperpolar izat ions greater than -77 mV ( E ^ p ) . The E ^ a p and E l h p v a l u e s a r e represented on the graph by where the ir respective l ines cross the membrane potent ia l (absc issa) . 80 mV 20 -7 1 5 -10 • / y -50 - 60/ J k i 1 H -90 • -5 -10 Membrane Potential (mV) • DAP o|_HP r = 0.996 r = 0.996 E D A P = " 6 l m V ^ =-77mV 81 observed which lasted 92 +/- 6.9 ms (mean +/- SEM; n = 14). The duration was dependent to a large extent on the amplitude of the preceding EPSP and the RMP of the granule c e l l (F ig . 3.10). The DAP was enhanced or reversed by hyperpolariz ing and depolar iz ing current i n j e c t i o n , respect ive ly . The equi l ibr ium potent ia l for the DAP (E^gp) was determined for six granule c e l l s and the resu l t s are shown in F i g . 3.11. The mean RMP for these c e l l s was -70 mV and the E^ a p exhibited a mean reversa l at -61 mV (or 9 mV pos i t ive to the RMP). Perforant path st imulat ion evoked an LHP with a peak amplitude between 150-200 ms and a duration of 0.4-2 s (F ig . 3.10, LHP; c f . Thalmann and Ayala , 1982). The LHP was always enhanced by membrane depo lar izat ion and could be reversed by membrane hyperpo lar iza t ion . The LHP equi l ibr ium potent ia l f.Elhp) was determined for six granule c e l l s to be -77 mV (or 7 mV negative to the mean RMP; F i g . 3.11). In a d d i t i o n , the changes in res istance associated with the DAP ( R d a p) and LHP (R^p) w e r e measured using a series of current pulses . The I-V p r o f i l e s are shown (A) before, (B) 20 ms, and (C) 400 ms fol lowing subthreshold s t imulat ion of the PP ( F i g . 3.12). Although the rap id ly changing DAP does not allow for an accurate descr ip t ion of R(j ap, i t i s obvious from the reduced voltage def lec t ions ( i . e . , compared to the pre-st imulus value (A)) that a decrease in the membrane resistance occurred during the DAP ( F i g . 3.12 B) . A reduction from R n was also exhibi ted during the LHP, 82 Figure 3.12. The a l t era t ions in membrane resistance associated with the DAP (R^p) and LHP (Rihp)* membrane slope resistance measurements were ca lcu la ted from the voltage amplitudes just pr ior to the termination of the current pulses . The res t ing membrane res istance (R n) was 28 Mohms for th i s p a r t i c u l a r granule c e l l with R,~iap = 23 Mohms and Rihp = 25 Mohms. 83 84 Figure 3.13. The effect of r a i s i n g e x t r a c e l l u l a r potassium from 4.25 to 8.0 mM on the input-output (I-O) r e l a t i o n s h i p of the MPP-evoked EPSP and membrane propert ies (n=3). The increase in [K]o caused a sh i f t in the RMP from -70 mV to -62 mV and was associated with a reduction in R n from 30 Mohms to 18 Mohms (cf . A, B) . The RMP returned to c o n t r o l values fol lowing the return to normal media ( i . e . , -72 mV); however,the R n remained s l i g h t l y lower at 25 Mohms (C) . (A) Stimulation of the perforant path at three d i f f eren t i n t e n s i t i e s ( i . e . , 7, 9, and 11 vo l t s ) evoked a t y p i c a l 1-0 EPSP r e l a t i o n s h i p . (B) The reduction of the RMP during 8 mM potassium resul ted in a depression of the 1-0 curve with lower stimulus i n t e n s i t i e s (4, 5, and 6 vo l t s ) evoking granule c e l l discharge (B). 85 A. B. C. 10 ms RMP(mV) A. Control - 7 0 B. 8 m M K + ( 4 5 m i n ) - 6 2 C. Wash(lhr) - 7 2 I I nA J l O m V R n ( M A ) 3 0 18 2 5 86 however, the change associated with R]_hp (3 Mohms) < R(jap (5 Mohms). Thus, both the DAP and LHP depend on the ac t iva t ion of an underlying conductance. 3.3.4 The effect of ionic subst i tu t ion on the membrane  c h a r a c t e r i s t i c s and perforant path evoked responses of  granule c e l l s Potassium Rais ing the e x t r a c e l l u l a r potassium concentration ( [K] 0 ) from the normal l e v e l of 4.25 mM to 8 mM resul ted in an average reduction of the RMP by 13 +/- 3 mV (mean +/-SEM, n=3) and an 8 +/- 3 Mohm decrease in R n values ( F i g . 3.13 A, B) . These changes amounted to a 16% and a 33% reduction from the ir contro l values , re spec t ive ly . The attenuation in R n may be a t t r ibuted to an increase in conductance associated with AR, since the I-V r e l a t i o n s h i p in the hyperpolar iz ing range was reduced in amplitude. This resu l t would be expected i f the AR observed in response to hyperpolar izat ion was dependent on potassium ions (Constanti and Galvan, 1983; Hagiwara et a l . , 1976). The e f fects of increasing [ K ] 0 were also observed on the PP evoked EPSP. During the perfusion with raised potassium, the same s t imulat ion to the PP e l i c i t e d reduced EPSPs, as shown by the changes in the 1-0 r e l a t i o n s h i p ( F i g . 3.13 A, B) . This a l t e r a t i o n in the 1-0 re la t ionsh ip probably resu l ted from the change in RMP (-75mV to -62mV) associated with the increased [ K ] c . 87 Figure 3.14. The effect of subs t i tu t ing 2mM B a 2 + for C a 2 + on the membrane propert ies of granule c e l l s . Ba* increased the membrane slope resistance from a value of 85 Mohms ( A i , i i ) to 191 Mohms (B i , i i ) . Note that B a 2 + d id not a l t e r the l i n e a r i t y of the current-voltage r e l a t i o n s h i p . The enhanced R n with B a 2 + was associated with a concomitant increase in the membrane time constant ( T c ; A i i i , B i i i ) . The untreated membrane was defined by a s ingle T c (2.5 ms) as shown by the l i n e a r i t y of the ln(dv/dt ) p lot (A i i i ) . However, perfusion with B a 2 + (B i i i ) a l t ered the membrane c h a r a c t e r i s t i c s so that the granule c e l l exhibi ted at least two T c ' s ( T e , 4 ms; T^, 21 ms). Because the T c measurements have been shown to be dependent on the membrane p o t e n t i a l , the T c ' s of the two condit ions were ca lcu la ted from voltage def lect ions with s imi lar amplitudes. 88 89 Table 3.2. The ' e f fec t s of B a 2 + on the membrane propert ies of granule c e l l s (mean (+/- SEM); n = 7). A l l values were obtained fol lowing 30 min. perfusion with B a 2 + . Control B a 2 + (1 mM) RMP (-mV) 74 (3.4) 60 (3.4) R n (Mohms) 33 (5.3) 68 (14.4) T e (ms) 4 (0.5) 6 (1.4) T i (ms) 5 ( 1 . 2 ) 27 (2.8) 90 Bar ium Subst i tut ion of barium ( B a 2 + ; 1 or 2 mM) for calcium in the medium resul ted in a very c h a r a c t e r i s t i c progression of a l t e r a t i o n s in granule c e l l responses (n=7). The i n i t i a l e f fect occurred within minutes of exposure to B a 2 + and consisted of a reduction in the RMP with a concomitant increase in R n and membrane T c in response to hyperpolar iz ing current in jec t ion ( F i g . 3.14; Table 3 .2) . An example of the effect B a 2 + had on the I-V r e l a t i o n s h i p of a granule c e l l is shown in F i g . 3.14. This p a r t i c u l a r granule c e l l exhibi ted a l inear I-V p r o f i l e which i n i t i a l l y gave an R n of 85 Mohms. In the presence of B a 2 + the I-V r e l a t i o n s h i p maintained i t s l i n e a r i t y while the R n more than doubled to 191 Mohms (F ig . 3.14 i , i i ) . Time constant analys i s of amplitude-matched voltage p r o f i l e s for the normal (second hyperpolar iz ing response) and the B a 2 + treated ( f i r s t hyperpolar iz ing response) granule c e l l , showed that the contro l response was f i t t e d by a s ing le exponential g iv ing a T c of 3 ms; whereas, the B a 2 + treated response exhibited at least two T c ' s , an ear ly ( T e = 4 ms) and a late (T^ = 21 ms) component ( F i g . 3.14, i i i ) . The increase in R n and T c may be p a r t i a l l y a t t r i b u t e d to B a 2 + attenuating a potassium ' leak' conductance which is normally ac t ive at the RMP (Constanti and Galvan, 1983; Schwindt and C r i l l , 1980). 91 Figure 3.15. The ef fect of 1 mM B a 2 + on the e x t r a c e l l u l a r and i n t r a c e l l u l a r responses evoked by MPP s t imulat ion . A comparison between the ex tra- and i n t r a c e l l u l a r potent ia l s showed that the number of PS's and AP's increased with longer perfusion of B a 2 + . The increase in the number of AP's appeared to be re lated to the development of a depo lar izat ion which had a peak latency of approximately 50 ms (30 min, see F i g . 3.16). 92 l m M B a 2 + ExtracelL RMP Intracell. Control 20min 30 min —• n A I II hr - 7 5 5ms •65 10 ms •68 ^ I2mV n - 6 8 1 1 1 J l m V 5ms J 2 0 m V 20 ms 93 Figure 3.16. In another granule c e l l s t imulat ion of the MPP in the presence of 2 mM B a 2 + evoked a s ingle act ion potent ia l which was followed by a graded depolar izat ion (arrow). The depo lar i za t ion , on reaching threshold amplitude, r e p e t i t i v e l y discharged the granule c e l l (C). A l l act ion potent ia l s were c l ipped by photography. 94 2 mM Ba ( 25 min ) A. B. C . 20 ms 95 In addi t ion to a l t e r i n g the membrane propert ies of granule c e l l s , B a 2 + a lso changed the PP evoked synaptic c h a r a c t e r i s t i c s . Pr ior to B a 2 + per fus ion , s t imulat ion of the PP evoked the t y p i c a l EPSP-AP-DAP and EPSP-PS sequences obtained with i n t r a c e l l u l a r and e x t r a c e l l u l a r recordings , respect ive ly ( F i g . 3.15). As the perfusion with B a 2 + continued, the e x t r a c e l l u l a r response was character ized by an increasing number of evoked PSs ( F i g . 3.15; c o n t r o l -1 h r . ) . I n t r a c e l l u l a r records taken over the same period showed the development of a depo lar iz ing po tent ia l which peaked at about 50 ms and i n i t i a t e d a burst of AP's ( F i g . 3.15, 30 m i n . ) . This depolar iz ing p o t e n t i a l was probably the resul t of an inward current c a r r i e d by B a 2 + and has been termed 1^  in sp inal motoneurons (Schwindt and C r i l l ; 1980)-. As shown in F i g . 3.16, the depo lar iz ing potent ia l was a graded response and only i n i t i a t e d a burst of APs when i t reached threshold . S i g n i f i c a n t l y , the i n i t i a l EPSP evoked a s ingle AP, whereas the Ba -dependent potent ia l was capable of inducing a burst discharge in the neuron. After 1 h r . perfusion of B a 2 + , PP s t imulat ion immediately evoked r e p e t i t i v e discharges from the granule c e l l which approximately corre la ted with the number of PSs recorded e x t r a c e l l u l a r l y (F ig . 3.15). Following prolonged exposure to B a 2 + , granule c e l l s exhibi ted rhythmical spontaneous burst discharges which are shown at d i f f erent time scales ( F i g . 3.17). The s ingle spontaneous burst (F ig . 3.17 A) i l l u s t r a t e s two features of 96 Figure 3.17, Prolonged exposure to Ba^ caused granule c e l l s to spontaneously and rhythmical ly burst discharge. The period fol lowing the burst response was followed by a post-burst a f terhyperpolar izat ion (PB-AHP) which appeared to determine the in ter -burs t i n t e r v a l . However, hyperpolar iz ing current i n j e c t i o n blocked the spontaneous bursts and uncovered a pers is tent slowly depo lar iz ing p o t e n t i a l (D). ,-J20mV 5s 98 Figure 3.18. The change in membrane resistance associated with the post-burst AHP was determined by i n j e c t i n g hyperpolar iz ing current pulses at d i f f erent times during the AHP. (A) S o l i t a r y burst i s shown that was immediately followed by a long hyperpolar izat ion (PB-AHP, arrow). (B, C) The PB-AHP was associated with a reduction in membrane resistance as indicated by the smaller voltage de f l ec t ions , i . e . , measured from membrane potent ia l to peak nega t iv i ty , produced at the on-set of the PB-AHP (cf. t h i r d and fourth voltage responses in (O). (D) Comparing the PB-AHP amplitude (ordinate) with the ir membrane res istance values (abscissa) showed a l inear c o r r e l a t i o n (r=0.9997). 99 100 the granule c e l l discharge: 1) each succeeding AP within the burst was i n i t i a t e d at progress ive ly hyperpolarized membrane p o t e n t i a l s , ind icat ing that e i ther a depolar iz ing potent ia l was inac t iva t ing or that an underlying hyperpolar izat ion developed during the discharge; and 2) termination of the discharge immediately resul ted in a pronounced AHP. These observations suggest that termination of the spontaneous burst discharge may be the resul t of an act ive i n h i b i t o r y mechanism. The nature of th i s hyperpolar izat ion was invest igated by in jec t ing a hyperpolar iz ing current pulse at varying times along the AHP ( F i g . 3.18 B, C ) . The resu l t s indicated that the amplitude of the AHP was corre la ted with an apparent decrease in membrane resistance ( F i g . 3.18 D) . The rhythmical nature of the spontaneous discharges are i l l u s t r a t e d in (F ig . 3.17 G) . In order to determine whether these discharges were generated by spontaneously occurring EPSPs or by i n t r i n s i c neuronal propert i e s , the ef fect of i n j e c t i n g a long hyperpolar iz ing current pulse on the frequency of spontaneous bursts was observed. As shown in F i g . 3.17 (D), hyperpolar izat ion of the granule c e l l arrested the spontaneous discharges and uncovered a slow d e p o l a r i z a t i o n . This implies that the mechanism subserving burst generation is i n t r i n s i c to the granule c e l l s ince no underlying EPSPs were observed. The slow depo lar iza t ion most l i k e l y represents a maintained inf lux of B a 2 + , which counterbalances the AHP and i n i t i a t e s burst discharges in granule c e l l s (Schwindt and C r i l l , 1982). 101 Figure 3 . 1 9 . Following the spontaneous bursts , continued exposure to B a 2 + resulted in augmented burst discharges (A, arrow) which led to a depolarized res t ing membrane potent ia l (B, f i l l e d arrow). Inject ing a small hyperpolar iz ing current (B, open arrow) returned the membrane potent ia l to i t s pre-burst value; whereas, upon termination of the current i n j e c t i o n , the granule c e l l r e p e t i t i v e l y discharged and returned to the depolarized res t ing membrane p o t e n t i a l . 102 103 Figure 3.20. The ef fect of lowering [Ca]o from 2 to 0.5 mM and increasing [Mg]o to 14 mM on MPP-evoked p o t e n t i a l s . A l t e r i n g the membrane potent ia l showed that the granule c e l l i n i t i a l l y produced a DAP ( f i l l e d arrow) and LHP (open arrow) to MPP s t imulat ion . Following the perfusion of the s l i c e with the experimental medium, the DAP could no longer be inverted by membrane depo lar izat ion and the LHP was replaced by a s l i g h t depolar izat ion at a l l membrane p o t e n t i a l s . 1 04 Control Membrane I.5 hr low Ca Potential (mV) JV. •75 j 5 m V QI s 105 With further exposure to B a 2 + , the granule c e l l s developed prolonged burst discharges ( F i g . 3.20 A, arrow), which led to the establishment of a depo lar iz ing res t ing p o t e n t i a l at -30 mV (F ig . 3.19 B, f i l l e d arrow; Fournier and Crepe l , 1984; Godfraind, 1985). When a granule c e l l was in t h i s depolarized state , i t would return to the o r i g i n a l RMP spontaneously or with hyperpolar iz ing current pulses (F ig . 3.19 B, open arrow; Godfraind, 1985). Termination of the hyperpolar iz ing current in j ec t ion promoted a long burst of APs which got progress ive ly smaller in amplitude u n t i l the membrane potent ia l s t a b i l i z e d at the depolarized l e v e l . Thus, in the presence of B a 2 + , granule c e l l s tend to f l i p between two rest ing po ten t ia l s : one at approximately -65 mV; and another at approximately -30 mV. Calc ium It has been hypothesized that the mechanism underlying the granule c e l l LHP may be a calcium-dependent potassium conductance (gK C a ) (Thalmann and Ayala , 1982). If th i s i s the case, then reducing e x t r a c e l l u l a r calcium ([Ca] Q ) should decrease the LHP. The T y p i c a l i n t r a c e l l u l a r PP evoked responses were exhibited p r i o r to a reduction in [ C a ] 0 from 2 to 0.5 mM and an increase in [Mg]Q to 14 mM ( F i g . 3.20). Following 1.5 hrs . perfusion with the low C a 2 + medium, the stimulus in tens i ty de l ivered to the PP had to be increased three - fo ld to evoke an EPSP of equivalent amplitude as the 106 Figure 3.21. The effect of 10 micromolar (+/-)baclofen on MPP evoked po ten t ia l s . A l t e r i n g the membrane po tent ia l showed that the granule c e l l i n i t i a l l y produced a DAP ( f i l l e d arrow) and LHP (open arrow) to MPP s t imula t ion . Following the perfusion of the s l i c e with the experimental medium, the DAP could no longer be inverted by membrane depolar izat ion and the LHP was e l iminated . 107 Control v-Membrane Baclofen Potential I hc IO pM (mV) - 5 0 - i ^ I ~L_ - 6 0 J V _ . - 7 0 (RMP) 8 0 - 1 108 contro l response. Although the EPSP was present, the DAP was a l t e r e d such that i t could no longer be reversed to an IPSP ( c f . , the DAP responses at -55 mV; f i l l e d arrows) ind icat ing that the d i - or po lysynapt ica l ly GABA-mediated response was p r e f e r e n t i a l l y blocked. The LHP was s i m i l a r l y reduced and replaced by a s l i g h t depolar izat ion (open arrow) suggesting that e i ther the LHP was dependent on C a 2 + or transmitter released from a mult i - synapt ic pathway. Baclofen Perfusion of the GABAB receptor agonist , baclofen (10 micromolar), reduced the perforant path evoked EPSP and resul ted in the loss of the DAP reversa l ( f i l l e d arrow) and the LHP ( F i g . 3.21; open arrow). Although increasing the stimulus in tens i ty evoked an EPSP of comparable amplitude to c o n t r o l , the other potent ia l s were s t i l l absent. The granule c e l l s d id not exhibi t measurable postsynaptic a l t e r a t i o n s in e i ther RMP or R n at th i s concentration of baclofen; therefore , baclofen appeared to exert i t s e f fect on presynaptic elements. The fact that both the DAP and LHP were s i m i l a r l y el iminated by baclofen suggests that a common mechanism was d i srupted . Since the GABA-mediated i n h i b i t i o n is thought to represent the a c t i v a t i o n of interneurons (Lomo, 1971b), i t seems l i k e l y that baclofen in the DG can s e l e c t i v e l y reduce d i s y n a p t i c a l l y mediated neurotransmitter 109 re lease . Thus i t i s poss ible that the LHP is dependent on the release of neurotransmitters from secondary neurons. 3.4. Discussion 3 .4 .1 . Membrane C h a r a c t e r i s t i c s Analys is of the i n t r a c e l l u l a r I-V re la t ionsh ips ra ises the p o s s i b i l i t y that there are two populations of granule c e l l s in the DG: 1) that exhibi ted non-ohmic behavior in response to both depolar iz ing and hyperpolar iz ing current i n j e c t i o n , s imi lar to that observed in hippocampal pyramidal neurons d i sp lay ing AR (Hotson et a l . , 1979); and 2) that displayed a l inear I-V r e l a t i o n s h i p over the range of injected currents (F ig . 3 .1) . The l a t t e r a lso maintained s i g n i f i c a n t l y lower RMP and SA values . Since these neuronal propert ies are part of the c r i t e r i a establ ished to judge the q u a l i t y of i n t r a c e l l u l a r impalements, i t is poss ible that the di f ference in I-V p r o f i l e s was due to var iab le neuronal penetrations (Constanti and Galvan, 1983; Schwindt and C r i l l , 1980). If th i s i s the case, then one would expect larger current leakage around the microelectrode causing a reduction in R n . This was not observed. This comparison may be inappropriate because of the method u t i l i z e d to determine the R n values . That i s , granule c e l l s exh ib i t ing AR would maintain apparently lower R n values since hyperpolar iz ing current i n j e c t i o n act ivated a process which decreased 110 associated voltage changes. Although these i n t r a c e l l u l a r data are i n s u f f i c i e n t to d i f f e r e n t i a t e c e l l u l a r populat ions, e x t r a c e l l u l a r recordings have d is t inguished three c lasses of putat ive granule c e l l s based on the half-widths of the e x t r a c e l l u l a r l y re f l ec ted AP and the number of evoked c e l l discharges (Ranck, 1973; Rose et a l . , 1983). Thus, the p o s s i b i l i t y remains that there are granule c e l l subpopulations, but further ana lys i s i s necessary to determine whether they may be i d e n t i f i e d by the i r current-voltage r e l a t i o n s h i p s . AR has been previous ly described in granule c e l l s (Barnes and McNaughton, 1980; C r u n e l l i et a l . , 1984) and i t was demonstrated in th i s study that the AR exhibi ted in response to hyperpolar iz ing current in jec t ion was enhanced or abolished by r a i s i n g [ K ] Q and subs t i tu t ing B a 2 + for C a 2 + in the perfusate, re spec t ive ly . These data are consistent with voltage-clamp analys i s from the o l fac tory c o r t i c a l neuron (Constanti and Galvan, 1883), s t a r f i s h egg c e l l (Hagiwara et a l . , 1976), and frog muscle (Leech and S t a n f i e l d , 1981) which indicated that AR resu l t s from voltage-dependent changes in the d i r e c t i o n of current flow through a K + -channel ( i . e . , r e c t i f i c a t i o n ) . It i s poss ible that the AR observed in granule c e l l s also depends on the behavior of a K + - channe l , however, voltage-clamp analys is and other pharmacological manipulations ( e . g . , the ef fects of TEA) w i l l be necessary to determine the processes underlying AR. 111 Regardless of the mechanism that subserves AR in granule c e l l s , the processes responsible for the non-l inear response to hyperpolar iz ing current in jec t ion in granule c e l l s appears to be d i s s i m i l a r from those in hippocampal pyramidal neurons. Pyramidal neurons' I-V p r o f i l e s are character ized by a 'sag' in the hyperpolar iz ing e lec trotonic po tent ia l which causes an overshoot of the RMP upon termination of the current in jec t ion ( F i g . 6 . 2 ; H a l l i w e l l and Adams, 1 9 8 2 ; Schwartzkroin, 1 9 7 7 ) . These responses have been a t t r i b u t e d to a B a 2 + - i n s e n s i t i v e potassium current , termed I Q ( H a l l i w e l l and Adams, 1 9 8 2 ) . Since the current -induced e l ec tro ton ic potent ia l s recorded from granule c e l l s decayed to the RMP ( i . e . , there was no overshoot of RMP upon termination of current in jec t ion) and because B a 2 + blocked the hyperpolar izat ion- induced AR, I Q probably does not contribute to the AR observed in these neurons. In addi t ion to i t s ef fect on the granule c e l l AR, B a 2 + produced the formation of a second stable membrane potent ia l approximately 4 0 mV depolarized from RMP (Godfraind, 1 9 8 5 ) . The genesis of th i s complex potent ia l has been shown by voltage-clamp techniques in other preparations to resu l t from two act ions of B a 2 + : 1 ) the reduction of outward K + currents ( e . g . , I ] . e a k ) ; and 2 ) the enhancement of a presumed pers i s tent inward C a 2 + current , termed 1^ by Schwindt and C r i l l ( 1 9 8 0 ) . Thus, the stable depolarized membrane p o t e n t i a l develops when I i e a k < Ij (Schwindt and C r i l l , 1 9 8 0 ) . Godfraind ( 1 9 8 5 ) concluded that an inward current 112 analogous to 1^  operates under phys io log ica l condit ions in granule c e l l s because the K + -channel blocker, tetraethylammonium (TEA), produced the stable depolarized membrane potent ia l and th i s could only occur i f an inward current was present. Moreover by showing that the soma, but not the dendrites , of granule c e l l s could support the stable depolarized membrane potent ia l in the presence of the N a + -channel blocker, tetrodotoxin (TTX), Godfraind (1985) demonstrated the p o s s i b i l i t y that granule c e l l s maintain a d i f f e r e n t i a l d i s t r i b u t i o n of Ca 2 + - channe l s with the dendrites having many fewer than the soma. B a 2 + a lso produced spontaneous rhythmical bursts in granule c e l l s (3.17). A s imi lar response has been observed in hippocampal pyramidal c e l l s where i t was hypothesized that Ba induces these bursts by attenuating a Ca* -dependent K + conductance (gK^g) and enhancing an inward current normally c a r r i e d by C a 2 + (Hotson and Pr ince , 1981). Although these same mechanisms may subserve granule c e l l burst generation, one s i g n i f i c a n t dif ference i s observed between the spontaneous bursts e l i c i t e d by these d i f f erent neuronal types. That i s , there i s a reduction of the post-burst a f terhyperpo lar iza t ion (PB-AHP) in pyramidal c e l l s (Hotson and Pr ince , 1980; 1981) but not in granule c e l l s (F ig . 3.17). The PB-AHP in granule c e l l s was associated with a conductance change and appeared to contro l the in ter -burs t i n t e r v a l ( F i g . 3.18). However, membrane hyperpolar izat ion uncovered a slowly depolar iz ing po tent ia l which most l i k e l y 113 contributed the p r i n c i p a l impetus for burst generation, as well as p a r t i a l l y determining the in ter -burs t i n t e r v a l . Furthermore, the fact that hyperpolar iz ing current i n j e c t i o n blocked burst discharges without uncovering spontaneously act ive EPSPs (F ig . 3.17 D), rules out the p o s s i b i l i t y that synaptic events i n i t i a t e granule c e l l burst ing behavior (Johnston and Brown, 1981). These data indicate that the spontaneous burst ing of granule c e l l s induced by B a 2 + depends on i n t r i n s i c membrane propert ies and that the processes contr ibut ing to th i s burst ing may be d i f f eren t between granule and pyramidal c e l l s . On the other hand, i t i s poss ible that s imi lar mechanisms are responsible for the burst ing ( e . g . , an augmentation of an inward current ; Schwartzkroin and Wyler 1979) and that d i s s i m i l a r i t i e s concerning B a 2 + e f fect on the PB-AHP are re lated to the contro l of in ter -burs t in terva l s and not to the burst mechanism per se. 3 .4 .2. Synaptic potent ia l s As previously shown, i n t r a c e l l u l a r records of subthreshold PP st imulat ion c h a r a c t e r i s t i c a l l y evoked a monosynaptic EPSP in granule c e l l s (Fournier and Crepe l , 1984; Fr i cke and Pr ince , 1984; Lomo, 1971a; Thalmann and Ayala , 1982). Hyperpolar iz ing or depo lar iz ing the membrane potent ia l enhanced or reduced the change in voltage associated with the EPSP, respect ive ly ( F i g . 3 .8) . This 1 14 response was expected from the work in the neuromuscular junc t ion , which demonstrated that the EPP amplitude d i r e c t l y var ied with the di f ference between the membrane potent ia l and the E epp ( F a t t and Katz, 1951). The E e p S p value could not be determined in granule c e l l s due to contaminating responses, though others have shown the PP-evoked EPSP to have an equi l ibr ium potent ia l of -6 mV ( C r u n e l l i et a l . , 1984). In agreement with McNaughton et a l . (1981), r e l a t i v e l y large EPSPs ( greater than 29 mV) were necessary to evoke an AP. Synaptic a c t i v a t i o n only evoked a s o l i t a r y AP in granule c e l l s (F ig . 3.7) unless i n t r a c e l l u l a r electrodes were f i l l e d with KC1, in which - case granule c e l l s discharged r e p e t i t i v e l y (Ol iver , unpublished observation; c f . , Deadwyler et a l . , 1975; Dudek et a l . , 1976; Fournier and C r e p e l , 1984). This effect of KC1 resul t s from a l t e r i n g the EQT_ to a more pos i t ive p o t e n t i a l , such that a c t i v a t i o n of the GABA-mediated C l ~ conductance w i l l now depolarize the granule c e l l above AP threshold causing mult ip le discharges (Eccles et a l . , 1977). The present study showed that granule c e l l EPSPs were followed by DAPs (Fournier and Crepe l , 1984; Misgeld et a l . , 1982; Thalmann and Ayala , 1982). The DAP appeared to be a depolariz ing-IPSP s imi lar to that found in o l fac tory c o r t i c a l neurons because i t s equi l ibr ium potent ia l was below AP threshold and acted to i n h i b i t neuronal discharge (Schof ie ld , 1978). The DAP was evoked with subthreshold PP 115 st imulat ion suggesting i t s a c t i v a t i o n v ia feed-forward processes (Buzsaki and Czeh, 1981; Douglas et a l . , 1983). Antidromic st imulat ion induced a s imi lar potent ia l (D-IPSP) ind ica t ing that i t may a lso be evoked in a recurrent or feed-back manner (Lomo, 1971b). Although DAPs were prominent features in the granule c e l l s from which we recorded, other studies have shown granule c e l l s exh ib i t ing an IPSP in response to PP st imulat ion (Andersen et a l . , 1966a; Fr icke and Pr ince , 1984; Lomo, 1971 a ) . S i g n i f i c a n t l y , both potent ia l s resu l t predominantly from a GABA-mediated increase in C l ~ conductance, since they were a l tered by reducing [ C 1 ] G and perfusing GABA antagonists (Thalmann and Ayala , 1982; Fr icke and Pr ince , 1984). Assuming that a l l recordings were obtained from granule c e l l s , the di f ference in the p o l a r i t y of these a f t erpotent ia l s must r e f l e c t some d i s s i m i l a r i t i e s in experimental methods. One p o s s i b i l i t y i s that parameters which effect EQI ( e . g . , [C1'] Q or i n t r a c e l l u l a r electrode e l e c t r o l y t i c so lut ions; Eccles et a l . , 1977), were var iable between studies; however, a comparison d id not uncover s i g n i f i c a n t d i f f erences . For the most p a r t , the contrast ing data regarding the PP-evoked DAP and IPSP may be explained by d i f ferences in granule c e l l RMP values between the various s tudies . For example, Fr i cke and Prince (1984) included granule c e l l s with RMP values greater than -50 mV in the i r inves t iga t ion , whereas the present study reported an average RMP greater than -70 mV. Given a E^gp value of 1 16 -61 mV (F ig . 3.11), granule c e l l s recorded by Fr icke and Prince (1984) would d isp lay an IPSP; however, based on the e l e c t r o p h y s i o l o g i c a l c r i t e r i a es tabl i shed for determining the qua l i ty of i n t r a c e l l u l a r impalement, the studies report ing a DAP must be considered the more p h y s i o l o g i c a l . Even though v a r i a b i l i t y in RMP values appears to be a v a l i d explanation for recording' e i ther a DAP or IPSP in response to PP s t imulat ion , Thalmann and Ayala (1982) recorded from a putative granule c e l l which exhibited a DAP and an IPSP in response to PP-st imulat ion and MF ac t iva t ion evoked from CA3, respec t ive ly . The IPSP maintained an E j p S p at -75 mV and was blocked by the GABA antagonist , p i c r o t o x i n , ind ica t ing that i t , too, i s a GABA-mediated C l ~ conductance. In contrast to th i s IPSP, MF a c t i v a t i o n from the h i lu s evoked a depolariz ing-IPSP (D-IPSP) which had an E d - i p s p approximately equal to E dap a t ~6 1 m V « T ^ e discrepancy between MF evoked responses may be re lated to the use of d i f f eren t s t imulat ion s i t e s ( i . e . , h i l a r vs. CA3). For example, i t i s poss ible that the hi lar-evoked D-IPSP was contaminated with an EPSP e l i c i t e d from the h i l a r polymorphic c e l l projec t ion that makes synaptic contacts with the proximal one- th ird of the granule c e l l dendrites (Laurberg and Sorensen, 1981; Swanson et a l . , 1978). Several re su l t s argue against th i s i n t e r p r e t a t i o n : 1) e x t r a c e l l u l a r p o t e n t i a l s , which were recorded simultaneously with the i n t r a c e l l u l a r D-IPSP, d id not indicate the presence of an EPSP; 2) the D-IPSP's latency to on-set indicated a 1 17 monosynaptic p o t e n t i a l ; 3) reversa l of the h i l a r act ivated D-IPSP indicated only the s ingle potent ia l at th i s latency; and 4) iontophoresis of GABA at the soma and/or dendrites of granule c e l l s produces a depolar izat ion and a mean E g a ^ a 17 mV pos i t i ve to the RMP (Assaf et a l . , 1981). These data suggest that ac t iva t ion of recurrent GABAergic interneurons depolarize granule c e l l s . Unfortunately, the present inves t igat ion did not assess the ef fect of GABA antagonists on the D-IPSP; thus, i t is poss ib le that th i s response is not GABA-mediated. Another explanation for the difference between the MF evoked GABA-mediated responses may be that the neuron which exhibited the IPSP was not a granule c e l l ; th i s seems un l ike ly because PP st imulat ion e l i c i t e d the c h a r a c t e r i s t i c DAP and LHP t y p i c a l of granule c e l l s (see F i g . 1 C of Thalmann and Ayala , 1982). Since the IPSP was antagonized by p i c r o t o x i n , i t is a lso improbable that the IPSP was due to an increase in K + permeabil i ty r e s u l t i n g from the ac t iva t ion of the GABA B -receptor (Gahwiler and Brown, 1985). Perhaps the best explanation for the contrast in the two ant idromica l ly evoked potent ia l s l i e s in the amount of GABA released by the d i f f eren t s t imulat ion s i t e s . That i s , hippocampal pyramidal neurons exhib i t both hyperpolar iz ing and depo lar iz ing potent ia l s in response to GABA that are predominantly due to an increase in Cl~-conductance (Alger and N i c o l l , 1982b; Andersen et a l . , 1980; Thalmann et a l . , 1981), though the depolar iz ing response e l i c i t e d by GABA 118 must also involve a cat ion (Alger and N i c o l l , 1982b). It i s bel ieved that the hyperpolar iz ing and depolar iz ing responses are coupled to e i ther a synaptic or extrasynaptic GABA-receptor, respect ive ly (Alger and N i c o l l , 1982b). Thus, given the neural c i r c u i t r y of the dentate, s t imulat ion of the PP or the h i lu s may act ivate s i g n i f i c a n t l y more GABAergic interneurons than MF ac t iva t ion v i a CA3. If t h i s i s the case, then the former would release a greater amount of GABA thereby increasing the chances of binding to the extrasynaptic GABA-receptor and evoking a D-IPSP ( c f . , Alger and N i c o l l , 1982b). In contras t , ac t iva t ion of the MFs from CA3 excites r e l a t i v e l y few interneurons which leads to the ac t iva t ion of mostly synaptic GABA A -receptors and an IPSP. This hypothesis can be tested by observing the i n t r a c e l l u l a r response to the app l i ca t ion of the GABA analogue 4 , 5 , 6 , 7 , -tetrahydroisoxazolo [5 ,4 , -c] p y r i d i n e - 3 - o l (THIP), which has been shown to p r e f e r e n t i a l l y bind synaptic GABA A -receptors in hippocampal pyramidal neurons (Alger and N i c o l l , 1982b). Thus, i f a hyperpolar izat ion of the granule c e l l s occurs in response to THIP, then i t i s l i k e l y that granule c e l l s maintain two GABA-receptor-ionophore complexes, a synaptic and an extrasynaptic which induce hyperpolar iz ing and depolar iz ing responses, r e spec t ive ly . In addi t ion to the Cl~-dependent component of the DAP, the p o s s i b i l i t y that C a 2 + contr ibutes to th i s potent ia l was indicated by the synapt i ca l ly evoked depolar iz ing po tent ia l which developed during the perfusion of B a 2 + ( F i g . 3.17). 119 Although the development of a B a 2 + po tent ia l i s not proof that under phys io log i ca l condit ions a C a 2 + - c u r r e n t d c a ^ e x i s t s , since B a 2 + passes more read i ly than C a 2 + through C a 2 + - c h a n n e l s (Schwindt and C r i l l , 1980). However, i f a I C a i s normally present in granule c e l l s , i t must be r e l a t i v e l y small because in normal ACSF the granule c e l l s do not exhib i t burst ing even with the a id of an apparent depolar iz ing-IPSP. Furthermore, the fact that a DAP-l ike po ten t ia l was maintained in a low [ C a ] 0 , high [Mg] 0 medium suggests the possible involvement of Na + in the DAP. The contr ibut ion these ions have in generating the DAP w i l l have to be assessed. St imulation of e i ther the PP or h i l u s a lso evoked a LHP in granule c e l l s which followed the DAP or D-IPSP. The LHP did not appear to be coupled to the EPSP, since subthreshold h i l a r st imulat ion which did not induce a s i g n i f i c a n t EPSP c h a r a c t e r i s t i c a l l y evoked the LHP ( F i g . 3.6; Thalmann and Ayala , 1982). Furthermore the LHP has been shown to be: 1) dependent on [ K ] Q ; 2) independent of GABA-mediated C l ~ conductances; and 3) to have an E ^ p close to the E K (F ig . 3.11; Thalmann and Aya la , 1982). The d i s p a r i t y in E ^ p values between the present study (-77 mV) and Thalmann and Ayala 's (-90 mV) probably resulted from the d i f f erent [ K ] 0 and the varying degrees of DAP contamination. That i s , normal [ K ] Q d i f f e r e d by 0.75 mM between the s tudies . Using the Nernst equation and an i n t r a c e l l u l a r K + concentration of 130 mM (Schof ie ld , 1978), the d i f ference in K + concentration 120 accounts for approximately a 5 mV sh i f t in E ^ p . Moreover the LHP apparently i s p a r t i a l l y contaminated With a GABA-mediated C l ~ conductance, since perfusion with p i cro tox in enhances the LHP amplitude (Thalmann and Ayala , 1982). Thus, i f the LHP recorded in the present study was more contaminated by the processes subserving the DAP, then a more pos i t i ve E i h p would be ca lcu la ted due to the depo lar iz ing influence of the C l ~ conductance. S imi lar ef fects of GABA antagonists and a l tered [ K ] Q on the LHP of hippocampal pyramidal neurons a lso have been observed which indicates that an increase in gK, and not g C l , underl ies the LHP (Alger,1984; Newberry and N i c o l l , 1984b; Thalmann, 1984). The granule c e l l LHP probably does not depend on a voltage-dependent gK, since i t may be evoked by subthreshold st imulat ion which does not produce an EPSP ( F i g . 3.6; Thalmann and Ayala , 1982). Furthermore the DAP and the LHP were more sens i t ive than the monosynaptic EPSP to a lowering of [Ca] Q and a r a i s i n g of [Mg]Q in the perfusate ( F i g . 3.21), suggesting that the DAP and LHP are generated by d i -or polysynaptic c i r c u i t r i e s (Alger, 1984; Newberry and N i c o l l , 1984a). However, s imi lar ionic manipulations a lso decrease the Q^ca * n hippocampal pyramidal neurons (Hotson and Pr ince , 1980; Wong and Pr ince , 1981). Thus, the data are consistent with the LHP being e i ther a 9KCa o r a polysynapt ical ly-mediated p o t e n t i a l (Thalmann and Aya la , 1982). 121 The LHP in hippocampal pyramidal neurons i s bel ieved to be a neurotransmitter-mediated increase in gK (Alger , 1984; Kehl and McLennan, 1983; Knowles et a l . , 1984; Lancaster and Wheal, 1984; Newberry and N i c o l l , 1984b; Thalmann, 1984). Although the neurotransmitter has not been i d e n t i f i e d several reports indicate that GABA, act ing on the B-p-chlorophenyl-GABA (baclofen)-sens i t ive GABAg receptor complex, act ivates a gK with s imi lar propert ies to those of the LHP (Gahwiler and Brown, 1985; Misgeld et a l . , 1982; Newberry and N i c o l l , 1984a). The perfusion of granule c e l l s with low concentrations of baclofen (10~^ M) reduced the EPSP, DAP and LHP evoked by PP s t imulat ion , without causing a hyperpolar izat ion of the membrane potent ia l or a change in R n ( F i g . 3.22; c f . , Ault and Nadler, 1983a; Lanthorn and Cotman, 1981). This effect of baclofen on granule c e l l s may be explained by a reduction in the presynaptic release of neurotransmitters (Ault and Nadler, 1983a; Davidoff and Sears, 1974; Lanthorn and Cotman, 1981). S i g n i f i c a n t l y , the EPSP could s t i l l be evoked in the presence of baclofen by increasing the stimulus in tens i ty de l ivered to the PP, even though the DAP and LHP remained abolished ( F i g . 3.22). These data suggest that baclofen decreases the release of GABA and poss ib ly other neurotransmitters from the DG interneurons which attenuates the DAP and LHP responses. Baclofen has a lso been shown to decrease GABA-mediated i n h i b i t i o n in the CA1 area when measured by paired pulse s t imulat ion (Ault and Nadler, 1983b). 1 22 Although the loss of the LHP in response to reduced [Ca] Q and elevated [Mg]Q^ can poss ibly be explained on the e l iminat ion of a gKfja' * s m o r e d i f f i c u l t to assign th i s act ion to baclofen since C a 2 + in f lux into pre- or postsynaptic elements i s unaffected by baclofen at concentrations less than 50 micromolar (Heinemann et a l . , 1984). Because the act ion of baclofen is putat ive ly mediated by synaptic receptors , i t seems un l ike ly that the LHP is a gK coupled to the release of i n t r a c e l l u l a r C a 2 + , as i s the case for sympathetic ganglion c e l l s (Kuba, 1980). Therefore, the data indicate that the LHP, l i k e the DAP, i s due to e i ther d i - or polysynaptic release of a neurotransmitter. The hyperpolar izat ion that baclofen produces in granule c e l l s i s very s l i g h t when compared to i t s e f fect in hippocampal pyramidal neurons (Misgeld et a l . , 1982). Hence, i t seems un l ike ly that GABA, act ing on the GABAg-receptor, induces the granule c e l l LHP as proposed for hippocampal pyramidal neurons (Gahwiler and Brown, 1985; Newberry and N i c o l l , 1984b). Although GABA may not induce the LHP, i t is poss ible that GABAergic interneurons release other neurotransmitters that are responsible for the LHP. In th i s respect , neuropeptides have been c o - l o c a l i z e d to the GABAergic interneurons (Kosaka et a l . , 1985) and i f they are released may account for the LHP. In conc lus ion , there may be two populations of granule c e l l s d i s t inguished by t h e i r e l e c t r o p h y s i o l o g i c a l responses to current i n j e c t i o n . Although the funct ional d i f ferences 123 for granule c e l l s maintaining a l inear versus a non-l inear I-V r e l a t i o n s h i p were not invest igated , the AR observed in response to depolar iz ing current pulses should enhance both the s p a t i a l and temporal summation propert ies of these neurons. Poss ibly th i s neuron may require add i t iona l synaptic inputs , at spec i f i c frequencies, to i n i t i a t e a granule c e l l AP. Furthermore PP or h i l a r s t imulat ion evoked depolar iz ing IPSPs which correspond in latency to on-set and duration of the putative GABA-mediated i n h i b i t i o n . Following the GABA-induced depolar izat ions a LHP was observed which may resul t from the a c t i v a t i o n of d i - or polysynaptic mechanisms. Although the mechanisms of the GABA-mediated depolar izat ions and LHP are unknown, i t i s c l ear that these potent ia l s may exert profound influence on granule c e l l behavior. In p a r t i c u l a r , the depolar izat ion associated with the DAP should have profound ef fects on the temporal and the spa t ia l summation propert ies of granule c e l l s . Thus, understanding the consequences of these a f t e r p o t e n t i a l s , in terms of their c e l l u l a r funct ion , w i l l be explored in the fol lowing chapter using the paired-pulse paradigm. 124 Chapter 4 Postsynaptic processes contribute to the e x c i t a b i l i t y changes exhibi ted by dentate gyrus in response to paired pulse st imulat ion 4.1. Introduction The c e l l u l a r substrates of l earning and memory are bel ieved to l i e in the regions of neuronal contact — the synapses. This hypothesis of learning and memory presumes that p r i o r synaptic a c t i v a t i o n induces temporary or permanent a l t e r a t i o n s in synaptic e f f i cacy which underl ies the formation of memories. The changes in synaptic strength may resul t from a c t i v i t y in a s ingle pathway (homosynaptic) or depend on the a c t i v i t y of d i f f erent pathways (heterosynaptic) . Perhaps the c l eares t example of how learning or memory may occur v ia modulation of synaptic function i s demonstrated by the s i p h o n - g i l l withdrawal ref lex of the mollusc, A p l y s i a . The s i p h o n - g i l l withdrawal re f l ex , an adapted behavior which contracts the g i l l in order to prevent damage, i s great ly enhanced fol lowing a noxious stimulus appl ied to the siphon of A p l y s i a . This s e n s i t i z a t i o n of the ref lex las t s 1 25 for hours (Pinsker et a l . , 1973) and resu l t s from presynaptic f a c i l i t a t i o n of transmitter release (Cas te l lucc i and Kandel, 1976). The enhancement of transmitter release i s complex and has both homo- and heterosynaptic components. St imulation of the head i s bel ieved to evoke a f a c i l i t a t o r y interneuron which synapses with the presynaptic terminals of the sensory neurons. The sensory neurons make synaptic contacts with the motor c e l l s responsible for the g i l l r e f l e x . Kle in et a l . (1980) proposed that the f a c i l i t a t o r y neuron ( L 2 9 ) releases a neurotransmitter, putat ive ly serotonin, onto the terminals of the sensory neuron a c t i v a t i n g adenylate cyc lase . The induction of adenylate cyclase increases cAMP leve l s (Bernier et a l . , 1982) which, in turn , causes a cAMP-dependent prote in kinase to phosphorylate K + -channe l s . The phosphorylation inact ivates the K + -channel which resu l t s in the prolongation of the sensory neuron's terminal act ion potent ia l and enhances C a 2 + i n f l u x . The augmented C a 2 + inf lux increases transmitter release to the motor neurons c o n t r o l l i n g the g i l l ref lex and produces the f a c i l i t a t i o n . Though the s i p h o n - g i l l withdrawal ref lex i s a simple form of learning in an invertebrate , the enhancement of transmitter release subserving th i s phenomenon has been observed in vertebrate per iphera l and centra l neurons. For example, a s ingle stimulus (conditioning) de l ivered to the neuromuscular junction enhances or depresses the end-plate p o t e n t i a l evoked by a second pulse (test) depending on the 1 26 i n t e r v a l between the successive pulses (for a review see E c c l e s , 1964). This increase and decrease in the end-plate po tent ia l (EPP) has been termed paired-pulse f a c i l i t a t i o n and depression, respec t ive ly . Furthermore, fol lowing repe t i t i ve s t imulat ion the EPP remains enhanced for several minutes. These phenomena appear to resu l t from a homosynaptic increase in the presynaptic release of neurotransmitter (del C a s t i l l o and Katz , 1954). The enhanced release is Ca 2 + -dependent and may occur from res idual C a 2 + binding to a prote in (X) in the presynaptic terminal (Katz and M i l e d i , 1968; Rahamimoff, 1968). This res idual CaX hypothesis i s supported by the fact that increasing external C a 2 + augments the EPP amplitude (Katz and M i l e d i , 1967) and decreases paired-pulse f a c i l i t a t i o n , presumably due to the deplet ion of transmitter (Katz and M i l e d i , 1968; Rahamimoff, 1968). A l t e r a t i o n s in neuromuscular synaptic e f f icacy appear to resu l t from pathway spec i f i c changes in the presynaptic release of neurotransmitter. However, neuronal p l a s t i c i t y in cer ta in CNS structures has been shown to depend on postsynaptic processes. Repet i t ive a c t i v a t i o n of the dentate gyrus perforant path-granule c e l l synapse resu l t s in both short-term and long-term changes in synaptic e f f i c a c y . The short-term a l t e r a t i o n s in e x t r a c e l l u l a r EPSP amplitude are apparently due to modulation of neurotransmitter release analogous to that found at the neuromuscular junct ion . That i s , paired pulse s t imulat ion of the perforant path (PP) 1 27 evokes a f a c i l i t a t i o n of the EPSP at condi t ion- tes t (C-T) in terva l s less than 0.1 s which is superimposed on a much longer period (less than 8 s) of transmitter deplet ion (Alger and T e y l e r , 1976; Lomo, 1971b; McNaughton 1980, 1982; Racine and Milgram, 1983; White et a l . , 1979). A unique feature of the perforant path-granule c e l l synapse i s the f a c i l i t a t i o n and depression of the EPSP which accompanies st imulat ion of the l a t e r a l (LPP) or medial^ (MPP) perforant path, respect ive ly (McNaughton, 1980; McNaughton and Barnes, 1977). Experimental manipulations that reduce the quantity of transmitter released on the f i r s t pulse can a l t e r the MPP to the extent that i t exhib i t s potent iat ion (McNaughton, 1980). Therefore, the di f ferences in the pathways may resul t from the amount of transmitter released by the i n i t i a l st imulus. Although stimulus-induced a l t era t ions in the perforant path-evoked EPSP may be accounted for by changes in the presynaptic release of transmit ter , they cannot explain changes in granule c e l l discharge. Paired pulse st imulat ion of the MPP is associated with a depressed EPSP at the same C-T in t erva l s that increase the number of granule c e l l s that f i r e , as measured by changes in the population spike (PS) (McNaughton and Barnes, 1977). These data, together with the fact that an antidromic condi t ioning pulse (Lomo, 1971b) or heterosynaptic s t imulat ion (Assaf and M i l l e r , 1981; Douglas et a l . , 1983) may lead to the potent iat ion of the PS without any s i g n i f i c a n t change in the EPSP, suggest that some 128 aspects of the short-term a l t e r a t i o n s in the dentate gyrus are not a t t r ibutab le to modulation of presynaptic events. In the previous chapter, i t was shown that PP s t imulat ion evokes both a depolar iz ing a f t e r p o t e n t i a l (DAP) and a la te hyperpolar izat ion (LHP) in granule c e l l s (Thalmann and Ayala , 1982). Since the DAP and the LHP are associated with changes in membrane conductance, i t i s poss ible that the uncoupling of the EPSP and PS is p a r t i a l l y dependent on these a f t e r p o t e n t i a l s . Thus, the object of the present study was to corre la te the i n t r a c e l l u l a r events in the granule c e l l s with the e x t r a c e l l u l a r p o t e n t i a l s , in response to paired pulse s t imulat ion of the PP. The ef fects of pa ired pulse s t imulat ion of the LPP were compared to the MPP in order to determine whether the di f ferences observed in the e x t r a c e l l u l a r responses have s imi lar i n t r a c e l l u l a r • c o r r e l a t e s . F i n a l l y , a ser ies of experiments using antidromic st imulat ion and i n t r a c e l l u l a r current in j ec t ion was undertaken to assess the poss ible role that postsynaptic mechanisms may play in the uncoupling of the EPSP and the somatic processes c o n t r o l l i n g granule c e l l discharge. 4.2. Methods The methods employed in th i s chapter are e s s e n t i a l l y the same as those discussed in Chapter 2. 129 Figure 4.1. Typica l e x t r a c e l l u l a r EPSPs and population spikes (PS) recorded from the middle molecular layer (MML) (A) and granule c e l l layer (GCL; B,C) in response to paired st imulat ion of the MPP at 20, 80, and 400 ms C-T i n t e r v a l s . The EPSPs (A,B) were depressed at a l l C-T i n t e r v a l s , whereas the PS (C) was inh ib i t ed at 20 and 400 ms and potentiated at an 80 ms C-T i n t e r v a l . The MML and GCL EPSPs were recorded simultaneously and the dashed l ines represent the amplitude of the condi t ioning EPSP. 130 Medial Cond. 20 80 400 C-T Interval (ms) 131 Figure 4.2. Histograms d i sp lay ing the mean percent change of the test EPSP and PS recorded from six s l i c e s in response to MPP s t imulat ion . E x t r a c e l l u l a r EPSPs recorded from (A) the middle molecular layer (MML) and (B) the granule c e l l layer (GCL) were reduced at a l l condi t ion- tes t i n t e r v a l s . The population spike (C) was inh ib i t ed at 20 ms and between 0.2-8 s and potentiated at C-T in terva l s from 40-100 ms . In th i s and a l l subsequent f igures , EPSP values were obtained using subthreshold s t imulat ion . A l l SEM values were within seven percent of the means and the l i n e indicates 100% of condi t ioning response. 1 32 Medial deno 8 "* a. «• • M l III! EPSP,. 1 Pop. Sp o-^ oJ** o.o* o°* o-\° 0 * ° 0 ? ° 0 > ° z & v°°l°° l°°»P° COND - T E S T I N T E R V A L (•) 133 4.3. Results 4 .3 .1 . I n t r a c e l l u l a r corre la tes of paired pulse s t imulat ion  in the l a t e r a l , and medial perforant paths E x t r a c e l l u l a r EPSP responses, evoked by s t imulat ion of the MPP and recorded from ei ther the middle molecular layer (MML) or the granule c e l l layer (GCL), were i n h i b i t e d by a condit ioning pulse at C-T in terva l s between 20ms - 8s ( F i g . ' s 4 .1, 4.2; A, B) . One can observe that the test EPSP was reduced at the dendr i t i c recording s i t e and that a p a r a l l e l i n h i b i t i o n was recorded at the GCL. Note that the EPSP was least inh ib i t ed at C-T in terva l s between 40 - 100ms (F ig . 4.2 A , B ) . When the stimulus in tens i ty was adjusted to evoke a PS, th i s response exhibited the c h a r a c t e r i s t i c i n h i b i t i o n - potent iat ion - i n h i b i t i o n sequence at C-T in terva l s of 20, 80, and 400 ms, respect ive ly ( F i g . ' s 4.1, 4.2; C ) . Uncoupling of the EPSP and PS was indicated by the fact that the PS exhibited potent iat ion during C-T in terva l s (40 - 100 ms) associated with depression of the EPSP. In contrast to MPP s t imulat ion , paired s t imul i de l ivered to the LPP resul ted in e i ther potent iat ion or i n h i b i t i o n of the EPSP when recorded from the outer molecular layer (OML) and GCL, respect ive ly ( F i g . ' s 4.3, 4.4; A , B ) . The attenuation of the test EPSP as i t propagated from the dendr i t i c to the somatic region indicated that postsynaptic processes were modifying the EPSP recorded at the GCL. Thus, the 1 34 Figure 4,3. T y p i c a l e x t r a c e l l u l a r EPSPs evoked by paired st imulat ion of the l a t e r a l perforant path (LPP). (A) The EPSP recorded from the outer molecular layer (OML) potentiated at a l l C-T in terva l s shown. (B) The EPSP recorded from the granule c e l l layer (GCL) was i n h i b i t e d at a l l C-T i n t e r v a l s . Recordings from the OML and GCL were taken simultaneously and^the dashed l ines indicate the amplitude of the condit ioning EPSP. 135 C-T Interval (ms) 1 36 Figure 4.4. Histograms d i sp lay ing the mean percent change of the test EPSP recorded from six s l i c e s in response to l a t e r a l perforant path (LPP) s t imulat ion . E x t r a c e l l u l a r EPSPs recorded from (A) the outer molecular layer (OML) were potentiated at condi t ion- tes t (C-T) in terva l s less than 2s. (B) The EPSPs recorded from the granule c e l l layer (GCL) were reduced at a l l condi t ion- tes t i n t e r v a l s . EPSP values were obtained using subthreshold s t imulat ion . A l l SEM values were within seven percent of the means and the l i n e indicates 100% of condit ioning response. 1 37 dend 0 p i 0 p " 0 t f > O J P 0jf» O 3 P 0>« 0jP v p 0 K & p^o c o n d . - t e s t i n t e r v a l ( » ) a a D a. 1 " EPSP soma o.^oP f c o<*oP* 0>° 0*° 0^0>° cond.-test i n t e r v a l (a) 138 i n t r a c e l l u l a r corre la tes of the EPSP depression and EPSP -PS uncoupling recorded at the GCL were invest igated . As shown in F i g . 4.5, s t imulat ion of e i ther the LPP or the MPP evoked an EPSP followed by a DAP ( f i l l e d arrow) and LHP (open arrow) with the MPP producing larger amplitude potent ia l s in response to i d e n t i c a l stimulus parameters. These data i l l u s t r a t e that at short (less than 100 ms) and long ( greater than 150 ms) C-T in terva l s test EPSPs were evoked during a DAP or LHP, respect ive ly ( F i g . 4.5, arrowheads). EPSPs evoked at C-T in terva l s during the DAP and LHP were recorded extra- and i n t r a c e l l u l a r l y . C h a r a c t e r i s t i c e x t r a c e l l u l a r dendr i t i c potent ia l s were observed following s t imulat ion of MPP and LPP ( F i g . 4.6 C, D) . The EPSP recorded i n t r a c e l l u l a r l y from a c t i v a t i o n of both pathways was potentiated at C-T in terva l s less than 80 ms and i n h i b i t e d at 400 ms ( F i g . ' s 4.6, 4.7 A, B) . Augmentation of the EPSP appeared to depend on summation with the DAP, since the change in voltage produced by the test EPSP ( V 2 ) was s i g n i f i c a n t l y reduced with reference to the condit ioning EPSP (V, ) ( F i g . 4.8 A , B ) . Current- induced depolar izat ions equivalent to the DAP, however, have been shown to reduce the PP evoked EPSP (see Chapter 3; F i g . 3 .8) . Thus, i t i s poss ible that the DAP masks the potent ia t ing ef fects of paired pulse s t imulat ion . In support of th i s i n t e r p r e t a t i o n , paired MPP st imulat ion of granule c e l l s which did not exhibi t a DAP resulted in 1 39 Figure 4.5. T y p i c a l i n t r a c e l l u l a r records obtained from a granule c e l l at d i f f erent membrane potent ia l s in response to l a t e r a l perforant path (LPP) and medial perforant path (MPP) s t imula t ion . These records were obtained from the same granule c e l l and the potent ia l s were evoked using the same st imulat ing electrode and st imulat ion i n t e n s i t y . When the granule c e l l was at res t ing membrane p o t e n t i a l (RMP) st imulat ion of e i ther the LPP or MPP evoked an EPSP, DAP and LHP (small arrows from l e f t to r i g h t , r e s p e c t i v e l y ) . Inject ing depolar iz ing current into the granule c e l l (-57) reversed the DAP ( f i l l e d arrow) and enhanced the amplitude of the LHP (open arrow). Inject ing hyperpolar iz ing current (-77, -87) increased the amplitude of the DAP and reversed the LHP. Note that the amplitude of the a f t erpotent ia l s were greater when act ivated by the MPP. 140 141 Figure 4.6. Typ ica l i n t r a c e l l u l a r (upper) and e x t r a c e l l u l a r (lower) records obtained from paired st imulat ion of the LPP (A, C) and the MPP (B, D) at C-T in terva l s of 20, 80, and 400 ms. The e x t r a c e l l u l a r and i n t r a c e l l u l a r records were recorded simultaneously with the e x t r a c e l l u l a r EPSPs being recorded from the OML (C) and MML (D). Note that s t imulat ion of e i ther MPP or LPP evoked i n t r a c e l l u l a r test EPSPs that were f a c i l i t a t e d at a C-T i n t e r v a l of 20ms. I n t r a c e l l u l a r test EPSPs evoked at 80 or 400 ms C-T in terva l s were s l i g h t l y a l tered and i n h i b i t e d , re spec t ive ly . Dashed l i n e indicates the amplitude of the condi t ioning EPSP. 1 42 -io J i i • • • • 1 • • • • — i r • • • • \ • • • • 1 o 100 o 1O0 Time(ms) 143 Figure 4.7. Histogram (mean +/- SEM; n=6) comparing the i n t r a c e l l u l a r and e x t r a c e l l u l a r test EPSPs evoked from (A) MPP and (B) LPP st imulat ion at 20, 80 and 400 ms C-T i n t e r v a l s . (A) The MPP evoked i n t r a c e l l u l a r EPSP (cross-hatched bars) exhibi ted potent iat ion at C-T in terva l s less than 100 ms; whereas, the e x t r a c e l l u l a r EPSPs (MML, open bar; GCL, f i l l e d bar) were i n h i b i t e d at a l l per iods . (B) Paired st imulat ion of the LPP resulted in a s imi lar f a c i l i t a t i o n of the i n t r a c e l l u l a r EPSP at test in terva l s less than 100 ms (cross-hatched b a r ) . The e x t r a c e l l u l a r EPSP recorded from the OML (open bar) was enhanced at in terva l s less than 1 s, even though, the e x t r a c e l l u l a r EPSP recorded from the GCL ( f i l l e d bar) at 20 ms and 400 ms C-T in terva l s were i n h i b i t e d . Note that at the i n t r a c e l l u l a r EPSP at the 400 ms period was also i n h i b i t e d . 1 44 E i 1 ( 1 r < C - T Interval (ms) 145 Figure 4.8. The effect of paired pulse s t imulat ion on the i n t r a c e l l u l a r EPSP when measured as an absolute change in vol tage . (A) Measuring the test EPSP as a change in voltage (V 2 ) reversed the potent iat ion ( F i g . 4.7) to i n h i b i t i o n when compared to the condit ioning EPSP (V 1 ) at a 20 ms C-T i n t e r v a l . (B) Histogram (mean, n = 6) of MPP and LPP evoked test EPSPs comparing the percent change of the i n t r a c e l l u l a r test EPSP at C-T in terva l s of 20, 80 and 400 ms. The test EPSPs at 20 and 400 ms were i n h i b i t e d , whereas, the 80ms test EPSP was s l i g h t l y potent iated. Note the s i m i l a r i t y in these values and those obtained for the e x t r a c e l l u l a r EPSP recorded from the GCL. A l l SEM values were within four percent of the means. 146 Lagend ta ucsui cvocco S 3 U T D I U . CVMCD 20 80 400 COND.-TEST INTERVAL (ms) 147 i n h i b i t i o n and potent iat ion of the i n t r a c e l l u l a r test EPSP at C-T in terva l s less than 40 ms and between 40-160 ms, respect ive ly ( F i g . 4 .9) . This enhancement occurred despite the fact that the EPSP was evoked during the LHP (F ig . 4.9, open arrow), ind ica t ing that summation of the test EPSP with the DAP was not a prerequis i te for augmentation. In contrast to the DAP, the hyperpolar izat ion and the conductance increase associated with the granule c e l l LHP apparently attenuated the i n t r a c e l l u l a r test EPSP as i t propagated from the dendrites to the c e l l body. Paired PP st imulat ion resulted in a decrease of the EPSP amplitude and in the p r o b a b i l i t y of granule c e l l discharge at a 400 ms C-T i n t e r v a l (F ig . 4.10 A ) . In order to determine i f postsynaptic processes contributed to the reduction of the EPSP, a depolar iz ing current pulse which induced a granule c e l l discharge ( F i g . 4.10 C) was de l ivered 400 ms fol lowing a MPP condit ioning st imulus. As shown in F i g . 4.10 (B), the MPP stimulus abolished the current- induced act ion potent ia l by reducing the depolar izat ion below threshold . Reversing the paradigm in another granule c e l l , so that the current -induced c e l l discharge preceded (400 ms) the MPP st imulus, resul ted in the i n h i b i t i o n of the EPSP to subthreshold values (4.11 B) . Hence the postsynaptic a c t i v a t i o n of granule c e l l s was s u f f i c i e n t to reduce the EPSP evoked from a previous ly unstimulated a f ferent . These data indicate that postsynaptic mechanisms contribute to the depression of both the EPSP and the subsequent f i r i n g of granule c e l l s . 1 48 Figure 4.9'. The effect of paired MPP st imulat ion on the i n t r a c e l l u l a r test EPSP in the absence of a DAP. I n t r a c e l l u l a r test responses exhibi ted an i n i t i a l i n h i b i t i o n at C-T in terva l s less than 40ms ( f i l l e d arrow) that reversed to potent ia t ion at periods between 40-160 ms (open arrow). This p a r t i c u l a r granule c e l l maintained an RMP of -62 mV and was impaled with a potassium methylsulfate f i l l e d microelectrode. A l l records represent the average of four sweeps. > 1 49 -10 I ' 200 0 TIME (ms) 1 50 Figure 4.10. T y p i c a l i n t r a c e l l u l a r records demonstrating that the perforant path evoked LHP may be p a r t i a l l y responsible for the paired pulse depression of the EPSP and granule c e l l discharge at a 400 ms C-T i n t e r v a l . Condit ioning the granule c e l l with MPP st imulat ion blocked the t r igger ing of another act ion potent ia l 400 ms la t er by e i ther (A) a second stimulus de l ivered to the MPP or (B) depo lar iz ing current i n j e c t i o n . (C) Unconditioned depolar iz ing current in jec t ion evoked the granule c e l l to discharge 5/6 t r i a l s p r i o r to MPP condi t ioning st imulus. A l l records represent six sweeps. 4 0 0 m s U n c o 152 Figure 4.11. I n t r a c e l l u l a r records demonstrating the postsynaptic nature of the granule c e l l i n h i b i t i o n at a C-T i n t e r v a l of 400 ms. The unconditioned response ( th ird response in e i ther group) shows that pr ior to a condi t ioning stimulus both (A) depolar iz ing current in jec t ion and (B) MPP st imulat ion evoked act ion p o t e n t i a l s . (A) Subthreshold MPP condi t ioning s t imul i e l iminated current- induced granule c e l l discharges at a 400 ms C-T i n t e r v a l . (B) Reversing the paradigm such that the current in jec t ion preceded suprathreshold MPP s t imulat ion , resul ted in the i n h i b i t i o n of the synapt i ca l ly evoked granule c e l l discharge at a 400 ms C-T i n t e r v a l . A l l records represent seven sweeps. 154 This p o s s i b i l i t y was examined further by comparing the s i m i l a r i t i e s in time course between the attenuation of spike a c t i v a t i o n evoked by paired MPP s t imul i and paired current pulses . MPP st imulat ion resul ted in the c h a r a c t e r i s t i c LHP which i n h i b i t e d the granule c e l l discharge for up to 3 s ( F i g . 4.12; upper t r a c e ) . Current- induced act ion potent ia l s were a lso abolished during the AHP for a 2 s per iod , ind ica t ing that membrane hyperpolarizat ions promote paired pulse depression ( F i g . 4.12, lower t race ) . 4 .3 .2 . The possible mechanisms underlying the potent iat ion  of the population spike and enhanced p r o b a b i l i t y of granule  c e l l discharge Stimulation of e i ther the LPP or MPP resul ted in i n h i b i t i o n and potent iat ion of granule c e l l discharge at C-T in terva l s of 20 and 400ms (F ig . 4.13 A) and 80ms (F ig . 4.13 B) , re spec t ive ly . The PS exhibi ted a s imi lar sequence of suppression and f a c i l i t a t i o n ( F i g . 4.14). The a l t e r a t i o n s in PS amplitude, however, d id not always corre la te with the changes of the i n t r a c e l l u l a r test EPSP. These data indicate that several mechanisms may modulate granule c e l l discharge. One possible explanation for the di f ference between the changes in EPSP and PS amplitude is that condi t ioning granule c e l l s a l t e r s the threshold for AP generation. The effect of paired MPP st imulat ion on AP threshold was checked in s ix granule c e l l s with a s l i g h t enhancement or reduction in threshold observed at 20 ms and 80 ms, re spec t ive ly . In 155 Figure 4.12. Comparison between the a b i l i t y of synaptic and current-evoked af terhyperpolar izat ions to i n h i b i t granule c e l l discharge. Suprathreshold st imulat ion of the MPP (top) resulted in the depression of granule c e l l discharge in response to a second MPP stimulus for a per iod of 3s. Paired current in jec t ion (lower trace) attenuated granule c e l l discharge for 2s. 1 56 Medial Per. Path 9 * \ J & \ > \ A A A * Current •I InA J lOmV 0.5 s 157 Figure 4.13. T y p i c a l i n t r a c e l l u l a r records showing the effect of C-T i n t e r v a l on the p r o b a b i l i t y of discharging a granule c e l l . Suprathreshold s t imulat ion of the MPP (A) depressed granule c e l l discharge when a test stimulus was de l ivered at in terva l s of 20 and 400 ms. (B) Subthreshold MPP st imulat ion of the same granule c e l l evoked a discharge at an 80 ms C-T i n t e r v a l . C o n d . 2 0 8 0 C - T Interval ( m s ) 4 0 0 159 F i g . 4.14. Histogram (mean +/- SEM; n = 6) showing the effect of paired PP st imulat ion on the i n t r a c e l l u l a r EPSP (cross-hatched bars) and e x t r a c e l l u l a r PS (open bars) . St imulation of LPP (A) and MPP (B) resulted in s imi lar a l t e r a t i o n s , that i s , the i n t r a c e l l u l a r test EPSP potentiated at C-T in terva l s less than 100 ms and depressed at 400ms. The PS exhibited i t s t y p i c a l i n h i b i t i o n , f a c i l i t a t i o n , and i n h i b i t i o n sequence at C-T in terva l s of 20, 80 and 400 ms, respec t ive ly . Uncoupling of the EPSP and PS occurred at 20ms and was indicated by the depression of the PS during a period of EPSP enhancement. 160 B. . „ 160 -j c 'c o 140 -c o o H— o • 0s-CD 120 -to c o CL _ CO CD cr est 100 -est h-<i— -o de 8 0 -Q. e < 6 0 -- l r Lateral Stirn. 0 Infra Soma • E x t r a - P S . (n=6) Medial Stim. E3 Infra Soma • E x t r a - P S . (n=6) 20 i r 8 0 4 0 0 2 0 C - T Interval (ms) 1 r i r 8 0 4 0 0 161 2/6 neurons examines MPP st imulat ion de l ivered at a 20ms C-T i n t e r v a l reached the i n i t i a l threshold value (dashed l ine) but f a i l e d to induce c e l l discharge ( F i g . 4.15 A ) . The EPSPs in the other four granule c e l l s were depressed at 20 ms probably as a resul t of the a c t i v a t i o n of GABA-mediated i n h i b i t i o n . Subthreshold st imulat ion given at an 80 ms C-T i n t e r v a l reduced (4/6) or did not change (2/6) the o r i g i n a l AP threshold value ( F i g . 4.15 B, C ) . The reduction in threshold noted with paired MPP st imulat ion was a lso observed in response to paired s t imulat ion of e i ther MPP-MF ( F i g . 4.16 A) or MF-MF ( F i g . 4.16 C ) . The decrease in threshold appeared to be corre la ted with the amplitude and the duration of the depo lar iz ing a f t erpotent ia l s evoked by PP or MF st imulat ion ( F i g . 4.16 A., C ) . Although these data imply that the reduction in AP threshold was due to the depo lar iz ing a f t e r p o t e n t i a l s , granule c e l l s occas ional ly displayed a lower discharge threshold in the absence of membrane depolar izat ion (F ig . 4.16 E ) . 4 .3 .3 . The ionic mechanisms underlying paired pulse  i n h i b i t i o n of granule c e l l a c t i v i t y The ef fect of ion ic manipulations on the i n h i b i t i o n of granule c e l l a c t i v i t y was determined for the e x t r a c e l l u l a r PS evoked at various C-T i n t e r v a l s . Although th i s method cannot d i f f e r e n t i a t e between pre- and postsynaptic e f fec t s , 162 Figure 4.15. The ef fect of a condi t ioning pulse on the spike a c t i v a t i o n threshold of granule c e l l s . (A) Suprathreshold st imulat ion increased the f i r i n g threshold at a C-T i n t e r v a l of 20 ms in 2/6 granule c e l l s . (B) Subthreshold st imulat ion reduced the spike threshold (arrow) at a C-T i n t e r v a l of 80 ms in 4/6 neurons. (C) Enlargement and comparison of the suprathreshold unconditioned contro l response ( s o l i d l ine) and the test evoked discharge at 80 ms (dotted l i n e ) . Threshold for act ion potent ia l generation marked by open arrow. Note that there was a 5mV reduction in the threshold potent ia l ( arrow) at the 80 ms response. Dashed l i n e (A, B) indicates act ion potent ia l threshold and a l l spikes were c l i p p e d . 163 1 64 Figure 4.16. The contr ibut ion of depolar iz ing a f t erpotent ia l s to changes of granule c e l l e x c i t a b i l i t y . E x t r a c e l l u l a r recordings from the GCL i l l u s t r a t e d the t y p i c a l f a c i l i t a t i o n of the antidromic spike (AS; unconditioned AS shown in lower trace of (B) and f i r s t response in (O) when paired with perforant path (B) or mossy f iber (MF; D) s t imula t ion . Simultaneous i n t r a c e l l u l a r records showed that i f an i n i t i a l l y subthreshold MF stimulus (A; lower trace) was evoked during the DAP (upper trace) the granule c e l l discharged. (C) I n t r a c e l l u l a r corre la te of (D) showing that s t imulat ion of the MF e l i c i t s a depo lar i z ing -i n h i b i t o r y potent ia l (D-IPSP; f i r s t response) and that subsequent MF s t imul i causes the granule c e l l to discharge when de l ivered during the D-IPSP. (E) I n t r a c e l l u l a r record showing that granule c e l l s exhibi ted a l t ered e x c i t a b i l i t y in response to paired MF st imulat ion in the absence of changes in membrane p o t e n t i a l . Act ion potent ia l s in (A, C, and E) were c l i p p e d . 1 65 166 i t does provide information about whether a p a r t i c u l a r treatment a l t e r s i n h i b i t o r y processes in the dentate. Attenuation of the PS in response to paired PP st imulat ion ( F i g . 4.14) may be due to the a c t i v a t i o n of GABAergic interneurons (Andersen et a l , 1966a; Lomo, 1971b) which have been shown to make synaptic contact with the dendri tes , somata and i n i t i a l segments of granule c e l l s (Kosaka et a l . , 1984). Perfusion with the GABAA antagonist , b i c u c u l l i n e , reversed the i n h i b i t i o n of the PS normally present fol lowing paired MF-PP st imulat ion at 20 ms ( F i g . 4.17). Lowering the e x t r a c e l l u l a r C l ~ concentration from 130 mM to 40 mM produced s imi lar resu l t s as b i c u c u l l i n e on the 20 ms PS i n h i b i t i o n (data not shown). S i g n i f i c a n t l y neither treatment reduced the i n h i b i t i o n of the PS exhibited at a 400 ms C-T i n t e r v a l . Therefore, the i n h i b i t i o n of the PS at 400 ms was not re lated to a GABA-mediated increase in C l ~ conductance. The fact that the PS i n h i b i t i o n las t s for seconds suggests that i t may be re lated to the sodium-potassium (Na/K) pump. Following 2 hours of perfusion with the Na/K pump i n h i b i t o r , ouabain (10~^ M), the i n h i b i t i o n of the PS at 20 ms was reduced, probably as a resul t of the depo lar iza t ion of the interneurons mediating th i s i n h i b i t i o n . On the other hand, the i n h i b i t i o n at 400 ms was only s l i g h t l y diminished ( F i g . 4.1.8). Thus the late i n h i b i t i o n of the PS was not dependent on a c t i v a t i o n of the Na/K pump. 167 Figure 4.17. The effect of the GABA antagonist , b i c u c u l l i n e (I0~k M), on PS i n h i b i t i o n . Antidromic a c t i v a t i o n of the granule c e l l s at a C-T i n t e r v a l of 20 ms t y p i c a l l y inh ib i t ed the PS (lower trace) when compared to i t s unconditioned (T, upper trace) amplitude. This i n h i b i t i o n was blocked by the perfusion of b i c u c u l l i n e (middle group) and returned in normal medium ( last group). 168 Recurrent Inhibition of Gronule Cells. 5msec Pre -Treatment Bicuculline (15 min,IO"6M) Wash (20 min) 169 It has been suggested that the LHP i s a calc ium-mediated potassium-dependent po tent ia l (Thalmann and Ayala , 1982; see chapter 3). To test th i s hypothesis TEA was added to the perfusion medium because i t has been shown to block both voltage-dependent and calcium-mediated potassium conductances in Aplys ia (Meech and Standen, 1975). Perfusion with 10 mM TEA (15 min) s l i g h t l y attenuated the i n h i b i t i o n of the PS at 400 ms and the e x t r a c e l l u l a r p o s i t i v i t y fol lowing the PS (F ig . 4.19? arrow). This second p o s i t i v i t y presumably i s the e x t r a c e l l u l a r r e f l e c t i o n of the voltage-dependent K + po tent ia l responsible for spike r e p o l a r i z a t i o n . In order to determine whether the attenuation of the PS was re lated to a decrease in a voltage-dependent or calc ium-mediated potassium conductance, the vo l tage- sens i t ive potassium channel blocker, cesium, was added to the perfusate . Following 70 min of 2 mM cesium, the pos i t ive potent ia l fol lowing the PS was reduced ( F i g . 4 . 1 9 , arrow) without any apparent effect on the PS i n h i b i t i o n at 400 ms. Therefore, the decrease in the PS i n h i b i t i o n at 400 ms in the presence of TEA probably resul ted from the attenuation of a calcium-mediated potassium conductance. 4.4 Discussion The experiments described in th i s chapter were designed to determine the ro le of postsynaptic mechanisms in the short-term a l t e r a t i o n s of granule c e l l e x c i t a b i l i t y observed 170 Figure 4.18. The ef fect of the sodium-potassium pump i n h i b i t o r , ouabain (10~5 M), on the i n h i b i t i o n of the PS at C-T in t erva l s of 20 and 400 ms. T y p i c a l i n h i b i t i o n of the PS (control) was observed p r i o r to ouabain perfus ion . Following 2 hours of perfusion with ouabain, the i n h i b i t i o n of the PS at 20 ms was reversed, whereas, the depression at a C-T i n t e r v a l of 400 ms was only s l i g h t l y reduced. C - T In terva l C o n t r o l 2 0 m s •i i1 4 0 0 m s -VlN-^N 1 0 p W \ O u a b a i n 172 Figure 4 . 1 9 . The ef fects of potassium channel blockers on the i n h i b i t i o n of the PS at a C-T i n t e r v a l of 400 ms. Perfusion with cesium (2 mM) reduced the voltage-dependent potassium conductance associated with spike r e p o l a r i z a t i o n (arrows) and s l i g h t l y enhanced the i n h i b i t i o n of the PS. Tetraethylammonium (TEA, lOrnM) attenuated the spike repo lar i za t ion (arrow) and the PS i n h i b i t i o n at a C-T i n t e r v a l of 400 ms. 173 2 m M Cs + (70min) IOmM TEA (I5min) 174 with the paired pulse paradigm. This work d i f f e r s from previous reports in that extra- and i n t r a c e l l u l a r potent ia l s of granule c e l l s were c o r r e l a t e d . In general , our e x t r a c e l l u l a r data on the e x c i t a b i l i t y changes observed with the paired pulse paradigm agree with previous reports . That i s , paired st imulat ion of the LPP and MPP resulted in potent iat ion or i n h i b i t i o n of the test EPSPs, respect ive ly ( F i g . ' s 4.2, 4.4; Harr i s and Cotman, 1983; McNaughton, 1980; McNaughton and Barnes, 1977). Based on e x t r a c e l l u l a r recordings th i s dif ference in response to paired st imulat ion between the pathways has been a t t r ibuted to a deplet ion of neurotransmitter in the MPP (McNaughton, 1980). The present i n t r a c e l l u l a r ana lys i s , however, showed that PP st imulat ion evoked a f t erpotent ia l s in granule c e l l s which appeared to modify e x t r a c e l l u l a r and i n t r a c e l l u l a r test EPSPs. Granule c e l l s exhibited a DAP fol lowing the EPSP which appeared to have opposing ef fects on the i n t r a c e l l u l a r and e x t r a c e l l u l a r EPSPs. P r i m a r i l y , the DAP summed with the i n t r a c e l l u l a r EPSP at C-T in terva l s less than 80 ms and promoted potent iat ion of the EPSP ( F i g . 4 .6) . The depo lar izat ion associated with the DAP also reduces the amplitude of the test EPSP. The l a t t e r i s analogous to the attenuation of the EPSP observed with depolar iz ing current i n j e c t i o n (Chapter 3; F i g . 3.8) and explains why the e x t r a c e l l u l a r EPSP recorded at the MML and GCL were depressed ( F i g . 4.7 A, B; f i l l e d bars) during periods of 175 i n t r a c e l l u l a r potent iat ion (20 ms; cross-hatched bars ) . That i s , the inward current flow associated with the EPSP d e p S p ) i s decreased by membrane depolar izat ion as the resu l t of being c loser to the equi l ibr ium potent ia l of the EPSP ( E e p S p ) . This reduction in I e p S p causes a smaller ex tra- and i n t r a c e l l u l a r EPSP. Since the presence of the DAP a l tered the EPSP amplitude, i t was not known i f the potent iat ion of the MPP-evoked EPSP resul ted from i t s summation with the DAP or the f a c i l i t a t i o n of transmitter re lease . In support of the l a t t e r , records obtained from a presumed granule c e l l which did not exhib i t a DAP displayed augmentation of the i n t r a c e l l u l a r test EPSP even when evoked on a hyperpolarized membrane potent ia l ( F i g . 4.9; c f . Lomo, 1971b). This observation indicates that the MPP does indeed f a c i l i t a t e with paired pulse s t imulat ion . Moreover, whether or not these data were recorded from a granule c e l l or one of the numerous interneurons found in the GCL (Lorente de No, 1934; Seress and Pokorny, 1981) i s i r re l evant to the functioning of the MPP, since i t is un l ike ly that a pathway w i l l maintain two d i f f erent mechanisms of EPSP enhancement depending on the neuronal type with which i t makes synaptic contact . One must conclude then that the DAP in granule c e l l s attenuates and conceals the potent ia t ion exhibi ted by the MPP and that e x t r a c e l l u l a r records are i n s u f f i c i e n t to determine the a l t e r a t i o n s of EPSPs produced by paired pulse s t imulat ion . 176 The f a c i l i t a t i o n of the MPP evoked EPSP suggests that t h i s afferent i s not d i f f erent from the LPP (McNaughton, 1980) and that di f ferences between the two inputs may be re la ted to the d i s t r i b u t i o n of the synapes mediating the DAP. The fact that the LPP-evoked EPSP was f a c i l i t a t e d in the OML and i n h i b i t e d at the GCL suggests that the DAP was not present in the d i s t a l dendrites of granule c e l l s . Furthermore the depression of the EPSP as i t propagated to the soma, coupled with the observation that heterosynaptic commissural s t imulat ion reduces perforant path act ivated EPSPs (Buzsaki and Czeh, 1981; Douglas et a l . , 1983; c f . F i g . 3 from Steward et a l . , 1977), indicates that DAPs reduce e x t r a c e l l u l a r EPSP amplitudes. L a s t l y , immunohistochemical evidence i l l u s t r a t e s that the GABAergic interneurons bel ieved to mediate the DAP response make synaptic contact with only the somata and proximal dendr i t i c shafts of granule c e l l s (Kosaka, 1983; Kosaka et a l . , 1984). Thus, the DAP appears to be l o c a l i z e d to the granule c e l l body and near dendr i t i c processes and is e f f ec t ive in attenuating EPSPs only in these regions . The presence of the DAP and the f a c i l i t a t i o n of the i n t r a c e l l u l a r EPSP at C-T in terva l s less than 80 ms p a r t i a l l y explains why the PS was enhanced during periods of e x t r a c e l l u l a r EPSP depression ( F i g . 4.4 and 4.7; Assaf and M i l l e r , 1981; Harr i s et a l . , 1979; McNaughton and Barnes, 1977). The augmentation of PSs evoked e i ther ant idromica l ly or heterosynapt ica l ly (Lomo, 1971b) i s also c l a r i f i e d by the 177 presence of depolar iz ing a f t e r p o t e n t i a l s . That i s , test EPSPs summate with the underlying depolar izat ions which promote granule c e l l discharge. Although the duration of the depolar iz ing potent ia l s coincided with the enhanced p r o b a b i l i t y of granule c e l l discharge (F ig . 4.16), i t appeared that several other mechanisms played a ro le in the changes of granule c e l l e x c i t a b i l i t y at C-T in terva l s less than 80 ms. F i r s t l y , i f a condi t ioning stimulus was of s u f f i c i e n t intens i ty to evoke a PS, then a test pulse given 20 ms l a t er would show a reduced PS ( F i g . 4.1) and have a lower p r o b a b i l i t y of discharging the granule c e l l ( F i g . 4.13 A ) . The fact that the GABA antagonist , b i c u c u l l i n e , blocked th i s effect i s consistent with the hypothesis that the dentate gyrus maintains GABA-mediated i n h i b i t i o n (F ig . 4.17; Andersen et a l . , 1966a; Fr icke and Pr ince , 1984; Lomo, 1971b; Thalmann and Ayala , 1982). Secondly, granule c e l l s exhibi ted an apparent reduction in the threshold p o t e n t i a l at C-T in terva l s between 40-100 ms ( F i g . 4.15). This decrease in threshold was independent of both orthodromic and pathway spec i f i c a c t i v a t i o n , since antidromic MF st imulat ion evoked the same reduction in threshold ( F i g . 4.16 A, C; c f . Lomo, 1971b). F i n a l l y , a condit ioning MF stimulus which d id not s i g n i f i c a n t l y a l t e r the res t ing membrane potent ia l increased the p r o b a b i l i t y of an i d e n t i c a l test pulse to discharge the granule c e l l up to 100 ms ( F i g . 4.16 E ) . These data suggest that the DAP, the changes in granule c e l l AP threshold l e v e l s , and the changes in the 178 external mi l i eu may have an important part in r e c r u i t i n g other granule c e l l s to discharge. In respect to the l a t t e r , a l t e r a t i o n s in [ K ] Q have been re lated to enhanced e x c i t a b i l i t y of hippocampal pyramidal neurons (Alger and T e y l e r , 1978; Creager et a l . , 1980) and repe t i t i ve s t imulat ion of the perforant path has been shown to increase [ K ] 0 and granule c e l l e x c i t a b i l i t y ( F r i t z and Gardner-Medwin, 1976). Therefore, several mechanisms may be re lated to the enhanced granule c e l l e x c i t a b i l i t y observed with paired pulse s t imulat ion and further inves t igat ion is required to determine the role that changes in the e x t r a c e l l u l a r ionic environment p lays . In contrast to the DAP, the LHP appeared to depress the i n t r a c e l l u l a r EPSP and granule c e l l discharge at C-T in terva l s greater than 100 ms ( F i g . 4.10). The LHP reduced current- induced depolar izat ions below the l e v e l of AP generation (F ig . 4.16). The depression of the current-evoked depolar izat ions was not dependent on the discharge of the granule c e l l , since subthreshold st imulat ion of the MPP also prevented the current evoked discharge ( F i g . 4.17 A ) . In a d d i t i o n , a previous ly unstimulated MPP evoked granule c e l l discharge was depressed at a 400 ms C-T i n t e r v a l by a condi t ioning current pulse that e l i c i t e d an AHP ( F i g . 4.17 B) . Hence, under condit ions where presynaptic deplet ion of transmitter was not present, the postsynaptic AHP s u f f i c i e n t l y depressed the EPSP so as not to discharge the granule c e l l . Therefore, i t appears that the granule c e l l 179 LHP and AHP contribute to the attenuation of EPSPs and granule c e l l discharge. Although the postsynaptic depression of the PP evoked EPSP i s considerable , i t cannot completely account for the attenuation of the EPSP observed with paired pulse s t imula t ion . The primary reason for th i s conclusion i s that a postsynaptic hyperpolar izat ion such as the LHP should enhance I e p S p , hence EPSP amplitude, by increasing the d i f ference between the membrane potent ia l and the EPSP equi l ibr ium p o t e n t i a l . This enhancement of synaptic dr ive should be detected as an augmented e x t r a c e l l u l a r EPSP recorded in the dendr i t i c reg ion. It has been shown, however, that paired MPP st imulat ion resul ts in EPSP depression ( F i g . 4.4; McNaughton, 1980; McNaughton, 1982).. Thus, both presynaptic i n h i b i t i o n of transmitter release (Lomo, 1971b; McNaughton, 1980; Racine and Milgram, 1983) and the LHP contribute to the e x c i t a b i l i t y changes observed with the paired pulse paradigm at C-T in terva l s greater than 100 ms. Given the complex in teract ions between the pre - and postsynaptic mechanisms c o n t r o l l i n g granule c e l l e x c i t a b i l i t y , i t would be advantageous to manipulate the granule c e l l a f t erpotent ia l s experimentally and observe the e f fects on the paired pulse phenomena. Thalmann and Ayala (1982) observed that the DAP increased in response to GABAA antagonists and reduced e x t r a c e l l u l a r C l ~ , ind ica t ing that the DAP i s p a r t i a l l y modulated by a GABA-mediated C l ~ 180 conductance. With regard to the LHP, Thalmann and Ayala (1982) showed that i t was a potassium-dependent p o t e n t i a l . Perfusion of the N a + / K + pump i n h i b i t o r ouabain and e x t r a c e l l u l a r recordings of the PS at C-T in terva l s greater than 100 ms showed that the LHP i s probably not re lated to the sodium/potassium pump ( F i g . 4.18). The best evidence concerning the nature of the LHP i s the observation that TEA s l i g h t l y attenuated the i n h i b i t i o n of the PS at 400 ms ( F i g . 4.19). This ef fect of TEA was probably due to i t s a b i l i t y to reduce a calcium-mediated potassium conductance (Meech and Standen, 1975), since perfusion of cesium, an antagonist of vo l tage - sens i t ive potassium channels, d id not have a s imi lar e f f ec t . Given the presynaptic mechanisms contr ibut ing to the EPSP depression, i t i s impossible to determine the basis of the DAP and LHP without i n t r a c e l l u l a r a n a l y s i s . Thus, further research is required to determine the extent to which the DAP and LHP influence the a l t e r a t i o n s in synaptic e f f i cacy induced by paired pulse s t imulat ion . In conclus ion , the data presented in th i s study showed that the f a c i l i t a t i o n and the depression exhibi ted in the dentate gyrus resulted from p r e - , post - , and poss ibly extrasynaptic a l t e r a t i o n s . Simultaneous recordings of extra-and i n t r a c e l l u l a r potent ia l s a lso revealed the shortcomings of in terpre t ing the e x t r a c e l l u l a r f i e l d potent ia l s in the dentate gyrus. Therefore, studies u t i l i z i n g only e x t r a c e l l u l a r techniques to determine the changes or the mechanisms responsible for the changes in e i ther the 181 potent ia t ion or i n h i b i t i o n of the EPSP should be viewed with caut ion . 182 Chapter 5 An extra- and i n t r a c e l l u l a r analys i s of the dentate gyrus during and fol lowing kindl ing- induced epi lepsy 5.1. Introduction The c e l l u l a r mechanisms of epilepsy have been invest igated fol lowing the app l i ca t ion of various pharmacological agents (Dingledine and Gjers tad , 1980; Prince 1978; Schwartzkroin and Wyler, 1980; Wood, 1975; Yamamoto and Kawai, 1968) and r e p e t i t i v e e l e c t r i c a l s t imulat ion known to induce convulsive a c t i v i t y (Ben-Ari et a l . , 1979; F u j i t a and Sakuranaga, 1981; Heinemann et a l . , 1977; Spencer and Kandel, 1969). While these studies have provided valuable information about the acute a l t e r a t i o n s in synaptic and i n t r i n s i c membrane propert ies of neurons which may contr ibute to the generation of ep i l ept i form a c t i v i t y , they have not addressed the question of what chronic or long-term changes in neuronal processes are associated with epi leptogenes is . One experimental model of epi lepsy which has been extensively u t i l i z e d in determining the chronic pathology associated with seizure disorders i s k ind l ing (for reviews see Goddard 1983; Kalichman, 1982; Racine, 1978). K ind l ing 183 is the term given by Goddard et a l . (1969) to the process whereby i n i t i a l l y subconvulsive e l e c t r i c a l s t imulat ion of s p e c i f i c forebrain structures leads to the progressive development of motor se izures . In order to induce k indl ing the stimulus parameters must evoke a focal se izure ( i . e . afterdischarge) in the primary target of the act ivated pathway (Racine, 1972b). Due to the depression of CNS e x c i t a b i l i t y fol lowing afterdischarges (AD) and se izures , k ind l ing s t imul i are more e f f ec t ive i f given at d a i l y in terva l s (Goddard et a l . , 1969; Mucha and P i n e l , 1977; Racine et a l . , 1973). Based on behavioral responses to s t imula t ion , the evolut ion of k indl ing has been c l a s s i f i e d into f ive stages: 1) f a c i a l clonus; 2) head nodding; 3) forelimb clonus; 4) rear ing; and 5) rearing and f a l l i n g (Racine, 1972b). Kindl ing animals past stage 5 resu l t s in the occurrence of spontaneous ( i . e . not evoked) convulsions ( P i n e l , 1981; P ine l et a l . , 1975; P ine l and Rouvner, 1978). Although the mechanisms underlying the behavioral manifestations of k indl ing are not yet known, a l t era t ions in the e l e c t r o p h y s i o l o g i c a l propert ies of both primary and secondary ( i . e . those CNS structures rece iv ing a d i r e c t projec t ion from the primary target) s i t e s have been reported. Electrographic records have shown that ADs in the primary s tructure increase in amplitude and duration as k ind l ing progresses (Goddard et a l . , 1969; Racine, 1972b). 184 Kindl ing a l so decreases the stimulus threshold required to e l i c i t " ADs (Pinel et a l . , 1976; Racine, 1972a). S i g n i f i c a n t l y , the k ind l ing of motor seizures depends on the a c t i v a t i o n of ADs (Racine, 1972b), whereas the lowering of AD threshold is independent of AD generation and may a c t u a l l y be enhanced with the use of low stimulus i n t e n s i t i e s (Pinel et a l . , 1976; Racine, 1972a). The lowering of AD thresholds and development of motor seizures appear to re f l ec t permanent a l t era t ions in neuronal processes, since these c h a r a c t e r i s t i c s were recorded six (Racine, 1972a) and twelve weeks (Goddard et a l . , 1969) fol lowing the las t kindled se izure . In contrast to the reduction of AD threshold observed in the primary s tructure , k ind l ing does not appear to have a s imi lar ef fect in secondary structures (Racine, 1972a; 1975); however, the number of s t imul i required to rekindle an animal from a secondary s i t e i s s i g n i f i c a n t l y decreased (Goddard et a l . , 1969; Mclntyre and Goddard, 1973; Racine, 1972b; 1975). This ' transfer* effect suggests that transynaptic a l t e r a t i o n s occur with k i n d l i n g . Other e lectrographic changes in secondary structures include: 1) the occurrence of large amplitude spikes ( i n t e r i c t a l spikes) ; 2) the increase in amplitude and number of i n t e r i c t a l spikes (IIS) as k ind l ing progresses (Racine, 1972b); and 3) the long-term enhancement of s ing le -pulse evoked responses (Chandler et a l . , 1982; Racine et a l . , 1975; Racine et a l . , 1983). 185 Because the presence of IISs i s considered a hallmark of e p i l e p t i c t i s sue , the ir appearance in secondary s tructures indicates the propagation of ADs to these s i t es (Racine, 1972b). In an attempt to discover the locat ion of the generator(s) of IISs, K a i r i s s et a l . (1984) found that the amygdala and the pyriform cortex were the f i r s t areas to produce IISs. The appearance of IISs in these areas occurred f i r s t even when they were not the primary s tructure being k ind led . The hippocampus rare ly exhibi ted IISs and th i s was interpreted as proof that the hippocampus is not required for the development of k ind l ing (Ka ir i s s et a l . , 1984). Given the evidence suggesting that IISs corre la te with i n h i b i t o r y events (Engel and Ackermann, 1980; F u j i t a et a l . , 1983) th i s las t assert ion seems unfounded. In fac t , k ind l ing through the hippocampus may be establ ished with less s t imul i fol lowing amygdala les ions (Mclntyre et a l . , 1982) and l e s ion ing the hippocampus reduces the a b i l i t y of a previous ly amygdala-kindled animal to respond with convulsions (Yoshida, 1984). Thus the hippocampus appears to support k ind l ing and may be necessary for i t s expression. As stated prev ious ly , k ind l ing produces long-term f a c i l i t a t i o n of synaptic responses evoked in secondary s tructures (Chandler et a l . , 1982; Racine et a l . , 1975; Racine et a l . , 1983). This increase in the synaptic po tent ia l s i s s imi lar to that observed with long-term potent ia t ion (Douglas and Goddard, 1975; Racine, 1975). Racine and coworkers (1983) examined the p o s s i b i l i t y that 186 long-term potent iat ion (LTP) underl ies k ind l ing and found that some aspects of LTP could not be evoked fol lowing k i n d l i n g . They concluded that s imi lar mechanisms may subserve both LTP and k i n d l i n g . In th i s respect , the use of amino-phosphonovalerate (APV) to antagonize the N-methyl-D-aspartate (NMDA) receptor, which act ivates a voltage-dependent C a 2 + conductance in hippocampal neurons (Dingledine, 1983), retards the development of both LTP (Col l ingr idge et a l . , 1983; Harr i s et a l . , 1984) and k ind l ing (Peterson et a l . , 1983). In contrast to the above f indings , i t has been reported that k indl ing v ia the entorhinal cortex occurred without the permanent potent iat ion of the perforant path-granule c e l l EPSP (Giacchino et a l . 1984; Maru et a l . , 1982). Though there i s s u f f i c i e n t support for the hypothesis that k ind l ing resu l t s from an increase in neuronal e x c i t a b i l i t y ( e . g . , the enhanced duration of ADs), i t has been suggested that attenuation of i n h i b i t o r y processes contributes to k i n d l i n g . In p a r t i c u l a r , a reduction in GABA-mediated i n h i b i t i o n is bel ieved to play an important ro le in the expression of seizures (for reviews see Kalichman, 1982a; Meldrum, 1975; Walker, 1983). In support of t h i s , the development of k ind l ing is f a c i l i t a t e d by GABA antagonists (Kalichman, 1982b) and reduced in the presence of GABA-receptor agonists (Joy et a l . , 1984) and c l i n i c a l l y used anticonvulsants ( e . g . , barbi turates and benzodiazepines) that potentiate GABA-mediated i n h i b i t i o n (Albertson et a l . , 187 1981; Racine et a l . , 1975). Another mo'del of epi lepsy ( i . e . , the app l i ca t ion of alumina gel to the cortex for a long period) was shown to decrease c o r t i c a l l eve l s of GAD and GABAergic neurons (Bakay and H a r r i s , 1981; Ribak et a l . , 1982). Thus, pharmacological and biochemical data indicate that a l t e r a t i o n s in GABA-mediated i n h i b i t i o n may p a r t i c i p a t e in k i n d l i n g , however, e l e c t rophys io log i ca l recordings from the hippocampus imply that inh ib i tory processes are augmented fol lowing k indl ing (Fuj i ta et a l . , 1983; Tuff et a l . , 1983a; Racine et a l . , 1983a). It has been hypothesized that potent iat ion of the GABA-mediated i n h i b i t o r y system is responsible for th i s enhancement (Tuff et a l . , 1983a); the f a c i l i t a t i o n is i n d i r e c t , though, and re lated to an increase in benzodiazepine receptors associated with the GABA-ionophore complex (McNamara et a l . , 1980; Niznik et a l . , 1984; Tuff et a l . , 1983b). Although the overwhelming number of studies on k indl ing have focused on the events which lead to epi leptogenesis , we bel ieve i t i s equally important to understand those processes which may prevent or retard the expression of k i n d l i n g . Therefore, we have examined the i n h i b i t o r y processes of the dentate gyrus and the CA1 region (Chapter 6) in hippocampal s l i c e s prepared from kindled r a t s . In a d d i t i o n , the i n t r a c e l l u l a r propert ies of granule c e l l s were compared between contro l and kindled preparat ions . The re su l t s of the study indicate that s i g n i f i c a n t a l t e r a t i o n s 188 occur in the i n h i b i t o r y processes and the membrane propert ies of granule c e l l s fol lowing commissural-kindl ing. 5.2 Methods Male Wistar rats were anesthetized with Nembutal (50 mg/kg, injected in traper i tonea l ly ) and placed into a stereotaxic apparatus. The s k u l l was exposed and by d e l i c a t e l y pressing down on the f r o n t a l and p a r i e t a l bones bregma was determined. A hole was then d r i l l e d into the s a g i t t a l suture 1.8 mm poster ior to bregma and a b ipolar st imulat ing electrode was posi t ioned 4.2 mm ventra l to the c o r t i c a l surface . A recording electrode was placed in the h i lu s of the dentate gyrus using the stereotaxic coordinates from bregma: AP, -3 .3 ; L , 1.8; V, 3.7 mm. Since s t imulat ion of the commissures t y p i c a l l y resu l t s in the a c t i v a t i o n of granule c e l l s in both the dorsal and ventra l blades of the dentate gyrus, the maximum potent ia l that can be recorded is c h a r a c t e r i s t i c a l l y midway between the two blades and centered in the h i l u s (McNaughton and Barnes, 1977). Therefore, the recording electrode pos i t i on was maximized in the h i l u s by s t imulat ing the commissures and f inding the locat ion which gave the largest pos i t ive p o t e n t i a l . Subsequent to the placement of both the recording and s t imulat ing e lectrodes , screws were inserted in to L the s k u l l at s tra teg ic points and posit ioned so that they made contact with the cortex . A small gauge s tee l wire was wrapped 189 around the screws to provide a ground reference e lectrode, and then the electrodes were secured by an a c r y l i c cement. Following one week of post-operative recovery, the rats were placed into an observation box and the s t imulat ing , recording, and ground leads were connected to the appropriate e lectrodes . The recording electrode was connected, v ia a preampl i f i er , to an osc i l loscope which was then fed to a chart recorder. The st imulat ing electrode was connected to a constant current d . c . st imulator adjusted to give a 10"^ A, 1 s pulse (60 Hz) . Pr ior to the de l ivery of the k i n d l i n g - s t i m u l i , a 30 s basel ine period was co l l ec t ed on the chart recorder and fol lowing the s t imulat ion the h i l a r a c t i v i t y was recorded for approximately 2-5 minutes. The k indl ing stimulus was given d a i l y and the occurrence of an afterdischarge (AD) was noted. An AD was defined as a large amplitude, high frequency discharge. Hippocampal s l i c e s were prepared from the c h r o n i c a l l y stimulated animals e i ther twenty-four hours or six weeks fol lowing the i r las t k ind l ing st imulus. The dentate gyrus i n h i b i t o r y processes of normal and kindled preparations were assessed by comparing e x t r a c e l l u l a r population spikes (PS) evoked by paired perforant path (PP) s t imula t ion . The development of the enhanced i n h i b i t i o n was corre la ted with the number of evoked ADs (0, 5, and 10 ADs) or stage 5 motor seizures (Racine, 1972b). In an attempt to determine the mechanism of the enhanced i n h i b i t i o n , a low C l ~ (40 mM) containing ACSF 190 F i g . 5.1. Typ ica l chart recordings of the a c t i v i t y in the dentate gyrus h i l a r region recorded from c h r o n i c a l l y implanted electrodes subsequent to a commissural-evoked k ind l ing st imulus. (A-D) The records i l l u s t r a t e the development of the prolongation of the afterdischarge (AD) as a function of the number of ADs that have been previously evoked (A = 0; B = 5; C = 10; D = 20). Following 30 ADs, a motor seizure (stage 5) was observed which las ted for the duration of the AD recorded in the h i lu s (E, open arrows). The f i l l e d arrowheads mark the appearance of wet-dog shakes. 191 1 92 medium was perfused. This manipulation reduces the effect of GABA-mediated i n h i b i t i o n found in the dentate gyrus. In add i t ion , granule c e l l membrane propert ies and synaptic potent ia l s were compared between the two groups using data obtained with i n t r a c e l l u l a r recordings (see Chapter 2 for a general descr ip t ion of the in v i t r o preparation and i n t r a c e l l u l a r recording techniques and a n a l y s i s ) . 5.3. Results 5 .3 .1 . E x 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 the a l t e r a t i o n s  produced during the development of k indl ing Afterdischarges Following one week of post-operat ive recovery, k ind l ing s t imul i de l ivered through an electrode placed in the hippocampal commissures evoked afterdischarges (AD) in the h i l u s of the dentate gyrus ( F i g . 5.1; Madryga et a l . , 1975). The f i r s t stimulus e l i c i t e d a br ie f ( less than 10 s) AD in forty-one of f o r t y - f i v e animals. In the other four animals, the i n i t i a l stimulus d id not a l t e r the e x t r a c e l l u l a r po tent ia l (F ig . 5.1 A ) . Addi t iona l t r i a l s resu l ted in the progressive lengthening of ADs ( less than 90 s) and the occurrence of observable behavioral changes ( e . g . , wet-dog shakes) c h a r a c t e r i s t i c of the development of k ind l ing ( F i g . 5.1, B-D). The wet-dog shakes appeared fol lowing an AD-induced depression of e l e c t r i c a l a c t i v i t y 193 F i g . 5.2. O s c i l l o s c o p e t r a c e s showing the p o p u l a t i o n spike responses evoked by s t i m u l a t i o n of the p e r f o r a n t path at C-T i n t e r v a l s of 20, 80 and 420 ms. C o n t r o l s l i c e s (A) e x h i b i t the c h a r a c t e r i s t i c i n h i b i t i o n , p o t e n t i a t i o n , and i n h i b i t i o n sequence, whereas hippocampal s l i c e s removed from r a t s that had a minimum of 5 motor s e i z u r e s (B) e x h i b i t e d only i n h i b i t i o n of the t e s t responses. Histograms (mean +/- SEM) compare the e f f e c t s of p a i r e d p e r f o r a n t path s t i m u l a t i o n between c o n t r o l (C) and k i n d l e d (D) p r e p a r a t i o n s at 13 C-T i n t e r v a l s (n > 25 f o r each C-T i n t e r v a l ) . ( >J'p< 0.05; T T p< 0.01; O p < 0.001; t w o - t a i l e d , t - t e s t ) 1 9 4 Control - ^ - A r| rV-B. Kindled I m V 5 mttt C 2 0 8 0 « 2 0 C-T Interval (msec) 150 100 50 o c o u a) IT c n * 150 or "5 a) J 100 " 5 . E < 50 -' I " i • • r " i • • i OO 200 500 I 4 o o o o u I " I • • I ft I • • I • 100 200 500 I 4 msec sec C-T Interval TJ" 195 which lasted for longer than 3 min ( F i g . 5.1; c f . , pre-stimulus basel ine to that fol lowing the AD). In order for a stage 5 motor seizure to be e l i c i t e d by the k ind l ing stimulus, an average of twenty-five t r i a l s was necessary. The e lectrographic corre la te of the behavioral seizure i s shown in F i g . 5.1 (E) . The duration of the convulsion (open arrows, E) coincided with the high amplitude sp ik ing . Chronic recordings from the h i lu s exhibi ted a prolongation of the AD as k ind l ing progressed, implying an enhanced e x c i t a b i l i t y of the neurons in th i s area. In v i t r o assessment of i n h i b i t o r y processes The f i r s t experiment tested the v a l i d i t y of using hippocampal s l i c e s to assess a l t e r a t i o n s induced from chronic manipulations. The resu l t s of th i s experiment were obtained from hippocampal s l i c e s taken from age-matched contro ls and kindled animals that had a minimum of 5 stage 5 motor seizures (range 5-26). Paired pulse s t imulat ion of the perforant path f ibers at 13 condi t ion- tes t (C-T) in terva l s gave a r e l i a b l e assessment of changes in granule c e l l e x c i t a b i l i t y ( F i g . 5 .2) . In non-kindled contro l s (n=3l), the pattern of a c t i v i t y was an i n h i b i t i o n - p o t e n t i a t i o n -i n h i b i t i o n (IPI) sequence for C-T in terva l s of 20-40 ms, 40-100 ms, and 0.1-8 s, respect ive ly ( F i g . 5.2 A, C; c f . McNaughton, 1980; Tuff et a l . , 1983a). The o v e r a l l pattern 196 F i g . 5.3. E x t r a c e l l u l a r potent ia l s recorded in the granule c e l l layer from the in v i t r o hippocampus in response to paired pulse st imulat ion of the perforant path at d i f f erent i n t e n s i t i e s (50 and 150 10" 6 A). The e x c i t a b i l i t y of the granule c e l l s i s depressed at 20 ms, enhanced at 80 ms, and depressed again after a 200 ms condi t ion- tes t (C-T) i n t e r v a l . Note that increasing stimulus in tens i ty does not a l t e r the.temporal r e la t i onsh ip of granule c e l l e x c i t a b i l i t y as measured by paired pulse . (These records and a l l subsequent records were taken from hippocampal s l i c e s ) . 197 C 20 80 420 C-T Interval (msec) . — 200 -i ' ' ' • I"'l • • I -0 100 200 500 I 4 msec sec C-T Interval 198 o f n e u r o n a l e x c i t a b i l i t y d i d n o t s h o w a d e p e n d e n c e o n t h e s t i m u l u s i n t e n s i t y e m p l o y e d ( F i g . 5 . 3 , c f . 5 0 a n d 1 5 0 m i c r o a m p s t i m u l a t i o n ) . S l i c e s p r e p a r e d f r o m c o m m i s s u r a l - k i n d l e d a n i m a l s e x h i b i t e d i n h i b i t i o n o f t h e t e s t r e s p o n s e a t a l l C - T i n t e r v a l s , w i t h s i g n i f i c a n t d i f f e r e n c e s f r o m t h e c o n t r o l p r e p a r a t i o n p r e s e n t b e t w e e n 2 0 - 4 0 0 ms ( F i g . 5 . 2 B , D ) . T h u s k i n d l i n g i s a s s o c i a t e d w i t h t h e r e v e r s a l o f t h e p o t e n t i a t i o n p h a s e i n t o o n e o f i n h i b i t i o n , r e s u l t i n g i n a c o n t i n u e d s u p p r e s s i o n o f g r a n u l e c e l l a c t i v i t y . T h e c h a n g e s i n i n h i b i t i o n a r e i n g o o d a g r e e m e n t w i t h T u f f e t a l . ( 1 9 8 3 a ) a n d i l l u s t r a t e t h e u s e f u l n e s s o f t h e i n v i t r o p r e p a r a t i o n i n a s s e s s i n g c h r o n i c a l t e r a t i o n s i n n e u r o n a l p h y s i o l o g y . D e v e l o p m e n t o f k i n d l i n g - e n h a n c e d i n h i b i t i o n T h e c h a n g e s i n t h e i n h i b i t i o n o f t h e P S i n d u c e d b y k i n d l i n g w e r e c o r r e l a t e d w i t h t h e n u m b e r o f e v o k e d A D s . T h e I P I s e q u e n c e o f a c t i v i t y w a s n o t a l t e r e d i n s l i c e s o b t a i n e d f r o m a n i m a l s r e c i p i e n t s o f 6 - 2 2 k i n d l i n g s t i m u l i t h a t f a i l e d t o e v o k e A D s ( F i g . 5 . 4 B ) . A l t h o u g h t h e g e n e r a l p a t t e r n o f I P I w a s u n c h a n g e d b y s t i m u l i t h a t w e r e u n s u c c e s s f u l i n e v o k i n g A D s , t h e r e w a s a s i g n i f i c a n t r e d u c t i o n i n t h e p o t e n t i a t i o n o b s e r v e d a t a C - T i n t e r v a l o f 8 0 ms ( F i g . 5 . 5 , ( 0 ) ) . F o l l o w i n g t h e a c t i v a t i o n o f f i v e A D s t h e e n h a n c e d i n h i b i t i o n w a s a l m o s t m a x i m a l l y i n d u c e d ( F i g . 5 . 4 C ) , e x h i b i t i n g a p p r o x i m a t e l y a 7 0 % r e d u c t i o n i n e x c i t a b i l i t y 199 F i g . 5.4. The development and prolongation of the AD was accompanied by an a l t e r a t i o n in the e x c i t a b i l i t y of granule c e l l s . Paired pulse s t imulat ion of the perforant path shows the t y p i c a l (A) i n h i b i t i o n , po tent ia t ion , i n h i b i t i o n sequence at 20, 80, and 400 ms C-T i n t e r v a l s , r e spec t ive ly . Kind l ing s t imul i that do not evoke ADs, have l i t t l e ef fect on the e x c i t a b i l i t y of granule c e l l s (B; 20 st imulat ions without producing a s ingle AD). However, the potent iat ion observed at an 80 ms C-T i n t e r v a l was depressed fol lowing 5 ADs (C) and was further depressed by 10 ADs (D). 200 Control No AD's 5 AD's 10 A D s A A _ A _ A. Cond. 20 8 0 C-T Interval (ms) 4 0 0 _TlmV 5 ms 201 Fi g . 5.5. Graphic representation of the data showing the changes in i n h i b i t i o n with an increasing number of evoked ADs. The depression of the potentiation observed at an 80 ms C-T in t e r v a l i s plotted with respect to the number of ADs. One can note an increase in the i n h i b i t i o n which appears to be maximal following 10 ADs and i s not altered further by the presence of motor seizures, (n = 6 for each group) 202 Number of Afterdischarges 203 when compared to contro l potentiated values ( F i g . 5 .5) . The further ac t iva t ion of ADs or the development of motor seizures d id not s i g n i f i c a n t l y increase the i n h i b i t i o n (F ig . 5.5 c f . 10 AD to s e i zure ) . Therefore, the reduction in neuronal e x c i t a b i l i t y exhibited by the dentate gyrus neurons would appear to occur rap id ly with the i n i t i a t i o n of ADs and p r i o r to and independent of motor se izures . Ef fect of reducing GABA-mediated i n h i b i t i o n In an attempt to determine the mechanism subserving the apparent enhanced i n h i b i t i o n in the dentate gyrus, the recurrent i n h i b i t i o n responsible for the i n i t i a l i n h i b i t o r y period ( i . e . , 20-40 ms) was reduced by decreasing the e x t r a c e l l u l a r chlor ide concentration ( [ C l ] 0 ) from 130 mM to 40 mM. It was reasoned that i f the augmented i n h i b i t i o n observed with k indl ing was the resul t of an increase in the recurrent inh ib i tory processes, then e l iminat ing th i s factor should restore the po tent ia t ion . In contro l s l i c e s (n=4), the perfusion of low [C1] Q medium resul ted in the loss of the i n i t i a l i n h i b i t o r y period at 20 ms and an augmentation of the potent iat ion phenomena ( F i g . 5.6 A, C ) . In a l l cases the greatest amount of potent ia t ion appeared at the shorter C-T i n t e r v a l s , demonstrating the potency of the recurrent i n h i b i t i o n under normal condi t ions . The late period of i n h i b i t i o n was 204 F i g . 5.6. The effects of low [C1] Q (40 mM) on the paired perforant path evoked i n h i b i t i o n in contro l and kindled preparations (n=4). Osc i l loscope records (A, B) and histograms (C, D) show a reversa l of the early i n h i b i t i o n (C-T i n t e r v a l , 20 ms) in both preparat ions , while the late i n h i b i t o r y period (greater than 200 ms) remains unchanged. Note that the potent iat ion phase fol lowing the perfusion of low [C1] Q l a s t s for C-T i n t e r v a l s less than 60 ms in the kindled preparat ion. ( P < 0.1; ^ T p <0 .05 ; Q P < ° - 0 1 two- ta i l ed , t - t es t ) 205 Low Cl " 300 n Control B. 200 <n | 100 co <D cr. c § 0 a. -r / /-r TJ" a Kindled 5 msec 80 msec 420 O , 5 0 100 50 -0 -r»-r-100 200 msec 500 J E T 1 12 3 4 sec C - T Interval 206 unaffected by the low [C1] Q medium, ind ica t ing that i t i s not a Cl~-dependent i n h i b i t i o n . In comparison, kindled s l i c e s showed only a reversal of the i n h i b i t i o n at the 20-40 ms C-T in terva l s in low [ C l ] 0 ( F i g . 5.6 B, D) . The fact that potent iat ion was present fol lowing the loss of the ear ly i n h i b i t i o n implied that at least one of the mechanisms subserving the potent iat ion phenomenon was maintained in the kindled preparat ion. Moreover, the continued i n h i b i t i o n at C-T in terva l s of 60-100 ms ( F i g . 5.6 B, D) demonstrated that the enhanced i n h i b i t i o n induced by k ind l ing was not due to augmentation of the Cl"-dependent, GABA-mediated i n h i b i t i o n . These data suggest that the increased i n h i b i t i o n observed in the dentate gyrus, subsequent to k i n d l i n g , was re lated to a shortening in onset latency of a la te C l ~ - i n s e n s i t i v e i n h i b i t o r y process or a reduction in po tent ia t ion . Duration of enhanced i n h i b i t i o n To determine whether the enhanced i n h i b i t i o n re f l ec ted a permanent a l t e r a t i o n in the i n h i b i t o r y processes of the dentate gyrus, paired pulse interact ions were recorded six weeks fol lowing the las t kindled se izure . In a l l cases examined (n=6), the magnitude and duration of the paired pulse i n h i b i t i o n were e s s e n t i a l l y the same as that observed 24 h r s . post -se izure ( F i g . 5.7, f i l l e d c i r c l e s ) . In a d d i t i o n , perfusion of a low [C1] Q medium produced resu l t s 207 F i g . 5.7. The graph i l l u s t r a t e s that even af ter s ix weeks without a k indl ing stimulus, pa ired pulse s t imulat ion of the perforant path resulted in i n h i b i t i o n at a l l C-T in terva l s tested ( f i l l e d c i r c l e ) . In a d d i t i o n , perfusion of a low [C1] Q (40 mM) containing media showed a reversa l of the ear ly i n h i b i t i o n (20 ms) but no effect on the other i n h i b i t o r y per iod . 208 200-1 O U ZUU 500 I 4 msec sec C-T Interval 209 i d e n t i c a l to those obtained 1 day after the las t se izure , i . e . , potent iat ion at a 20 ms C-T and i n h i b i t i o n at a l l other in terva l s ( F i g . 5.7, open c i r c l e s ) . 5 .3 .2 . I n t r a c e l l u l a r comparison of the membrane propert ies  and synaptic potent ia l s between contro l and kindled granule  c e l l s The resu l t s of the i n t r a c e l l u l a r study are based on the recordings obtained from 13 contro l and 12 ' k i n d l e d ' granule c e l l s . This small sample s ize was caused in large part by the d i f f i c u l t y of impaling granule c e l l s which met the minimal e l ec t rophys io log i ca l c r i t e r i a . St imulation of the PP c h a r a c t e r i s t i c a l l y evoked an EPSP - ac t ion potent ia l (AP) - depolar iz ing a f t e r p o t e n t i a l (DAP) sequence in both the contro l and kindled preparations ( F i g . 5.8 A ) . The late hyperpolar izat ion was not examined in the present study. Since mult ip le spike a c t i v a t i o n was never evoked from contro l or kindled s l i c e s , even when the stimulus in tens i ty was increased up to f ive times that required for threshold a c t i v a t i o n ( e . g . , 400 microamps), the mechanisms subserving AP generation appeared to be unaltered by k i n d l i n g . Granule c e l l s from the contro l and the kindled preparations t y p i c a l l y maintained high RMP (range: -65 to -82 mV) and SA (92 to 110 mV) values which d id not s i g n i f i c a n t l y d i f f e r between groups (Table 1). Examination of current-vol tage (I-V) p r o f i l e s revealed that the average 210 Table 5.1. A comparison of granule c e l l membrane propert ies recorded from contro l and kindled preparations (mean +/-SEM) . (n) RMP (-mV) SA (mV) R n (Mohms) T e (ms) Ti (ms) Control (13) 74 +/- 2 98 +/- 2 28 +/- 2 4 +/- 0.5 4 +/- 0.5 Kindled (12) 71 +/- 3 94 +/- 2 40 +/- 3* 4 +/- 0.3 6 +/- 0.5* Note: RMP = rest ing membrane p o t e n t i a l ; SA = spike amplitude; R n = input res is tance; T e = membrane time constant measured during early (< 3 ms) port ion of charging p r o f i l e ; T^ = membrane time constant measured af ter 3 ms of charging p r o f i l e and ; * = p < 0.01 as determined by a two-t a i l e d Student t - t e s t . 21 1 F i g . 5.8. I n t r a c e l l u l a r records from a contro l and 'k ind led ' granule c e l l show that no s i g n i f i c a n t di f ferences occurred in the monosynaptic EPSP evoked by the perforant path. Furthermore, the spike amplitudes and depolar iz ing a f t erpotent ia l s were the same (A). The increase in R n associated with the kindled preparation is shown by the lower currents required in the kindled granule c e l l (42 Mohms) to reach the equivalent voltages of the contro l neuron (28 Mohms). The contro l granule c e l l displayed a s ingle membrane time constant (4 ms), whereas two time constants were required to define the kindled granule c e l l ( T e = 4 ms; TT_ = 8 ms). The time constants were determined for the most hyperpolarized voltage responses. 212 Control Kindled A. j20mV 10 ms B. I nA JlOmV 10 ms 5-1 *0 5-1 r o ms -5-< ~ - i r ico o ms 100 213 (+/- SEM) input resistance (R n ) of 'k ind led ' granule c e l l s (40 +/- 3 Mohms) was s i g n i f i c a n t l y (p < 0.01) greater than that of controls (28 +/- 2 Mohms). Analys is of the charging p r o f i l e s a l so indicated a s i g n i f i c a n t a l t e r a t i o n in the membrane time constant. Control neurons exhibited a s ingle T c (4 ms) as shown by the l i n e a r i t y of the T c p lot (Fig 5.8 C ) . In contras t , 'k ind led ' granule c e l l s exhibi ted two time constants ( T e , 4 ms; T j , 6 ms; F i g . 5.8 C ) . The T]_ appeared to be both voltage- and time-dependent, but these aspects were not character ized . 5.4. Discussion The present study demonstrates that commissural-k ind l ing enhances the i n h i b i t o r y processes of the dentate gyrus, as measured by paired-pulse s t imulat ion (Tuff et a l . , 1983a). In add i t i on , k ind l ing apparently a l t ered the input resistance and the time constants of granule c e l l s . L a s t l y , th i s study i l l u s t r a t e d the usefulness of the in v i t r o preparation in studying the pathology of chronic disease s tates . The f inding that dentate gyrus ADs show a progressive lengthening and reduction in threshold with the development of k ind l ing points out the enhanced e x c i t a b i l i t y of th i s CNS structure (Racine, 1972a). It i s paradoxica l , therefore , that the paired pulse data indicate an augmentation of the 214 i n h i b i t i o n in the dentate gyrus (Racine et a l . , 1983; Tuff et a l . , 1983). Paired-pulse s t imulat ion of the perforant path in normal preparations evokes a population spike that responds with an IPI sequence at C-T in terva l s 20, 80, and 400 ms (McNaughton and Barnes, 1977; Racine et a l . , 1983; Tuff et a l . , 1983a). Following k ind l ing the PS exhibited i n h i b i t i o n at a l l C-T in terva l s ( F i g . 5 .2) . The increase in i n h i b i t i o n depended on the evocation of ADs, but not on the presence of motor seizures (F ig . 5 .5) . When k indl ing s t imul i f a i l e d to induce ADs a reduction in paired pulse f a c i l i t a t i o n was observed at a C-T i n t e r v a l of 80 ms. This attenuation was always less than that evoked with ADs, but i s s imi lar to the decrease in paired pulse potent iat ion noted af ter the induction of LTP in the dentate gyrus (Berger et a l . , 1984). Given our k indl ing stimulus parameters (10~ 4 A, 60 Hz, 1 s) i t i s l i k e l y that LTP was present in the animals not exh ib i t ing ADs. Thus, i t i s poss ible that k ind l ing and LTP potentiate i n h i b i t o r y systems in the DG, and that k indl ing has a greater effect than LTP because i t involves more neurons in the process. In order to assess th i s p o s s i b i l i t y , i t i s e s sent ia l to understand the mechanism(s) subserving the a l t ered i n h i b i t i o n . Based on the s imi lar time courses of the GABA-mediated and the k indl ing- induced i n h i b i t i o n s , i t was postulated that k ind l ing produced i t s ef fect by augmenting the GABA-mediated Cl~-dependent i n h i b i t i o n (Tuff et a l . , 1983a). In support of t h i s , the number of hippocampal benzodiazepine receptors 215 increase subsequent to k ind l ing (McNamara et a l . , 1980; Niznik et a l . , 1984; Tuff et a l . , 1983b), and i t has been previously shown that diazepam f a c i l i t a t e s GABA-mediated i n h i b i t i o n in the dentate gyrus (Adamec et a l . , 1981). Our resu l t s d id not substantiate t h i s hypothesis , since reducing the GABA-mediated i n h i b i t i o n with perfusion of a low [ C l ] c containing medium did not completely reverse the ef fect of k i n d l i n g . Hence, the increased i n h i b i t i o n in the dentate gyrus fol lowing k indl ing may be due to e i ther an enhancement of a chloride-independent i n h i b i t i o n or a decrease in the a b i l i t y of the perforant path-granule c e l l synapse to potent ia te . In support of the former, the increase in benzodiazepine receptors found to occur with k indl ing may enhance a calcium-mediated potassium conductance (gK^ a) in granule c e l l s , since perfusing low concentrations (nM) of the benzodiazepine, midazolam, increased th i s conductance in hippocampal CA1 pyramidal c e l l s (Carlen et a l . , 1983). Furthermore, the kindl ing- induced progressive loss of a neuron-speci f ic calc ium-binding protein (CaBP) l o c a l i z e d to the dentate gyrus granule c e l l s (Baimbridge and M i l l e r , 1984; M i l l e r and Baimbridge, 1983) may contribute to an increase in the g K ^ a . That i s , the kindl ing- induced loss of CaBP has been associated with enhanced i n t r a c e l l u l a r l eve l s of C a 2 + (Mody and M i l l e r , 1983). Since the g K C a in molluscan neurons is dependent on the i n t r a c e l l u l a r concentration of C a 2 + ([Ca]^) (Gorman and Thomas, 1980), the e l iminat ion of 216 an i n t r a c e l l u l a r C a 2 + buffer (CaBP) may increase the [Ca]^ re su l t ing in an augmented gKfj a . Therefore, the k i n d l i n g -induced increase in paired pulse i n h i b i t i o n may be re lated to an e a r l i e r a c t i v a t i o n of the gK^ a in response to enhanced [Ca]j caused by the increase in benzodiazepine receptors and the loss of CaBP. One argument against the role of CaBP in the enhanced i n h i b i t i o n is that the i n h i b i t i o n increases p r i o r to a s i g n i f i c a n t decrease in CaBP leve ls (Baimbridge and M i l l e r , 1984; Mody and O l i v e r , unpublished observat ion) . As an a l t ernat ive explanation the increased i n h i b i t i o n may resu l t from the augmentation of the mechanisms mediating the Cl~-independent late a f terhyperpolar izat ion (LHP) exhibited by granule c e l l s (Thalmann and Aya la , 1982, see Chapters 3, 4) . Although a l t era t ions in the above processes may explain the enhanced i n h i b i t i o n , a reduction in the mechanisms underlying f a c i l i t a t i o n in the dentate gyrus cannot be excluded. The observation that the kindled preparation exhibi ted paired pulse potent iat ion of the PS in low [ C l ] 0 medium suggests that the mechanisms subserving f a c i l i t a t i o n were s t i l l present fol lowing k i n d l i n g . Normally th i s observation would provide s u f f i c i e n t proof that a depression in the potent iat ing mechanisms d id not occur; however, potent iat ion of the dentate gyrus PS appears to depend on several processes, one being heterosynaptic (Lomo, 1971b; Chapter 4). Thus, c l a r i f i c a t i o n of the role that e i ther reduced potent iat ion or enhanced i n h i b i t i o n plays in the 217 a l t ered e x c i t a b i l i t y of the dentate gyrus fol lowing k indl ing w i l l have to wait u n t i l a l l the processes contr ibut ing to the potent iat ion and i n h i b i t i o n of the granule c e l l s are understood and can be independently manipulated. Another f inding in th i s study was that k ind l ing a l t ered the granule c e l l membrane such that increases in both the R n and T c were observed. These changes in membrane propert ies are not general ized to a l l hippocampal neurons, since CA1 pyramidal c e l l s d id not show s imi lar a l t e r a t i o n s (Ol iver and M i l l e r , 1985b; Chapter 6). In a d d i t i o n , the morphological changes in granule c e l l s which could account for these membrane e f fects do not occur with k indl ing (Crandal l et a l . , 1979; Goddard and Douglas, 1975; Racine et a l . , 1975). The experimental protocol used in th i s study does not allow one to determine whether the changes in the membrane propert ies are re lated to a l t e r a t i o n s in passive or act ive (e .g . voltage-dependent) events; therefore , further i n t r a -c e l l u l a r charac ter i za t ion of 'k ind led ' granule c e l l s w i l l be required . While the funct ional s ign i f i cance of the a l t e r a t i o n s in i n h i b i t o r y processes and granule c e l l membrane propert ies remains unclear , i t i s paradoxical that neuronal t i ssue predisposed to seizure a c t i v i t y exhib i t s an enhanced e f f i cacy to suppress granule c e l l d ischarge. This change may r e f l e c t a compensatory mechanism which prevents further or continuous ep i l ept i form discharge of the hippocampus proper. From a t e l e o l o g i c a l viewpoint, i t makes sense to have the 218 primary pathway into a s tructure prone to ep i l ept i form a c t i v i t y , such as the hippocampus, pass through a system which acts to l i m i t or f i l t e r the high-frequency bursts which would promote seizure a c t i v i t y . It may be equally important in t h i s scheme to have t h i s f i l t e r i n g structure enhance i t s dampening ef fect as the other s i t e becomes increas ingly susceptible to epi leptogenic behavior. This argument may be supported by the 2-deoxyglucose study which showed that ep i l ept i form a c t i v i t y e l i c i t e d in the medial entorhinal cortex did not necessar i ly propagate past the dentate gyrus into the hippocampus (Co l l ins et a l . , 1983), and by the present study which i l l u s t r a t e d that the augmented i n h i b i t i o n occurs ear ly in the development of k i n d l i n g . It i s poss ible that th i s function of the dentate gyrus also makes necessary the greater number of s t imul i required to kindle an animal v ia the hippocampus, as compared with other s tructures (Goddard et a l . , 1969; Madryga et a l . , 1975), as well as suppressing i n t e r i c t a l spikes within the hippocampus ( K a i r i s s et a l . , 1984). Further research is necessary to uncover the mechanism(s) subserving the enhanced i n h i b i t i o n and i t s role in regulat ing or promoting epi leptogenes is . 219 Chapter 6 Inhib i tory processes of hippocampal CA1 pyramidal neurons fol lowing kindl ing- induced epi lepsy in the rat 6.1. Introduction A loss of i n h i b i t i o n in the centra l nervous system has been suggested to be a major factor contr ibut ing to the generation of ep i l ept i form a c t i v i t y (Meldrum, 1975; Walker, 1983). In the hippocampal formation, one of the most seizure prone regions of the brain (Liberson and Akert , 1953), neuronal i n h i b i t i o n is mostly due to a recurrent ly ac t ivated GABA-mediated increase in ch lor ide conductance (Alger and N i c o l l , 1982a; Al l en et a l . , 1977; Andersen et a l . , 1982; Curt i s et a l . , 1970; Dingledine and Langmoen, 1980). S i g n i f i c a n t l y , in the presence of GABA antagonists which diminish or block the i n h i b i t o r y postsynaptic po ten t ia l (IPSP), hippocampal pyramidal neurons exhibi t large spontaneous depolar izat ions and mult ip le spike discharges ( i . e . , paroxysmal depo lar iza t ion sh i f t s ) which are considered the hallmarks of ep i l ept i form a c t i v i t y (Dingledine and Gjers tad , 1980; Schwartzkroin and Pr ince , 1980; Yamamoto and Kawai, 1968). Furthermore i t has been shown that r e p e t i t i v e s t imulat ion of cer ta in hippocampal pathways reduces the chloride-dependent IPSP p r i o r to 220 inducing seizure a c t i v i t y (Ben-Ari et a l . , 1979; Sawa et a l . , 1963; Spencer and Kandel , 1969). Conversely, c l i n i c a l l y used anticonvulsants ( e . g . , barbi turates and benzodiazepines) potentiate GABA-mediated i n h i b i t i o n (Alger and N i c o l l , 1982b; Costa et a l . , 1976; Wolf and Haas, 1977), as well as retard or block the development of kindled seizures (Albertson et a l . , 1981; Racine et a l . , 1975). Although i t i s evident from these data that an acute reduction in the e f f i cacy of synaptic i n h i b i t i o n plays some role in the t r igger ing of seizure responses, i t i s not c lear whether th i s i s a contr ibut ing factor to the chronic pred i spos i t ion of neuronal t i ssue to ep i l ept i form a c t i v i t y . Several studies have indicated that with the establishment of chronic seizure foc i there i s a s i g n i f i c a n t decrease in cerebra l c o r t i c a l l eve l s of glutamic ac id decarboxylase, an enzyme required for the synthesis of GABA, and in the number of GABA-containing nerve terminals which synapse on c o r t i c a l pyramidal neurons (Bakay and H a r r i s , 1981; Ribak et a l . , 1982). However, s imi lar a l t era t ions have not been observed when using k ind l ing as an experimental model of epi lepsy (Kalichman, 1982). In fac t , recent e l e c t r o p h y s i o l o g i c a l evidence suggests that GABA-mediated and poss ib ly other i n h i b i t o r y processes are enhanced, rather than diminished, fol lowing k ind l ing (Fuj i ta et a l . , 1983; O l i v e r and M i l l e r , 1985; Tuff et a l . , 1983a). In order to examine more c l o s e l y the poss ib le long-term a l t e r a t i o n s in i n h i b i t i o n which may be associated with 221 F i g . 6.1. I l l u s t r a t i o n of the transverse hippocampal s l i c e showing the placements of the s t imulat ing electrodes in the stratum radiatum (SR) and alveus (Alv) which evoked the c h a r a c t e r i s t i c i n t r a c e l l u l a r EPSP-IPSP and IPSP responses recorded from CA1 pyramidal neurons, re spec t ive ly . 222 223 epi leptogenes is , we have invest igated the membrane propert ies and i n h i b i t o r y processes of CA1 pyramidal neurons using hippocampal s l i c e s prepared from commissural-kindled r a t s . Moreover, since recent evidence suggests the presence of a feed-forward i n h i b i t i o n in the CA1 (Alger, 1984; Alger and N i c o l l , 1982a; Buzsaki and E i d e l b e r g , 1982; Frotscher et a l . , 1984; Knowels and Schwartzkroin, 1981; Lynch et a l . , 1981), we compared the frequency response c h a r a c t e r i s t i c s of the GABA-mediated recurrent IPSP with those of the feed-forward i n h i b i t o r y mechanism(s). 6.2. Methods Hippocampal s l i c e s were prepared from age-matched contro l and commissural-kindled rats which had a minimum of 5 motor seizures (range, 5-55). Bipolar s t imulat ing e lectrodes were pos i t ioned within the CA1 hippocampal region in e i ther the stratum radiatum (SR), to act ivate pyramidal neurons v ia the Schaffer c o l l a t e r a l s , or the alveus (Alv) , to evoke the recurrent i n h i b i t o r y pathway putat ive ly mediated by basket c e l l interneurons ( F i g . 6 . 1 . ) . Standard i n t r a c e l l u l a r techniques were u t i l i z e d to obtain res t ing membrane potent ia l (RMP), input res is tance ( R n ) , and spike amplitude (SA). The change in neuronal res is tance associated with the IPSP ( R i p S p ) » was ca lcu la ted from current-vol tage (I-V) p r o f i l e s in which the IPSP was evoked during the current i n j e c t i o n . Since the IPSP has 224 Table 6.1 Membrane propert ies of CA1 pyramidal neurons recorded from contro l and kindled preparations (mean +/-SEM) . RMP (-mV) R n (M ohms) SA (mV) T c (ms) Control 67 +/- 1 29 +/- 1 91 +/- 1 12 +/- 1 (n) (25) (25) (18) (11) Kindled 66 +/- 1 31 +/- 1 87 +/- 2 13 +/- 1 (n) (19) (22) (20) (7) Note : RMP = rest ing membrane p o t e n t i a l ; R n = input res is tance; SA = spike amplitude; T c = membrane time constant. 225 been shown to reverse asymmetrically (Dingledine and Langmoen, 1980), a l l voltage measurements for ^ipsp were taken at a f ixed latency defined by the peak of the hyperpolar iz ing p o t e n t i a l . Membrane time constants (T c ) were ca lcu la ted from voltage p r o f i l e s generated at a s ingle current in tens i ty (-0.2 to -0.6 nA). D i f f e r e n t i a t i o n of the voltage p r o f i l e at 10~6 s in terva l s was done by computer; the natural logarithm of each sampling period was then p lo t ted versus time and the negative slope of the best f i t t i n g l i n e , as ca lcu la ted by l inear -regres s ion a n a l y s i s , was taken as the T c . Inhib i tory processes inf luencing CA1 pyramidal neurons of contro l and kindled preparations were examined using two procedures: 1) depolar izat ion- induced pyramidal a c t i v i t y was observed during and fol lowing s ingle pulse or frequency (5,-10 and 20 Hz; 35-400 pulses) s t imulat ion of the SR and Alv ; and 2) changes in the IPSP amplitudes were monitored during and subsequent to frequency st imulat ion of the recurrent i n h i b i t o r y pathway. In order to determine R i p S p during frequency s t imula t ion , current pulses were injected during the l a t t e r port ion of the stimulus t r a i n (10-15 pulses ) , to minimize electrode a r t i f a c t s produced by continued and rapid current passage. 6.3. Results 226 F i g . 6.2. Comparison between contro l and kindled CA1 pyramidal c e l l membrane proper t i e s . (A) Threshold s t imulat ion of SR evokes a s imi lar EPSP, spike discharge and a f t e r p o t e n t i a l sequence in both preparat ions . (B) Corresponding current-voltage p r o f i l e s and time constant p lo ts (C) indicate that k ind l ing does not induce a l t e r a t i o n s in the membrane propert ies of CA1 pyramidal c e l l neurons (see Table 6.1 . ) . 227 227 228 I n t r a c e l l u l a r recordings were obtained from 25 contro l and 22 'k ind led ' CA1 pyramidal neurons. No s i g n i f i c a n t di f ferences in membrane propert ies ( i . e . , RMP, R n , T c ) or SA were observed between these two neuronal populations (Table 6 . 1 . ) . St imulation of SR evoked the c h a r a c t e r i s t i c EPSP, spike discharge, and a f t e r p o t e n t i a l sequence of CA1 pyramidal c e l l s and in no case were mult ip le spikes present in e i ther preparation ( F i g . 6.2. A ) . T y p i c a l I-V p r o f i l e s and T c p lots are shown for both contro l and 'k ind led ' neurons ( F i g . 6.2. B, C ) . Subthreshold st imulat ion of SR resul ted in s imi lar periods of i n h i b i t i o n on depolar izat ion- induced neuronal discharge for contro l (241 +/- 70 ms, n=5) and kindled (238 +/- 43 ms, n=5) preparations (mean +/- SEM; F i g . 6.3. A).. The recurrent IPSP's, evoked by alvear s t imulat ion , were comparable between the groups ( contro l , 173 +/- 50 ms, n=5; k indled , 140 +/- 39 ms, n=7; F i g . 6.3. B) . Repet i t ive s t imulat ion (5, 10 and 20 Hz) de l ivered to e i ther SR or Alv f a i l e d to demonstrate any c h a r a c t e r i s t i c d i f ferences between the i n h i b i t o r y processes modulating pyramidal c e l l discharge in contro l and kindled preparat ions . Repet i t ive a c t i v a t i o n of SR p a r t i a l l y or completely i n h i b i t e d depolar izat ion- induced neuronal discharge for the duration of the stimulus t r a i n (range, 0.5-20 s; n=8) in both preparat ions . During the stimulus t r a i n (20 Hz, 10 s) the SR-evoked hyperpolar izat ion was not reduced in amplitude, since termination of the stimulus 229 F i g . 6.3. Inh ib i t i on evoked by subthreshold s t imulat ion of SR or Alv on depolar izat ion- induced discharge of CA1 pyramidal c e l l s from contro l and kindled preparat ions . The duration of i n h i b i t i o n for SR and Alv st imulat ion was: (mean +/- SEM) c o n t r o l , 241 +/- 70 ms; 173 +/- 50 ms; k indled , 238 +/- 43 ms; 140 +/- 39 ms, respec t ive ly . No s i g n i f i c a n t changes were found between the two experimental groups. 230 231 t r a i n uncovered a prolonged hyperpolar izat ion capable of i n h i b i t i n g c e l l u l a r spiking for periods of up to 3 s ( F i g . 6 .4 .pA, B) . Therefore, the burst of act ion potent ia l s observed towards the end of the stimulus t r a i n ( F i g . 6.4. A; arrow), must have resulted from the frequency potent iat ion of the EPSP (Alger and Tey ler , 1976; Creager et a l . , 1980). In contrast , r epe t i t i ve s t imulat ion of the A l v , regardless of experimental group, reduced the amplitude of the recurrent IPSP (F ig . 6.4. C, D) . In some instances the diminished IPSP resulted in the discharge of the pyramidal c e l l s . Furthermore, the termination of the Alv st imulat ion d id not produce a prolonged hyperpo lar iza t ion; rather , the pyramidal neurons immediately discharged with an increased f i r i n g rate over baseline values ( i . e . , rebound exc i ta t i on ; F i g . 6.4. D). In order to examine more c lo se ly the reduction in the amplitude of the recurrent IPSP associated with repe t i t i ve Alv a c t i v a t i o n , ind iv idua l sweep records taken before and during the la s t few pulses of a 100 pulse t r a i n were analyzed from non-discharging pyramidal c e l l s . As shown in F i g . 6 .5 . , two types of responses were observed from both contro l and kindled preparat ions . The f i r s t showed no s i g n i f i c a n t s h i f t in IPSP amplitude; however, there was a 2-3 mV hyperpolar izat ion (arrow) superimposed on the response during the stimulus t r a i n ( F i g . 6.5. A ) . This response was observed more commonly when lower s t imulat ion frequencies were used and the l eve l of membrane hyperpolar izat ion 232 F i g . 6 . 4 . Inh ib i t ion of the depolar izat ion- induced discharge of CA1 pyramidal c e l l s by 20 Hz st imulat ion of SR and Alv in contro l and kindled preparat ions . I n h i b i t i o n of the neuronal discharge occurred during r e p e t i t i v e a c t i v a t i o n of SR (A,B) . However, in some instances an increase in discharge rate was observed during the course of the frequency t r a i n (A, arrow), even though, a maintained hyperpolar izat ion (< 5 s) was present fol lowing cessat ion of the te tani (A,B) . 20 Hz st imulat ion of the Alv reduced the amplitude of the IPSP but does not necessar i ly cause an increase in the c e l l discharge rate (C,D) . A l s o , note that termination of Alv s t imulat ion resul ted in an immediate increase in discharge rate of the pyramidal c e l l ( i . e . , rebound e x c i t a t i o n ) . 233 234 appears to depend on the stimulus in ter -pulse i n t e r v a l (cf . A l l e n et a l . , 1 977) . The second response exhibi ted by the IPSP was character ized by a s imi lar membrane hyperpolar izat ion (arrow) as described above. In th i s case, the IPSP amplitude was diminished to the l e v e l of the maintained hyperpolar izat ion ( F i g . 6.5. B) . The net ef fect of the r e p e t i t i v e st imulat ion was to 'clamp' the membrane potent ia l in a hyperpolarized state (cf . Andersen and Lomo, 1969). A comparable analys is of the SR-evoked i n h i b i t i o n could not usual ly be made due to frequency potent iat ion of the EPSP. However, in the few cases where th i s d id not appear to be a problem, the l ong- la s t ing hyperpolar izat ion remained unaffected or was s l i g h t l y enhanced by r e p e t i t i v e a c t i v a t i o n . To determine whether a change in res istance was associated with the reduced Alv-evoked IPSP, values for R i p s p w e r e ca lcu la ted before, during and fol lowing frequency s t imulat ion in both contro l and kindled preparat ions . Although i n d i v i d u a l neurons from both experimental groups exhibi ted reproducible a l t e r a t i o n s in R ; _ c _ ( i . e . , increased, decreased or unchanged), there was no s i g n i f i c a n t d i f ference in the grouped data between contro l R i p S p values and those measured during r e p e t i t i v e s t imula t ion . In a d d i t i o n , no di f ferences were detected in the IPSP response between contro l and kindled preparations (Table 6 . 2 . ) . An example of a CA1 neuron whose RjnsD remained unaltered 235 F i g . 6.5. Two t y p i c a l responses of the Alv-evoked IPSP to 20 Hz st imulat ion are shown for contro l and kindled preparat ions . Records are i n d i v i d u a l sweeps from d i f f erent pyramidal c e l l s before and during (arrow) frequency s t imulat ion . (A) i l l u s t r a t e s no s i g n i f i c a n t reduction in the IPSP amplitude (measured from pre-st imulus base l ine) ; whereas, (B) showed a reduced IPSP that 'clamps' the membrane at a hyperpolarized l e v e l . The s h i f t in membrane potent ia l during the r e p e t i t i v e ac t iva t ion of the Alv i s dependent on the inter-s t imulus i n t e r v a l . 236 Control Kindled A. B. ,_J 2 m V 1 0 ms j 5 m V 10 ms 237 Table 6.2. The e f f e c t s of 10 Hz (100 p u l s e s ) Alv s t i m u l a t i o n on the amplitude and r e s i s t a n c e of the IPSP. IPSP (-mV) Ripsp (M ohms) PRE DURING PRE DURING C o n t r o l 4 +/- 1 0.6 +/- 1 16 +/- 1 15 +/- 1 (n) (9) (5) (14) (7) K i n d l e d 4 +/- 1 0.5 +/- 1 16 +/- 1 15 +/- 1 (n) (12) (6) (13) (7) Note: IPSP = i n h i b i t o r y post s y n a p t i c p o t e n t i a l ; R^ = neuronal r e s i s t a n c e a s s o c i a t e d with IPSP. 238 during frequency (10 Hz) s t imulat ion is shown in F i g . 6.6. Control s t imulat ion (0.1 Hz) of the alveus evoked an IPSP that was associated with a 38% reduction in membrane res is tance (A; R n , 32 M ohms; Ripgp, 20 M ohms) and further increases in the s t imulat ion frequency did not a l t e r the R i p s p v a l u e (B)• 6.4 Discussion The resu l t s of the present study indicate that commissural-kindling is not corre la ted with any s i g n i f i c a n t long-term a l t e r a t i o n s in e i ther membrane propert ies or i n h i b i t o r y processes associated with the CA1 pyramidal neurons. These data are inconsistent with the hypothesis that a chronic reduction of i n h i b i t o r y mechanisms may be a contr ibut ing factor to the pred i spos i t ion of neuronal t issue to ep i l ept i form a c t i v i t y . The p o s s i b i l i t y that transient reductions of i n h i b i t i o n , associated with the k ind l ing st imulus, are required for the i n i t i a t i o n of a seizure cannot be excluded. In th i s respect , we examined the frequency response of the A l v - and SR-evoked i n h i b i t o r y processes from both experimental groups and found that k i n d l i n g d id not change the short-term a l t e r a t i o n s produced by r e p e t i t i v e a c t i v a t i o n . It was observed, however, that Alv and SR i n h i b i t o r y mechanisms responded d i f f e r e n t i a l l y to frequency s t imula t ion . 239 F i g . 6.6. Representative example of current-vol tage p r o f i l e s with an Alv-evoked IPSP act ivated during the current in j ec t ion and associated p lots from a CA1 pyramidal neuron (A) before, (B) during 10 Hz st imulat ion of the alveus, and (C) 1 min fol lowing termination of the r e p e t i t i v e s t imulat ion . Note that no s i g n i f i c a n t a l t e r a t i o n s in R n or ^ipsp occurred during the frequency st imulat ion (see Table 6 . 2 . ) . 240 241 Repet i t ive ac t iva t ion of the Alv progress ive ly reduced the amplitude of the recurrent IPSP (cf. A l l e n et a l . , 1977; Andersen and Lomo, 1969; McCarren and Alger , 1985), but, the expected concommitant reduction in the conductance of the IPSP was not cons i s tent ly observed (Ben-Ari et a l . , 1979; McCarren and Alger , 1985). In add i t i on , the reduction of the Alv-evoked IPSP never resul ted in the i n i t i a t i o n of ep i l ept i form po ten t ia l s . These re su l t s d i f f e r from those of Ben-Ari et a l . (1979), who found that when the entorhinal cortex or fornix were r e p e t i t i v e l y stimulated the re su l t ing IPSP, recorded in CA3 pyramidal neurons, exhibi ted a reduced amplitude and conductance which led to seizure generation. While these c o n f l i c t i n g resu l t s may r e f l e c t a general di f ference between the i n h i b i t o r y processes modulating CA1 and CA3 pyramidal c e l l a c t i v i t y , i t i s poss ible that the di f ference may be a t t r ibuted to the contamination of the CA3 pyramidal neuron's recurrent IPSP with other potent ia l s (Andersen and Lomo, 1967; Spencer and Kandel, 1961). In support of the l a t t e r , the repe t i t i ve a c t i v a t i o n of the CA3 recurrent i n h i b i t i o n , in the absence of contaminating potent ia l s ( i . e . , deafferentat ion of the f o r n i x ) , led to an ' i n h i b i t o r y ' seizure which was character ized by the membrane po tent ia l of the CAS pyramidal c e l l remaining hyperpolarized throughout the duration of the stimulus t r a i n (Kandel and Spencer, 1961b; and c f . Andersen and Lomo, 1969). This i s in marked contrast to the resu l t s of Ben-Ari et a l . (1979), but i s s imi lar to the 'clamping' 242 of the membrane potent ia l we observed in CA1 neurons during frequency st imulat ion of the A l v . Thus, i t appears that a reduction in the recurrent , GABA-mediated IPSP is by i t s e l f i n s u f f i c i e n t to produce ep i lept i form a c t i v i t y . A l t e r n a t i v e l y , one must postulate that a f a i l u r e in other i n h i b i t o r y processes occurs or that a l t e r a t i o n s in both i n h i b i t o r y and exc i tatory events, associated with orthodromic a c t i v a t i o n , play a role in epi leptogenesis (Dingledine and Gjers tad , 1980; Schwartzkroin and Wyler, 1980). In th i s context, the dif ferences in the frequency response c h a r a c t e r i s t i c s of the A l v - and SR-evoked i n h i b i t o r y mechanisms may be s i g n i f i c a n t . In contrast to Alv s t imulat ion , r e p e t i t i v e ac t iva t ion of SR resulted in a prolongation of i n h i b i t i o n , as indicated by the l ong- la s t ing hyperpolar izat ion (LHP) fol lowing the stimulus t r a i n ( F i g . 6 .5 . ) . Although we d id not invest igate the nature of th i s i n h i b i t i o n , the duration (3 s) indicates that i t may be e i ther the calcium-dependent potassium conductance (Brown and G r i f f i t h , 1983a) or the slow hyperpolar iz ing synaptic po tent ia l (Alger, 1984) c h a r a c t e r i s t i c of these c e l l s . In add i t i on , an increase in neuronal e x c i t a b i l i t y was occas ional ly observed during frequency s t imulat ion ( F i g . 6 .5 . , arrow) despite the presence of the LHP. These observations suggest that e i ther frequency potent iat ion of an EPSP occurred or that the recent ly demonstrated feed-forward dendr i t i c i n h i b i t i o n , inf luenc ing CA1 pyramidal neurons, was reduced during 243 r e p e t i t i v e st imulat ion (Alger and N i c o l l , 1982a,b; Wong and Watkins, 1982). In view of the observation by Wong and Watkins (1982) that prolonged frequency st imulat ion of SR reversed the GABA-mediated feed-forward i n h i b i t i o n to a depo lar iz ing p o t e n t i a l , the. increase in neuronal e x c i t a b i l i t y i l l u s t r a t e d during the l a t t e r phase of the r e p e t i t i v e t r a i n , may r e f l e c t th i s same change ( F i g . 6.5) . It i s noteworthy that the LHP was incapable of preventing the neuronal discharge during the r e p e t i t i v e s t imulat ion , implying that a p h y s i o l o g i c a l l y potent exc i tatory event i s i n i t i a t e d by prolonged synaptic a c t i v i t y . In conclus ion, the resu l t s of the present study are inconsistent with the hypothesis that a chronic reduction of i n h i b i t o r y processes underl ie the enhanced s e n s i t i v i t y to ep i l ept i form behavior associated with k i n d l i n g . In a d d i t i o n , frequency st imulat ion of the Alv or SR did not induce s e i z u r e - l i k e a c t i v i t y in CA1 pyramidal neurons recorded from the kindled preparat ions , ind ica t ing that the mechanisms which subserve the k ind l ing phenomenon are located outside the CA1 region. In th i s regard, biochemical (Baimbridge and M i l l e r , 1984; M i l l e r and Baimbridge, 1983) and e l e c t r o p h y s i o l o g i c a l (Fuj i ta et a l . , 1983; O l i v e r et a l . , 1983; O l i v e r and M i l l e r , 1985; Tuff et a l . , 1983a) corre la t e s of k ind l ing indicate s i g n i f i c a n t a l t e r a t i o n s in other hippocampal regions, p a r t i c u l a r l y the dentate gyrus. Paradox ica l ly , these e l e c t r o p h y s i o l o g i c a l studies provide evidence of enhanced i n h i b i t i o n ; th i s may r e f l e c t a 244 compensatory change in neuronal processes which serve to suppress, rather than augment, the pred i spos i t ion of these c e l l s to epi leptogenesis . 245 Chapter 7 Summary and Discussion 7.1 Summary The importance of presynaptic modulation of neurotransmitter release in a l t e r a t i o n s of synaptic e f f i cacy produced by r e p e t i t i v e afferent s t imulat ion i s well documented. In the mammalian CNS most studies have u t i l i z e d e x t r a c e l l u l a r techniques to examine the poss ible mechanisms that may subserve synaptic p l a s t i c i t y . Unfortunately, th i s method of analys i s i s unre l iab le in determining the contr ibut ion that e i ther pre- or postsynaptic processes make to the changes in c e l l u l a r e x c i t a b i l i t y . Therefore, the present ser ies of experiments were designed to: 1) corre la te e x t r a c e l l u l a r synaptic potent ia l s with i n t r a c e l l u l a r events; 2) assess the extent to which postsynaptic mechanisms p a r t i c i p a t e in the a l t e r a t i o n of synaptic and granule c e l l e x c i t a b i l i t y during paired-pulse s t imulat ion; and 3) determine i f the kindl ing- induced enhancement in granule c e l l e x c i t a b i l i t y modifies i n t r a c e l l u l a r propert ies or i n h i b i t o r y processes in the dentate granule c e l l s and CA1 pyramidal neurons. 246 I n t r a c e l l u l a r analys i s determined that two populations of granule c e l l s may ex is t which may be d i s t inguished by the ir I-V r e l a t i o n s h i p . Approximately f i f t y percent of granule c e l l s exhibited AR in response to both depo lar iz ing and hyperpolar iz ing current i n j e c t i o n , whereas the other c e l l s displayed a l inear I-V p r o f i l e . The AR induced by membrane hyperpolar izat ion appeared to be mediated by a potassium channel, since increasing [ K ] Q or adding [ B a ] c enhanced and attenuated th i s phenomenon, re spec t ive ly . Neurons d i sp lay ing AR also maintained s i g n i f i c a n t l y greater RMP and AP amplitude values when compared to the granule c e l l s which exhibited a l i n e a r I-V p r o f i l e . These l a t t e r di f ferences suggest that the absence of AR in some granule c e l l s may be related to poor electrode impalement; thus, further experiments w i l l be necessary to c l a r i f y the existence of subpopulations of these neurons. Perforant path st imulat ion evoked a t y p i c a l monosynaptic EPSP followed by a DAP and LHP. The DAP has been shown to be p a r t i a l l y GABA-mediated and Cl~-dependent; whereas, the LHP is most l i k e l y K +-dependent (Thalmann and Ayala , 1982). Based on the attenuation of the DAP and LHP during perfusion of e i ther a reduced [Ca] Q and ra ised [Mg]Q medium or baclofen, i t was postulated that these a f t e r p o t e n t i a l s were d i - s y n a p t i c a l l y ac t ivated v i a interneurons. The fact that baclofen decreased the LHP without inducing a postsynaptic hyperpolar izat ion in granule c e l l s implies that GABA, act ing on granule c e l l GABAB 247 receptors , i s not re la ted to the processes subserving the LHP. Given the evidence for c o - l o c a l i z a t i o n of neuropeptides in dentate GABAergic interneurons (Kosaka et a l . , 1985), i t i s poss ib le that the LHP depends on the release of these transmit ters ; however, experimental evidence i s lacking on th i s po int . The contr ibut ion that the DAP and the LHP make to paired-pulse phenomena of the PP-granule c e l l unit was examined in Chapter 4. The DAP was shown to have c o n f l i c t i n g e f fects on the test EPSP. F i r s t l y , homo- or heterosynaptic EPSPs summed with the DAP to promote f a c i l i t a t i o n . Secondly, by v i r tue of i t s membrane d e p o l a r i z a t i o n , the DAP attenuated the test EPSP by reducing the di f ference between the membrane potent ia l and the E e p S p . This l a t t e r ef fect may be responsible for the apparent depression of the e x t r a c e l l u l a r EPSP during periods of i n t r a c e l l u l a r augmentation. The fact that i n t r a c e l l u l a r EPSPs potent iated, explains the increase in the p r o b a b i l i t y of granule c e l l discharge during periods of e x t r a c e l l u l a r EPSP attenuation ( i . e . , uncoupling) . In contrast to the f a c i l i t a t i n g influence of the DAP, the LHP was shown to i n h i b i t propagating EPSPs and granule c e l l APs. Although both the DAP and LHP played s i g n i f i c a n t roles in the expression of the paired-pulse phenomenon in granule c e l l s , there was evidence that presynaptic regulat ion of transmitter release contributed to the a l t e r a t i o n s in granule c e l l e x c i t a b i l i t y . For example, in the absence of a DAP the test EPSP exhibited potent iat ion 248 from a hyperpolarized membrane p o t e n t i a l , suggesting that the DAP was not e ssent ia l for the i n t r a c e l l u l a r augmentation of test EPSPs. Therefore, the present study demonstrated the importance of pre- and postsynaptic processes in modulating the a l t e r a t i o n s in granule c e l l e x c i t a b i l i t y in response to paired-pulse a c t i v a t i o n . The e f fects of k ind l ing on both neuronal and i n h i b i t o r y mechanisms of dentate granule c e l l s and CAT pyramidal neurons were examined in Chapters 5 and 6, re spec t ive ly . Kind l ing apparently a l t ered the membrane propert ies of granule c e l l s such that they maintained greater input resistances and time constants. In a d d i t i o n , the i n h i b i t o r y processes measured with paired-pulse s t imulat ion appeared to be enhanced in the dentate gyrus fol lowing k i n d l i n g , although changes in potent iat ion could not be discounted. This a l t e r a t i o n depended on the k indl ing s t imul i to tr igger hippocampal ADs and did not require the presence of motor se izures . The increase in i n h i b i t i o n d id not appear to be caused by augmentation of the Cl~-dependent conductance, because manipulation of [ C l ] 0 d id not reverse th i s e f f ec t . Furthermore, the k indl ing- induced enhancement of i n h i b i t i o n was shown to las t at least six weeks with only a s l i g h t decrease. A l l of the above a l t e r a t i o n s in granule c e l l propert ies are , „ so f a r , l o c a l i z e d to these neurons since s imi lar modif icat ions d id not occur in CA1 pyramidal c e l l s . The s p e c i f i c i t y of these changes indicate that the dentate gyrus may play a c r i t i c a l role in the development of 249 k i n d l i n g , though, further research is required to e s tab l i sh the exact nature of the changes and whether these a l t e r a t i o n s serve to protect against or promote epi leptogenes is . 7.2 Implications of the present study The present study bears d i r e c t l y on two points of dentate granule c e l l e lectrophys io logy: 1) the p o l a r i t y of the GABA-mediated p o t e n t i a l ; and 2) the mechanism subserving paired-pulse heterosynaptic i n t e r a c t i o n s . Previous i n t r a c e l l u l a r studies have not agreed on what response follows the perforant path evoked EPSP or the p o l a r i t y of the potent ia l ac t ivated by mossy f iber s t imulat ion . I n i t i a l invest igat ions using acute preparations observed an IPSP in response to both PP or MF st imulat ion ( e . g . , Lomo, 1971a). Further experimentation u t i l i z i n g the hippocampal s l i c e has given equivocal resu l t s with some studies report ing e i ther IPSPs ( e . g . , Fr icke and Pr ince , 1984) or DAPs in response to perforant path s t imulat ion ( e . g . , Thalmann and Ayalya, 1982). S i g n i f i c a n t l y , both the IPSP and the DAP are sens i t ive to manipulations that a l t e r a GABA-mediated C l ~ conductance (Fricke and Pr ince , 1984; Thalmann and Aya la , 1982). In the present in v i t r o study, PP or h i l a r s t imulat ion evoked a DAP in a l l granule c e l l s . It i s poss ible that the DAP reported in th i s and some of the other in v i t r o studies resulted from the loss of i n h i b i t o r y 250 neurons during the s l i c i n g procedure (Dunwiddie, 1981). If th i s was the case, though, then exogenously appl ied GABA to the denervated postsynaptic receptors should produce a hyperpo lar i za t ion . Assaf et a l . (1981) have shown that iontophoretic app l i ca t ion of GABA, i rrespec t ive of the locat ion injected along the somato-dendritic ax i s , induces a depolar izat ion in granule c e l l s . In a d d i t i o n , granule c e l l s exh ib i t ing an IPSP maintained low rest ing membrane potent ia l s which is ind ica t ive of poorly impaled neurons. Thus, perforant path st imulat ion produces an EPSP that i s followed by a depolar iz ing p o t e n t i a l . This DAP was shown to have a reversa l po tent ia l below that for granule c e l l discharge and, hence, represents a depolar iz ing i n h i b i t o r y p o t e n t i a l . The s ign i f i cance of the depo lar iz ing a f t erpoten t ia l in synaptic integrat ion became evident when one compared the extra- and the i n t r a c e l l u l a r responses to paired-pulse s t imulat ion . Paired-pulse s t imulat ion of the LPP and the MPP has been shown to resu l t in potent ia t ion and depression of the e x t r a c e l l u l a r EPSPs, respect ive ly ( e . g . , McNaughton and Barnes, 1977). It was la ter hypothesized, again based on e x t r a c e l l u l a r recordings, that the d i f ference between the two pathways ( i . e . , LPP and MPP) occurred as a function of the amount of transmitter released on the i n i t i a l s t imulus . Thus the MPP was bel ieved to release more of i t s a v a i l a b l e neurotransmitter on the f i r s t pulse , such that subsequent s t imul i l i b e r a t e d q u a n t i t a t i v e l y less and produced a 251 r e l a t i v e depression of the EPSP (McNaughton, 1980). I n t r a c e l l u l a r analys i s of paired LPP or MPP s t imulat ion , however, i l l u s t r a t e d that the EPSPs summed with the underlying DAP and exhibited potent ia t ion . Furthermore the DAP ac tua l ly depressed the EPSP amplitudes when recording ex tra - or i n t r a c e l l u l a r l y by reducing the di f ference between the membrane potent ia l and the EPSP equi l ibr ium p o t e n t i a l . Paired MPP st imulat ion evoked potent iat ion of the i n t r a c e l l u l a r test EPSP in the absence of a DAP (Lomo, 1971b; Chapter4), ind ica t ing that the MPP maintains the processes responsible for synaptic f a c i l i t a t i o n , but the DAP normally suppresses i t s expression. The l a t t e r in terpre ta t ion is also supported by the paired-pulse attenuation of a heterosynapt ica l ly evoked EPSP (Steward et a l . , 1977). These data are in d i rec t contrast to the mechanism proposed by McNaughton (1980) and suggest that: 1) MPP and LPP both have the capacity to potent iate; and 2) granule c e l l postsynaptic processes contribute to the paired-pulse phenomena recorded in the dentate gyrus. In addi t ion to i t s e f fect on the EPSP, the presence of the DAP explains the f a c i l i t a t i o n of granule c e l l discharge to heterosynaptic inputs (Assaf and M i l l e r , 1981; Douglas et a l . , 1983; Lomo, 1971b; Racine and Robinson, 1986). That i s , i n i t i a l s t imul i which evoke a DAP w i l l allow any subsequent EPSP to sum with th i s p o t e n t i a l and bring more granule c e l l s to act ion potent ia l threshold . This las t property of granule c e l l s has a s i g n i f i c a n t effect on the processing of 252 information and may define the function of the dentate gyrus (see below). Although the DAP played an important ro le in pa ired-pulse potent iat ion under normal condi t ions , the loss of the population spike f a c i l i t a t i o n fol lowing k indl ing d id not appear to be re lated to a l t e r a t i o n s in the DAP. Tuff et al.(1983b) have previously suggested that a k indl ing- induced increase in benzodiazepine receptors enhances the GABA-mediated C l " conductance and causes the i n h i b i t i o n of the PS at a l l C-T i n t e r v a l s . This does not appear to be the case, though, since lowering the [C1] Q which should have reduced the i n h i b i t o r y contr ibut ion of the GABAergic p o t e n t i a l d id not restore the normal pattern of PS po tent ia t ion . Further inves t igat ion incorporat ing both i n t r a - and e x t r a c e l l u l a r recording w i l l be e s sent ia l to determine the processes subserving th i s change. 7.3 The possible s ign i f i cance of the depo lar iz ing a f t e r p o t e n t i a l in hippocampal funct ioning The hippocampal formation has been described as the assoc ia t ion cortex for the neocortex (Swanson, 1979). Impl i c i t in th i s statement is the b e l i e f that somewhere in the HF, multimodal information ( e . g . , sensory) i s integrated and then passed on in i t s a l t ered ( i . e . , associated) s tate . A prerequ i s i t e for the integrat ion of disparate inputs i s the a b i l i t y of a target neuron to somehow 'sample and hold' the incoming s ignals for a per iod , al lowing temporally 253 re lated events to sum. Dentate granule c e l l s , with the ir DAP, appear to be i d e a l l y suited for such a r o l e . Assuming that normal background a c t i v i t y of the various inputs to the dentate gyrus increases during condit ions required to form assoc ia t ions , a hypothesis may be put forward regarding how granule c e l l s incorporate the d i f f erent s ignals and produce a modified output. The focal point of the hypothesis emphasizes the a b i l i t y of granule c e l l s to f a c i l i t a t e heterosynaptic inputs . The primary assumption in the model i s that the granule c e l l s receive tonic a c t i v i t y from a var ie ty of s tructures and that the l e v e l of exc i ta t ion r e f l e c t s the degree of p a r t i c i p a t i o n each structure contributes to the on-going behavior. For example, in the presence of strong odors the o l fac tory projec t ion into the hippocampus would maintain the highest l e v e l of e x c i t a t i o n . Thus, as spec i f i c pathways become more involved in the on-going pattern of behavior, t h e i r a c t i v i t y would be enhanced and the p r o b a b i l i t y for EPSP summation and the evocation of the DAP would increase proport ionate ly . The DAP appears to be evoked at subthreshold stimulus i n t e n s i t i e s , i n d i c a t i n g that the mechanisms generating th i s po tent ia l operate in a feed-forward manner. The presence of the DAP further augments the chances of EPSP summation by increasing the capacity for heterosynaptic i n t e r a c t i o n s . F i n a l l y , when the t o t a l afferent input maintains a frequency greater than 10 Hz 254 ( i . e . , when the inputs occur during the DAP) the EPSPs w i l l add with preceding DAPs and discharge the granule c e l l s . S i g n i f i c a n t l y , granule c e l l f i r i n g w i l l ac t ivate the h i l a r polymorph neurons which give r i s e to the associat ional /commissural system projec t ing back onto the granule c e l l s . Since st imulat ion of these neurons evokes a depo lar iz ing potent ia l (D-IPSP), t h i s i s a form of recurrent e x c i t a t i o n . The D-IPSP maintains an equi l ibr ium p o t e n t i a l below granule c e l l act ion potent ia l threshold and ,as such, may only par t i c ipa te in the heterosynaptic f a c i l i t a t i o n of EPSPs to promote the further discharge of granule c e l l s . This anatomical arrangement tends to keep granule c e l l s in a hyperexcitable state and may allow for the production of long-term potent iat ion at some of the afferent synapses in the dentate gyrus. The maintainance of such a depo lar izat ion may create the condit ions necessary to produce cooperative long-term potent ia t ion . It has been shown that the induction of LTP is enhanced when heterosynaptic inputs are simultaneously act ivated ( e . g . , Lee, 1983); though, the LTP tends to occur only in those pathways which are more strongly ac t iva ted . Thus, the connections that are d i r e c t l y involved in increasing the p r o b a b i l i t y of EPSP summation in granule c e l l s may be s e l e c t i v e l y potent iated. The important aspect of th i s model is the a b i l i t y of the neuronal c i r c u i t r y to u t i l i z e short-term mechanisms ( i . e . , heterosynaptic potent iat ion) to induce long-term a l t e r a t i o n s in synaptic 255 e f f i c a c y . In support of t h i s h y p o t h e s i s , Robinson (1986) has r e c e n t l y shown that p e r f o r a n t path induced LTP i s enhanced when preceded by s t i m u l a t i o n of the medial s e p t a l nucleus. To summarize, the model presented here suggests that granule c e l l s may i n c r e a s e t h e i r p r o b a b i l i t y of d i s c h a r g i n g to a f f e r e n t input by promoting h e t e r o s y n a p t i c i n t e r a c t i o n s at s p e c i f i c f r e q u e n c i e s which, i n t u r n , leads to the long-term augmentation of syn a p t i c e f f i c a c y . In c o n c l u s i o n , the present r e s u l t s demonstrate the n e c e s s i t y of r e c o r d i n g both e x t r a - and i n t r a c e l l u l a r l y when a s s e s s i n g the events a s s o c i a t e d with neuronal p l a s t i c i t y . L i k e many other s t u d i e s , the answers obtained l e a d to many more q u e s t i o n s . S p e c i f i c a l l y , i t becomes obvious that the DAP and LHP of granule c e l l s must be f u l l y understood i f we are to comprehend the a l t e r a t i o n s i n s y n a p t i c e f f i c a c y e x h i b i t e d by such d i v e r s e paradigms as p a i r e d - p u l s e s t i m u l a t i o n and k i n d l i n g . Although the experiments demanded to achieve t h i s goal are made more d i f f i c u l t by the small s i z e of the granule c e l l s , the rewards f o r c o n t i n u i n g such r i g o r o u s i n v e s t i g a t i o n s c o u l d l e a d to a b e t t e r comprehension of the mechanisms subserving memory or the pathogenesis of many CNS d i s o r d e r s . •NT 256 REFERENCES Adamec, R . E . , McNaughton, B . , K . E . 1981. Ef fec t s of e x c i t a b i l i t y in the ra t : E p i l e p s i a , 22: 205-215. Racine, R . , and L iv ings ton , diazepam on hippocampal act ion in the dentate area. Albert son , T . E . Peterson S . L . and Stark L . G . 1981. The anticonvulsant e f fects of diazepam and phenobarbital in prekindled and kindled seizures in r a t s . Neuropharmacol., 20: 597-603. A lger , B . E . 1984. C h a r a c t e r i s t i c s of a slow hyperpolar iz ing synaptic po tent ia l in rat hippocampal pyramidal c e l l s in v i t r o . J . Neurophys io l . , 52: 892-910. A lger , B . E . and N i c o l l , R .A. 1979. GABA-mediated biphasic i n h i b i t o r y responses in hippocampus. Nature, 281: 315-317. A lger , B . E . and N i c o l l , R .A. 1982a. Feed-forward dendr i t i c i n h i b i t i o n in rat hippocampal pyramidal c e l l s studied in v i t r o . J . P h y s i o l . (Lond.) , 328: 105-123. A lger , B . E . and N i c o l l , R .A . 1982b. Pharmacological evidence for two kinds of GABA receptor on rat hippocampal pyramidal c e l l s studied in v i t r o . J . P h y s i o l . (Lond.) , 328: 125-141. Alger , B . E . and T e y l e r , T . J . 1976. Long-term and short-term p l a s t i c i t y in the CA1, CA3, and dentate region of the hippocampal s l i c e . Brain Res . , 110: 463-480. Alger , B . E . and T e y l e r , T . J . 1978. Potassium and short-term p l a s t i c i t y in the hippocampal s l i c e . Brain Res . , 159: 239-242. 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 Rubia, F . J . 1977. The ionic mechanisms concerned in generating the i . p . s . p s of hippocampal pyramidal c e l l s . Proc. R. Soc. L o n d . , 198: 363-384. Andersen, P . , B ie , B . , and Ganes, T . 1982. D i s t r i b u t i o n of GABA sens i t ive areas on hippocampal pyramidal neurons. Exp. Brain Res . , 45: 357-363. Andersen, P. and Lomo, T . 1967. Control of hippocampal output by afferent vo l l ey frequency. Prog. Brain Res . , 27: 400-412. 257 Andersen, P. and Lomo, T. 1969. Organization and frequency dependence of hippocampal i n h i b i t i o n . In: Basic  Mechanisms of the E p i l e p s i e s . Eds. H .H. Jasper, A . A . Ward, A. Pope. L i t t l e , Brown & C o . , Boston. , 604-609. Andersen, P . , Dingledine, R . , Gjerstad, L . , Langmoen, I . A . , and Mosfeldt-Laursen, A. 1980. Two responses of hippocampal pyramidal c e l l s to app l i ca t ion of gamma-aminobutyric a c i d . J . P h y s i o l . (Lond.) , 305: 279-296. Andersen, P . A . , E c c l e s , J . C . , and Loyning, Y. 1964. Pathway of postsynaptic i n h i b i t i o n in the hippocampus. J . Neurophys io l . , 27: 608-619. Andersen, P . , Holmqvist, B . , and Voorhoeve, P . E . 1966a. Entorhinal ac t iva t ion of dentate granule c e l l s . Acta P h y s i o l . Scand., 66: 448-460. Andersen, P . , Holmqvist, B . , and Voorhoeve, P . E . 1966b. Exc i ta tory synapses on hippocampal a p i c a l dendrites act ivated by entorhinal s t imulat ion . Acta P h y s i o l . Scand. , 66: 461-472. Ashwood, T . J . , Lancaster, B . , and Wheal, H .V. 1984. In vivo and in v i t r o studies on putative interneurones in the rat hippocampus: poss ible mediators of feed-forward i n h i b i t i o n . Brain Res . , 293: 279-291. Assaf, S .Y. and M i l l e r , J . J . 1981. Commissural potent iat ion of perforant path evoked responses in the dentate gyrus of the r a t . Can. J . P h y s i o l . Pharmacol. , 59: 1117-1121. Assaf, S . Y . , C r u n e l l i , V . , and K e l l y , J . S . 1981. Depolar iz ing postsynaptic actions of GABA in the rat dentate gyrus. . In: Amino Acid Neurotransmitters, eds. F . V . DeFeudis and P. Mandel, Raven Press, New Y o r k . , 239-248. A u l t , B. and Nadler, J . V . 1983a. Ef fects of baclofen on synapt ica l ly - induced c e l l f i r i n g in the rat hippocampal s l i c e . Br . J . Pharmacol. , 80: 211-219. A u l t , B. and Nadler, J . V . 1983b. Ef fects of baclofen on synapt ica l ly - induced c e l l f i r i n g in the rat hippocampal s l i c e . Br . J . Pharmacol. , 80: 211-219. Baimbridge, K . G . and M i l l e r J . J . 1984. Hippocampal ca lc ium-binding prote in during commissural k indl ing- induced epi leptogenesis: progressive decl ine and e f fects of ant iconvulsants . Brain Res . , 324: 85-90. 258 Bakay, R.A. and H a r r i s , A . B . 1981. Neurotransmitter, receptor and biochemical changes in monkey c o r t i c a l e p i l e p t i c f o c i . Brain Res . , 206: 387-404. Barnes, C . A . and McNaughton, B . L . 1980. Phys io log ica l compensation for loss of afferent synapses in rat hippocampal granule c e l l s during senescence. J . P h y s i o l . ( L o n d . ) , 309: 473-485. Barrionuevo, G. and Brown T . H . 1983. Associat ive long-term potent iat ion in hippocampal s l i c e s . Proc. N a t l . Acad. S c i . USA, 80: 7347-7351. Baudry, M. Ol iver M. Creager R. Wieraszko A. and Lynch G. 1980. Increase in glutamate receptors fol lowing r e p e t i t i v e e l e c t r i c a l s t imulat ion in hippocampal s l i c e s . L i f e S c i . , 27: 325-330. B e n - A r i , Y . , K r n j e v i c , K . , and Reinhardt, W. 1979. Hippocampal seizures and f a i l u r e of i n h i b i t i o n . Can. J . P h y s i o l . Pharmacol. , 57: 1462-1466. Berger, T.W. Balzer J . R . Eriksson J . L . and Sclabass i R . J . 1984. Long-term potent iat ion a l t e r s nonlinear c h a r a c t e r i s t i c s of hippocampal perforant path-dentate synaptic transmiss ion. Abs. Soc. Neurosc i . , 10: 1047. Bernier , L . C a s t e l l u c c i V . F . Kandel E . R . and Schwartz J . H . 1982. F a c i l i t a t o r y transmitter causes a se lec t ive and prolonged increase in adenosine 3':5'-monophosphate in sensory neurons mediating the g i l l and siphon withdrawal ref lex in a p l y s i a . J . Neurosc i . , 2: 1682-1691 . B l i s s , T . V . P . and Gardner-Medwin A.R. 1973. Long- las t ing potent iat ion of synaptic transmission in the dentate area of the unanesthetized rabbit fol lowing st imulat ion of the perforant path. J . P h y s i o l . , 232: 357-374. B l i s s , T . V . P . and Lomo T. 1973. Log- la s t ing potent iat ion of synaptic transmission in the dentate area of the anesthetized rabbit fol lowing st imulat ion of the perforant path. J . P h y s i o l . , 232: 331-356. Brown, D.A. and G r i f f i t h , W.H. 1983. Pers is tent slow inward calcium current in voltage-clamped hippocampal neurones of the guinea-pig . J . P h y s i o l . (Lond.) , 337: 303-320. Brown, T . H . and Johnston D. 1983. Voltage-clamp analys i s of mossy f iber synaptic input to hippocampal neurons. J . Neurophys io l . , 50: 487-507. Buzsaki , G. 1984. Feed-forward i n h i b i t i o n in the hippocampal formation. Prog. N e u r o b i o l . , 22: 131-153. 259 Buzsaki , G. and Czeh, G. 1981. Commissural and perforant path interact ions in the rat hippocampus: f i e l d potent ia l s and unitary a c t i v i t y . Exp. Brain Res . , 43: 429-438. Buzsaki , G. and E ide lberg , E . 1982. Convergence of a s soc ia t iona l and commissural pathways on CA1 pyramidal c e l l s of the rat hippocampus. Brain Res . , 237: 283-295. Car len , P . L . , Gurevich, N . , and Pole , P. 1983. Low-dose benzodiazepine neuronal i n h i b i t i o n : enhanced calc ium-mediated potassium-conductance. Brain Res . , 271: 358-364. C a s t e l l u c c i , V . F . and Kandel E . R . 1976. Presynaptic f a c i l i t a t i o n as a mechanism for behavioral s e n s i t i z a t i o n in A p l y s i a . Science, 194: 1176-1178. Chandler, D .B . Woolley D . E . and Overman S.R. 1982. Amygdaloid k indl ing progress ive ly increases posttetanic potent iat ion and long-term potent iat ion of responses evoked in the hippocampus and dentate gyrus. Proc. West. Pharmacol. S o c , 443-447. C o l l i n g r i d g e , G . L . C r u n e l l i V. Forda S.R. and K e l l y J . S . 1982. Gamma-D-glutamylglycine as a spec i f i c antagonist of synaptic transmission in the medial perforant pathway in the rat hippocampal s l i c e preparation-. J . P h y s i o l . , 327: 83-84P. C o l l i n g r i d g e , G . L . Kehl S . J . and McLennan H. 1983. Exc i ta tory amino acids in synaptic transmission in the schaffer co l latera l -commissural pathway of the rat hippocampus. J . P h y s i o l . , 334: 33-46. C o l l i n g r i d g e , G . L . , Gage, P.W., and Robertson, B. 1984. Inhib i tory post-synaptic currents in rat hippocampal CA1 neurones. J . P h y s i o l . (Lond.) , 356: 551-564. C o l l i n s , R . C . Tearse R . G . and Lothman E.W. 1983. Funct ional anatomy of l imbic se izures: foca l discharges form medial entorhinal cortex in r a t . Brain Res. , 280: 25-40. Constant i , A. and Galvan, M. 1983. Fast inward-rect i fy ing current accounts for anomalous r e c t i f i c a t i o n in o l fac tory cortex neurones. J . P h y s i o l . (Lond.) , 385: 153-178. Costa, E . A . Guidot t i A. Mao C . C . and Suria A. 1976. New concepts on the mechanism of act ion of benzodiazepines. L i f e S c i . , 17: 167-186. 260 C r a n d a l l , J . E . , Bernste in , J . J . , Boast, C . A . , and Zornetzer, S . F . 1979. Kindl ing in the rat hippocampus: absence of dendr i t i c a l t e r a t i o n s . Behav. Neur. B i o l . , 27: 516-522. Creager, R . , Dunwiddie, T . , and Lynch, G. 1980. Paired-pulse and frequency f a c i l i t a t i o n in the CA1 region of the in v i t r o rat hippocampus. J . P h y s i o l . (Lond. ) , 299: 409-424. C r u n e l l i , V . , Forda, S . , and K e l l y , J . S . 1983. Blockade of amino acid- induced depolar izat ions and i n h i b i t i o n of exc i tatory post-synaptic potent ia l s in rat dentate gyrus. J . P h y s i o l . (Lond.) , 341: 627-640. C r u n e l l i , V . , Forda, S . , and K e l l y , J . S . 1984. The reversal po tent ia l of exc i tatory amino ac id act ion on granule c e l l s of the rat dentate gyrus. J . P h y s i o l . (Lond.) , 351: 327-342. C u r t i s , D . R . , F e l i x , D . , and McLennan, H. 1970. GABA and hippocampal i n h i b i t i o n . BR. J . Pharmacol. , 40: 881-883. Davidoff , R. and Sears E . S . 1974. The effects of L i o r e s a l on synaptic a c t i v i t y in the i so la ted sp ina l cord . Neuorology, 24: 957-963. Deadwyler, S . A . , Dudek, F . E . , Cotman, C.W. , and Lynch, G. 1975. I n t r a c e l l u l a r responses of rat dentate granule c e l l s in v i t r o : posttetanic potent iat ion to perforant path s t imulat ion . Brain Res . , 88: 80-85. Del C a s t i l l o , J . and Katz B. 1954. S t a t i s t i c a l factors involved in neuromuscular f a c i l i t a t i o n and depression. J . P h y s i o l . , 124: 574-585. Dingledine , R. 1983. N-methyl aspartate ac t ivates voltage-dependent calcium conductance in rat hippocampal pyramidal c e l l s . J . P h y s i o l . (Lond. ) , 343: 385-405. Dingledine , R. and Gjers tad , L . 1980. Reduced i n h i b i t i o n during ep i l ept i form a c t i v i t y in the in v i t r o hippocampal s l i c e . J . P h y s i o l . (Lond.) , 305: 297-313. Dingledine , R. and Langmoen, I . A . 1980. Conductance changes and i n h i b i t o r y act ions of hippocampal recurrent IPSPs. Brain Res . , 185: 277-287. Dolphin , A . C . Err ington M . L . and B l i s s T . V . P . 1982. Long-term potent iat ion of the perforant path in vivo i s associated with increased glutamate re lease . Nature, 297: 496-498. 261 Douglas, R.M. and Goddard G.V. 1975. Long-term potent iat ion of the perforant path-granule c e l l synapse in the rat hippocampus. Brain Res . , 86: 205-215. Douglas, R . M . , McNaughton, B . L . , and Goddard, G .V . 1983. Commissural i n h i b i t i o n and f a c i l i t a t i o n of granule c e l l discharge in fasc ia dentata. J . Comp. N e u r o l . , 219: 285-294. Dudek, F . E . , Deadwyler, S . A . , Cotman, C.W. , and Lynch, G. 1976. I n t r a c e l l u l a r responses from granule c e l l layer in s l i c e s of rat hippocampus: perforant path synapse. J . Neurophys., 39: 384-393. Dunwiddie, T . and Lynch G. 1978. Long-term potent iat ion and depression of synaptic responses in the rat hippocampus: l o c a l i z a t i o n and frequency dependency. J . P h y s i o l . , 276: 353-367. Dunwiddie, T . V . 1981. Age-related di f ferences in the in v i t r o rat hippocampus. Dev. Neurosc i . , 4: 165-175. Ecc l e s , J . C . 1964. v The physiology of synapses ' . Springer-Ver lag ; B e r l i n , Heidelberg, New York. Ecc l e s , J . C . 1983. Calcium in long-term potent iat ion as a model for memory. Neurosc i . , 10: 1071-1081. Ecc l e s , J . , N i c o l l , R . A . , Oshima, T . , and Rubia, F . J . 1977. The anionic permeabil i ty of the i n h i b i t o r y postsynaptic membrane of hippocampal pyramidal c e l l s . Proc. R. Soc. L o n d . , 198: 345-361. Engel , J . and Ackermann R . F . 1980. I n t e r i c t a l EEG spikes corre la te with decreased, rather than increased, ep i l eptogen ic i ty in amygdaloid kindled r a t s . Brain Res . , 190: 543-548. F a t t , P. and Katz, B. 1951. An analys i s of the end-plate p o t e n t i a l recorded with an i n t r a - c e l l u l a r e lectrode . J . P h y s i o l . (Lond.) , 115: 320-370. F i fkova , E . and Van Harreveld A. 1977. Long- las t ing morphological changes in d e n d r i t i c spines of dentate granular c e l l s fol lowing s t imulat ion of the entorhinal area . J . Neurocyto l . , 6: 211-230. Fournier , E . and Crepe l , F . 1984. E l e c t r o p h y s i o l o g i c a l propert ies of dentate granule c e l l s in mouse hippocampal s l i c e s maintained in v i t r o . Brain Res . , 311: 75-86. 262 F r i c k e , R .A. and Pr ince , D.A. 1984. Electrophysiology of dentate gyrus granule c e l l s . J . Neurophys io l . , 51: 195-209. F r i t z , L . C . and Gardner-Medwin, A .R . 1976. The effect of synaptic ac t iva t ion on the e x t r a c e l l u l a r potassium concentration in the hippocampal dentate area, in v i t r o . Brain Res . , 112: 183-187. Frotscher , M . , Leranth, C . , Lubbers, K . , and O e r t e l , W.H. 1984. Commissural afferents innervate glutamate decarboxylase immunoreactive non-pyramidal neurons in the auinea pig hippocampus. Neurosci . L e t t . , 46: 137-143. F u j i t a , Y. and Sakuranaga, M. 1981. Spontaneous hyperpolarizat ions in pyramidal c e l l s of c h r o n i c a l l y stimulated rabbit hippocampus. Jap. J . P h y s i o l . , 31: 879-889. F u j i t a , Y . , Hatada, H . , Takeuchi, T . , Sato, H . , and Minami, S. 1983. Enhancement of EEG spikes and hyperpolar izat ions of pyramidal c e l l s in the kindled hippocampus of the r a b b i t . Jap. J . P h y s i o l . , 33: 227-238. Gaarskjaer, F . B . 1981. The hippocampal mossy f iber system of the rat s tudied. Corre la t ion between topographic organizat ion and neurogenetic gradients . J . Comp. N e u r o l . , 203: 717-735. Gaarskjaer, F . B . and Laurberg . , S. 1983. Ecotopic granule c e l l s of h i l u s fasciae dentatae projec t ing to the i p s i l a t e r a l regio i n f e r i o r of the rat hippocampus. Brain Res . , 274: 11-16. Gahwiler, B . H . and Brown, D .A. 1985. GABAb-receptor-act ivated K+ current in voltage-clamped CA3 pyramidal c e l l s in hippocampal c u l t u r e s . Proc. N a t l . Acad. S c i . USA., 82: 1558-1562. Giacchino, J . L . , Somjen, G . G . , Frush, D . P . , and McNamara, J . O . 1984. L a t e r a l entorhinal c o r t i c a l k ind l ing can be es tabl i shed without potent iat ion of the en torh ina l -granule c e l l synapse. Exp. N e u r o l . , 86: 483-492. Gloor , P . , Vera , C . L . , and S p e r t i , L . 1964. E l e c t r o p h y s i o l o g i c a l studies of hippocampal neurons. I I I . Responses of hippocampal neurons to repe t i t i ve perforant path v o l l e y s . Electroenceph. C l i n . Neurophys io l . , 17: 353-370. Goddard, G .V . 1983. The k indl ing model of ep i lepsy . TINS, 6(7): 275-279. 263 Goddard, G.V. and Douglas, R.M. 1975. Does the engram of k ind l ing model the engram of normal long-term memory. Can. J . Neurol . S c i . 2:385-394 Goddard, G .V . Mclntyre D . C . and Leech C . K . 1969. A permanent change in brain function resu l t ing from d a i l y e l e c t r i c a l s t imulat ion . Exp. N e u r o l . , 25: 295-330. Godfraind, J . M . 1985. Intrasomatic and i n t r a d e n d r i t i c recordings of plateau potent ia l s in s l i c e s of the dentate gyrus maintained in v i t r o . Exp. Brain Res . , 57: 233-238. Gorman, A . L . F . and Thomas, M.V. 1980. Potassium conductance and in terna l calcium accumulation in a molluscan neurone. J . P h y s i o l . (Lond.) , 308: 287-313. Hagiwara, S . , Miyazaki , S . , and Rosenthal, N.P. 1976. Potassium current and the ef fect of cesium on th i s current during anomalous r e c t i f i c a t i o n of the egg c e l l membrane of a s t a r f i s h . J . Gen. P h y s i o l . , 67: 621-638. H a l l i w e l l , J . V . and Adams, P.R. 1982. Voltage-clamp analys i s of muscarinic exc i ta t ion in hippocampal neurons. Brain Res . , 250: 71-92. H a r r i s , E.W. and Cotman, C.W. 1983. Ef fec ts of a c i d i c amino ac id antagonists on paired-pulse potent iat ion at the l a t e r a l perforant path. Exp. Brain Res . , 52: 455-460. H a r r i s , E.W. Ganong A . H . and Cotman C.W. 1984. Long-term potent iat ion in the hippocampus involves a c t i v a t i o n of N-methyl-D-aspartate receptors . Brain Res. , 323: 132— 137. H a r r i s , E . W . , Lasher, S .S. and Steward, 0. 1979. Analys i s of the h a b i t u a t i o n - l i k e changes in transmission in the temprodentate pathway of the r a t . Brain Res . , 162:21-32 Heinemann, U . , Hamon, B . , and Konnerth, A. 1984. GABA and baclofen reduce changes in e x t r a c e l l u l a r free calcium in area CA1 of rat hippocampal s l i c e s . Neurosci . L e t t . , 47: 295-300. Heinemann, U . , Lux, H . D . , and Gutnick, M . J . 1977. E x t r a c e l l u l a r free calcium and potassium during paroxysmal a c t i v i t y in the cerebral cortex of the ca t . Exp. Brain Res . , 27: 237-243. Hjorth-Simonsen, A. and Jeune B. 1972. O r i g i n and termination of the hippocampal perforant path in the rat studied by s i l v e r impregnation. J . Comp. N e u r o l . , 144: 215-232. 264 Hotson, J . R . and Prince , D.A. 1980. A calc ium-act ivated hyperpolar izat ion follows repe t i t i ve f i r i n g in hippocampal neurons. J . Neurophys io l . , 43(2): 409-419. Hotson, J . R . and Pr ince , D.A. 1981. P e n i c i l l i n - and barium-induced ep i l ept i form burst ing in hippocampal neurons: act ions on Ca++ and K+ p o t e n t i a l s . Ann. N e u r o l . , 10: 11-17. Hotson, J . R . , Pr ince , D . A . , and Schwartzkroin, P .A . 1979. Anomalous inward r e c t i f i c a t i o n in hippocampal neurons. J . Neurophys io l . , 42: 889-895. Johnston, D. and Brown T . H . 1981. Giant synaptic po ten t ia l hypothesis for ep i lept i form a c t i v i t y . Sc ience . , 211: 294-297. Johnston, D. Habl i t z J . J . and Wilson W.A. 1980. Voltage clamp disc loses slow inward current in hippocampal burst f i r i n g neurones. Nature, 286: 391-393. Joy, R.M. Albertson T . E . and Stark L . G . 1984. An analys i s of the act ions of progabide, a spec i f i c GABA receptor agonist , on k indl ing and kindled se izures . Exp. N e u r o l . , 83: 144-154. K a i r i s s , E.W. Racine R . J . and Smith G.K. 1984. The development of the i n t e r i c t a l spike during k ind l ing in the r a t . Brain Res . , 322: 101-110. Kalichman, M.W. 1982a. Neurochemical corre lates of the k ind l ing model of ep i lepsy . Neurosci . Biobehav. Rev. , 6: 165-181. Kalichman, M.W. 1982b. Pharmacological inves t igat ion of convulsant gamma-aminobutyric ac id (GABA) antagonists in amygdala-kindled r a t s . E p i l e p s i a , 23: 163-171. Kalichman, M.W. Liv ingston K . E . and Burnham W.M. 1981. Pharmacological inves t igat ion of gamma-aminobutyric ac id (GABA) and the development of amygdala-kindled seizures in the r a t . Exp. N e u r o l . , 74: 829-836. Kandel , E . R . and Spencer, W.A. 1961. Electrophysio logy of hippocampal neurones. I I . Af t er -po ten t ia l s and r e p e t i t i v e f i r i n g . J . Neurophys io l . , 24: 243-259. Kandel , E . R . and Spencer, W.A. 1961a. Exc i ta t ion and i n h i b i t i o n of s ingle pyramidal c e l l s during hippocampal se izures . Exp. N e u r o l . , 4: 162-179. 265 Kandel , E . R . , Spencer, W.A. , and B r i n l e y , F . J . 1961. Electrophysiology of hippocampal neurons. I . Sequential invasion and synaptic organizat ion . J . Neurophys io l . , 24: 225-242. Katz , B. and M i l e d i R. 1967. A study of synaptic transmission in the absence of nerve impulses. J . P h y s i o l . , 192: 407-436. Katz , B. and Mi l ed i R. 1968. The role of calcium in neuromuscular f a c i l i t a t i o n . J . P h y s i o l . , 195: 481-492. Kehl , S . J . and McLennan, H. 1983. Evidence for a b i c u c u l l i n e - i n s e n s i t i v e l ong- la s t ing i n h i b i t i o n in the CA3 region of the rat hippocampal s l i c e . Brain Res . , 279: 278-281. K l e i n , M. Shapiro E . and Kandel E . R . 1980. Synaptic p l a s t i c i t y and the modulation of the Ca2+ current . J . Exp. B i o l . , 889: 117-157. Knowles, W.D. and Schwartzkroin,; P . A . 1981. Local c i r c u i t synaptic interact ions in hippocampal brain s l i c e s . J . Neurosc i . , 1: 318-322. Knowles, W.D. , Schneiderman, J . H . , Wheal, H . V . , Stafstrom, C . E . , and Schwartzkroin, P .A . 1984. Hyperpolariz ing potent ia l s in guinea pig hippocampal CA3 neurons. C e l l . Molecular N e u r o b i o l . , 4: 207-230. Koerner, J . F . and Cotman C.W. 1981. Micrcromolar L-2-amino-4-phosphonobutyric ac id s e l e c t i v e l y i n h i b i t s perforant path synapses from l a t e r a l entorhinal cortex . Brain Res . , 216: 192-198. Kohler , C. Chan-Palay V. and Wu J - Y . 1984. Septal neurons containing glutamic ac id decarboxylase immunoreactivity project to the hippocampal region in the rat b r a i n . Anat. Embryol . , 169: 41-44. Kosaka, T . 1983. Axon i n i t i a l segments of the granule c e l l in the rat dentate gyrus: synaptic contacts on bundles of axon i n i t i a l segments. Brain Res . , 274: 129-134. Kosaka, T . , Hama, K . , and Wu, J . 1984. GABAergic synaptic boutons in the granule c e l l layer of rat dentate gyrus. Brain Res . , 293: 353-359. Kosaka, T . , Kosaka, K . , T a t e i s h i , K . , Hamaoka, Y . , Yanaihara, N . , Wu, J . Y . and Hama, K. 1985. GABAergic neurons containing CCK-8- l ike and /or V I P - l i k e immunoreactivities in the rat hippocampus and dentate. J . Comp. N e u r o l . , 239:420-430. 266 Kuba, K. 1980. Release of calcium ions l inked to the a c t i v a t i o n of potassium conductance in a ca f fe ine -treated sympathetic neuron. J . P h y s i o l . (Lond.) , 298: 251-269. Kuhnt, U. Mihaly A. and Joo F . 1985. Increased binding of calcium in the hippocampal s l i c e during long-term potent ia t ion . Neurosci . L e t t . , 53: 149-154. Laatsch, R . H . and Cowan W.M. 1966. Electron microscopic studies of the dentate gyrus of the r a t . I . Normal s tructure with spec ia l reference to synaptic organizat ion . J . Comp. Neur . , 128: 359-396. Lancaster, B. and Wheal, H .V . 1984. The synapt i ca l l y evoked late hyperpolar izat ion in hippocampal CA1 pyramidal c e l l s i s res i s tant to i n t r a c e l l u l a r EGTA. N e u r o s c i . , 12: 267-275. Lanthorn, T . H . and Cotman, C.W. 1981. Baclofen s e l e c t i v e l y i n h i b i t s exci tatory synaptic transmission in the hippocampus. Brain Res . , 225: 171-178. Laurberg, S. 1979. Commissural and i n t r i n s i c connections of the rat hippocampus. J . Comp. N e u r o l . , 184: 685-708. Laurberg, S. and Sorensen, K . E . 1981. Assoc ia t iona l and commissural c o l l a t e r a l s of neurons in the hippocampal formation (hi lus fasciae dentatae and subf ie ld CA3). Brian Res . , 212: 287-300. Lee, K . S . 1983. Cooperat iv i ty among afferents for the induction of long-term potent iat ion in the CA1 region of the hippocampus. J . N e u r o s c i . , 3: 1369-1372. Lee, K . S . , O l i v e r M.W., Schott ler F . and Lynch G . s . 1981. Electron microscopic studies of brain s l i c e s : the ef fects of high-frequency s t imulat ion on dendr i t i c u l t r a s t r u c t u r e . In: Electrophysio logy of Isolated Mammalian CNS Preparat ions . Edited by G.A. Kerkut and H.V. Wheal, Academic Press I n c . , London. , 189-211. Lee, K . S . Schott ler F . O l i v e r M. and Lynch G. 1980. Br ie f bursts of high-frequency s t imulat ion produce two types of s t r u c t u r a l change in rat hippocampus. J . Neurophys io l . , 44: 247-258. Leech C . A . and S t a n f i e l d , P.R. 1981. Inward r e c t i f i c a t i o n in frog s k e l e t a l muscle f ibres and i t s dependence on membrane potent ia l and external potassium. J . P h y s i o l . (Lond. ) , 319: 295-309. 267 Liberson , W.T. and Akert , K. 1953. Observations on e l e c t r i c a l a c t i v i t y of the hippocampus, thalmus, s tr iatum, and cortex under res t ing condit ions and during experimental seizure states in guinea p igs . Electroenceph. C l i n . Neurophys io l . , 5: 320. Lomo. T . , 1971a. Patterns of a c t i v a t i o n in a monosynaptic c o r t i c a l pathway: the perforant path input to the dentate area of the hippocampal formation. Exp. Brain Res . , 12: 18-45. Lomo. T . , 1971b. Potent iat ion of monosynaptic EPSPs in the perforant path-dentate granule c e l l synapse. Exp. Brain Res . , 12: 46-63. Lorente de No, R. 1934. Studies on the s tructure of the cerebral cortex I I . Continuation of the study of the ammonic system. J . Psychol . N e u r o l . , 46: 113-177. Lynch, G. Larson J . Kelso S. Barrionuevo G. and Schott ler F . 1983. I n t r a c e l l u l a r in jec t ions of EGTA block induction of hippocampal long-term potent ia t ion . Nature, 305: 719-721. Lynch, G.S . and Baudry M. 1984. The biochemical intermediates in memory formation: a new spec i f i c hypothesis . Science, 224: 1057-1063. Lynch, G.S . Jensen R .A . McGaugh J . L . Davi la K. and Ol iver M.W. 1981. Ef fec t s of enkephalin, morphine and naloxone on the e l e c t r i c a l a c t i v i t y of the in v i t r o hippocampal s l i c e preparat ion . Exp. N e u r o l . , 71: 527-540. MacVicar, B .A . and Dudek, F . E . 1982. E lec tro ton ic coupling between granule c e l l s of rat dentate gyrus: phys io log i ca l and anatomical evidence. J . Neurophys io l . , 47: 579-592. McCarren, M. and Alger B . E . 1985. Use-dependent depression of IPSPs in rat hippocampal pyramidal c e l l s in v i t r o . J . Neurophys io l . , 53: 557-571. Mclntyre , D . C . and Goddard G.V. 1973. Transfer , interference and spontaneous recovery of convulsions kindled from the rat amygdala. Electroenceph. C l i n . Neurophys io l . , 35: 533-543. Mclntyre , D . C . Stuckey G.N. and Stokes K . A . 1982. Ef fec ts of amygdala les ions on dorsal hippocampus k ind l ing in r a t s . Exp. N e u r o l . , 75: 184-190. 268 McNamara, J . O . Peper A . M . and Patrone V. 1980. Repeated seizures induce long-term increase in hippocampal benzodiazepine receptors . Proc. N a t l . Acad. S c i . USA, 77: 3029-3032. McNaughton, B . L . 1980. Evidence for two p h y s i o l o g i c a l l y d i s t i n c t perforant pathways to the fac ia dentata. Brain Res . , 199: 1-19. McNaughton, B . L . 1982. Long-term synaptic enhancement and short-term potent iat ion in rat fasc ia dentata act through d i f ferent mechanisms. J . P h y s i o l . , 324: 249-262. McNaughton, B . L . Douglas R.M. and Goddard G .V . 1978. Synaptic enhancement in fasc ia dentata: cooperat iv i ty among coactive a f ferents . Brain Res . , 157: 277-293. McNaughton, B . L . and Barnes, C . A . 1977. Phys io log i ca l i d e n t i f i c a t i o n and analys is of dentate granule c e l l responses to s t imulat ion of the medial or l a t e r a l perforant pathways in the r a t . J . Comp. Neur . , 175: 439-454. McNaughton, B . L . , Barnes, C . A . , and Andersen, P. 1981. Synaptic e f f icacy and epsp summation in granule c e l l s of rat fasc ia dentata studied in v i t r o . J . Neurophys., 46 (5): 952-956. McNaughton, N. and M i l l e r J . J . 1984. Medial septal project ions to the dentate gyrus of the ra t : e l e c t r o p h y s i o l o g i c a l analys i s of d i s t r i b u t i o n and p l a s t i c i t y . Exp. Brain Res . , 56: 243-256. Madison, D . V . and N i c o l l R .A . 1983. Noradrenaline blocks accommodation of pyramidal c e l l discharges in the hippocampus. Nature, 299: 636-638. Madison, D . V . and N i c o l l R .A. 1984. Control of the r e p e t i t i v e discharge of rat CA1 pyramidal neurones in v i t r o . J . P h y s i o l . (Lond. ) , 354: 319-331. Madryga, F . J . , Goddard, G . V . , and Rasmusson, D.D. 1975. The k ind l ing of motor seizures from hippocampal commissure of r a t . P h y s i o l . P s y c h o l . , 3(4): 369-373. Maru, E . Tatsuno J . Okamoto J . and Ashida H. 1982. Development and reduction of synaptic potent iat ion induced by perforant path k i n d l i n g . Exp. N e u r o l . , 78: 409-424. Meech, R.W. and Standen, N.B. 1975. Potassium a c t i v a t i o n in Hel ix Aspersa neurones under voltage clamp: a component mediated by calcium i n f l u x . J . P h y s i o l . , 249: 211-239. 269 Meldrum, B .S . 1975. Epi lepsy and gamma-aminobutyric a c i d -mediated i n h i b i t i o n . I n t e r n a t l . Rev. N e u r o b i o l . , 17: 1-36. M i l l e r , J . J . and Baimbridge, K . G . 1983. Biochemical and immunohistochemical corre la tes of k indl ing- induced epi lepsy: ro le of calcium binding pro te in . Brain Res . , 278: 322-326. Misge ld , U . , Klee , M . R . , and Zeise , M . L . 1982. Differences in burst c h a r a c t e r i s t i c s and drug s e n s i t i v i t y between CA3 neurons and granule c e l l s . In Physiology and Pharmacology of Epi leptogenic Phenomena. Eds. M.R. Klee et a l . , Raven Press, New Y o r k . , 131-139. Mody, I . and M i l l e r J . J . 1985. Levels of hippocampal calcium and zinc following kindl ing- induced ep i lepsy . Can. J . P h y s i o l . Pharmacol. , 63: 159-161. Mucha, R . F . and Pine l J . P . 1977. Postseizure i n h i b i t i o n of kindled se izures . Exp. N e u r o l . , 54: 266-282. Newberry, N.R. and N i c o l l , R .A. 1984a. Direct hyperpolar iz ing act ion of baclofen on hippocampal pyramidal c e l l s . Nature, 308: 450-452. Newberry, N.R. and N i c o l l , R . A . 1984b. A b i c u c u l l i n e -res i s tant i n h i b i t o r y post-synaptic potent ia l in rat hippocampal pyramidal c e l l s in v i t r o . J . P h y s i o l . (Lond.) , 348: 239-254. N i c o l l , R .A. and Alger , B . E . 1981. Synaptic exc i ta t ion may act ivate a calcium-dependent potassium conductance in hippocampal pyramidal c e l l s . Science, 212: 957-959. N i c o l l , R . A . , E c c l e s , J . C , Oshima, T . , and Rubia, F . 1975. Prolongation of hippocampal i n h i b i t o r y postsynaptic potent ia l s by barb i turates . Nature, 258: 625-627. Nizn ik , H . B . , Burnham, W.M. , and Ki sh , S . J . 1984. Benzodiazepine receptor binding following amygdala-kindled convulsions: d i f f e r i n g resul ts in washed and unwashed brain membranes. J . Neurochem., 43: 1732-1736. O l i v e r , M.W. and M i l l e r J . J . 1985. A l t era t ions of i n h i b i t o r y processes in the dentate gyrus following k i n d l i n g -induced ep i lepsy . Exp. Brain Res . , 57: 443-447. O l i v e r , M.W. and M i l l e r J . J . 1985. Inhibi tory processes of hippocampal CA1 pyramidal neurons fol lowing k i n d l i n g -induced epi lepsy in the r a t . Can. J . P h y s i o l . Pharmacol. , 63: 872-878. 270 Olney, J . W . , DeGubareff, T . , and S l o v i t e r , R .S . 1983. "Epi l ept i c" brain damage in rats induced by sustained e l e c t r i c a l s t imulat ion of the perforant path. I I . i n t r a c e l l u l a r analys i s of acute hippocampal pathology. Brain Res. B u l l . , 10: 699-712. Peterson, D.W. C o l l i n s J . F . and Bradford H . F . 1983. The kindled amygdala model of epi lepsy: anticonvulsant act ion of amino ac id antagonists . Brain Res . , 275: 169-172. P i n e l , J . P . 1981. Spontaneous kindled motor seizures in r a t s . In: Kindl ing 2. Edited by J . A . Wada, Raven Press , New Y o r k . , 179-192. P i n e l , J . P . and Rovner L . I . 1978. Electrode placement and kindl ing- induced experimental ep i lepsy . Exp. N e u r o l . , 58: 335-346. P i n e l , J . P . and Rovner L . I . 1978. Experimental epi leptogenesis: k indl ing- induced epi lepsy in r a t s . Exp. N e u r o l . , 58: 190-202. P i n e l , J . P . Mucha R . F . and P h i l i p s A . G . 1975. Spontaneous seizures generated in rats by k i n d l i n g : a prel iminary report . P h y s i o l . P s y c h o l . , 3: 127-129. P i n e l , J . P . Skelton R. and Mucha R . F . 1976. K i n d l i n g - r e l a t e d changes in afterdischarge "thresholds". E p i l e p s i a , 17: 197-206. Pinsker, H.M. Hening W.A. Carew T . J . and Kandel E . R . 1973. Long-term s e n s i t i z a t i o n of a defensive withdrawal ref lex in A p l y s i a . Science, 182: 1039-1042. Pr ince , D .A . 1978. Neurophysiology of ep i lepsy . Ann. Rev. N e u r o s c i . , 1: 395-415. Racine, R. 1972a. Modi f i ca t ion of seizure a c t i v i t y by e l e c t r i c a l a c t i v i t y . I . After-discharge threshold . Electroenceph. C l i n . Neurophys io l . , 32: 269-279. Racine, R. 1972b. Modi f icat ion of seizure a c t i v i t y by e l e c t r i c a l s t imulat ion: I I . Motor se izures . Electroenceph. C l i n . Neurophys io l . , 32: 281-294. Racine, R. 1975. Modi f i ca t ion of seizure a c t i v i t y by e l e c t r i c a l s t imulat ion: c o r t i c a l areas. Electroenceph. C l i n . Neurophys io l . , 38: 1-12. Racine, R. 1978. K i n d l i n g : the f i r s t decade. Neurosurgery, 3: 234-252. 271 Racine, R . J . Burnham W.M. Gartner J . G . and Levitan D. 1973. Rates of motor seizure development in rats subjected to e l e c t r i c a l brain s t imulat ion: s t r a i n and i n t e r -st imulat ion e f fec t s . Electroenceph. C l i n . Neurophys io l . , 35: 553-556. Racine, R . J . K a i r i s s E . and Smith G. 1981. Kindl ing mechanisms: the evolution of the burst response versus enhancement. In: Kindl ing 2. Edi ted by J . A . Wada, Raven Press, New Y o r k . , 15-29. Racine, R . J . Milgram N.W. and Hafner S. 1983. Long-term potent iat ion phenomena in the rat l imbic forebra in . Brain Res . , 260: 217-231. Racine, R . J . Newberry F . and Burnham W.M. 1975. Post-ac t iva t ion potent iat ion and the k indl ing phenomenon. Electroenceph. C l i n . Neurophys io l . , 39: 261-271. Racine, R . J . and Milgram, N.W. 1983. Short-term potent iat ion phenomena in the rat l imbic forebra in . Brain Res . , 260: 201-216. Racine, R. Liv ingston K. and Joaquin A. 1975. Ef fec t s of procaine hydrochloride , diazepam, and diphenylhydantoin on seizure development in c o r t i c a l and subcor t i ca l s tructures in r a t . Electroenceph. C l i n . Neurophys io l . , 38: 355-365. Rahamimoff, R. 1968. A dual e f fect of calcium ions on neuromuscular f a c i l i t a t i o n . J . P h y s i o l . , 195: 471-480. Ranck, J . B . 1973. Studies on s ingle neurons in dorsal hippocampal formation and septum in unrestrained r a t s . Part I . Behavioral corre la tes and f i r i n g reper to i res . Exp. N e u r o l . , 41: 462-531. Ribak, C . E . Bradburne M. and Harr i s A . B . 1982. A p r e f e r e n t i a l loss of GABAergic, symmetrical synapses in e p i l e p t i c f o c i : a quant i ta t ive analys i s of monkey neocortex. J . Neurosc i . , 2: 1725-1735. Ribak, C . E . Vaughn J . E . and Saito K. 1978. Immunocytochemical l o c a l i z a t i o n of glutamic ac id decarboxylase in neuronal somata fol lowing co l ch i c ine i n h i b i t i o n of axonal t ransport . Brain Res . , 140: 315-332. Robinson, G .B . 1986. Enhanced long-term potent iat ion induced in the rat dentate gyrus by coact ivat ion of septal and entorhinal inputs: temporal cons tra in t s . Brain Res . , 379: 56-62. 272 Robinson, G . B . and Racine, R. 1986. Interactions between septal and entorhinal inputs: f a c i l i t a t i o n e f f e c t s . Brain Res . , 379: 63-67. Rose, G . , Diamond, D . , and Lynch, G .S . 1983. Dentate granule c e l l s in the rat hippocampal formation have the behavioral c h a r a c t e r i s t i c s of theta neurons. Brain Res . , 266: 29-37. Savage, D.D. Werling L . L . Nadler J . V . and McNamara J . O . 1982. Se lect ive increase in L-3H-glutamate binding to a qu i squalate - sens i t ive s i t e in hippocampal synaptic membranes after angular bundle k i n d l i n g . Eur . J . Pharmacol. , 85: 255-256. Sawa, M . , Maruyama, N . , and K a j i , . S. 1963. I n t r a c e l l u l a r po tent ia l during e l e c t r i c a l l y induced se izures . Electroenceph. C l i n . Neurophys io l . , 15: 209-220. Scharfman, H . E . and Sarvey J . M . 1985. Postsynaptic f i r i n g during repe t i t i ve st imulat ion i s required for long-term potent iat ion in hippocampus. Brain Res . , 331: 267-274. Schof i e ld , C . N . 1978. A depolar iz ing i n h i b i t o r y po tent ia l in neurones of the o l fac tory cortex in v i t r o . J . P h y s i o l . (Lond.) , 275: 559-566. Schwartzkroin, P .A. 1975. C h a r a c t e r i s t i c s of CA1 neurons recorded i n t r a c e l l u l a r l y in the hippocampal in v i t r o s l i c e preparat ion . Brain Res . , 85: 423-436. Schwartzkroin, P .A. .1977. Further c h a r a c t e r i s t i c s of hippocampal CA1 c e l l s in v i t r o . Brain Res . , 128: 53-68. Schwartzkroin, P .A . 1981. To s l i c e or not to s l i c e . In: Electrophysio logy of Isolated Mammalian CNS Preparat ions . Edited by G.A. Kerkut and H.V. Wheal, Academic Press I n c . , London., 15-50. Schwartzkroin, P .A . and Mathers L . H . 1978. Phys io log ica l and morphological i d e n t i f i c a t i o n of a nonpyramidal hippocampal c e l l type. Brain Res . , 157: 1-10. Schwartzkroin, P . A . and Prince D .A. 1980a. Changes in exc i ta tory and i n h i b i t o r y synaptic potent ia l s leading to epi leptogenic a c t i v i t y . Brain Res . , 183: 61-76. Schwartzkroin, P .A . and Prince D.A. 1980b. Ef fects of TEA on hippocampal neurons. Brain Res . , 185: 169-181. Schwartzkroin, P .A. and Stafstrom, C E . 1980. Ef fec t s of EGTA on the ca lc ium-act ivated a f terhyperpo lar iza t ion in hippocampal CA3 pyramidal c e l l s . Science, 210: 1125-1 126. 273 Schwartzkroin, P .A. and Wyler, A .R. 1980. Mechanisms underlying ep i lept i form burst discharge. Ann. N e u r o l . , 7: 95-107. Schwindt, P . C . and C r i l l , W.E. 1982. Factors inf luencing motoneuron rhythmic f i r i n g : resu l t s from a voltage-clamp study. J . Neurophys io l . , 48: 875-890. Schwindt, S . C . and C r i l l , W.E. 1980. Ef fec ts of barium on cat sp ina l motoneurons studied by voltage clamp. J . Neurophys io l . , 44: 827-846. Seress, L . and Pokorny, J . 1981. Structure of the granular layer of the rat dentate gyrus. A l i g h t microscopic and Golgi study. J . Ana't., 133: 181-195. Seress, L . and Ribak, C . E . 1983. GABAergic c e l l s in the dentate gyrus appear to be l o c a l c i r c u i t and project ion neurons. Exp. Brain Res . , 50: 173-182. Seroogy, K . B . , Seress, L . , and Ribak, C . E . 1983. U l t ras truc ture of commissural neurons of the h i l a r region in the hippocampal dentate gyrus. Exp. N e u r o l . , 82: 594-608. Skrede, K . K . and Malthe-Sorenssen D. 1981. Increased rest ing and evoked release of transmitter fol lowing r e p e t i t i v e e l e c t r i c a l t e tanizat ion in hippocampus: a biochemical corre la te to l o g - l a s t i n g synaptic po ten t ia t i on . Brain Res . , 208: 436-441. S l o v i t e r , R .S . 1983. "Epi l ept i c" brain damage in rats induced by sustained e l e c t r i c a l s t imulat ion of the perforant path. I . Acute e l e c t r o p h y s i o l o g i c a l and l i gh t microscopic s tudies . Brain Res. B u l l . , 10: 675-697. Spencer, W.A. and Kandel, E . R . 1969. Synaptic i n h i b i t i o n in se izures . In Basic Mechanisms of the E p i l e p s i e s . Eds. H .H. Jasper, A . A . Ward, A. Pope, L i t t l e , Brown & C o . , Boston. , 575-603. Steward, 0. 1976. Topographic organizat ion of the project ions from the entorhinal area to the hippocampal formation of the r a t . J . Comp. N e u r o l . , 167: 285-314. Steward, 0 . , White, W . F . , and Cotman, C.W. 1977. Potent iat ion of the exc i ta tory synaptic act ion of commissural, a s soc ia t iona l and entorhinal afferents to dentate granule c e l l s . Brain Res . , 134: 551-560. 274 Steward, 0 . , White, W . F . , Cotman, C.W. , and Lynch, G. 1976. Potent iat ion of exc i tatory synaptic transmission in the normal and in the reinnervated dentate gyrus of the r a t . Exp. Brain Res . , 26: 423-441. Storm-Mathisen, J . 1977. L o c a l i z a t i o n of transmitter candidates in the b r a i n : the hippocampal formation as a model. Prog. N e u r o b i o l . , 8: 119-181. Swanson, L.W. 1979. The hippocampus - new anatomical i n s i g h t s . TINS, 2: 9-12. Swanson, L . W . , Sawchenko, P . E . , and Cowan, W.M. 1981. Evidence for c o l l a t e r a l project ions by neurons in Ammon's horn, the dentate gyrus, and the subiculum: a mult ip le retrograde labe l ing study in the r a t . J . N e u r o s c i . , 1: 548-559. Swanson, L . W . , Tey ler , T . J . , and Thompson, R . F . 1982. Hippocampal long-term potent ia t ion: mechanisms and impl icat ions for memory. Neurosci . Res. Prog. B u i . , 20: 613-771. Swanson, L . W . , Wyss, J . M . , and Cowan, W.M. 1978. An autoradiographic study of the organizat ion of intrahippocampal assoc iat ion pathways in the r a t . J . Comp. Neur. , 181: 681-716. Takeuchi, A. 1958. The l ong - la s t ing depression in neuromuscular transmission of f rog . Jap. J . P h y s i o l . , 8: 102-113. Thalmann, R . H . 1984. Reversal propert ies of an EGTA-res i s tant late hyperpolar izat ion that follows synaptic s t imulat ion of hippocampal neurons. Neurosci . L e t t . , 46: 103-108. Thalmann, R . H . and Aya la , G . F . 1982. A late increase in potassium conductance follows synaptic s t imulat ion of granule neurons of the dentate gyrus. Neurosci . L e t t e r s , 29: 243-248. Thalmann, R . H . Peck E . J . Ayala G . F . 1981. Biphasic response of hippocampal pyramidal neurons to GABA. Neurosci . L e t t . , 21: 319-324. T u f f , L . P . , Racine, R . J . , and Adamec, R. 1983a. The ef fects of k ind l ing on Gaba-mediated i n h i b i t i o n in the dentate gyrus of the ra t : paired-pulse depression. Brain Res . , 277: 79-90. 275 Tuf f , L . P . Racine R . J . and Mishra R .K. 1983b. The ef fects of k ind l ing on GABA-mediated i n h i b i t i o n in the dentate gyrus of the r a t . I I . Receptor binding. Brain Res . , 277: 91-98. ' Turner, R.W. and M i l l e r J . J . 1982. Ef fec t s of e x t r a c e l l u l a r calcium on low frequency induced potent iat ion and habituat ion in the in v i t r o hippocampal s l i c preparat ion . Can. J . P h y s i o l . Pharmacol. , 60: 266-275. Voneida, T . J . , V a r d a r i s , R . M . , F i s h , S . E . , and Reihe ld , C . T . 1981. The o r i g i n of the hippocampal commissures in the r a t . Anat. Rec. , 201: 91-103. Voronin, L . L . 1983. Long-term potent iat ion in the hippocampus. Neurosc i . , 10: 1051-1069. Wadman, W . J . , Heinemann, U . , Konnerth, A . , and Neuhaus, S. 1985. Hippocampal s l i c e s of kindled rats reveal calcium involvement in epi leptogenes is . Exp. Brain Res . , 57: 404-407. Walker, J . E . 1983. Glutamate, GABA, and CNS disease: a review. Neurochem. Res . , 8: 521-550. Wheal, H .V . and M i l l e r J . J . 1980. Pharmacological i d e n t i f i c a t i o n of acety lchol ine and glutamate exc i tatory systems in the dentate gyrus of r a t . Brain Res . , 182: 145-155. White, W.F. Nadler J . V . Hamberger A. Cotman C.W. and Cummins and J . T . , 1977. Glutamate as transmitter of hippocampal perforant path. Nature, 270: 356-357. White, W . F . , Nadler, J . V . , and Cotman, C.W. 1979. Analys is of short-term p l a s t i c i t y at the perforant path-granule c e l l synapse. Brain Res . , 178: 41-53. Wigstrom, H. and Gustafsson B. 1983a. Heterosynaptic modulation of homosynaptic l ong - la s t ing potent iat ion in the hippocampal s l i c e . Acta P h y s i o l . Scand. , 119: 455-458. Wigstrom, H. and Gustafsson B. 1983b. Large long- la s t ing potent ia t ion in the dentate gyrus in v i t r o during blockade of i n h i b i t i o n . Brain Res . , 275: 153-158. Wigstrom, H. and Gustafsson B. 1983c. F a c i l i t a t e d induction of hippocampal l ong - la s t ing potent iat ion during blockade of i n h i b i t i o n . Nature, 301: 603-604. 276 Wigstrom, H. and Gustafsson B. 1985. On long- la s t ing potent iat ion in the hippocampus: a proposed mechanism for i t s dependence on coincident pre- and postsynaptic a c t i v i t y . Acta P h y s i o l . Scand. , 123: 519-522. Wolf, P. and Haas, H . L . 1977. Ef fec t s of diazepines and barbiturates on hippocampal recurrent i n h i b i t i o n . Naunyn-Schmiedeberg's Arch. Pharmacol. , 299: 211-218. Wolf, P. and Haas, H . L . 1981. Af t erpo ten t ia l generation in hippocampal pyramidal c e l l s . J . Neurophys io l . , 45: 86-97. Wong, R . K . S . and Watkins, D . J . 1982. C e l l u l a r factors inf luencing GABA response in hippocampal pyramidal c e l l s . J . Neurophys io l . , 48: 938-951. Wood, J . D . 1975. The ro le of gamma-aminobutyric ac id in the mechanism of se izures . Prog. N e u r o b i o l . , 5: 79-95. Wyss, J . M . 1981. An autographic study of the efferent connections of the entorhinal cortex in the r a t . J . Comp. N e u r o l . , 199: 495-512. Yamamoto, C. 1982. Quantal analys i s of exc i tatory postsynaptic potent ia l s induced in hippocampal neurons by ac t iva t ion of granule c e l l s . Exp. Brain Res . , 46; 170-176. Yamamoto, C. and Kawai, N. 1968. Generation of the seizure discharge in thin sections from the guinea p ig brain in ch lor ide - f ree medium in v i t r o . Jap. J . P h y s i o l . , 18: 620-631. Yamamoto, C. and Mcl lwain , H. 1966. Potent ia l s evoked in v i t r o in preparations from the mammalian b r a i n . Nature, 210 (5040): 1055-1056. Yoshida, K. 1984. Influences of b i l a t e r a l hippocampal les ions upon kindled amygdaloid convulsive seizure in r a t s . P h y s i o l . Behav., 32: 123-126. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0097484/manifest

Comment

Related Items