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Action potential discharge in somata and dendrites of CA1 pyramidal neurons of mammalian hippocampus.. Turner, Ray William 1985

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ACTION POTENTIAL DISCHARGE IN SOMATA OF  AND DENDRITES  CA1 PYRAMIDAL NEURONS OF MAMMALIAN  HIPPOCAMPUS:  AN ELECTROPHYSIOLOGICAL ANALYSIS  by  RAY W. B.Sc,  University  TURNER of B r i t i s h  Columbia  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in  THE  FACULTY OF GRADUATE STUDIES  (Department o f P h y s i o l o g y )  We a c c e p t to  THE  this  thesis  the required  as  conforming  standard  UNIVERSITY OF BRITISH COLUMBIA A u g u s t , 1985 ©  Raymond W i l l i a m  Turner, 1985  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may department or by h i s or her  be granted by the head o f representatives.  my  It i s  understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be  allowed without my  permission.  Department o f The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6  (.3/81)  written  ABSTRACT  The  electrophysiological  d e n d r i t i c membranes o f using  the  rat  in  analysis  current-source  density  served  CA1 p y r a m i d a l  vitro  comprehensive  of  suprathreshold synaptic  radiatum  (SR). Laminar  extracellular along  the  inputs  field  slice  preparation. field  intracellular  A  potentials, a c t i v i t y has (AP)  neurons. of CAl pyramidal  stimulation  afferent  and  of o r i g i n of action p o t e n t i a l  1) A c t i o n p o t e n t i a l d i s c h a r g e by  and  somatic  were i n v e s t i g a t e d  extracellular  (CSD)  i n CAl pyramidal  of  neurons  hippocampal  to i d e n t i f y the s i t e  discharge  properties  in  of  stratum  profiles  potentials  dendro-somatic  the  alveus oriens  of  the  were r e c o r d e d  axis  of  c e l l s was  the  (antidromic) or (SO) o r  "stimulus at  evoked  stratum evoked"  25jum i n t e r v a l s  pyramidal  cell  and a  1 - d i m e n s i o n a l CSD a n a l y s i s a p p l i e d . 2) The s h o r t e s t  latency population  s i n k was r e c o r d e d  i n stratum  oriens, a region  corresponding  CAl  pyramidal  potential  neurons.  (current  A  spike response  pyramidale  or the proximal  t o somata  biphasic  source/sink)  and c u r r e n t stratum  and a x o n h i l l o c k s  positive/negative  was  recorded  in  of  spike  dendritic  r e g i o n s , w i t h b o t h c o m p o n e n t s i n c r e a s i n g i n peak l a t e n c y  through  the d e n d r i t i c  stratum  field  with  d i s t a n c e from  the border  of  pyramidale. 3) A c o m p a r a t i v e somatic  intracellular  and d e n d r i t i c membranes  t h e p a t t e r n o f AP d i s c h a r g e axis. while  analysis revealed  o f evoked  activity in  a basic s i m i l a r i t y  at a l l levels of the  in  dendro-somatic  S t i m u l a t i o n o f t h e a l v e u s , SO, o r SR e v o k e d a s i n g l e s p i k e injection  of  depolarizing  current  evoked a r e p e t i t i v e  iii train  of  spikes  grouped  b a s i c p a t t e r n s o f AP 4) B o t h  current  displayed a  for  comparative purposes into  discharge.  and  stimulus  progressive decline  evoked  intracellular  i n amplitude  and  h a l f w i d t h w i t h d i s t a n c e from the border of stratum 5) The  was  found i n  the  region  of the  apparent t h r e s h o l d f o r spike a c t i v a t i o n Stimulus  cell  spike recorded  i n stratum  biphasic extradendritic f i e l d  through  profile  laminar  l a t e n c y from the c e l l The  analysis  to  pyramidale.  body,  spike  with  pyramidale,  generation  cells  arborizations.  the and  conduct w i t h  increasing  body l a y e r .  are  of a s p i k e i n the  subsequent  no  p o t e n t i a l shown  evoked c h a r a c t e r i s t i c s of a c t i o n p o t e n t i a l d i s c h a r g e  pyramidal  in  in dendritic locations.  a l i g n e d w i t h the  a  increase  e v o k e d i n t r a d e n d r i t i c s p i k e s were e v o k e d b e y o n d  peak o f t h e p o p u l a t i o n  CAl  spikes  only consistent voltage threshold for i n t r a c e l l u l a r  discharge  6)  three  retrograde  interpreted  to i n d i c a t e the  initial  r e g i o n o f t h e soma-axon h i l l o c k spike  invasion  of  in  and  dendritic  iv ACKNOWLEDGEMENTS  For support  and g u i d a n c e  over t h e course o f  p r o g r a m I am i n d e b t e d t o D r . James J . M i l l e r . t o e x p r e s s my  sincere thanks t o  my g r a d u a t e  I would a l s o  Thomas L. R i c h a r d s o n  a n a l y s i s programs, r e c o r d i n g equipment and t e c h n i c a l f o r t h e endless hours effort  that  made  of discussion,  this  work  s u p p o r t and  possible.  Thanks  like  f o r data  a d v i c e , and  collaborative also t o other  members o f t h e l a b o r a t o r y and d e p a r t m e n t whose c o n t r i b u t i o n s and comments were g r e a t l y I Henze  gratefully and  figures.  I  Rob also  appreciated.  acknowledge  Andersen wish  the  expert  i n photography to  thank  assistance of Kurt and  preparation  of  t h e members o f t h e a d v i s o r y  c o m m i t t e e a n d D r . P. C a r l e n , t h e e x t e r n a l e x a m i n e r f o r r e v i e w i n g this thesis. Finally,  I w o u l d l i k e t o a c k n o w l e d g e t h e award o f a  S t u d e n t s h i p by t h e M e d i c a l R e s e a r c h C o u n c i l o f Canada.  V  TABLE OF  CONTENTS  C e r t i f i c a t e of Examination  i  Abstract  i i  Acknowledgements  iv  Table of Contents List  of Figures  List  of Tables  1-0.  INTRODUCTION  1-1.  v viii x i 1  Anatomy  1  - The H i p p o c a m p a l  Formation  1  - L a m i n a e and N e u r o n a l E l e m e n t s o f Regio S u p e r i o r (CAl) - A f f e r e n t S y n a p t i c Inputs t o Regio S u p e r i o r  1-2.  2 6  1) S c h a f f e r C o l l a t e r a l s  7  2) C o m m i s s u r a l  Projections  7  Slice Preparation  8  The H i p p o c a m p a l  1- 3. 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 o f CAl  P y r a m i d a l Neurons  9  1) E v o k e d E x t r a c e l l u l a r F i e l d P o t e n t i a l s  9  2) E v o k e d A c t i o n P o t e n t i a l D i s c h a r g e  13  1- 4. THE PRESENT STUDY  18  2- 0. METHODS.  20  2- 1. S u r g i c a l P r o c e d u r e  20  2-2.  21  R e c o r d i n g Chamber  2- 3. S t i m u l a t i n g and R e c o r d i n g T e c h n i q u e s 3- 0. CURRENT-SOURCE  DENSITY ANALYSIS OF ACTION POTENTIAL  DISCHARGE I N THE C A l REGION OF THE HIPPOCAMPUS 3- 1. I n t r o d u c t i o n  22  27 27  vi 3-2. M e t h o d s  31  - Current-Source  Density Calculations  - S t i m u l a t i n g and R e c o r d i n g  31  Procedures  35  3-3. R e s u l t s  40  - Morphological C h a r a c t e r i s t i c s of the Rat C A l P y r a m i d a l Neuron  40  - Laminar P r o f i l e s o f Evoked A c t i v i t y  i n CAl  P y r a m i d a l Neurons  42  - A n t i d r o m i c P o p u l a t i o n S p i k e Response  42  - Subthreshold Excitatory Synaptic Potentials  47  - Orthodromic P o p u l a t i o n Spike Responses  52  3- 4. D i s c u s s i o n  65  4- 0. COMPARATIVE INTRACELLULAR A N A L Y S I S OF SOMATIC AND DENDRITIC ELECTROPHYSIOLOGY OF THE  4-1.  C A l PYRAMIDAL NEURON  73  Introduction  73  4-2. M e t h o d s  76  4-3. R e s u l t s  80  - Membrane C h a r a c t e r i s t i c s  80  - C u r r e n t Evoked S u p r a t h r e s h o l d Responses  85  - S t i m u l u s Evoked S u b t h r e s h o l d  93  Synaptic Potentials..  - S t i m u l u s Evoked S u p r a t h r e s h o l d Responses - Comparison  of  Suprathreshold  Stimulus  96 and  C u r r e n t Evoked S p i k e s  105  - Spike P r e - P o t e n t i a l s  110  4-4. D i s c u s s i o n - Membrane P r o p e r t i e s and E v o k e d Potentials - C u r r e n t Evoked S p i k e s  117 Synaptic 117 120  vii - s t i m u l u s Evoked S p i k e s  124  - S t i m u l u s Vs. C u r r e n t Evoked S p i k e s  126  - Spike Pre-Potentials  127  5-0. EVOKED CHARACTERISTICS OF ACTION POTENTIAL  DISCHARGE  ALONG THE DENDRO-SOMATIC A X I S OF THE  5-1.  C A l PYRAMIDAL NEURON  131  Introduction  131  5-2. M e t h o d s  134  5-3.  139  Results - S p i k e A m p l i t u d e and H a l f w i d t h  139  - Voltage Threshold of Orthodromic Spike Discharge..  149  - S t i m u l u s Evoked S p i k e L a t e n c y  152  - I n t r a d e n d r i t i c Spike F r a c t i o n a t i o n  156  - Isolation Knife  of  Apical  Dendritic  Elements  by  Cuts i n t h e C A l Region  160  5- 4. D i s c u s s i o n  165  - Evoked C h a r a c t e r i s t i c s o f S p i k e D i s c h a r g e  Along  the Dendro-Somatic A x i s - Dendritic  165  Spike F r a c t i o n a t i o n  169  - Evoked A c t i v i t y o f I s o l a t e d A p i c a l D e n d r i t e s 6- 0. GENERAL SUMMARY AND DISCUSSION - The  Site  Dendritic  of  Pyramidal  Cell  Possible  174  o f t h e Present Study Significance  to Pyramidal C e l l REFERENCES  of  174  Spikes  - Implications - The  Origin  171  Function  of Dendritic  175 Spikes 179 182  viii L I S T OF  FIG.  1.1:  FIGURES  Schematic i l l u s t r a t i o n  of the  intrinsic  s y n a p t i c o r g a n i z a t i o n of the hippocampal s l i c e . . . FIG.  1.2:  Evoked c h a r a c t e r i s t i c s o f e x t r a c e l l u l a r potentials  FIG.  3.1:  i n t h e CA1  3  field  region  10  Schematic diagram of the r a t pyramidal  cell  and p l a c e m e n t o f s t i m u l a t i n g e l e c t r o d e s i n t h e CA1 FIG.  3.2:  region  Laminar p r o f i l e s of a l v e a r a n t i d r o m i c extracellular source  FIG.  3.3:  36  field  p o t e n t i a l s and c u r r e n t -  d e n s i t y along the pyramidal  Laminar p r o f i l e s of e x t r a c e l l u l a r p o t e n t i a l s and c u r r e n t - s o u r c e through subthreshold oriens or stratum  FIG.  3.4:  evoked  cell  axis  field  d e n s i t y evoked  s t i m u l a t i o n of  stratum  radiatum  49  Laminar p r o f i l e s of e x t r a c e l l u l a r p o t e n t i a l s and c u r r e n t - s o u r c e  field  density  through suprathreshold s t i m u l a t i o n of  evoked stratum  oriens FIG.  3.5:  53  Laminar p r o f i l e s of e x t r a c e l l u l a r p o t e n t i a l s and c u r r e n t - s o u r c e through suprathreshold  field  density  s t i m u l a t i o n of  evoked stratum  radiatum FIG.  3.6:  57  A comparison of the t i m i n g r e l a t i o n s h i p evoked e x t r a c e l l u l a r current-source  field  3.7:  between  p o t e n t i a l s and  d e n s i t y at the somatic  d e n d r i t i c l e v e l of the pyramidal FIG.  44  and  apical  cell  Peak l a t e n c y o f t h e n e g a t i v e v o l t a g e  60 potential  ix and  current sink  following  along the pyramidal  suprathreshold stimulation  alveus or stratum FIG.  4.1:  cell  axis  of the  radiatum  62  S o m a t i c and a p i c a l d e n d r i t i c membrane r e s p o n s e t o a s e r i e s o f s q u a r e wave  potential  intracellular  current pulse injections FIG.  4.2:  "Type 1" c u r r e n t e v o k e d a c t i o n discharge  FIG.  4.3:  81  i n somatic  potential  and d e n d r i t i c membranes  "Type 2" and "Type 3" c u r r e n t e v o k e d potential discharge  i n somatic  86  action  and d e n d r i t i c  membranes FIG.  4.4:  90  S t i m u l u s evoked s u b t h r e s h o l d s y n a p t i c in pyramidal  FIG.  4.5:  4.6:  soma and a p i c a l d e n d r i t e  cell  Characteristics action  soma and a p i c a l d e n d r i t e of stratum  potential discharge  radiatum  97  evoked  i n pyramidal  cell  soma and a p i c a l d e n d r i t e FIG.  4.7:  afferent 4.8:  100  S o m a t i c and a p i c a l d e n d r i t i c repetitive stimulation  FIG.  r e s p o n s e t o 10Hz  of stratum  radiatum  synaptic inputs  103  C o m p a r i s o n o f s t i m u l u s and c u r r e n t e v o k e d action  potentials  i n the pyramidal  cell  apical dendrite FIG.  4.9:  Characteristics discharge and  FIG.  94  S t i m u l u s evoked a c t i o n p o t e n t i a l d i s c h a r g e i n pyramidal  FIG.  cell  potentials  106 of fast  i n pyramidal  pre-potential cell  soma  apical dendrite  I l l  4.10: A l v e a r a n t i d r o m i c a c t i v a t i o n o f segment" s p i k e s i n p y r a m i d a l  cell  "initial soma  X  and a p i c a l d e n d r i t e F I G . 5.1:  Representative  114  photographs of stimulus  spikes at various l o c a t i o n s along  the  dendro-somatic a x i s of the pyramidal F I G . 5.2:  P l o t s o f s t i m u l u s evoked  evoked  cell  spike amplitude  the dendro-somatic a x i s of the pyramidal F I G . 5.3:  P l o t s o f s t i m u l u s evoked along  spike amplitude  and  the dendro-somatic a x i s of  cell  147  P l o t of voltage threshold f o r orthodromic discharge  along  of the pyramidal Comparison  potentials  the dendro-somatic  spike  axis  cell  150  of s t i m u l u s evoked  spike discharge  F I G . 5.7:  spike halfwidth  145  P l o t s o f c u r r e n t evoked  the pyramidal  F I G . 5.6:  cell....142  cell  h a l f w i d t h along  F I G . 5.5  along  the dendro-somatic a x i s of the  pyramidal F I G . 5.4:  140  intradendritic  to extracellular  i n stratum  pyramidale  F r a c t i o n a t i o n of stimulus  field and  radiatum....154  evoked  intradendritic spikes F I G . 5.8:  Stimulus  evoked  from the c e l l the CAl region  activity  157 of d e n d r i t e s  isolated  body by a k n i f e c u t i n 162  xi L I S T OF TABLES  TABLE 1:  A v e r a g e r e s t i n g membrane p o t e n t i a l r e s i s t a n c e of somatic  and a p i c a l  impalements o f t h e pyramidal TABLE 2:  Average amplitude s t i m u l u s evoked pyramidal  cell  and i n p u t dendritic  cell  83  and h a l f w i d t h o f c u r r e n t and intracellular  spikes  i n the  soma and a p i c a l d e n d r i t e  108  - 1 1-0.  INTRODUCTION  1-1.  Anatomy  The  Hippocampal The  a  hippocampus i s a f o r e b r a i n c o r t i c a l  small  gyrus  ventricle. as  Formation  the  inferior  horn  of  the  as  lateral  P r i m i t i v e h o m o l o g u e s o f t h e h i p p o c a m p u s c a n be t r a c e d  f a r back  progressive  as  the  cyclostomes  development  phylogeny. mammalian  beneath  s t r u c t u r e found  Although  the  structure  comprising  a  through vertebrate portion  instance,  in  the r a t ,  advanced  t h e hippocampus  of  lower  size  i n more  expansion  of  development o f t h e corpus c a l l o s u m has reduced t h e r e l a t i v e hippocampus  the  major  with  and  the  tissue,  a  1975),  neocortex  of  cortical  of  (Angevine  vertebrate forms  a  forms. For  long  tube-like  s t r u c t u r e from a d o r s o - a n t e r i o r s e p t a l p o l e t o a p o s t e r o - l a t e r a l temporal pole  beneath  the  floor of  the l a t e r a l  ventricle. In  man, t h e h i p p o c a m p u s i s c o n f i n e d p r i m a r i l y t o t h e t e m p o r a l l o b e , forming the f l o o r of the i n f e r i o r  horn o f t h e l a t e r a l  ventricle  and e x t e n d i n g f r o m w i t h i n a f e w c e n t i m e t e r s o f t h e t e m p o r a l to  pole  t h e splenium o f t h e corpus callosum. In  coronal  a r i s e a s an itself  to  (hereafter  section, the  extension of the f o r m two  Cajal  hippocampus),  (1911) d i v i d e d  s u p e r i o r and r e g i o i n f e r i o r , the p r i n c i p l e c e l l (Fig  type  1.1). Lorente  de  formation  b a c k on  t h e hippocampus  proper  and d e n t a t e g y r u s ( F i g  t h e hippocampus i n t o  regio  i n reference to the organization of  of the structure, the No  appears t o  entorhinal cortex folded  interlocking gyri,  r e f e r r e d t o as  1 . 1 ) . Ramon y  hippocampal  (1934)  further  pyramidal subdivided  neuron the  - 2 hippocampus  into  (Cornu Ammonis). into  regions  four  Of t h e s e  The  CA1  region  cells,  CA2  and  CA3  aspect  of  the  hippocampus,  i s t a k e n as  inferior. in  the  CA1  L a m i n a e and  region  parallel  be  epithelial  the  d o r s a l aspect  CA4  the  r e g i o n . The  can  be  stratum  is  pyramidal  cells  appearance.  into  extending  stratum  (CAD  six strata  from  the  ( C a j a l 1911).  ( F i g 1.1).  basal  occasionally  apical dendrite projects radially l a c u n o s u m and  moleculare,  stratum lacunosum-moleculare  The  a layer  t o f o u r c e l l s deep w i t h  One  dendrites  into through  the l a t t e r  two  of the  extending  o r more d e n d r i t e s  r e g i o n , g i v i n g the c e l l The  running  ventricular  pyramidale,  a s h o r t d i s t a n c e from t h e i r o r i g i n and  anatomical  s h a f t s of pyramidal c e l l s layer  CA2 regio  further  Superior  divided  somata t h r e e  d e n d r i t e from the a p i c a l  oriens  pyramidal  to regio superior.  the  apical dendritic  ventral  and  a r i s e from the b a s a l p o r t i o n of the pyramidal c e l l  stratum  pyramidal  boundary between  hippocampus,  layer  perpendicular to the c e l l  branching  of  i n the  confined to  Elements of Regio  cell  packed pyramidal c e l l  "pyramidal"  cytological  "modified"  surface to the hippocampal f i s s u r e  obvious  b a s a l and  the  restricted  superior to  to  neurons i n each  pyramidal c e l l s and  CA  a r e a s were d i v i d e d  of pyramidal  s t u d y was  of  Neuronal  Regio  CA3  the l e t t e r s  t h e l i m i t between r e g i o s u p e r i o r  As t h e p r e s e n t  description w i l l  most  the g i a n t  by  c according  d e f i n e s the  l o c a t e d w i t h i n the h i l a r  CA1  and  b, and  efferent projections  field.  and  abbreviated  t h e CA1  d e s i g n a t e d a,  f e a t u r e s or  cells  fields  and  and  one  large  a triangular undergo extend  or  profuse into  the  the alvear region.  The  the stratum  radiatum,  often referred to  i n t h e r a t ( F i g 1.1;  Cajal  1911).  as  - 3 FIG.  1.1  slice.  Ultrastructural Recordings  Superior.  were  anatomy o f t h e t r a n s v e r s e restricted  Notations:  S.Ori. = stratum oriens S.Pyr. = stratum  pyramidale  S.Rad. = s t r a t u m  radiatum  S.Lac. = s t r a t u m  lacunosum  S.Mol. = s t r a t u m  moleculare  S.C.  = Schaffer  B.C.  = basket  collaterals  cell  to  hippocampal  the CAl f i e l d  of  Regio  CA I (Regio Superior)  Area Dentata  - 5 The a p i c a l d e n d r i t e i s c h a r a c t e r i z e d by a t h i c k in  stratum  occurring  radiatum  with  i n the m i d - d i s t a l  hippocampus, branching  many  at  apical  the  the hippocampal  greater  degree  stratum  radiatum.  dendrites  border  although the maximal  on  a  of  proximal s h a f t  exhibit  stratum  of a r b o r i z a t i o n In  the r a t  profuse  lateral  lacunosum-moleculare,  l i m i t of d e n d r i t i c  e x t e n s i o n i s found  f i s s u r e . D e n d r i t i c spines are r e l a t i v e l y  the proximal  apical  d i s t r i b u t e d on d e n d r i t i c  dendritic branches,  stratum lacunosum-moleculare  shaft  but  at  sparse  are p r o f u s e l y  i n c r e a s i n g i n number  toward  (Westrum and B l a c k s t a d 1962).  The axons o f CA1 pyramidal neurons course r a d i a l l y  through  stratum o r i e n s and c o n s o l i d a t e w i t h i n the a l v e a r white matter to project  to  the  subicular  pyramidal  e f f e r e n t f i b e r s o f the f i m b r i a  cell  layer  or  to join  (Knowles and Schwartzkroin 1981b;  Lorente de No 1934). Ramon y C a j a l least thirteen  (1911) and Lorente de No (1934) d e s c r i b e d  neuronal c e l l  from the pyramidal neuron. the  basket  recurrent  cells,  a  inhibition  types i n  at  regio superior d i s t i n c t  However, the most common of these are  s e t of interneurons b e l i e v e d t o mediate of  pyramidal  cells  ( A l l e n e t a l . 1977;  Andersen e t a l . 1964,1969; D i n g l e d i n e and Langmoen 1980; Knowles and  Schwartzkroin  1981a;  Tombol  et  al.  1979).  Although  c y t o l o g i c a l f e a t u r e s can be used to d i s t i n g u i s h between  several  subtypes  certain  of  basket  cells  (Lorente  de  No  1934),  c h a r a c t e r i s t i c s can be c o n s i d e r e d common t o each c l a s s . The c e l l bodies  of  basket  pyramidal neurons, stratum  oriens.  extending p a r a l l e l  cells  are  generally  larger  than adjacent  and are l o c a t e d w i t h i n stratum pyramidale Most  have  a  to pyramidal  b a s a l and a p i c a l d e n d r i t i c c e l l dendrites  or tree  w i t h i n stratum  - 6 -  o r i e n s and stratum radiatum, stratum  radiatum  several  c o l l a t e r a l s back to form h o r i z o n t a l p r o j e c t i o n s i n the  proximal  radiatum,  basket-like  with  plexus  dendrites  (Lorente  synaptic  input  lacunosum-moleculare,  into  sending  stratum  and  r e s p e c t i v e l y . The axon extends  final  around de No  to  termination  pyramidal 1934). A  these  cells  p r o j e c t i o n s of CAl pyramidal c e l l Basket c e l l  in  the  form of a  c e l l somata and  major source is  derived  proximal  of e x c i t a t o r y  from  collateral  axons.  interneurons are  thought to  be i n h i b i t o r y i n  nature, each p r o v i d i n g a potent r e c u r r e n t i n h i b i t i o n of proximal d e n d r i t e s and axon h i l l o c k s of as many as 500 neurons (Andersen  et a l . 1969;  cells  physiological have  also  i n h i b i t i o n of N i c o l l 1982;  characteristics been  pyramidal  Knowles and Schwartzkroin  Somogyi et a l . 1983). In a d d i t i o n , i n t e r n e u r o n s with and  somata,  proposed  pyramidal neurons Andersen et a l . 1969;  similar  to  to b r i n g about i n the  1981a;  anatomical  that of basket a feed-forward  CAl r e g i o n  (Alger and  Ashwood et a l . 1984;  Knowles  and Schwartzkroin 1981a; Schwartzkroin and Mathers 1978). through formation of neurons,  basket  extensive synaptic contact  c e l l s are  b e l i e v e d to  Thus,  with pyramidal  be r e s p o n s i b l e  f o r the  major balance of i n h i b i t o r y i n f l u e n c e i n the r e g i o n .  A f f e r e n t Synaptic Inputs t o Regio S u p e r i o r A c h a r a c t e r i s t i c f e a t u r e of the hippocampus i s the l a m e l l a r organization  of  afferent  synaptic  distribution  of  terminal  projections  dendritic  structures  (Andersen  1965). For  i n s t a n c e , the  enter  hippocampus  the  et  inputs onto  al.  m a j o r i t y of approximately  and  the  basal  laminar and  apical  1971a; Raisman et a l .  afferent synaptic inputs perpendicular  to  the  - 7 -  s e p t o - h i p p o c a m p a l p o l e , and p r o j e c t i n a l a m i n a r synaptic contact axis  (Raisman  along et  discrete  a l . 1965).  regions  of  Although  a  f a s h i o n t o form  the dendro-somatic wide  variety  a n a t o m i c a l l y and c h e m i c a l l y d e f i n e d p r o j e c t i o n s y s t e m s t h e CA1 major  region, the afferents,  present  the  study  Schaffer  was c o n f i n e d  collateral  of  innervate  t o two o f t h e  and t h e c o m m i s s u r a l  inputs to regio superior. 1) S c h a f f e r  Collaterals  A m a j o r i n p u t f r o m r e g i o i n f e r i o r o f t h e h i p p o c a m p u s t o CA1 was f i r s t  described  by S c h a f f e r  i n 1892. T h i s system  as an a x o n c o l l a t e r a l p r o j e c t i o n o f CA3 p y r a m i d a l before  the efferent fiber enters  through stratum  de  cells  shortly  t h e f i m b r i a . These course  o r i e n s and p y r a m i d a l e  to p r o j e c t through stratum  originates  radiatum  back  o f CA2 and t h e n t u r n  of regio superior  (  down  Lorente  No 1 9 3 4 ) . T h e r e , e n - p a s s a g e e x c i t a t o r y s y n a p t i c c o n t a c t s  formed  on  the  apical  dendrites  of  CA1  pyramidal  ( A n d e r s e n and Lomo 1 9 6 6 ; Westrum and B l a c k s t a d  are  neurons  1962).  2) C o m m i s s u r a l P r o j e c t i o n s Commissural hippocampus  arise  inputs in  to large  c o n t r a l a t e r a l hippocampus f i b e r s enter  t u r n and p r o g r e s s  regio  region  from  proceed  the  of  the  dorsal  CA3 f i e l d  a l . 1965).  of the  E f f e r e n t CA3  i n a rostral direction  p s a l t e r i u m . There t h e  to  superior  contact  upon t h e b a s a l and a p i c a l d e n d r i t e s o f t h e CA1  to  fibers  through the c o n t r a l a t e r a l f i m b r i a  alveus  neuron  enter  part  i n the ventral caudad  CA1  (Raisman e t  t h e f i m b r i a and  cross the midline  the  and  and f o r m e x c i t a t o r y s y n a p t i c  ( B l a c k s t a d 1956; Raisman e t a l . 1965).  pyramidal  - 8 1-2. The H i p p o c a m p a l S l i c e  The  laminar  anatomical make  Preparation  organization  of  s p e c i f i c i t y of synaptic  this  system  an  ideal  the  hippocampus  and  inputs onto pyramidal  model  f o r the  the  neurons  study  of  the  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 c o r t i c a l neurons i n the mammalian  CNS.  electrical  activity  stereotaxic in  the  Original  investigations  of the  into  hippocampus were  the  performed  i m p l a n t a t i o n o f s t i m u l a t i n g and r e c o r d i n g  acute,  information  anesthetized  has  been  animal.  gained  in  through  electrodes  Although a great  this  evoked  way, t h e r e  deal of  are several  drawbacks t o t h i s approach, i n c l u d i n g t h e e f f e c t s o f a n e s t h e t i c s on  e v o k e d p o t e n t i a l s , and d i f f i c u l t i e s  site  of  recording  and  stimulating  i n determining  the  e l e c t r o d e s . Many o f t h e s e  p r o b l e m s have been overcome t h r o u g h development o f t h e i n slice  preparation  technique, in vitro  (Skrede  small sections of  Westgaard  neuronal  observed  in  the  intact  technique.  well For  longitudinal afferent  the  suited  example,  axis of the  synaptic  electrodes  of  animal  to a  the  slice  application taken  inputs,  and placed  become  of  the  slice  accessible  exhibit  to  those  structure the  slice to the  the majority and  observation.  preparation to  of  w i t h i n t h e major  r e g i o s u p e r i o r under d i r e c t m i c r o s c o p i c  region  maintained  perpendicular  stimulating  this  1 9 7 5 , 1 9 7 7 ) . The  h i p p o c a m p u s makes t h i s  hippocampus p r e s e r v e s  c a n be a c c u r a t e l y  use  comparable  (Schwartzkroin  vitro  With  t i s s u e c a n be  characteristics  organization  particularly  through  1971).  i n t h e a b s e n c e o f a n e s t h e t i c , and a r e f o u n d t o  electrophysiological  laminar  and  exact  of  recording strata of Therefore,  a l l s t r a t a of the CAl  electrophysiological  analysis,  - 9 permitting  a  characterization  e n t i r e dendro-somatic  axis of  of  evoked  activity  the pyramidal  along the  neuron.  For these  r e a s o n s , t h e h i p p o c a m p a l s l i c e t e c h n i q u e was u s e d i n t h e p r e s e n t study.  1-3.  Electrophysiological  Characteristics  of  CAl  Pyramidal  Neurons  1)Evoked  Extracellular Field  The CAl  characteristics  pyramidal  efferent  investigations and  of extracellular  neurons  pathways  Potentials  evoked  have  been  (Andersen  by  field  stimulation  described  potentials  of  of afferent or  indetail  i n previous  1960; A n d e r s e n e t a l . 1966a,b; A n d e r s e n  Lomo 1 9 6 6 ; C r a g g and  Hamlyn 1 9 5 5 ; G l o o r e t a l .  1 9 6 3 ; Leung  1979a,b,c; S p e r t i e t a l . 1967). Electrical alvear  stimulation of  region  neurons, potential  evokes  an  pyramidal c e l l  a n t i d r o m i c response  in  stratum  pyramidale  neurons,  and  is  " p o p u l a t i o n s p i k e " (Andersen Activation afferent  of  inputs  in  ( F i g 1.2A).  referred  1 . 2 B ) . The i n i t i a l of  extracellular  stratum  triphasic  potential  in  This  waveform  to  as  an  of  antidromic  c o l l a t e r a l / c o m m i s s u r a l (Sch/Comm) radiatum  negativity  component i s g r a d e d  calcium  negative-going  e t a l . 1971b).  Schaffer  longer duration  pyramidal  discharge of a population  evokes a s m a l l b i p h a s i c  n e g a t i v e / p o s i t i v e d e f l e c t i o n superimposed a larger,  within the  within  c h a r a c t e r i z e d by a s h a r p , s h o r t l a t e n c y  r e p r e s e n t s t h e summed s y n c h r o n o u s pyramidal  axons  (arrows low  on t h e r i s i n g  in  edge  stratum radiatum  i n n a t u r e and  of (Fig  independent  i n F i g 1.2B,C), e v i d e n t as  calcium  a  medium ( e x p a n d e d i n F i g  - 10 FIG.1.2 the  Characteristics  CA1 r e g i o n .  stratum  pyramidale  pyramidal c e l l intensities potential of  through  B.  stimulation  region, The  (EPSP) and p o p u l a t i o n s p i k e synaptic  inputs  in  potential  stimulus  associated  Ca+2  with  from  spike,  revealing  i n the apical dendritic of the  fiber potential  region  fiber potential  higher stimulus i n t e n s i t y , i l l u s t r a t i n g the  are  axonal  lowering t o .ImM  ( S t . P y r . ) and a p i c a l  and  expanded v i e w  ( S t . Rad.)  1.6mM  the  D. An  Both the  C. The e f f e c t o f  ( S t . Rad.). Note t h e g r a d u a l d e c l i n e  fiber potential  stimulation  of afferent  potentials  population  increasing  radiatum.  activity  concentration  s t r a t u m radiatum evoked somatic  EPSP  efferent  postsynaptic  evoked t h r o u g h  response  in  i n t e n s i t i e s . The a r r o w d e n o t e s t h e  i n stratum radiatum.  extracellular  of  shown f o r  stratum  in  recorded  excitatory  ( S t . P y r . ) and a p i c a l d e n d r i t i c  projections the  evoked  stimulation.  shown f o r i n c r e a s i n g fiber  antidromic population spike  axons i n t h e a l v e a r  of  afferent  somatic  A. An  o f evoked e x t r a c e l l u l a r p o t e n t i a l s  on  dendritic  i n amplitude  of  a Ca+2-independent (arrow). in C  shown a t  the t r i p h a s i c nature  i n t h e a b s e n c e o f an e x t r a c e l l u l a r EPSP.  a of  1.2D). T h i s  w a v e f o r m i s t h e compound a c t i o n p o t e n t i a l  w i t h t h e a c t i v i t y o f Sch/Comm r e f e r r e d t o as  a x o n s i n s t r a t u m r a d i a t u m and i s  a "fiber potential".  The s e c o n d component  smooth g r a d e d n e g a t i v e w a v e f o r m w i t h region al.  o f Sch/Comm a f f e r e n t  associated  inputs  isa  a maximal n e g a t i v i t y i n t h e (Andersen 1960; Andersen  1 9 6 6 a ; A n d e r s e n and Lomo 1 9 6 6 ; Leung 1 9 7 9 c ) . T h i s  et  potential  i s calcium-dependent  ( F i g 1.2C) and r e p r e s e n t s  thee x t r a c e l l u l a r  reflection  of  excitatory  potential  associated  with  dendrites The  the  synaptic  postsynaptic  (EPSP)  depolarization of pyramidal c e l l  ( A n d e r s e n 1 9 6 0 ; A n d e r s e n and Lomo 1 9 6 6 ; L e u n g  apical 1979c).  EPSP c o n d u c t s e l e c t r o t o n i c a l l y t h r o u g h t h e d e n d r i t i c  declining to the  i n a m p l i t u d e and  cell  layer  t o appear  stratum pyramidale a sharp  reversing  "population  positive-going  synaptic  reflecting  synchronous  following  positive-going  ( F i g 1.2B). W i t h h i g h e r  negative-going  the  as a  i n polarity just  synaptic  stimulus  spike"  waveform  in  discharge  depolarization  of  proximal  potential i n intensities,  i s e v o k e d upon stratum  of  field,  pyramidale,  pyramidal  the  cell  the  neurons population  ( A n d e r s e n e t a l . 1971b; F i g 1 . 2 B ) . Activation evokes  a  dendritic  of  similar  sequence  of  afferents potentials  a x i s o f t h e pyramidal neuron  of stratum oriens extracellular 1966;  commissural  in  along  ( n o t shown).  e v o k e s a f i b e r p o t e n t i a l and a  EPSP i n t h e  basal  stratum  d e n d r i t i c region  the  basal  Stimulation  negative-going (Gessi  et a l .  L e u n g 1 9 7 9 c ) . The EPSP n e g a t i v i t y i s m a x i m a l i n t h e r e g i o n  of a f f e r e n t  synaptic  inputs,  declines  i n amplitude through  d e n d r i t i c t r e e , and i n v e r t s t o a p o s i t i v e p o t e n t i a l near pyramidale then  oriens  bring  stratum  (Leung 1 9 7 9 c ) . H i g h e r i n t e n s i t i e s o f s t i m u l a t i o n about  neuronal  discharge  the  and t h e g e n e r a t i o n  can of a  13  -  population  -  s p i k e at the c e l l l a y e r .  2) Evoked A c t i o n P o t e n t i a l Discharge According  to  the t r a d i t i o n a l  a l l - o r - n o n e a c t i o n p o t e n t i a l (AP)  model of discharge  neuronal  function,  c o u l d occur o n l y  in  the region of the soma-axon h i l l o c k . The  dendritic  of  s i t e for termination  a  cell  afferent  served  as  excitatory  the  synaptic  e l e c t r o t o n i c conduction hillock  region.  modified  in  principle  of s y n a p t i c  However,  recent  inputs,  this  years  and  for  the  c u r r e n t s to  view  with  arborization  has  the  been  passive  the soma-axon substantially  knowledge t h a t d e n d r i t i c  membrane can a l s o e x h i b i t e l e c t r o r e s p o n s i v e p r o p e r t i e s , and regional variations  i n the  d i s t r i b u t i o n of  ( L l i n a s 1975; The  L l i n a s and  Llinas  (Ekerot and and  of  spikes in  the  axon  also  within  the  evoke  L l i n a s and  Nicholson  1980a,b, 1984;  Nicholson  and L l i n a s  voltage-dependent Na+ give  dendritic  all-or-none  arborization  i n t r i n s i c voltage-dependent Ca+2  a r i s e at one  the  presumed  dendritic  tree  membrane,  at  and  can  the  afferent  d e n d r i t i c tree  Ca+2-dependent  through  channels  1980a,b). Ca+2 s p i k e s may dendritic  climbing f i b e r  d e p o l a r i z a t i o n of the  prolonged  channels i n  r i s e to f a s t Na+-dependent  s t i m u l a t i o n of  i n p u t s . However, s y n a p t i c  of  1981;  hillock  response to  examined i n the P u r k i n j e c e l l  Oscarrson  Sugimori  1971). In these neurons,  can  i n the d e n d r i t i c  evoked a c t i v i t y of somatic and d e n d r i t i c membranes of a  the cerebellum  region  can  Sugimori 1980b; Wong et a l . 1979).  neuron has been most thoroughly  1971;  that  i o n i c channels  lead to r e g e n e r a t i v e a c t i o n p o t e n t i a l d i s c h a r g e tree  of  the  action  ( L l i n a s and  "hot s p o t s " of low the  of  Sugimori  or more l o c a t i o n s  determine  spikes  within  threshold  pattern  of  - 14 Na+-dependent s p i k e d i s c h a r g e i n t h e P u r k i n j e c e l l 1975;  soma  L l i n a s and N i c h o l s o n 1 9 7 1 ) . F o r i n s t a n c e , Ca+2 s p i k e s  p r o p a g a t e t o t h e s o m a t i c r e g i o n , summating w i t h i n t h e tree  to  form  a  r e p e t i t i v e Na+ way,  (Llinas  the  prolonged  depolarization  spike discharge  dendritic spike  afferent synaptic  inputs  at the  can serve  dendritic  capable of evoking  somatic l e v e l .  t o "boost  by e n s u r i n g a  In this  the weight"  reliable  A  hillock  1975). differential  confer a the  of  dendro-somatic  t r a n s f e r o f s y n a p t i c c u r r e n t s t o t h e r e g i o n o f t h e axon (Llinas  can  distribution  non-uniformity to  of  ionic  channels can thus  the e x c i t a b i l i t y  o f membrane a l o n g  dendro-somatic a x i s o f a neuron. In t h e case o f t h e P u r k i n j e  cell,  v o l t a g e - d e p e n d e n t Ca+2 c h a n n e l s g i v e r i s e t o  Ca+2  spikes  i n the  c h a n n e l s and  dendrites,  Na+-dependent s p i k e  the  somatic region.  are  i n f a c t so pronounced  cannot  while  actively  electrotonically dendritic tree  The d i s t r i b u t i o n o f  reinvade decay  (Llinas  the  through  the  level  of  Na+  restricted to  Na+ a n d Ca+2  channels  t h a t s o m a t i c Na+ s p i k e s  dendritic  arborization,  proximal  but  portion  of the  1975; L l i n a s and S u g i m o r i 1980b).  Active,  a l l - o r - n o n e r e s p o n s e s c a n t h u s be e v o k e d dendritic  voltage-dependent  activation are  i n t h i s neuron  regenerative  a  cell,  a t b o t h t h e s o m a t i c and  but  the  exact  form  and  c h a r a c t e r i s t i c s o f t h e e v o k e d p o t e n t i a l depend upon t h e s p e c i f i c distribution  and p r o p e r t i e s o f i n t r i n s i c v o l t a g e - d e p e n d e n t  ionic  channels. A second c o r t i c a l neuron membrane  excitability  i s the  h i p p o c a m p u s . The p r o p e r t i e s o f the  pyramidal c e l l  thought t o e x h i b i t a pyramidal  neuron  non-uniform o f mammalian  spike discharge at the  soma w e r e f i r s t  characterized  i n vivo  level of through  - 15 the  analysis of  extracellular  field  potentials  ( A n d e r s e n 1960;  A n d e r s e n e t a l . 1 9 6 6 a b ; A n d e r s e n and Lomo 1966; Leung  1979a,b;  f  Sperti  et  (Andersen Euler  a l . 1977),  e t a l . 1971b; G r e e n  and  vicinity  extracellular  Green  of the  1960)  intracellular  cell  soma  A n d e r s e n and Lomo 1966; K a n d e l 1961;  Spencer  and K a n d e l  the  in  vitro  intracellular  and  pyramidal  cell  axons  invasion  of  latency the  Stimulation basal  of  superimposed  latency  action  As  orthodromic  and  as  spike  stratum pyramidale,  intracellular  spike  discharge  1980;  efferent spike  by a s h o r t i n time  in  pyramidale.  stratum  to  upon  either  evokes a s i n g l e  spike  cell  somata  antidromic responses, the  intracellular  a close  spike  i s found t o  population  spike  correspondence  between  the e x t r a c e l l u l a r  waveform  ( R i c h a r d s o n e t a l . 1984a; S c h w a r t z k r o i n et  of  EPSP i n p y r a m i d a l  and  and  corresponding  also  with  revealing  Prince  indicated  coincide with the negative-going e x t r a c e l l u l a r in  examined  extra-  inputs impinging  structures  1975,1977).  the  Spencer  e v o k e s an a n t i d r o m i c  potential  upon t h e u n d e r l y i n g  of  1966a,b;  e t a l . 1984a;  Stimulation  somata,  synaptic  both  (Richardson  1977).  population  dendritic  (Schwartzkroin  through  Schwartzkroin  cell  afferent  or a p i c a l  i n the  and  somata have a l s o b e e n  i n the alveus  pyramidal  extracellular  cell  Slawsky  intracellular  a l . 1961;  et a l .  e t a l . 1961; K a n d e l  techniques  1975,1977;  Schwartzkroin  et  recordings  (Andersen  preparation  recording  Schwartzkroin  discharge  1 9 6 1 a , b ) . The e v o k e d c h a r a c t e r i s t i c s o f  spike discharge i n pyramidal in  unit  e t a l . 1961; K a n d e l  and  pyramidal  single  and P r i n c e 1980;  Turner  a l . 1984). Given  the  laminar  electrophysiological  organization  analysis  of  of  evoked  the  h i p p o c a m p u s , an  activity  c a n a l s o be  - 16 carried out at  the dendritic level  characteristics vivo  through  of an  extracellular  analysis  field  the C A l pyramidal Hamlyn  dendritic  of  cell  and  possible  pyramidal  cell  apical dendrite  synaptic  inputs.  cell  axis  Na+-dependent)  t o be  of  spikes  and  were  cell  With  preparation,  the a  action  along  of  number  region  to  actively  the  pyramidal  intracellular  recordings  t h e presence  dendritic spike  discharge  1977; Spencer and  of  microelectrode  afforded  of  thought  Kandel  by t h e s l i c e  investigators  have  impalements o f  now  pyramidal  t h e p r e s e n c e o f e v o k e d Na+ a n d  potentials  i n the  dendritic  region  ( B e n a r d o e t a l . 1 9 8 2 ; Masukawa and P r i n c e 1 9 8 4 ; Wong and 1979;  i n the  and Hamlyn 1 9 5 5 ; F u j i t a a n d  stability  d e n d r i t e s , and h a v e r e p o r t e d  Ca+2-dependent  and  as a s h a r p  " f a s t p r e - p o t e n t i a l s " (FPPs),  recording  succeeded i n o b t a i n i n g cell  thought  somata r e v e a l e d  representation  limited  detected  origin  ( A n d e r s e n and Lomo 1 9 6 6 ; S c h w a r t z k r o i n 1961b).  of  early studies  i n the dendritic  were  1960; Cragg  Na+-dependent  These  spike generation  agreement w i t h t h i s ,  t h e somatic  evoked  following stimulation of afferent  dendritic s i t e of  in t h e region of pyramidal all-or-none  of  the dendro-somatic axis  extracellular potential  Sakata 1962). I n  profiles  1962).  occurrence  Dendritic  (Andersen  were f i r s t e x a m i n e d i n  laminar  Sakata  the  propagate from a  c e l l . The  (Andersen 1959; Andersen 1960; Cragg  reported  (presumably  activity  p o t e n t i a l s along  1955; F u j i t a  negative-going  of the pyramidal  Prince  Wong e t a l . 1 9 7 9 ) . The g e n e r a t i o n  of dendritic spikes  thought t o exert considerable discharge  i n both normal  the p r e v a i l i n g hypothesis,  i n pyramidal  neurons  i n f l u e n c e on t h e p a t t e r n  of c e l l  and p a t h o l o g i c a l s t a t e s . A c c o r d i n g synaptic  activation of the  is  to  pyramidal  - 17 cell  under  normal  conditions  gives  (Na+-dependent) d e n d r i t i c s p i k e t h a t  rise  to  a  single fast  conducts t o t h e c e l l  t o appear  as a s m a l l  f a s t p r e - p o t e n t i a l i n the somatic  Through  summation  with  potentials,  the  dendritic  body  region.  underlying  dendritic  Na+  may s e r v e t o " b o o s t t h e  spike  postsynaptic  weight" of synaptic  i n p u t s b y i n c r e a s i n g t h e p r o b a b i l i t y f o r AP  discharge  axon  at  Schwartzkroin activation  the  1977; Spencer  of  pharmacological  hillock  excitatory agents  and  (Andersen  and  Lomo  1966;  Kandel 1961). I n c o n t r a s t ,  synaptic known  to  the  inputs  i n t h e presence of  reduce  the  i n h i b i t o r y feedback networks can evoke  efficacy  of  a burst discharge  of both  Na+ a n d C a + 2 - d e p e n d e n t d e n d r i t i c s p i k e s  (Wong and P r i n c e  1979).  The  i n this  dendritic  depolarization  r e p e t i t i v e Na+ s p i k e d i s c h a r g e proposal  that  dendritic  multiple spike discharge i n t h e hippocampus and W y l e r  evoked  i n the c e l l  spike  way c a n e l i c i t  body, l e a d i n g t o t h e  activation  may  underly  the  characteristic of epileptiform a c t i v i t y  (Schwartzkroin  and P r i n c e 1 9 8 0 ; S c h w a r t z k r o i n  1 9 7 9 ; Wong and P r i n c e 1 9 7 9 ; Wong e t a l . 1 9 7 9 ) .  - 18 1-4.  THE  PRESENT STUDY  Previous investigations activity  in  of the  the hippocampus  the CAl pyramidal neuron. important  the  role  and  pathology  of  epilepsy  of  the  spikes  understanding mammalian  play  an  thus  be  an  pyramidal  even c o n t r i b u t e t o  responsible  has  Na+-dependent under  et  al.  1962;  been  gained  dendritic the  structures.  f o r generation of important  (Andersen  1966a,b;  Masukawa  step  in  1960; and  Prince  Kandel  1 9 6 1 b ) . B a s e d on  the somatic  level,  the s i t e  t e n t a t i v e l y proposed t r e e at "hot  to exist  extensive  analysis  pyramidal  cell,  neuron  of  the  not y e t been  c o n d i t i o n s of  A n d e r s e n and  Lomo  Hamlyn 1955;  1984;  Schwartzkroin  intracellular  exact  site  1977;  spike  at was  dendritic and  Kandel  f o r Ca+2 s p i k e g e n e r a t i o n  potential  and  recordings  membrane ( S p e n c e r  ( L l i n a s 1975).  action  1966;  Fujita  at branchpoints of the  t o t h a t proposed  the c e r e b e l l a r P u r k i n j e  as t h e s e s p i k e s a r e  f o r g e n e r a t i o n o f t h e Na+  spots" of d e n d r i t i c  1961b), analagous  the p r o p e r t i e s of  physiological  Cragg  and  about  spike,  more  S p e n c e r and  The  of  the d i s c h a r g e p a t t e r n s of a c o r t i c a l neuron i n the  evoked  s p i k e has  output of the  mammalian c o r t i c a l  factors  synaptic depolarization  Sakata  level  CNS.  fast  Andersen  for action  dendritic  d i s c h a r g e may in  would  Much i n f o r m a t i o n  reliably  and  evidence  evoked  D e n d r i t i c s p i k e a c t i v a t i o n may  i n determining the f i n a l  Identification  the  the somatic  aberrant d e n d r i t i c  dendritic  c h a r a c t e r i s t i c s of  have r e p o r t e d  p o t e n t i a l discharge at both  cell,  -  in  However, d e s p i t e  discharge  of g e n e r a t i o n of a  in  the  dendritic  identified.  p r e s e n t s t u d y was  t h e r e f o r e undertaken  to  characterize  - 19 and  compare t h e  dendritic  electrophysiological properties  membranes  of  CA1  pyramidal  of  neurons  in  i d e n t i f y t h e s i t e o f o r i g i n o f evoked f a s t a c t i o n pyramidal c e l l The  apical  described are divided  o f e x p e r i m e n t a t i o n . The f i r s t evoked e x t r a c e l l u l a r  field  potentials  dendro-somatic  axis  second  intracellular  properties, the  of  synaptic  potentials  in  the  into  three  i n the  cell  investigation  potentials,  and  CAT region,  relationships  pyramidal  set  intracellular  spike  axis,  activity general derived  and  and  assess  and  the  the  membrane  a l l levels  of  (Chapter  parameters of  of the  pyramidal  t h e r e l a t i o n s h i p between i n t r a c e l l u l a r  extracellular  summary  along  characteristics  o f e x p e r i m e n t s examine v a r i o u s discharge at  and  ( C h a p t e r 3 ) . The of  spike  stages  analysis of  soma and a p i c a l d e n d r i t e s o f C A l p y r a m i d a l n e u r o n s  4 ) . The f i n a l  cell  to  i s a laminar p r o f i l e  determination of current-source density  an  order  dendrites.  r e s u l t s t o be  is  s o m a t i c and  field  discussion  potentials of  the  from these experiments a r e presented  (Chapter  5). A  d a t a and c o n c l u s i o n s i n C h a p t e r 6.  - 20 2-0.  Methods  2-1.  S u r g i c a l Procedure  -  E x p e r i m e n t s were p e r f o r m e d on t h e h i p p o c a m p a l f o r m a t i o n male W i s t a r in v i t r o skin  r a t s (150-250g;  Charles  s l i c e p r e p a r a t i o n . The  and  connective  tissue  s c a l p e l . C r a n i a l b o n e s were  River, Montreal) using  animals  were d e c a p i t a t e d  overlying  the  cold  (1-4  degrees  centigrade)  c o n s i s t i n g o f 124mM N a C l , 1.2mM  MgS04, 24mM  w i t h 95% and  02 / 5%  oxygenate  between  the  3mM  KC1,  was  the  tissue.  occipital  cortex  brain  lifted  out  with  filled  with  cold  separated  The  surface  from  discarded  remaining the  the c o r t i c a l  and  and  The  the  right  from the  c o r t e x c u t away. The stage  and  s c a l p e l cut  leaving  subicular cortex,  o f a S o r v a l l t i s s u e c h o p p e r and  then placed  one for onto  midbrain  placed  and  dish  ventro-medial  fornix to c a r e f u l l y  h i p p o c a m p u s was  and  the  the  the  left  t i p p e d up  c o r t e x and  cool  remaining  in a petri  h e m i s p h e r e was  tissue,  across  c o l d o x y g e n a t e d medium  frontal  o f t h e f i m b r i a and  h i p p o c a m p u s away  CaC12,  w e r e made  and  s u r f a c e o f t h e h i p p o c a m p u s e x p o s e d . A s p a t u l a was the curvature  solution  1.6mM  cuts  placed  a sagittal  a beaker of  of  coronal  medium.  by  dura  (Sigma) p r e o x y g e n a t e d  cerebellum  spatula  separated  later dissection.  was  oxygenated  h e m i s p h e r e immersed i n  coronal  a  and  by  forceps.  Ringer's  0.75mM KH2P04,  Complete  cerebellum  h e m i s p h e r e s were  the  poured over the exposed c o r t e x to  f r o n t a l c o r t e x . The  the  modified  the  removed  removed w i t h  NaHC03, lOmM D - g l u c o s e C02  skull  the  and  removed w i t h r o n g e u r s and  m a t e r c u t w i t h f i n e s u r g i c a l s c i s s o r s and A  of  "roll"  the  on t h e  s l i c e s o f 400um  beneath the  remaining cutting thickness  - 21 were c u t  10 d e g r e e s  hippocampus. Each  oblique to  s l i c e was  brush, placed i n a small to  a nylon  recording bath  lifted  from  axis of  the blade  were t h e n  r e c o v e r y and e q u i l i b r a t i o n  i t was f o u n d  that the time  critical  the  health  required f o r dissection of  a  slice  dissection. Therefore, following a  to  slice  electrophysiological  2-2. R e c o r d i n g  w i t h no  (as  min,  apparent  determined  characteristics).  resembling the i n in a viable  d e s i g n e d t o c r e a t e an v i v o c o n d i t i o n so as  artificial to maintain  s t a t e f o r extended p e r i o d s of  removal from the a n i m a l . B r i e f l y ,  this  artificial  fluid  an a t m o s p h e r e o f  warmed t o  b o d y t e m p e r a t u r e and  h u m i d i f i e d oxygen/carbon  cerebrospinal warmed,  dioxide gas.  The chamber i s c o n s t r u c t e d f r o m p l e x i g l a s s and c o n s i s t s c i r c u l a r water  j a c k e t and  r e c o r d i n g w e l l . The o u t e r w a t e r j a c k e t distilled inner  water  recording  and warmed chamber  centigrade. Plastic  time  i s accomplished  by p r o v i d i n g s l i c e s w i t h an o x y g e n a t e d  an o u t e r  by  Chamber  neuronal tissue following  was n o t a s  d i s s e c t i o n t i m e o f 5-7  viability  The r e c o r d i n g chamber i s environment  medium,  as t h e c a r e t a k e n d u r i n g  s l i c e s c o u l d be c u t f r o m t h e s e c o n d h e m i s p h e r e detriment  to the  environment.  Once t h e t i s s u e had b e e n p l a c e d i n c o l d o x y g e n a t e d  to  OOO  transferred  r e c o r d i n g chamber. S l i c e s  45 m i n f o r  the  with a  p e t r i d i s h o f medium, and  net within the  allowed at least  the l o n g i t u d i n a l  by a  a separate  is partially  heating c o i l  temperature  tubing connected  inner  of  35  circular  filled  to maintain +/-  0.5  to the ouside of  of  with an  degrees the bath  - 22 runs  through  the  outside  water  jacket  and d e l i v e r s warmed,  p r e o x y g e n a t e d medium t o t h e i n n e r chamber v i a g r a v i t y f e e d r a t e o f 2-3 m l / m i n E x c e s s medium i s p l a s t i c tubing Media o f  ( D i a l - a - F l o , S o r e n s o n R e s e a r c h Co., t h e n drawn o f f b y s u c t i o n t h r o u g h  in a  w e l l connected  altered ionic  interruption of flow  to the  c o n s t i t u e n c y can  a  Utah ).  a piece of  recording  chamber.  be i n t r o d u c e d  through a three-way  at  without  valve outside of  the  chamber. Slices chamber  are  and  on  illuminated  c h a m b e r . A 95% passed  placed  a  by  o x y g e n / 5%  through  the  nylon  net w i t h i n the recording  reflected  light  from  carbon d i o x i d e gas  outer  chamber  below t h e  mixture  to superfuse  i s then  slices with a  warm, h u m i d i f i e d g a s m i x t u r e .  2-3.  S t i m u l a t i n g and R e c o r d i n g  Bipolar 62um  nichrome  (Narishige) slice  wire  and  electrodes  were  mounted  positioned i n  (Carl  Zeiss).  constructed on  a  major s t r a t a  under d i r e c t o b s e r v a t i o n w i t h  microscope and  stimulating  Techniques  micro-manipulator of the hippocampal  t h e a i d o f a 4X  S q u a r e wave p u l s e s  generator  M o d e l DS2)  (Digitimer,  delivered  at  a baseline  minimal stimulus long-term  All  Medical  Systems  rate of  a four Corp.).  stimulation of  units  (Medical  channel  pulse  Stimuli  were  1/7 s e e s  and  i n t e n s i t i e s used whenever p o s s i b l e t o a v o i d any  alterations  potentials  c o n t r o l l e d by  dissecting  o f 0.1 msec d u r a t i o n  1-70V i n t e n s i t y w e r e d e l i v e r e d f r o m i s o l a t i o n  Systems Corp.,  from t w i s t e d  (Turner  recordings  in  the  characteristics  of  evoked  e t a l . 1982). i n the present  work were c a r r i e d o u t i n t h e  - 23 CAlb  region  recordings  of  the mid-dorsal  were  obtained  along  pyramidal  cell  axis, while  elements  were  restricted  to  electrodes  were  r a t hippocampus. the  entire  intracellular  Extracellular  extent  of  the  impalements o f neuronal  stratum  pyramidale  and s t r a t u m  radiatum. Recording  constructed  N a r i s h i g e o r F r e d e r i c k Haer m i c r o e l e c t r o d e  using  puller.  either  a  Extracellular  e l e c t r o d e s o f 2-6 megohm i m p e d a n c e were p u l l e d f r o m e i t h e r g l a s s c a p i l l a r y tubing  ( F r e d e r i c k H a e r , Omega D o t T u b i n g  o r f r o m 2.0mm g l a s s t u b i n g and f i l l e d e l e c t r o d e s were  backfilled with  while  the tips  under  microscopic  diameter  of  Intracellular constructed O.D.)  of  observation  l-2um  from g l a s s  and  e l e c t r o d e s w e r e mounted intracellular  electrodes  to  electrodes  i n reference  Extracellular  field  bandpass  and  Recorded  electrical  storage PDP  were  to  manipulator  connected  Haer,  1.5mm  Extracellular ( N a r i s h i g e ) and  equipped  with  a  to the preamplifier  (WPI, M o d e l KS700) a n d p o t e n t i a l s  responses was  (Tektronix)  11/23 c o m p u t e r f o r s t o r a g e  were  ( B u r l e i g h Inchworm PZ-550) .  c h l o r i d e bath  p o t e n t i a l s were f i l t e r e d  oscilloscope  electrolyte.  (Frederick  a silver-silver  activity  needle  impedance  K+-acetate.  advance  intracellular  Microelectrodes  megohm  1M  a  filled  Germany) t o a  with  on a m i c r o - m a n i p u l a t o r  headstage o f a dual microprobe recorded  30-80  with  v a r i a b l e speed p i e z o e l e c t r i c Recording  Wetzler,  c a p i l l a r y tubing  backfilled  O.D.)  e l e c t r o d e s were b r o k e n back  backfilling  of  Fiber  gauge h y p o d e r m i c  (Leitz  before  electrodes  w i t h 2M N a C l .  a 31  non-capillary  , 1.5mm  a  using  and  a O.lHz-lOkHz  DC-lOkHz  displayed  on  ground.  bandpass. a  photographed o r  d u a l beam led to a  and subsequent a n a l y s i s .  o f 30-80 megohm i m p e d a n c e were f o u n d t o  be  - 24 satisfactory apical  f o r obtaining  dendrites  of  pyramidal  c a p a b i l i t y o f an e l e c t r o d e tip  of the  chamber.  impalements cells.  either  The  pulses  to  custom-made " p r o b e - d r i v e r " ,  probe  were  passing  immersing t h e  a r t i f i c i a l CSF o f the  somata o r  current  was f i r s t e x a m i n e d b y  electrode into the  Command  of  the recording  provided  and e l e c t r o d e s b a l a n c e d  by  through  use  of a conventional  Wheatstone b r i d g e  electrode voltage  r e s p o n s e t o a s q u a r e wave c u r r e n t p u l s e o f  to  1.5  nA.  Electrodes  i n t r a c e l l u l a r work  c i r c u i t while monitoring the  were  i f capable  considered  of passing  pyramidale  or  observation,  stratum  of  radiatum  cell  high-frequency  sharp drop o f t h e v o l t a g e a  hyperpolarizing  applied t o a i d recovery The  cell  maintained  then  current  microelectrode  5-15min  impalement.  with  minimal  change  Impalements (0.5 s e c ) the  membrane was s i g n i f i e d  by a  o f -30 t o -60mV,  r e s t i n g membrane  immediately potential.  stable i fthe resting  gradual  to  removal cell  recover  Pyramidal  i n t h i s manner c o u l d be m a i n t a i n e d 4hrs,  microscopic  o f 0.25-0.75nA was  of the c e l l  following  allowed  feedback  stratum  through  current over l-2min f o l l o w i n g i n i t i a l was  direct  response i n t h e order  p e n e t r a t i o n was c o n s i d e r e d  was  within  by a p p l y i n g b r i e f  capacitance  for  ( < 2.0mV ) .  placed  under  were o b t a i n e d  electrode. Penetration of the  and  were  up  o f inward o r  and advanced s l o w l y t h r o u g h t h e s l i c e .  of t h e pyramidal bursts  electrodes  acceptable  .75 nA  outward c u r r e n t w i t h minimal v o l t a g e d e f l e c t i o n Intracellular  a  cell  potential  of hyperpolarizing  p e n e t r a t i o n . The c e l l from  the procedure of  impalements  obtained  i n a s t a b l e s t a t e f o r up  i n membrane  to  characteristics (ie.  r e s t i n g p o t e n t i a l ) o r evoked p o t e n t i a l s over t h e d u r a t i o n o f t h e recording  period.  - 25 The  q u a l i t y o f e l e c t r o d e p e n e t r a t i o n and e x t e n t  recovery  was  estimated  by  calculating  the  o f membrane  input resistance of  t h e membrane, a s d e t e r m i n e d b y t h e membrane r e s p o n s e t o a s e r i e s o f s q u a r e wave c u r r e n t p u l s e balanced, current  and pulse  amplitude  corresponding through the  inward  duration)  t h e membrane. The membrane r e s p o n s e was  recorded,  of  of  up  and o u t w a r d s q u a r e wave  t o l.OnA (100 msec  a current/voltage  the  twelve  injections  applied across and  s i x to  i n j e c t i o n s . The e l e c t r o d e was f i r s t  (I/V) graph l a t e r constructed  t h e membrane  voltage  current pulse. A b e s t - f i t linear  the slope  of the  resistance  (Ri).  estimated  by  range  o f membrane  l i n e taken  the  amplitude  against  of  of  t h r o u g h c o n s t r u c t i o n o f an e n t i r e  manner was n o t  commonly  used  impalements as t h e a p p l i c a t i o n o f a f u l l injections  input  c o u l d be  current pulse.  that obtained  was  drawn  t h e membrane v o l t a g e s h i f t i n  calculated i n this  method  the c e l l  input resistance  the R i value  this  that of the  v o l t a g e d e f l e c t i o n s , and  r e s p o n s e t o a s i n g l e 0.5 o r l.OnA i n w a r d  plot,  plotting  s t r a i g h t l i n e was  as t h e v a l u e  Alternatively, the  by  Although  as a c c u r a t e as current/voltage  in distal  dendritic  range o f c u r r e n t  pulse  ( p a r t i c u l a r l y o v e r 1.5nA) c o u l d l e a d t o a d e c l i n e  in  t h e q u a l i t y o r s u d d e n l o s s o f t h e d e n d r i t i c i m p a l e m e n t . D a t a was collected  from c e l l s  exhibiting  an i n p u t r e s i s t a n c e o f a t  18 megohm o r more, w i t h t h e R i v a l u e present The  study  ranging  o f impalements used i n t h e  f r o m 18-37 megohm.  membrane p o t e n t i a l b a s e l i n e was c o n t i n u o u s l y  using a d i g i t a l voltage d i s p l a y ,  monitored  and t h e a b s o l u t e v a l u e  r e s t i n g p o t e n t i a l taken  as  upon w i t h d r a w a l  e l e c t r o d e from t h e i n t r a c e l l u l a r  extracellular  least  of the  compartment.  the baseline voltage s h i f t  of the observed to the  - 26  -  Further i n f o r m a t i o n on r e c o r d i n g arrangements f o r experiments chapters.  are  provided  in  the  specific  METHODS s e c t i o n of r e l e v a n t  - 27 3-0.  CURRENT-SOURCE DENSITY ANALYSIS OF ACTION POTENTIAL  DISCHARGE IN THE  3-1.  CAl REGION OF THE  HIPPOCAMPUS  Introduction  Of paramount function i s  importance to  the understanding  the i d e n t i f i c a t i o n  potential  (AP)  generation,  following  integration  of  of the  the  actual s i t e  final  excitatory  of  output and  neuronal  for action  of  a  neuron  inhibitory  synaptic  p o t e n t i a l s . In most c e l l s , the conductance mechanism r e s p o n s i b l e f o r spike g e n e r a t i o n  i s thought to e x i s t  i n the axon h i l l o c k ,  a  region under the i n f l u e n c e of c u r r e n t s p a s s i v e l y conducting  from  the p o i n t  1958;  Kado  of s y n a p t i c  1973;  Ringham  However, i t i s now  termination 1971;  Smith  to  action  dendritic  potential  level.  f a c t o r i n the  This  discharge  characteristic  summate with  Kandel  and  Ottoson  1982;  Smith 1983).  membrane can d i s p l a y  at  could  1961;  inputs i f the and  dendritic  increase  the  (Andersen  and  L l i n a s 1975;  L l i n a s and  1979) discharge  i n both somatic and d e n d r i t i c l o c a t i o n s i s the pyramidal of mammalian hippocampus. Evidence  into evoked  the  cell  a p i c a l dendrite  characteristics  through  stimulation  and  be a s i g n i f i c a n t  neuron thought to e x h i b i t Na+-dependent s p i k e  pyramidal  a  giving  both the somatic  at the axon h i l l o c k  Spencer  Sugimori 1980b; Wong et a l . One  al.  synaptic currents  p r o b a b i l i t y f o r AP d i s c h a r g e 1966;  and  the dendro-somatic a x i s ,  i n t e g r a t i o n of s y n a p t i c  spike should  Lomo  et  thought t h a t neuronal  non-uniform e x c i t a b i l i t y along rise  (Edwards  of  f o r spike g e n e r a t i o n  came from  afferent  in  the  early investigations  extracellular of  neuron  field  potentials  excitatory  synaptic  - 28 inputs  (Andersen  Fujita  and  negative  1959; Andersen  Sakata  spike  cell  in  both  recordings "fast  in  directions  pyramidal  pre-potentials"  (Andersen  and Lomo  apical dendrite  finally  cell  cell  actual  Sakata  inferred  from  of  small  t o represent  confirmed  an  from w i t h i n t h e d e n d r i t i c 1 9 7 7 ; S p e n c e r and  impalements o f t h e pyramidal that stimulation  d e n d r i t i c membrane  s i t e f o r generation  spike i s presently Since  cell  of afferent  (Wong e t a l . 1 9 7 9 ) . of  unknown.  extracellular  generation  characteristics pyramidal  cell  in of  potentials are  the  dendrite  extracellular  axis. Several  potentials  However,  t h e Na+-dependent d e n d r i t i c  a c t i v a t i o n of i o n i c channels i n neuronal  field  the  i n p u t s g a v e r i s e t o s p i k e a c t i v a t i o n (Na+-dependent) i n  pyramidal  spike  cell  along  somata  1966; S c h w a r t z k r o i n  Kandel 1961b). M i c r o e l e c t r o d e  the  pyramidal  and Hamlyn 1 9 5 5 ; F u j i t a and  e l e c t r o t o n i c a l l y decayed s p i k e a r i s i n g  synaptic  of the  an e v o k e d  s u p p o r t f o r d e n d r i t i c s p i k e a c t i v a t i o n came  Na+-dependent  tree  reported  1955;  ( A n d e r s e n 1 9 6 0 ; A n d e r s e n and Lomo 1 9 6 6 ;  Andersen e t a l . 1966; Cragg  intracelluar  a l l levels  propagated  structure  1962). Further  studies  and Hamlyn  t h e a c t i v a t i o n o f a s p i k e i n d e n d r i t i c membrane  subsequently  pyramidal  These  potential at  axis, suggesting that  1962).  1960; Cragg  have  been  generated through  the  membrane, t h e s i t e  for  might field  laminar  be  potentials  profile  carried  detected  analyses  out  in  i n the  along the of  evoked  the CAl region.  However, t h e s i t e o f o r i g i n o f s p i k e s e v o k e d t h r o u g h s t i m u l a t i o n of a f f e r e n t synaptic along  the  pyramidal  investigators generation  i n p u t s has been  to  have  cell  assigned  proximal  placed  structure. the  dendritic  lowest  at various  For  instance,  threshold  membrane  points some  f o r spike  (Andersen  1959;  - 29 A n d e r s e n 1 9 6 0 ; F u j i t a and S a k a t a 1 9 6 2 ) , w h i l e o t h e r  s t u d i e s have  reported  (Andersen e t  al.  initial  1961)  or  spike activation distal  i n either  dendritic  regions  somatic  of the pyramidal  cell  ( A n d e r s e n 1 9 6 0 ; A n d e r s e n e t a l . 1 9 6 6 a , b ; A n d e r s e n and Lomo 1 9 6 6 ; C r a g g and Hamlyn 1 9 5 5 ) . The the  v a r i a b i l t y i n these  majority  of  extracellular  characteristics of relied  r e s u l t s may r e l a t e t o t h e f a c t investigations  spike discharge  i n the  s o l e l y upon t h e i n t e r p r e t a t i o n  waveforms. A l t h o u g h  much i n f o r m a t i o n  way, t h e l o w r e s i s t i v i t y  the  locate the potential  accuracy  true s i t e (Gloor  Nicholson  by  which  the  cell  have  potential  gained  in  this  field  potential  a l . 1963;  Leung  1979b;  resolution  a n a l y s i s can t h e evoked  M i t z d o r f 1985;  of neuronal  analysis, i n  which the i o n i c channels responsible f o r generation  o f an e v o k e d  can  be s p a t i a l l y  (sink) or accumulation  density  activity  (CSD)  potential  through current-source  in  conduction,  channels underlying  1 9 7 3 ) . A more a c c u r a t e  is obtained  has been  over space t h r o u g h volume  of ionic  et  of e x t r a c e l l u l a r  into  o f t h e e x t r a c e l l u l a r medium r e s u l t s  a d i s s i p a t i o n of voltage reducing  pyramidal  that  localized in  (source)  terms of  of current with respect  e x t r a c e l l u l a r medium ( P i t t s 1 9 5 2 ) . U n l i k e f i e l d density  of  localized,  current with  the  superior estimate transmembrane field  as  that  CSD  to the  potentials,  space  the  c a n be w e l l  p r o f i l e s provide  afar  compared  to  that obtained  of  through  a n a l y s i s (Freeman and S t o n e 1 9 6 9 ; M i t z d o r f 1 9 8 0 ;  1973; N i c h o l s o n  discharge  extracellular  loss  o f t h e l o c a t i o n , m a g n i t u d e and t i m e c o u r s e  At t h e present spike  the  result  currents  potential  Nicholson  in  a net  time,  in  the  and Freeman  1975).  a current-source CAl  pyramidal  density analysis cell  has  only  of been  - 30 performed  for  pyramidal for  cell  initial  axons  evoked  by  antidromic  sink  pyramidale.  soma-axon h i l l o c k , in  Both  proximal  as  and  oriens  apical  s u b s e q u e n t l y i n v a d e d by  a short duration  strong  a  conduct  in  that  a  retrograde  a r b o r i z a t i o n of pyramidal Comparable s t u d i e s discharge  in  spike  the  c e l l s was  i n d i c a t e d by  stratum  the basal  evidence  at  have not cell  y e t been  latency  the  stratum  the  were  providing layer  can  dendritic  performed f o r  following  density analysis  extracellular  o r i g i n of the  a short  the c e l l  through  the  neurons.  pyramidal  of evoked the CAl  found i n  source/sink,  inputs. Therefore,  in  site  d e n d r i t i c regions  evoked manner  or  afferent excitatory synaptic  c a r r i e d out  s t i m u l a t i o n of  (Leung 1 9 7 9 a , b ) . I n t h e s e s t u d i e s , t h e  spike a c t i v a t i o n i n pyramidal  region of the current  potentials  -  region  spike  stimulation a  field  f o r the purpose  evoked d e n d r i t i c s p i k e of p y r a m i d a l  of  current-source potentials  was  of l o c a t i n g the neurons.  - 31 3-2.  Methods  Current-Source  Density Calculations  Current-source  density analysis i s  a means b y w h i c h  ionic  movements a c r o s s t h e membranes o f c e l l s w i t h i n a p o p u l a t i o n be  localized  in  terms o f c u r r e n t  movement w i t h r e s p e c t  extracellular  space  extracellular  to the i n t r a c e l l u l a r  net  loss or  neuronal  as  membrane.  distribution  of of  density  the  to the extracellular  compartment  current  determined  by  the  (Freeman and  c a n be  flow  current i nthe  anatomical  Stone 1969;  i n a t l e a s t two p l a n e s can  be  attributed  elements running  transverse to  (ie.  fibers;  myelinated 1973)  or  dendrites) p a r a l l e l 1969; in  to  an  of  the  extracellular  is  i n large part  neuronal  Nicholson  tissue  and Freeman values of  of a rectangular  coordinate  to  the presence of neuronal  and  axis of  of current  flow  S t o n e 1 9 6 9 ; H a b e r l y and of  core conductors  current flow  Nicholson  of a  and  exhibit different  alignment  1985;  the structure  The  the direction  Freeman  to the  Mitzdorf  medium.  M i t z d o r f 1985; found t o  the s p a t i a l  deflections  structure  conductivity This  from  (conductivity)  tissues are  Variations  of  estimated  voltage  1975). Most  Stone  or source  extracellular  to  Shepherd  a  the flow of  extracellular  resistance  system.  often signifying  a  space.  Current-source  resistivity  as  In contrast,  a net accumulation  extracellular  compartment i s d e t e c t e d  a r e s u l t most  c u r r e n t from t h e i n t r a c e l l u l a r i s observed  to the  ( P i t t s 1 9 5 2 ) . A movement o f c u r r e n t f r o m t h e  sink of current,  depolarization of  can  and  cellular  (Freeman and  Freeman  aggregate  l e a d t o a l a c k o f homogeneity o f e x t r a c e l l u l a r  (ie.  1975). can a l s o  resistance  along  - 32 a  principle  coordinate  axis,  observed at the border of c e l l and  S h e p h e r d 1973;  Mitzdorf  Experimentally, the  a  simultaneously of  recording  electrodes,  taking  extracellular Nicholson current  1975;  ox,  az  are  V(t)  tissue displaying c a l c u l a t i o n of  t for  as  Under  is  at  ideal  obtained  by array  c o n d u c t i v i t y of  dimensions 1969). x,y,z  the  (Freeman  and  In t h i s case, of a  the  rectangular  by:  the  e x t r a c e l l u l a r voltage  and  a high  conductivity values for  degree of  current-source  commonly  found  few  principle cell  In  this  case, a  arranged  laminar  relatively  be  f o r time  cortical  1973).  the a  Such a of  c o m p r i s e d o f one  organization  at  neuronal  structures  in a serial  high  t  s i m p l i f i e d to  Shepherd  neuronal t i s s u e i s  types  the  can  and  in  x,y,z  laminar organization,  density  condition  where  the  Freeman 1 9 7 5 ) . However, i n  (Haberly  in  CSD  the  point  analysis  results  approximated  from a 3-dimensional  Stone  1-dimensional  mammalian CNS,  of  three  and  the  (Nicholson  is  (Haberly  =  ay,  x,y,z  is  tissue.  account  all  at time  d i m e n s i o n s , and point  into  system i s given  S(t,x,y,z)  neuronal  potentials  in  conductivity  f i b e r bundles  density  analysis  Freeman  density  coordinate  where  space  change i n  of evoked p o t e n t i a l s recorded  in  real-time  a  1985).  current-source  locations  conditions,  with  l a y e r s or  second s p a t i a l d e r i v a t i v e  equidistant  -  the or  a  laminar  fashion.  of neuronal  elements  conductivity  along  the  - 33 dendro-somatic  axis  of  the  principle  synchronous a c t i v a t i o n of the e x t r a c e l l u l a r current the  tissue  current into  (Haberly  flow  the  flow  (Haberly  and  of  of  estimates  density  s i n k s and  sources are  1980,1985; N i c h o l s o n  and  Therefore, given large scale equidistant  the  is  of evoked  absolute  1-dimensional a n a l y s i s given  ( V  and  a  the time  as a  a minor and  the  constant  S h e p h e r d 1973;  Mitzdorf  of: structure  potentials parallel  density  can  l -V - 2 - V  = (AX)  to  the  type  where  (V  r dx  of  measured  profiles,  by:  ^  S(tyx) = a  use  v a l u e of  polarity  treated  principle cell  current-source  a  are  1975).  a homogeneous c o n d u c t i v i t y t e n s o r ,  ,rt-V  excluding  the  synchronous a c t i v a t i o n of a l a m i n a t e d  c a l c u l a t i o n of  introduced  by  words,  CSD  usually  conditions  recording  of  little  a l s o been found t o e x e r t  Freeman  dendro-somatic a x i s of the 3)  errors  p r e s e r v e d . The  H a b e r l y and  the  laminar plane  q u a l i t a t i v e nature  the  q u a l i t a t i v e aspects of  S t o n e 1969;  of  c o n d u c t i v i t y along the p r i n c i p l e a x i s  conductivity  (Freeman and  majority  these dimensions  other  a f f e c t the  of a l a m i n a t e d s t r u c t u r e have  extracellular  In  during  Relatively  than  measurement, but  v a l u e s of v a r i a t i o n i n the  the  the  density  in  rather  1973).  a n a l y s i s may  course of c u r r e n t  2)  1973).  current-source  a quantitative Shepherd  current-source  1)  population,  Shepherd  conductivity  1 - d i m e n s i o n a l CSD  i n f l u e n c e on  t y p e , and  i s f o u n d n o r m a l t o t h i s a x i s , and  extracellular be  cell  cell  i s r e s t r i c t e d to the  and  calculation  found to  -  ax  =  ay  =  az  be  reduced to  a  - 34  V  +  l  V  2 V  ~  3  -  2  (AX)'  where  VI,  three  consecutive  separated  V2,  by  V3  the  CAl  of  cell  of the hippocampus  of s t i m u l a t i n g  v i s u a l l y confirmed, stimulation  and  of  large voltage pyramidal reducing of the  and  or  cell  (up  to  of the s l i c e  cell  axis  mV/mm;  to  found  dendritic  in  the  region  resistivity;  conductivity  will  a slight  also gives rise  Richardson  only  density  extracellular  conductivity  1-dimensional  stratum  J e f f e r y s 1984), t h i s  current-source  synchronous  of  increase  activation  affect  the  measurements,  of  can the  current-source  a p p r o p r i a t e l y a p p l i e d i n the CAl  be  in  density  to  a x i s of  the  et a l . 1984b), plane  resistivity  pyramidale  (1.5  has -  3X  slight variation  in  absolute  of  and  a  value  homogeneous  assumed. T h e r e f o r e , pyramidal  be  activated  the degree of c u r r e n t f l o w normal t o the laminar t i s s u e . Although  the  projections.  the dendro-somatic  120  symmetric  e l e c t r o d e s can  fiber  population  satisfies  preparation  population  efferent  of the c e l l  gradients along  perpendicular  recording  the pyramidal  afferent  Synchronous d i s c h a r g e  analysis, with  structures  body l a y e r . Through use  the placement  dendro-somatic  at  _x.  region  dendritic  p o t e n t i a l s recorded  the  r e q u i r e m e n t s o f a 1 - d i m e n s i o n a l CSD  pyramidal  been  along  A  a d i s t a n c e of  organization  by  the f i e l d  locations  Anatomically, the  represent  cell  given  a  population,  a  analysis  r e g i o n of the  can  hippocampus.  be  - 35 S t i m u l a t i n g and R e c o r d i n g  Procedures  Synchronous a c t i v a t i o n o f t h e p y r a m i d a l c e l l achieved through  stimulation of  hippocampal s l i c e . pyramidal  cell  known t o r e s u l t  The f i r s t  efferent  t h r e e d i s t i n c t pathways i n t h e  was s u p r a t h r e s h o l d  fibers  in  s t r a t u m p y r a m i d a l e . The s e c o n d inputs  to  the  pyramidal c e l l s through 3.1).  A  activation of  the alveus  (SI; F i g 3.1),  i n a n t i d r o m i c i n v a s i o n o f t h e p y r a m i d a l c e l l and  recurrent activation of inhibitory  excitatory  p o p u l a t i o n was  third  interneurons i n the region of  employed s t i m u l a t i o n o f  basal  dendritic  stimulation  stimulating  arborization  of  o f stratum o r i e n s (S2; F i g  electrode  was  placed  i n stratum  radiatum t o a c t i v a t e Schaffer c o l l a t e r a l / c o m m i s s u r a l inputs synapsing d i r e c t l y  afferent  upon p y r a m i d a l c e l l  excitatory  apical dendrites  (S3; F i g 3 . 1 ) . Laminar p r o f i l e s o f from each o f slice) 25um  extracellular  t h e above pathways  field  potentials  ( u s u a l l y one p r o f i l e  evoked f o r each  were c o n s t r u c t e d by r e c o r d i n g e x t r a c e l l u l a r p o t e n t i a l s a t intervals  parallel  p y r a m i d a l c e l l . To e n s u r e along a s t r a i g h t l i n e  to  the  dendro-somatic  axis  of the  t h a t r e c o r d i n g p o s i t i o n s were  p a r a l l e l to the dendritic  recording electrode manipulator  was  secured  located  structure, the  i n a position  with  the h o r i z o n t a l t r a v e l of t h e e l e c t r o d e o r i e n t e d p e r p e n d i c u l a r t o stratum pyramidale. t h e n be made and cell  Consecutive  without v i s u a l estimation  the distance of measured  manipulator.  movements o f t h e e l e c t r o d e c o u l d  the recording l o c a t i o n along  directly Evoked  withdrawal  of  electrode  track  of the recording  the  from  the  potentials electrode  in  micrometer  were from  order t o  always the  avoid  axis,  the pyramidal scale  of  recorded  maximum  depth  any a l t e r a t i o n  the during  of the of the  FIG.  3.1  strata  Schematic  of the CAl  placement  of  pyramidal  cell  used  evoke  to  pyramidal was  used  dendritic  cell to  diagram  of  region of the  stimulating  cell  and  hippocampus, i l l u s t r a t i n g  the  electrodes  population. an  the  Stimulation  antidromic  tree,  and  a  synaptic  activation  of  Stimulus  evoked  afferent third the  potentials  for  pyramidal  activation  of the  alveus  of  the  (SI)  was  response through s t i m u l a t i o n of  axons. A second e l e c t r o d e stimulate  rat  synaptic  in  stratum  apical were  i n stratum  inputs to the radiatum  dendritic  recorded  oriens  basal for  arborization.  parallel  dendro-somatic a x i s at a l l l e v e l s of the pyramidal  (S3)  (S2)  to  neuron.  the  - 37 -  S| — Alveus  St. Oriens  St. Pyramidale  St. Radiatum  - 38 extracellular tissue during  waveform forward  Profiles  of  that  might  electrode  field  within  t h a t t h e peak  potential  could  vary  with  depth  which  peak  amplitude  various p o i n t s along  the  the c e l l  compression  of  advancement.  potentials  electrode track revealed  at  accompany  a single recording  amplitude  o f an  evoked  r e c o r d i n g d e p t h , and t h a t t h e was  axis.  attained could d i f f e r at Since minor v a r i a t i o n s i n  w a v e f o r m c h a r a c t e r i s t i c s c o u l d be o f s e r i o u s c o n s e q u e n c e t o t h e accuracy  of  current-source  density  measurements,  evoked  p o t e n t i a l s w e r e c o l l e c t e d a t 20jum i n t e r v a l s o f d e p t h up t o 100/am above  and  below  the  (determined  prior  to serial  along to  approximate  depth  of  maximal waveform  p r o f i l e measurements).  Potentials  e a c h e l e c t r o d e t r a c t were t h e n a v e r a g e d b y c o m p u t e r  eleven  sweeps), a v o i d i n g  the problem of p o t e n t i a l  variation  w i t h r e c o r d i n g d e p t h and p r o v i d i n g t h e e q u i v a l e n t o f an value  of  current-source  perpendicular The  density  over  of f i e l d  p o t e n t i a l s were  w i t h a second r e c o r d i n g e l e c t r o d e p l a c e d and  laminar  any  change i n t h e c o n t r o l p o p u l a t i o n  the  experiment.  p r o f i l e s omitted  Current-source  interval  as along  current-source  "column"  average  of tissue  t o the dendro-somatic a x i s of the pyramidal  stability  dimension  a  (four  a  density continuous  the density  d e l t a X used i n each voltage gradient along  from data  continuously i n stratum  spike during the course  of  function  of  was o p t i m i z e d  axis.  performed time The  by v a r y i n g  c a l c u l a t i o n according different  pyramidale, of  was  cell  monitored  a n a l y s i s i n t h e event  analysis  pyramidal  cell.  regions  in  one  f o r e a c h 25 um (  r e s o l u t i o n of the value  to the slope  of the pyramidal  of  of the cell.  I n g e n e r a l , a d e l t a X o f 75um was u s e d i n d e n d r i t i c r e g i o n s , b u t  was such  shortened as  in  increase  vicinity  signal-noise  a sampling  a v e r a g e d and density  25rtm i n a r e a s  the  the  o n - l i n e at  to  digitally  calculations.  calculated  i n one  conductivity  w a v e f o r m s c a n n o t be  not  of  up t o  current-source the value of the  determined  in  the  were  could  density  present  study,  of major e f f e r e n t or  CSD  estimates profiles.  q u a l i t a t i v e c h a r a c t e r i s t i c s of s i n k - s o u r c e r e l a t i o n s h i p s  slice.  was  extracellular  c u r r e n t - s o u r c e d e n s i t y p r o f i l e s a r e u s e d t o compare  pathways i n the hippocampal  be  current-source  u n i t s a r e n o t p r o v i d e d on CSD  CAl region f o l l o w i n g s t i m u l a t i o n  to  collected  25kHz, and  before applying  Since and  potentials  In order  considered absolute q u a n t i t a t i v e  o f c u r r e n t d e n s i t y , and Rather,  ratio,  filtered  voltage gradient,  stratum pyramidale.  frequency  dimension,  was  of  with a steep  in  the the  afferent  - 40 3-3.  -  Results  M o r p h o l o g i c a l C h a r a c t e r i s t i c s o f t h e Rat C A l P y r a m i d a l In to  order to c o r r e l a t e current-source density  specific  anatomical  locations  of  the  Neuron  relationships  pyramidal  d e t a i l e d k n o w l e d g e o f b o t h t h e s t r u c t u r e and  cell,  r e l a t i o n s h i p of  a  the  cell  to v a r i o u s s t r a t a w i t h i n the CAl region i s r e q u i r e d . Almost  all  measurements  pyramidal c e l l  of  Maxwell  1980).  An  length  structures  guinea-pig or r a b b i t and  the  1961;  branching  have been c a r r i e d  (Cajal  Lorente  exception i s  and  1911, de No  out  C r a g g and  1934;  Westrum and  pattern i n the  Hamlyn  Turner  and  of  mouse,  1955;  Green  Schwartzkroin  B l a c k s t a d ' s (1962)  electron  m i c r o s c o p i c study of CAl pyramidal neurons i n the r a t .  Although  the g e n e r a l morphology of the p y r a m i d a l c e l l species,  the  exact  c o n s i d e r a b l y . For dimension  dimension  of  neuronal  elements  "considerably  smaller"  than  1961).  according  The  to  of  the  size  the  septo-temporal  violet  vary  the in  rat the  are rabbit  pyramidal c e l l  hippocampal  subfield  c e l l s of  can and  and  said to (Green also  be and  vary  along  the  r e g i o n c o n t a i n i n g some  the hippocampal  formation  were t h u s made t o o b t a i n q u a n t i t a t i v e e s t i m a t e s  cell  accomplished  can  1934).  t h e w i d t h o f s t r a t a and pyramidal  those  of the  smallest pyramidal  Attempts  in  p o l e , w i t h the d o r s a l CAlb  ( L o r e n t e de No  processes  between  i n s t a n c e , the l e n g t h of the proximal s h a f t  of  Maxwell  cell  is similar  o f t h e l e n g t h and  dendrites  through  light  in  the  CAl  microscopic  branching  of  p a t t e r n of  region.  This  examination  of  s t a i n e d hippocampal s l i c e s or of pyramidal c e l l s  was cresyl  in  s e c t i o n s of hippocampal t i s s u e s t a i n e d immunohistochemically  thin for  - 41 calcium-binding  protein  (CaBP;  Since subsequent i n t r a d e n d r i t i c apical  dendrite,  dendritic  attention  elements  d e n d r i t i c diameter  -  in  Baimbridge  1982).  Miller  r e c o r d i n g s were c o n f i n e d t o  was  f o c u s e d p r i m a r i l y upon  stratum  taken  and  the  apical  r a d i a t u m , w i t h measurements o f  f r o m t h e s t u d y o f Westrum and B l a c k s t a d  (1962). Measurements o f t h e w i d t h o f s t r a t a i n r a t hippocampus gave values f o r the  a l v e u s o f 70  s t r a t u m o r i e n s 160  +/  -  18.1  +/-  17.8  spanning  102  the d i s t a n c e  +/-  would  i n golgi-stained pyramidal  and  Schwartzkroin  15-20rim,  (n=18).  edge o f s t r a t u m  +/-  stratum  Pyramidal  cells  oriens to  therefore exhibit a  maximal  c e l l s of the guinea-pig  boundary between s o m a t i c  difficult  apical dendritic cell  44  up  (Turner  somata  as t h e  s h a f t , but  fall  respectively.  cell  within  Although  and  body t a p e r s  the width the  the  dendritic gradually depth  of  r a n g e o f 10-15pm  and  length  and  of  the  proximal  a p i c a l d e n d r i t i c s h a f t c a n v a r y c o n s i d e r a b l y , most a r e w i t h i n range  of  dendrite  25-100pm. is  The  greatest i n  diameter  of  the proximal  the  rat pyramidal  s h a f t (3-4pm)  t a p e r i n g t o a w i d t h o f 2-3jum a t t h e f i r s t b r a n c h and  B l a c k s t a d 1962).  slight diameter  angle  to  1980).  D e l i n e a t i o n of the  pyramidal  sem),  c o n t r a s t s w i t h a l e n g t h of  1mm  into the  mean +/-  7. 8jum (n=18) , and  4.4pm  l e n g t h o f 600jum. T h i s  membrane i s o f t e n  +/-  from t h e deep  stratum lacunosum-moleculare overall  (n=5;  jam (n=5) , s t r a t u m p y r a m i d a l e  2.9jum (n=34) , s t r a t u m r a d i a t u m 303 lacunosun-moleculare  pm  Secondary  towards  the  branches  hippocampal  o f 0.5-lpm (Westrum and  progress fissure,  B l a c k s t a d 1962).  t r a n s v e r s e spread of d e n d r i t i c branching  point  a  cell  gradually (Westrum  radially  at  a  tapering to a The  degree  from the r e g i o n of  of the  - 42 soma c a n  be as  g r e a t as  -  225um, b u t  i s more  usually within  a  r a n g e o f 50-100/jm.  Laminar P r o f i l e s o f Evoked A c t i v i t y The  characteristics  p o t e n t i a l s and  of  i n CAl Pyramidal  evoked  sink-source relationships  Neurons  extracellular  field  i n the CAl region  were  examined over the e n t i r e a x i s of the p y r a m i d a l neuron. most a t t e n t i o n was current-source radiatum,  f o c u s s e d upon e v o k e d e l e c t r i c a l  density  regions  in  stratum  corresponding  d e n d r i t e s of the pyramidal c e l l  Antidromic Population Spike The  laminar  (voltage)  and  F i g 3.2.  point  for  neuronal  the axis  pyramidale The found  In t h i s  distance is  taken  somata  of  extracellular  stratum  and  current-source  field  apical  potentials  d e n s i t y (CSD)  CAl pyramidal c e l l s  a l l subsequent f i g u r e s ,  evoked  are  the  shown  reference  of  the r e c o r d i n g e l e c t r o d e along  as  the  ventral  border  c h a r a c t e r i s t i c s of the antidromic f i e l d  to  vary  pyramidale,  according structure  alvear  negative-going  response  1967). A s i m i l a r  to  of  the  stratum  (Fig  3.2A). At  the l e v e l  evoked  characteristic (Fig  potential  the r e c o r d i n g p o s i t i o n  stimulation  potential  population spike  on e i t h e r  the  and  (Gjum) .  pyramidal c e l l  al.  and  pyramidale  and  population, respectively.  through antidromic a c t i v a t i o n of in  activity  Response  profiles  associated  to  However,  3.2A;  r e s p o n s e was  Leung  recorded  were  along of  a  short  of  an  stratum latency  antidromic  1979a,b; S p e r t i approximately  et  lOGjum  s i d e of s t r a t u m p y r a m i d a l e , but the n e g a t i v i t y of  p o t e n t i a l was  the  o f s h o r t e s t peak l a t e n c y i n s t r a t u m p y r a m i d a l e  the or  - 43 the  proximal  stratum  progressively  declined  along  the  stratum spike  apical  was  oriens. in  with  In stratum  continuous  positive/negative  The  population  amplitude  dendrite  pyramidale.  -  increased  distance  radiatum,  with  the  dendritic  and  spike  then  in latency  from the border  the n e g a t i v i t y of  negative  potential  the  component  a The  p o t e n t i a l s of  the  d e n d r i t i c waveform c o n t i n u e d  to increase with distance along  the  dendritic tree, giving rise  in distal  monophasic p o s i t i v e f i e l d  field  axis  amplitude pyramidale  (Fig  dendritic locations  d e n s i t y from laminar  p o t e n t i a l s also revealed  c h a r a c t e r i s t i c s of c u r r e n t sources cell  negative  3.2B).  Alvear  and  stratum  of c u r r e n t  region of stratum from the  inward  stratum  flow  radiatum,  of c u r r e n t  dendrites.  Both  the gradual  response  displayed  a  distance  from  border  stratum  the  radiatum,  progressively  the  source  increased  The negativity  the  indicating  intracellular  c o u l d be  observed  r e v e a l i n g an o u t w a r d  i n the source  of  evoked  oriens,  e x t r a - to  and  stratum  in  followed  region of pyramidal  increase  cell  s i n k of the d e n d r i t i c in  peak l a t e n c y  pyramidale.  In  with  distal  component o f t h e d e n d r i t i c w a v e f o r m  i n d u r a t i o n c o i n c i d e n t w i t h a d e c l i n e of  the current s i n k , e v e n t u a l l y forming in d i s t a l  pyramidal  c u r r e n t s i n k w i t h s h o r t e s t peak l a t e n c y w i t h i n or the p r o x i m a l  stimulation  the  i n the  large  the proximal-mid  apical  profiles  a transition  sinks along  compartment. A b i p h a s i c c u r r e n t s o u r c e / s i n k  an  a  a  t h e movement  by  to  potential.  C a l c u l a t i o n of current-source of antidromic  3.2A,  of  150pm).  peak l a t e n c y o f b o t h t h e p o s i t i v e and  (Pig  of  a monophasic c u r r e n t  source  apical dendritic locations. shift and  in  peak  latency  c u r r e n t sink along  of  the  field  the pyramidal  potential cell  axis i s  - 44 FIG.  3.2  potentials through  A,B.  Laminar  (voltage)  and  were  dendro-somatic  collected  extracellular  current-source  measurements comparison  at  25um  axis of the pyramidal  density calculated  i n 1 dimension  are displayed of  sink-source  diagram of the r a t pyramidal with the borders  field  d e n s i t y (CSD) e v o k e d  stratum pyramidale potentials  with  steps cell,  a  to  the  and B, c u r r e n t - s o u r c e  line A  CSD  to facilitate  scaled  schematic  i s i n t e r p o s e d between  a x i s i s taken  (Oum).  zero  relationships. cell  parallel  Evoked  f o r e a c h 25pm i n t e r v a l .  o f stratum pyramidale  Distance along the c e l l  profiles  d e n o t e d by d o t t e d  lines.  from t h e v e n t r a l border o f  C,D. A c o m p a r i s o n  of extracellular  (C) and a s s o c i a t e d CSD m e a s u r e m e n t s (D) a t t h e  l e v e l of pyramidal (160jum ) i n r e s p o n s e taken  of  s u p r a t h r e s h o l d a l v e a r a n t i d r o m i c s t i m u l a t i o n . A.  potentials  field  profiles  cell  somata (  -20pm ) and  apical  dendrites  t o a l v e a r a n t i d r o m i c s t i m u l a t i o n (waveforms  from laminar p r o f i l e s  i n A,B). Voltage p o l a r i t y  down i n t h i s a n d a l l s u b s e q u e n t  figures.  negative  - 45 -  A.  Voltage  B.  CSD  200-  v_ -—  O  -100  -  0  -  CD  2 S CO  + 100 -  >^  QCL 200  o o  10 300 -  1  C. Soma  D.  I Source Sink  Apical Dend.  1  5 mV 5 msec  - 46 more c l e a r l y  d i s t i n g u i s h e d by  response at the 3.2C,D). The  comparing the  l e v e l of stratum  n e g a t i v i t y of  evoked  p y r a m i d a l e and r a d i a t u m  the antidromic  field  e v o k e d a t a peak l a t e n c y o f 1.1msec i n t h e c e l l  of t h e evoked c u r r e n t  sink  (Fig  p o t e n t i a l was  body l a y e r  3.2C) and 1.96msec i n t h e a p i c a l d e n d r i t i c r e g i o n border of stratum pyramidale  antidromic  ( F i g 3.2C). A s h i f t  (Fig  160um f r o m t h e i n peak  i s a l s o a p p a r e n t , w i t h peak  latency latency  m e a s u r e m e n t s o f .9msec i n s t r a t u m p y r a m i d a l e and 2.1msec a t 160^um d e n d r i t i c r e c o r d i n g Therefore, indicate  activation of cell  body  alvear  and c u r r e n t - s o u r c e  stimulation  an a n t i d r o m i c  somata, as  population  l o c a t i o n ( F i g 3.2D).  both voltage  that  shown  spike  by  the  s p i k e and c u r r e n t  source/sink  through  stratum  subsequent  antidromic  spike  often  be  In  fact,  observed  stimulation,  analyses  cell.  presence  of  i n the  evidence  supporting  radiatum  short-latency  invasion  further  stratum  current  suggests  a  of the a p i c a l d e n d r i t i c  oriens  of p o t e n t i a l s following  could alvear  and a p i c a l d e n d r i t i c t r e e o f t h e  results  were  obtained  Leung 1 9 7 9 a , b ; the  interpretation that  p o t e n t i a l can  activation  positive-going  potential  evoked  previous  hippocampus  a l . 1967), a l l  an a n t i d r o m i c a l l y  s e q u e n t i a l l y invade  also  in  i n vivo  Sperti et  a r b o r i z a t i o n s of the CAl pyramidal  Antidromic (7-15msec)  of pyramidal  a b i p h a s i c p o t e n t i a l and  the basal  Similar  a l . 1966;  evoked a c t i o n dendritic  a  initial  v i c i n i t y of the c e l l  o f a l v e a r evoked p o t e n t i a l s i n t h e  (Gessi e t  the  the region  s i m i l a r sequence  in  in  profiles  i n d i c a t i n g t h a t an a n t i d r o m i c a l l y e v o k e d s p i k e c a n  conduct through both pyramidal  a  density  results in  sink  l a y e r . The c o n d u c t i o n o f  arborization.  the  t h e soma  and  neuron. a  long  duration  immediately f o l l o w i n g the  - 47 population spike i n stratum of  stratum  o r i e n s and  p o t e n t i a l was the c e l l  -  pyramidale  radiatum  (Fig  and  the proximal  3.2B;  -90  a s s o c i a t e d w i t h a current source  layer  (Fig  3.2B,  mid-dendritic locations.  -25 The  - 25jum)  sink  and  - 60pm).  comparison of  a  current sinks along  this  150jum) .  This  present  study,  activation stratum  potential but  of  pyramidale  Subthreshold  was  most  not  potential  et  al.  Excitatory Synaptic  stratum  oriens  characteristics  cell  stimulation  of at  pyramidal proximal to often  Fig cell  (Fig  graded  in  the  region of  Dingledine  radiatum  activation  and  Stratum  the region at  layer  to  invert  in  amplitude  in  (SO) of  of pyramidal  cell  the l e v e l  amplitude  of  The  the  negativity (3.5mV  towards  to a p o s i t i v e potential  stratum pyramidale.  the  potential  maximal i n s t r a t u m o r i e n s  decreasing  of  oriens  a p i c a l d e n d r i t e s ( F i g 3 . 3 A ) . The  3.3A),  in  excitatory  dendritic locations 3.3).  of  displayed  of  negative-going  duration in  or w i t h i n  largest  examined i n the  subthreshold stimulation  stratum  (EPSPs) i n  e v o k e d p o t e n t i a l was  -100/jm,  -  to the recurrent  1969:  a positive-going potential  b o d y l a y e r and  t h e SO  a  20msec  d e n d r i t e s and cell  50  Potentials  the  population  evoked  approximately  or  reflecting  postsynaptic potentials pyramidal  potential  Leung 1979a,b,c; S p e r t i e t a l . 1 9 6 7 ) .  F i e l d p o t e n t i a l s evoked through either  inputs  spike,  ( F i g 3.2B,  corresponds  synaptic  (Andersen  axis  extensively  likely  inhibitory  Langmoen 1980;  of a c t i o n  the a p i c a l d e n d r i t i c  of  current sink i n  component o f  the amplitude  This  i n the region  could thus o v e r l a p w i t h t h a t of the d e n d r i t i c antidromic preventing a  region  positivity  the proximal a p i c a l  the just was  dendritic  - 48 region,  progressively  positive  polarity  3 . 3 A ) . I n some  throughout  slices,  followed  by  increased  i n amplitude  3.3A).  a  This  deflection  both  amplitude  but r e t a i n i n g a  apical dendritic field (Fig  evoked d e n d r i t i c n e g a t i v i t y  potential  of  positive  with proximity t o stratum  potential just  in  the  t h e SO  second  beyond  waveform i n p r o x i m a l with  declining  was the  stratum  potentials  observed peak  of  oriens  evident  as  positive-going  negative d e n d r i t i c  ( -75pm - -125pm, F i g 3 . 3 A ) ,  in  case,  that  pyramidale ( F i g  a  the  the  form  n e g a t i v e / p o s i t i v e waveform i n t h e v i c i n i t y (-50pm, F i g 3 . 3 A ) . I n t h i s  polarity  of  a  biphasic  of stratum  pyramidale  t h e peak l a t e n c y o f  the basal  d e n d r i t i c n e g a t i v i t y c o u l d appear t o d e c r e a s e w i t h p r o x i m i t y the  pyramidal  increased  cell  i n amplitude  Current-source -going  The  as  the  towards stratum  region  of  current  pyramidal  s i n k was  decreased  in  amplitude  inverting  to  a  proximal  positive  pyramidale.  an i n w a r d cell  basal dendrites  maximal i n with  current  the stratum  proximity  source  movement o f  to  i n stratum  positive-going potential  l a t t e r was  ( F i g 3.3B). and  cell  layer,  pyramidale  and t h e  associated with  Subthreshold  s t i m u l a t i o n of stratum  negative-going  suggesting  t h e development cell  potential  of a  late  of the that the current  layer.  radiatum  with  region of the  i n amplitude  ( F i g 3.3B),  i n the region of the pyramidal  duration  current  oriens  the  found t o d e c l i n e i n t h e p r o x i m a l  basal dendrite coincident with the increase  source  long  a p i c a l d e n d r i t i c r e g i o n . The d u r a t i o n o f t h e d e n d r i t i c  c u r r e n t s i n k was  late  potential  o r i e n s was a s s o c i a t e d w i t h a g r a d e d  current sink, indicating  the  late  to  density analysis indicated that the negative  wave i n s t r a t u m  lasting in  layer  was  evoked a  long  maximal amplitude i n  FIG.  3.3  (voltage)  Laminar p r o f i l e s o f e x t r a c e l l u l a r and  subthreshold radiatum  current-source stimulation  (C,D) .  of  density stratum  oriens  evoked  e a c h 25/am i n t e r v a l .  CSD  line to  comparison  facilitate  diagram  (A,B) o r s t r a t u m  v e n t r a l border  measurements a r e d i s p l a y e d w i t h of sink-source  Distance along the c e l l  cell,  a zero A  i s interposed denoted  a x i s i s t a k e n from  ( Ojum ) .  25/im  relationships.  of the r a t pyramidal c e l l  of stratum pyramidale  at  i n 1 dimension f o r  between p r o f i l e s w i t h t h e b o r d e r s o f s t r a t u m p y r a m i d a l e by d o t t e d l i n e s .  through  axis of the pyramidal  B,D, c u r r e n t - s o u r c e d e n s i t y c a l c u l a t e d  scaled schematic  potentials  A,C. E v o k e d p o t e n t i a l s were c o l l e c t e d  steps p a r a l l e l t o the dendro-somatic and  (CSD)  field  the  Stim.Site  Stratum Radiatum  Stratum Oriens A. Voltage  B.  CSD  C.  Voltage  D.  CSD  200 -,  I  Sink Source 5mV  10 msec  51  -  stratum  radiatum  pyramidale Fig  250jum  This potential  increased  pyramidale, the  -  (3.8mV i n a m p l i t u d e  3.3C).  and  150  cell  in  peak  The  the  from  and  with  i n the proximal-mid  dendritic  negativity stratum  Current-source density analysis  long  in  stratum  duration  found  oriens, a region  in  current  the l e v e l of  of  pyramidal neurons Therefore,  200jum,  in  amplitude  to  stratum  i n the region of  similar  sink  was in  associated with a  the  3.3D). The  negative  region  of  sink  inputs,  current  or p r o x i m a l  stratum  positive-going potential  proximal basal  dendritic  extensions  ( F i g 3.3D).  activation  basal  restricted  indicating and  amplitude  apical of  were  of  synaptic  afferents  The  dendrites, and  fact  that  EPSP p a s t t h e s o m a t i c  zone  i n stratum  respectively. s i n k and  pyramidale  suggest  synaptic further and  dendritic  towards  pyramidal  The  decline  the presence  the  cell source  i n d i c a t e a conduction of at l e a s t the  in  EPSPs  pyramidal to  cell  of a  t h a t evoked  c u r r e n t s were f o u n d  through  and  of s t i m u l a t e d a f f e r e n t  current  region  p o r t i o n of the opposing late  the  electrotonically  beyond s t r a t u m p y r a m i d a l e  The  to  synaptic d e p o l a r i z a t i o n of  i n stratum  conducted  layer.  a  t h e EPSP  current source  the  m a j o r i t y of  o r i e n s o r r a d i a t u m g a v e r i s e t o an e x t r a c e l l u l a r n e g a t i v i t y current  of  a t t a i n e d a maximal  revealed t h a t the  stratum pyramidale  s o m a t a and  stratum  oriens.  corresponding to the  at  and  radiatum  mid-distal apical dendrite (Fig s o u r c i n g was  of  p o s i t i v e - g o i n g p o t e n t i a l was  amplitude  graded,  proximity  a positive polarity  as  evoked  border  declined progressively  duration  potential  the  22msec i n d u r a t i o n a t  latency  i n v e r t i n g to layer.  -  the  proximal  tree.  positive-going potential  and  current  source  - 52 observed SO  i n t h e p r o x i m a l s t r a t u m o r i e n s and  stimulation likely  evoked i n the  Dingledine supported  and by  correlates  the c e l l  finding  time  to  means  determined,  and  by w h i c h the l a t e  examined i n the p r e s e n t  Orthodromic  and  extracellular  pyramidal  positivity  recorded  neuron  is  from  (data  not  a c t i v a t e d was  not  inhibitory potential  was  not  further  Responses  evoked  field  through  i n F i g 3.4A,B. To  dendritic  activity  p o t e n t i a l s and  i n stratum pyramidale  potentials  suprathreshold  SO  a i d comparison  of  of the pyramidal  c o r r e s p o n d i n g CSD  and  and  radiatum  cell  measurements  (125pm) a r e shown i n  3.4C,D. The  p r o p e r t i e s of  were found  to vary  t h e SO e v o k e d p o p u l a t i o n  stratum  pyramidale,  p o p u l a t i o n s p i k e upon waveform most  SO  oriens, displaying  response  s t i m u l a t i o n evoked  to  a x i s . At the a sharp  the  level  negative  the u n d e r l y i n g p o s i t i v i t y of the s y n a p t i c  ( F i g 3.4A,C). The  o f t e n found  spike  i n a c h a r a c t e r i s t i c manner a c c o r d i n g  r e c o r d i n g p o s i t i o n along the a p i c a l d e n d r i t i c of  This  f  study.  density  apical  et  1979a,b c).  laminar p r o f i l e s of e x t r a c e l l u l a r  population, f i e l d  Fig  a l . 1969;  i n t e r n e u r o n s were  s t i m u l a t i o n are i l l u s t r a t e d  recorded  (Andersen  inhibitory potential  Population Spike  current-source  somatic  inhibitory  the  of the  following  the a c t i o n of  Leung  that  an  impalements  s h o w n ) . The  through  1980;  pyramidale  inhibitory synaptic potential  body l a y e r  Langmoen the  in  intrasomatic  r e p r e s e n t an  pyramidal c e l l  i n t e r n e u r o n s near  The  -  shortest latency population spike  i n stratum a gradual  pyramidale  or the p r o x i m a l  increase i n  proximal a p i c a l dendrite. Approximately  was  stratum  l a t e n c y through  150pm f r o m t h e b o r d e r  the of  FIG.  3.4  Laminar p r o f i l e s  (voltage)  and  current-source  suprathreshold potentials  of extracellular  stimulation  were  dendro-somatic  of  collected axis  of  density  at the  current-source density calculated i n t e r v a l . CSD facilitate schematic  measurements a r e  comparison diagram  of  of  (CSD)  stratum 25pm  steps  the  through  A.  parallel cell,  displayed with  to  cell  border  comparison  of  stratum  of extracellular  CSD m e a s u r e m e n t s -25pm ) and  (D) a t  (  potentials  the level  dendritic  on t h e r i s i n g  Opm  125pm ) i n  the  C,D. A  (C) and  associated  cell  response  s p i k e response  r e c o r d e d 125pm f r o m s t r a t u m  denoted  ).  of pyramidal  edge o f t h e  line to  a x i s i s taken from  (waveforms t a k e n from p r o f i l e s  t h a t the onset o f the  potential  field  apical dendrites (  oriens stimulation,  as a b r e a k  pyramidale  25pm  i s interposed  between p r o f i l e s w i t h t h e b o r d e r s o f s t r a t u m p y r a m i d a l e  ventral  B,  f o r each a zero  the  relationships. A scaled  r a t pyramidal  by d o t t e d l i n e s . D i s t a n c e a l o n g t h e c e l l  Evoked  and  1 dimension  sink-source  potentials  evoked  oriens.  pyramidal in  field  somata (  to stratum  i n A,B).  c a n be  Note  detected  positive-going dendritic pyramidale.  Recording Distance from Border of St. Pyramidale (pm)  -  $g  -  - 55 stratum  pyramidale,  biphasic  the p o p u l a t i o n spike response c o n s i s t e d o f a  positive/negative  underlying  waveform  by  potential,  a  break  on  positive-going synaptic potential and to  negative  distinguished the  rising  from t h e  edge o f t h e  (see F i g 3.4C).  The p o s i t i v e  component o f t h e d e n d r i t i c p o t e n t i a l t h e n  increase  i n latency  the n e g a t i v i t y "dropping  through the out"  continued  mid-dendritic region,  i n distal dendritic locations  with to  l e a v e a pure monophasic p o s i t i v e - g o i n g p o t e n t i a l . Calculation  of  current-source  c u r r e n t s i n k o f s h o r t e s t peak pyramidale  superimposed  density  uncovered a sharp  latency i n the region of  upon t h e  source  of  current  with synaptic activation of the basal dendrite Fig  3.4D).  The  current  through the proximal  sink  125jum;  dendritic stratum  F i g 3.4D).  response  pyramidale,  give rise  to a  ( F i g 3.4B, -25pm;  increased  Both  increased  the in  source  latency  and with  of the  distance  from  with the current sink gradually d e c l i n i n g to  monophasic c u r r e n t  source  in  distal dendritic  of the d e n d r i t i c sink  s p i k e response a t t h e c e l l  layer i s  by  following particularly  i n F i g 3.4B (50 - 125pm).  A population levels  with the  sink  c u r r e n t flow a s s o c i a t e d w i t h t h e l a r g e c u r r e n t source  evident  i n latency  i n the mid-dendritic region ( F i g  membrane. The p o s s i b l e " c o n t a m i n a t i o n "  the p o p u l a t i o n  associated  a p i c a l d e n d r i t e and was c o n t i n u o u s  s i n k component o f a s o u r c e / s i n k 3.4B,  gradually  stratum  the  pyramidal  cell  c o u l d a l s o be d e t e c t e d axis  ata l l  following  suprathreshold  stimulation of afferent synaptic inputs i n stratum  radiatum ( F i g  3.5).  of  spike response  In the region of the pyramidal  evoked a l a r g e p o p u l a t i o n dendritic synaptic  cell  l a y e r , SR  stimulation  s p i k e upon t h e p o s i t i v e - g o i n g  p o t e n t i a l ( F i g 3.5A, -50pm;  apical  F i g 3 . 5 C ) . The  - 56 l a r g e s t amplitude  and  shortest latency population  by SR s t i m u l a t i o n was most o f t e n f o u n d i n s t r a t u m proximal spike  stratum  then  o r i e n s . The  increased  through the proximal from  the  response  ventral  in  apical  continued  latency  of  of  through  of  dendritic  the  both  spike  converting  in  dendritic  region  increased  radiatum.  This  to  a  monophasic  t h e peak o f  the  that  SR  short latency current  sink  ( F i g 3.5A, -50pm) t h a t  waveform c o n t i n u e d with  increasing i n  as t h e both  potentials  the  source  latency through the  and  current sink i n the  current  f o l l o w i n g s t i m u l a t i o n of stratum  source/sink(s)  radiatum  of a sink  dendritic tree  ( F i g 3.5B, 200pm; F i g 3.5D). and  stratum  s i n k component  found t o conduct through t h e b a s a l d e n d r i t i c r e g i o n o f cells  a  radiatum,  i n peak l a t e n c y t h r o u g h t h e p r o x i m a l  of synaptic termination of  to  revealed  pyramidale  s u p e r i m p o s e upon t h e l o n g l a s t i n g  sequence  as  components o f t h e  through stratum  profiles  stratum  source/sink,  progressively  radiatum  f  a l a t e n c y beyond  density  of  gradually  dendritic  spike  ( F i g 3 . 5 A C ) . The  locations  s t i m u l a t i o n evoked a l a r g e amplitude the  the  EPSP.  Current-source  in  EPSP  - 100pm  the extracellular  and n e g a t i v e  increased  positive-going deflection at extracellular  stratum  dendritic  positive  i n amplitude  pyramidale,  mid  or  population  B e y o n d 50  s u p e r i m p o s e d upon  response distal  stratum  the  apical  the  decreased  dendritic field.  p o s i t i v e / n e g a t i v e waveform negativity  and  evoked  pyramidale  peak n e g a t i v i t y o f t h e latency  border  spike  region  A similar were a l s o pyramidal  afferent  inputs  (Fig 3.5). T h u s , t h e c h a r a c t e r i s t i c s o f t h e SR e v o k e d p o p u l a t i o n response  along  the  pyramidal  cell  axis are not unlike  spike those  - 57 F I G . 3.5  Laminar  (voltage)  and  current-source  suprathreshold potentials  stratum  were  dendro-somatic  profiles of extracellular  radiatum  collected axis  density  at  of  the  current-source density calculated interval.  CSD  facilitate schematic  measurements a r e  comparison diagram  of  of  (CSD)  25pm  steps  through  A.  parallel  pyramidal in  potentials  evoked  stimulation.  cell,  1 dimension  displayed with  sink-source  the  field  to and  cell  border  comparison measurements and  of  of  stratum  extracellular  stimulation  (  Opm  ).  potentials  (D) a t t h e l e v e l o f p y r a m i d a l c e l l  a p i c a l d e n d r i t e s ( 200pm  line to  denoted  a x i s i s taken from  pyramidale field  25pm  i s interposed  between p r o f i l e s w i t h t h e b o r d e r s o f s t r a t u m p y r a m i d a l e  ventral  B,  f o r each a zero  the  relationships. A scaled  r a t pyramidal  by d o t t e d l i n e s . D i s t a n c e a l o n g t h e c e l l  Evoked  ) i n response  (C)  C,D. and  the A CSD  somata  ( -50pm )  t o stratum  radiatum  (waveforms t a k e n from l a m i n a r p r o f i l e s  i n A,B).  - 58 -  A. 200-i  Voltage  B.  CSD  —  &> "D Q Q E -  0 0 -  E  Q _ <1>  OH o 2 to  >>  +100  O>C0 200-1  o o a> ^  20 3 0 0  J  c.  D.  Soma Sink 1  Apical Dend. ~ i  Source  J 5mV 5 msec  evoked through a n t i d r o m i c of the b a s a l  a c t i v a t i o n or synaptic  dendritic tree  clearly  illustrated  stratum  pyramidale  ( c f 3.2 and 3 . 4 ) . T h i s p o i n t  i n F i g 3.6, where e v o k e d f i e l d and  radiatum  current-source  density  comparison  responses  of  depolarization  are  evoked  to  by  potentials in  aligned with  measurements  i s more  provide  associated a  SR and a l v e a r  direct  stimulation  (records  t a k e n f r o m F i g s 3.2 and 3 . 5 ) . I n s t r a t u m p y r a m i d a l e t h e -  falling  edge  population evoked  and  spike  current  peak  of  a l i g n with sink  both  potential  correspond and  alvear  the r i s i n g  ( F i g 3.6A,C).  r a d i a t u m , t h e p o s i t i v e and n e g a t i v e spike  the  evoked  through  i n o n s e t and d u r a t i o n  edge and  SR  evoked  peak o f  Similarly,  in  components o f t h e  the  stratum  dendritic  s t i m u l a t i o n o f e i t h e r pathway to the d e n d r i t i c current  source  s i n k , r e s p e c t i v e l y ( F i g 3.6B). The s i m i l a r i t y b e t w e e n t h e a l v e a r  spike  response  latency  of  various  points  of the f i e l d  is  the  measurements  SR  and  further  negative along  of  pyramidale, progressively  of the  spike  pyramidal c e l l  of the current result  of  cell  current  (Fig  3.7;  peak  latency  in  or  stratum  increasing i n latency through both the  and c u r r e n t  Although the  towards  latency of the  s i n k were s i m i l a r a l o n g  a x i s , some d e v i a t i o n  flow  axis  sink at  s i n k e v o k e d by a l v e a r  s i n k was f o u n d i n p r o x i m a l  following population 3.7).  shortest  and a p i c a l d e n d r i t i c r e g i o n s .  evoked p o p u l a t i o n  c o m p a r i n g t h e peak  and 3 . 5 ) . B o t h t h e n e g a t i v i t y  p o t e n t i a l and t h e c u r r e n t were  by  population  p o t e n t i a l and c u r r e n t  pyramidal  t a k e n f r o m F i g s 3.2  stimulation  basal  emphasized field  the  and SR e v o k e d  the  spike discharge  i n t h e peak  latency  d e n d r i t i c regions late  source of  most  as a  current  i n stratum pyramidale ( F i g  - 60 FIG.  3_,_6_  evoked  A  comparison  extracellular  of  the  field  potentials  (CSD)  measurements  current-source  density  pyramidal  cell  s o m a t a (pop s p i k e )  dend).  A B. Somatic f  suprathreshold alveus.  suprathreshold stratum  Somatic  facilitate  of  (C)  stimulation  radiatum.  and  and at  of  pyramidal and  associated  the  level  apical dendrites  (A) and d e n d r i t i c r e s p o n s e  stimulation  C,D.  t i m i n g r e l a t i o n s h i p between  cell  dendritic  afferent  of  (extra-  ( B , 160pm ) t o axons  response  synaptic  i n the (D)  to  inputs  in  Z e r o l i n e s a r e i n c l u d e d on CSD m e a s u r e m e n t s t o  comparison of sink-source r e l a t i o n s h i p s .  Stim.Site  Alveus  Stratum Radiatum  A Pop Spike  -• Sink 1  Source  5mV i — i  2 msec  5 msec  FIG.  3.7  Peak  latency  population spike negativity 25pm  i n t e r v a l s along  cell  in  response  measurements (voltage)  of  and c u r r e n t s i n k  the dendro-somatic  to  alvear  the e x t r a c e l l u l a r (CSD) a t  axis of the pyramidal  antidromic  stimulation  (A)  or  s t i m u l a t i o n of a f f e r e n t synaptic inputs i n stratum radiatum (B). Latency  measurements  laminar p r o f i l e s of pyramidal c e l l  were  calculated  F i g s 3.2 and  axis taken  ( Opm  cell  t o s c a l e i s shown f o r  drawn  stratum pyramidale  ). A  schematic  f o r each s l i c e  computer  from  3.5, w i t h d i s t a n c e  from the  pyramidale  by  v e n t r a l border  diagram  the  along the of stratum  of the r a t pyramidal  comparison are denoted  and t h e b o r d e r s o f by d o t t e d  lines.  Stim. Site  Stratum Radiatum  Alveus Voltage  Voltage  CSD  CSD  B.  A.  o  4^  4o  J  200 •o • •  -loo  •  A  •  o  o  o o •  c* 0»  o •  m  •  • o  + 100  o o  •  A  o •  o •  200  o  •o  A  • o •  300  o  J  I  T"  2  3  4  7  8  Latency (msec)  I  9  1  1  10  -  Both the a l v e a r and  -  64  orthodromic evoked response were  found to d i s p l a y a non-uniform  increase  i n the peak l a t e n c y  the s p i k e waveform through the d e n d r i t i c f i e l d c e l l population. the p o p u l a t i o n the  initial  peak region  of  spike displayed  of  the  transition  little  degree  The g r e a t e s t found i n negative  pyramidal  negativity of  of l a t e n c y  shift  shift  i n peak l a t e n c y over  from stratum pyramidale. However, the  the  p o s i t i v e / n e g a t i v e p o t e n t i a l , with greater  of the  of  In proximal stratum radiatum, the n e g a t i v i t y of  100pm d i s t a n c e  latency  also  increased  spike  i n the approximate  waveform  to  both components e x h i b i t i n g  s h i f t through  regions,  a  mid stratum radiatum.  i n peak l a t e n c y of the negative  distal dendritic  a biphasic  p o t e n t i a l was  just  p r i o r to  component o f the d e n d r i t i c s p i k e  response.  l o s s of the  - 65 3-4.  Discussion  Current-source field  potentials  identify  the  density along  site  p o t e n t i a l s and  the  of  analysis  origin  spike discharge  of  known  of the  distribution  superior. current  the  case  sink  was  maximal  current  cell  of  SO in  cell  R a i s m a n e t a l . 1 9 6 5 ) . SR sink i n  a x i s has  synaptically declined was  and  region of the The  cell  electrotonic termination  the  regio  stratum  oriens,  a  region  of commissural tree  and  inputs  (Blackstad  1956;  stratum  radiatum,  (Lorente  de  No  1962).  dendritic  1934;  In  collateral Raisman e t  either  negativity  an  and  case,  and al. the  current  sink  pyramidale,  c u r r e n t w i t h i n and  area  and  beyond  the  layer.  amplitude,  synaptic  with  s t i m u l a t i o n , the n e g a t i v i t y  the m i d - d i s t a l  a source of  synaptic potentials active  localized  synaptic projections to  i n amplitude with proximity to stratum  associated with  evoked  s t i m u l a t i o n evoked a maximal n e g a t i v i t y  Blackstad  evoked  (SR)  structure consistent  the  to  neurons.  sink s p a t i a l l y  basal dendritic  commissural a f f e r e n t inputs Westrum  served  excitatory synaptic  encompassing the t e r m i n a l p r o j e c t i o n s of S c h a f f e r  1965;  extracellular  or radiatum  to the p o i n t of t e r m i n a t i o n  pyramidal  current  (SO)  afferent  In  corresponding upon t h e  of  cell  evoked  oriens  pyramidal  evoked  i n CAl pyramidal  an e x t r a c e l l u l a r n e g a t i v i t y and to regions  of  pyramidal  S t i m u l a t i o n of stratum  and  -  spatial distribution along  the  pyramidal  depolarization  conduction towards  synaptic potentials  the  of  and  of  EPSPs  pyramidal  were f o u n d  p o l a r i t y of  cell  a x i s i n d i c a t e an  dendritic from cell  to conduct  evoked  membrane  the p o i n t of layer.  and  synaptic  In a d d i t i o n ,  beyond the  somatic  -  level  and  through  a r b o r i z a t i o n , as positivity  some  through  along  the  decline  recording  neuron  (as  with  structure,  l e n g t h of the  a  pyramidale  now  the generation  cell  spike response  s t i m u l a t i o n of the  layer. This  generation  positive/negative  radiatum.  T h i s w a v e f o r m was  a n a l y s i s to correspond the  cell  layer  was  a l v e u s , as shown  by  spike i n the  r e s p o n s e w o u l d be conducting  also  the  apical  region  associated an  inward  of  e v o k e d an  CAl  the  extracellular  mid  as  field.  action  l o c a t i o n , where a l o c a l  density from Such a  potential circuit  s p i k e a c t i v a t i o n would g i v e r i s e t o  o f c u r r e n t f o l l o w e d by  a  stratum  conducting  dendritic  t h e c a s e o f an  flow  pyramidal  region, observed in  with  p o t e n t i a l s i n the  found through c u r r e n t - s o u r c e  towards a r e c o r d i n g  source  action  potential  expected f o r  d e p o l a r i z a t i o n preceding initial  of  to a current source/sink  through  Turner  response  r e s p o n s e was  apical dendritic  biphasic  5).  pyramidal  1981;  spike  axon h i l l o c k  stimulation  i n the  the  (Brown e t a l .  population  t h e soma and  Antidromic  (Chapter  the short e l e c t r o t o n i c  current  neurons.  confirmed  to  of  presumed r e g i o n o f  SO  s t i m u l a t i o n ) are found  immediate onset of a c u r r e n t s i n k , i n d i c a t i n g the  the  of  the  and  In  dendritic tree  of a short l a t e n c y population  of the pyramidal  .  1980).  antidromic  suprathreshold  of  been  the m a j o r i t y of  neuron  Schwartzkroin  synchronous  e v o k e d by  has  emphasizing  CAl pyramidal  T u r n e r and A  result  extracellular  procedures  SO  dendritic  i n amplitude  apical dendrite  conduct e l e c t r o t o n i c a l l y through  1984;  opposing  s y n a p t i c c u r r e n t s e v o k e d w i t h i n one  pyramidal  cell  the  beyond s t r a t u m  gradual  intradendritic  Therefore, the  and  of  t h e p r e s e n c e o f an  current source  f a c t , the presence e v o k e d EPSP  portion  i n d i c a t e d by  and  -  66  a c u r r e n t s i n k at the  an time  -  of s p i k e generation  67  -  at the r e c o r d i n g  site.  Thus, the evoked c h a r a c t e r i s t i c s of the antidromic i n d i c a t e the region  initial  of  the  generation pyramidal  of an  response  action potential in  cell  body  and  the  a  sequential  d e p o l a r i z a t i o n of d e n d r i t i c membrane, r e s u l t i n g i n a  retrograde  i n v a s i o n of the s p i k e through the a p i c a l d e n d r i t i c a r b o r i z a t i o n . As  mentioned,  a  similar  r e l a t i o n s h i p s was  observed i n  s t i m u l a t i o n , suggesting b a s a l and those  apical  of  in  of  an antidromic  in  vivo  source/sink  following  alvear  s p i k e i n v a s i o n of both above r e s u l t s agree  investigators,  the  current  stratum o r i e n s  d e n d r i t i c t r e e . The  previous  potentials  sequence  in  which  hippocampus  alvear  were  the with  evoked  i n t e r p r e t e d to  i n d i c a t e a s e q u e n t i a l s p i k e i n v a s i o n of axon h i l l o c k , soma, b a s a l and  apical dendritic structures  (Gessi et a l . 1966;  and Leung  1979a,b; S p e r t i et a l . 1967). Activation either  the  population  of  basal  afferent or  evoked  a  positive/negative progressively dendritic  at a l l l e v e l s  spike  were  fields.  The  or  case, the  for antidromically shortest latency  region of the pyramidal  evoked  or  cell  cell  proximal stratum o r i e n s . with  current  of  a  radiatum  a  biphasic  source/sink  i n l a t e n c y through both b a s a l and pattern  upon  sink of s h o r t e s t peak  continuous  s t i m u l a t i o n of a f f e r e n t s y n a p t i c t h a t found  also  of the pyramidal  current  or the  potential  increased  tree  of stratum o r i e n s  and  stratum pyramidale  waveforms  inputs t e r m i n a t i n g  dendritic  stimulation  population  latency in These  apical  s p i k e response  a x i s . Suprathreshold  synaptic  spike generation  inputs was  apical  following  thus very s i m i l a r to  evoked s p i k e  population  that  discharge.  spike  l a y e r , with a n t i -  was  found  and  In each i n the  orthodromic  - 68  -  s p i k e responses e x h i b i t i n g a s h i f t from stratum  pyramidale. In  i n peak l a t e n c y with d i s t a n c e  addition, a  d i r e c t comparison  a l v e a r and SR evoked p o t e n t i a l s r e v e a l e d a s i m i l a r between  extracellular  field  potentials  and  relationship current-source  d e n s i t y waveforms at both the somatic and a p i c a l d e n d r i t i c of  pyramidal  of  level  neurons.  Therefore, pyramidal c e l l would appear  the  sequence  of  spike  generation  in  f o l l o w i n g a c t i v a t i o n of a f f e r e n t s y n a p t i c i n d i s t i n g u i s h a b l e from  p o t e n t i a l evoked  that found  through a n t i d r o m i c  inputs  f o r an  stimulation.  action  Specifically,  s y n a p t i c d e p o l a r i z a t i o n of e i t h e r d e n d r i t i c t r e e r e s u l t s i n initial cell  generation  of a  spike i n  l a y e r , with a subsequent  the r e g i o n  the  of the pyramidal  r e t r o g r a d e s p i k e i n v a s i o n of  the b a s a l and a p i c a l d e n d r i t i c  the  both  arborizations.  O c c a s i o n a l l y , s h o r t l a t e n c y c u r r e n t s i n k s c o u l d be found i n both  the  proximal  stratum  oriens  and  stratum  pyramidale  f o l l o w i n g s u p r a t h r e s h o l d a c t i v a t i o n of any of the t h r e e s t i m u l u s pathways.  These  discharge  in  the  spinal  relative stratum  sinks  might  axon  hillock  and  the  pyramidal c e l l , soma-dendritic  current  corresponding to (SD)  components  motoneuron  size  and  pyramidale  does  cells  site for i n i t i a l  through  the i n i t i a l  et  packing not  region segment  of  the  (IS) and  proposed f o r  a l . 1957a,b). However, the of pyramidal neurons w i t h i n  permit  between the a c t i v i t y of i n i t i a l pyramidal  somatic  of s p i k e d i s c h a r g e  (Coombs  tight  represent s e q u e n t i a l AP  a  reliable  dissociation  segment and somatic membranes of  current-source  density analysis.  s p i k e g e n e r a t i o n i n the pyramidal c e l l  The  can thus  only be r e f e r r e d to the r e g i o n of the soma - axon h i l l o c k . Regardless of  the form  of a c t i v a t i o n ,  conduction of  the  - 69  -  spike through the a p i c a l d e n d r i t i c non-uniform dendritic minimal (peak  shift  velocity  the e x t r a c e l l u l a r  i n l a t e n c y and  latency  to occur  In  taken the  i n c r e a s e d markedly  in  lOOum o f  s p i k e response  from  the  dendritic  the  m/sec  antidromic  field  m i d - d i s t a l d e n d r i t e , the spike  i n l a t e n c y and  slowed  i n conduction  (0.1 m/sec) t o e v e n t u a l l y t e r m i n a t e a t a v a r i a b l e  in the d i s t a l  r e g i o n as  level  a monophasic p o s i t i v i t y  and  c u r r e n t s o u r c e . T h e s e v a l u e s compare t o a c o n d u c t i o n v e l o c i t y .36m/sec f o r t h e p r o j e c t i o n and  f i b e r s of the  1.2m/sec f o r  the a l v e a r region (Tielen There  would  change i n spike  waveform.  correlate the r a t the  s h a p e and  range  myelinated pyramidal c e l l  appear the  pyramidal c e l l shaft  of  25-100um  extensive  secondary  is  the  from  the  in  fast  the t h i c k  fact  initial  extracellular  earlier,  in distal  s t r a t u m r a d i a t u m may  conduction v e l o c i t y correspond  of a monophasic  region  i n the  soma, w i t h more  further of  to  or in  spike  shaft, with a  i n the r e l a t i v e l y t h i n  potential  of  stratum radiatum  proximal dendritic  presence  the  the  that these p o i n t s  cell  might then  The  in  by  As d e s c r i b e d  distal  branches.  conduction  of the  pyramidal  branching The  t r a n s i t i o n p o i n t s of  apical dendrite bifurcates  dendritic  spike  in  l o c a t i o n of major b r a n c h p o i n t s  the  slowing of conduction v e l o c i t y  of  axons  a p i c a l d e n d r i t e , as j u d g e d  stratum radiatum  conduction through  be two  apical dendrite.  of  lacunosum-moleculare. the proximal  to  interest  to the approximate  proximal  Schaffercollateral/commissural  conduction v e l o c i t y  Of  of  et a l . 1981).  thus  spike conduction through  a  displayed a  a c o n d u c t i o n v e l o c i t y o f 0.33  measurements  negativity).  potential  found  manner. Over a p p r o x i m a t e l y t h e i n i t i a l  field,  potential  t r e e was  secondary  positive-going indicate  secondary  a  loss  dendritic  -  70  -  branching. Previous afferent initial  i n v e s t i g a t i o n s have  synaptic activation  1955;  Hamlyn  of a  latency of f i e l d  potential  shortest  located  in  radiatum. revealed in  AP  hillock  components a l o n g  pyramidale  spatial  in  the proximal  dendritic origin 1962).  spike stratum  f o r the  evoked  In the  present  in  the  proximal  region  current-source  of stratum  density analysis  or  proximal  stratum  oriens. Since  through current-source  generation  can  a g a i n be  region of pyramidal  potentials.  interpretation of  to  density analysis,  taken  as  a more  to illustrate  the d i f f i c u l t i e s  that  identify the s i t e of o r i g i n  the  current density  different  the s i t e  neurons.  the s p a t i a l d i s t r i b u t i o n Indeed,  can  w i t h i n t h e soma-axon  ambiguity  of  was r e v e a l e d  s i t e of  origin  o f an  of extracellular field potential i n one  where peak l a t e n c y m e a s u r e m e n t s o f t h e p o p u l a t i o n s p i k e indicate a  found  l o c a l i z a t i o n o f transmembrane c u r r e n t f l o w  evoked response from  to  cell  an e v o k e d n e g a t i v e  latency  subsequent  a r i s e when a t t e m p t i n g  field  the pyramidal  s p i k e o f s h o r t e s t peak l a t e n c y was i n f a c t  The a b o v e r e s u l t s e r v e s can  profile  t h a t t h e s h o r t e s t l a t e n c y c u r r e n t s i n k was t o be  be a c h i e v e d for  Lomo 1 9 6 6 ; C r a g g  the  peak  slices  However,  stratum  accurate  some  cell  The f i r s t e v i d e n c e f o r  F u j i t a and S a k a t a  the population  pyramidal  p o t e n t i a l s , and c o n s i d e r a t i o n o f  a proximal  1960;  (Andersen  i nthe  of  studies reported  suggesting  results  of  analyses  potential  these  study,  within the  came f r o m l a m i n a r  field  a x i s . Some o f  spike  radiatum  1962).  F u j i t a and S a k a t a  evoked e x t r a c e l l u l a r  radiatum,  stratum  s p i k e from  dendritic spike discharge  of  in  that stimulation  ( A n d e r s e n 1 9 6 0 ; A n d e r s e n and  apical dendrite and  inputs  reported  f o r spikes  slice, appeared  evoked  from  -  each o f  the three  current-source origin  s t i m u l u s pathways.  density  spikes  i n the  a common  region  pyramidal  cell  a distal  dendritic  stratum  sink.  dendritic s i t e of origin  spike  (Andersen e t a l . 1966a,b; A n d e r s e n  Lomo  Hamlyn  Cragg  extracellular taken  as  levels study,  representation  the  of  and  peak  the  1955). of  negativity  pyramidal  a  cell  In  these  dendritic  of  stratum  radiatum,  conducting  s p i k e was o f t e n  t h e evoked response a t a l l  axis.  However,  i n the present  the  positive/negative spike peak o f t h e EPSP i n negative  synaptic  amplitude  than  cell  body  stratum  radiatum,  w a v e f o r m was  the negative  negativity  evoked  that the  cases greater of  i n peak  the d e n d r i t i c of a  potentials  short  would  the  beyond t h e  with the result  Measurement  dendritic  components  Furthermore,  often f a l l  i n many  component  225 a n d 250um).  represent  layer.  p o t e n t i a l would  ( s e e F i g 3.5A, on  response  by a p o s i t i v e p o t e n t i a l i n m i d - d i s t a l  w i t h b o t h t h e p o s i t i v e and n e g a t i v e  from  and  studies, the  t h e n e g a t i v i t y o f t h e SR e v o k e d d e n d r i t i c s p i k e  was f o u n d t o be p r e c e d e d  for  following stimulation of  afferent synaptic inputs 1966;  of  site of  of  as i n d i c a t e d by a s h o r t l a t e n c y c u r r e n t  O t h e r s have r e p o r t e d the  However, e x a m i n a t i o n  p r o f i l e s demonstrated  f o r a l l evoked  pyramidale,  -  71  spike latency  therefore  t h e peak l a t e n c y o f t h e s y n a p t i c p o t e n t i a l , g i v i n g t h e  impression  of  initial  spike  generation  i n distal dendritic  regions. One f a c t o r  contributing to  the d i f f i c u l t y  i n identifying  the e x t r a d e n d r i t i c s p i k e i s t h e f a c t t h a t t h e n e g a t i v e of  the  spike  distinguish EPSP.  In  potential  from this  the case,  could  underlying the  at  times  be  negativity  dendritic  spike  component  difficult  to  o f t h e SR e v o k e d would appear as a  monophasic p o s i t i v e  p o t e n t i a l , even  though s t i m u l a t i o n  alveus  oriens  same  or  stratum  distinct positive/negative recording  site.  present time, results  in  dendrite,  an  slice  would evoke a  p o t e n t i a l a t t h e same d e n d r i t i c  relate to  active  form  through s t i m u l a t i o n invading  spike  the  This r e s u l t i s not completely understood at the b u t may  a  in  of the  synaptic  of  the fact  that  SR  depolarization  membrane  activation  of the  alveus or  the apical dendrite  following  stimulation  of the apical  not  brought  stratum oriens.  A  spike  SR s t i m u l a t i o n may  thus  e n c o u n t e r membrane o f r e l a t i v e l y h i g h c o n d u c t a n c e i n t h e of s y n a p t i c  termination,  of t h e d e n d r i t i c s p i k e . fact that could  the  also  the  basal  of t h e  SO  the basal  to distinguish  stimulation  conducted  waveform  spike  confirm  and s y n a p t i c  body  spike  depolarization of of the exact  the positive-going  cell  the  extradendritic  potential, current-source  that  from the  dendritic  on t h e  d e n d r i t i c t r e e . However, r e g a r d l e s s  extradendritic  region  T h i s i n t e r p r e t a t i o n i s s u p p o r t e d by  be d i f f i c u l t  a n a l y s i s would had  d i s t o r t i n g the e x t r a c e l l u l a r  n e g a t i v e component o f  p o t e n t i a l during  about  layer through  spike  form  density response  the dendritic  field. T h e r e f o r e , t h e r e s u l t s o f t h e p r e s e n t s t u d y do n o t the  contention  of  a dendritic origin  the  apical dendritic field  the  initial  f o r evoked s p i k e s  o f the CAl pyramidal neuron.  s i t e f o r generation of a spike  current-source density pyramidal c e l l  analysis  population.  The  to the c e l l  soma-axon  hillock.  Rather,  body l a y e r o f  pyramidal c e l l  initiated  within  was l o c a l i z e d t h r o u g h  dendritic  would thus appear t o a r i s e t h r o u g h a r e t r o g r a d e i n v a s i o n d e n d r i t i c a r b o r i z a t i o n by a s p i k e  support  i n the region  the spike  of  the  of the  -  4-0.  COMPARATIVE  73  INTRACELLULAR ANALYSIS OF  DENDRITIC ELECTROPHYSIOLOGY OF  4-1.  of  ionic  channels  non-uniform  cell  is  one  regional  in  neuronal  neuron  of  evoked  activation  and  of the in  cell  1975,1977;  cell  by  varying  al.  thought  Ca+2  dendritic  The  l e v e l of  et a l .  Slawsky  potentials  s o m a t i c membrane,  differential  1977;  are  level  1 9 6 1 ; Schwartzkroin  more  (Benardo  at  the pyramidal  both  i n the pyramidal current  (Benardo 1979).  Wong e t a l .  w i t h the  of  found  (AP)  predominant  generation of  in large  e t a l . 1 9 8 2 ; Wong e t  e l e c t r o r e s p o n s i v e p r o p e r t i e s of  membranes  consequence t o the f i n a l  dendritic  a  .  dendritic  the  to exhibit  recording sites.  i n j e c t i o n of d e p o l a r i z i n g  s p i k e s at the d e n d r i t i c 1979)  cell  In c o n t r a s t , a combination of  S c h w a r t z k r o i n and  d e n d r i t i c than  a  pyramidal  afferent synaptic inputs i s  1979).  Ca+2-dependent  However,  to  and  1 9 8 2 ; Kandel  Wong e t a l .  1982;  axis of a  (Na+-dependent) a c t i o n p o t e n t i a l  et a l .  in  a c c o r d i n g to the form  apical dendritic  intracellular  result  hippocampal  and C a + 2 - d e p e n d e n t p o t e n t i a l s c a n be e v o k e d  Na+  and  The  distribution  e l e c t r o r e s p o n s i v e p r o p e r t i e s , w i t h the  between s o m a t i c  s o m a t i c and  (Benardo  can  mammalian CNS  For i n s t a n c e , s t i m u l a t i o n of  both the  membrane  1979).  activity  evoke a s i n g l e f a s t  spatial  along the dendro-somatic  T r a u b and L l i n a s  variation  pattern  al.  AND  C A l PYRAMIDAL NEURONS  i n t h e d e n s i t y and  excitability  1975;  (Llinas  et  SOMATIC  Introduction Regional variations  to  -  results  of  membrane  are  thought  to  be  of  important  output of the pyramidal c e l l .  previous exhibit  studies, a  low  selective  threshold  for  somatic  According  regions Na+  of  spike  - 74 activation precede  ( h o t s p o t s ) , and s p i k e g e n e r a t i o n  that  at  the  afferent synaptic Llinas  1979).  body,  appearing  inputs  The  pre-potential  axon  hillock  (Spencer  dendritic  at  the  following  and K a n d e l  spike  somatic  superimposed  level  upon  the  summate w i t h  originating  synaptic currents,  p r o b a b i l i t y f o rgeneration However, a m a j o r q u e s t i o n s i t e f o rgeneration  of  s t u d i e s have r e p o r t e d  and  neurons  Hamlyn  of  cell  fast  depolarization  1977; Spencer and  from w i t h i n t h e and i n t h i s way  evidence t o suggest  Kandel  d e n d r i t e can increase the hillock. actual  evoked  of  i n the apical d e n d r i t i c region  of  1962; S c h w a r t z k r o i n  Traub and  extracellular  site  i s i n the region  field  (Chapter  f o rspike  Cragg 1977;  L l i n a s 1 9 7 9 ; Wong e t a l .  through current-source  t h i s hypothesis  the i n i t i a l  Most  a dendritic site  and S a k a t a  K a n d e l 1961b;  spike discharge  synaptic  small  ( A n d e r s e n 1 9 6 0 ; A n d e r s e n and Lomo 1 9 6 6 ;  appear t o support study,  a  t h e Na+-dependent d e n d r i t i c s p i k e .  1 9 7 9 ) . However, r e s u l t s o b t a i n e d analysis  as  t h a t remains unanswered i s t h e  1955; F u j i t a  Spencer and  1961b; T r a u b and  o f a s p i k e a t t h e soma-axon  o r i g i n f o r the spike recorded pyramidal  stimulation of  then conducts t o t h e c e l l  ( A n d e r s e n and Lomo 1 9 6 6 ; S c h w a r t z k r o i n 1961b). Thus, a s p i k e  i n the dendrite can  density  p o t e n t i a l s would n o t 3 ) . According  generation  i n the  o f t h e soma-axon h i l l o c k , w i t h  occurring through a retrograde  to this pyramidal  dendritic  spike invasion of  the d e n d r i t i c a r b o r i z a t i o n . Given the would  seem  c o n f l i c t i n g nature necessary  to  understand the f a c t o r s responsible and  dendritic  level.  above h y p o t h e s e s , i t  further  electroresponsive properties of the  somatic  of the  The  pyramidal  characterize cell  i n order  f o r spike generation present  study  the to  at the  therefore  - 75 represents  a  comparative  properties,  synaptic  intracellular  potentials,  analysis  and a c t i o n p o t e n t i a l  i n t h e soma and a p i c a l d e n d r i t e s o f C A l p y r a m i d a l  o f membrane discharge  neurons.  - 76 4-2.  Methods Intracellular  restricted CAlb  to  impalements  stratum pyramidale  subfield  of  regio  recorded  i n these s t r a t a  somata  or  neurons  neuronal  and s t r a t u m  superior.  elements  extensions  of  potentials  to activity either  or  However,  that the results to  be  p r e s e n t e d were o b t a i n e d f r o m t h e s o m a t a a n d a p i c a l d e n d r i t e s  of  pyramidal  neurons.  Firstly, cell  type  radiatum  suggest  ofthe  pyramidal  ( C a j a l 1 9 1 1 ; L o r e n t e de No 1 9 3 4 ) .  s e v e r a l l i n e s o f evidence would  were  radiatum of t h e  Intracellular  could correspond  dendritic  non-pyramidal  of  the  pyramidal neuron  i n the comprised  CAl  region,  largely  i s by with  of  f a rthe  stratum  tightly  most common  p y r a m i d a l e and  packed pyramidal  cell  somata and a p i c a l d e n d r i t e s , r e s p e c t i v e l y . 'Non-pyramidal neurons are mainly found  localized  scattered  above s t r a t u m p y r a m i d a l e ,  through  numbers o f n o n - p y r a m i d a l (Cajal  1911; Lorente  the  pyramidal c e l l  neurons de  No  a r e found 1934).  l a y e r . Even i n stratum  Therefore,  abundance and d i s t r i b u t i o n o f p y r a m i d a l c e l l that i n t r a c e l l u l a r correspond  radiatum  suggest  probability  cells.  p r e v i o u s i n v e s t i g a t o r s h a v e c o m p a r e d t h e membrane  p r o p e r t i e s and d i s c h a r g e and  fewer  the relative  structures  r e c o r d i n g s i n C A l would i n h i g h  t o impalements o f p y r a m i d a l  Secondly,  a l t h o u g h some a r e  Schwartzkroin  patterns of pyramidal  1981a;  Turner  and  somata  (Knowles  S c h w a r t z k r o i n 1980) and  a p i c a l d e n d r i t e s (Benardo e t a l . 1982; S c h w a r t z k r o i n and M a t h e r s 197 8;  Wong  (Ashwood  et  et  a l . 197 9)  a l . 1984;  to  that  Knowles  of and  S c h w a r t z k r o i n and M a t h e r s 1 9 7 8 ; T u r n e r These  studies  indicate  that  non-pyramidal  neurons  Schwartzkroin  1981a;  and S c h w a r t z k r o i n  1980).  p y r a m i d a l and n o n - p y r a m i d a l  cell  - 77 elements  can  be  distinguished  electrophysiological impaled  neuronal  activity  characteristics.  structures  characteristic  of  dendrites, while c e l l u l a r non-pyramidal  on In  displayed pyramidal  elements  the the  present  evoked cell  basis  patterns  with properties  encountered.  i n stratum pyramidale  w i t h evoked c h a r a c t e r i s t i c s v e r y s i m i l a r correspond  to  1980)  apical dendrites  and  impalements o f  somata  t o those  (Turner  of pyramidal  of  ascribed to  F i n a l l y , p r e v i o u s i n v e s t i g a t o r s have a n a t o m i c a l l y impaled  study,  s o m a t a and a p i c a l  neurons were v e r y i n f r e q u e n t l y  t h a t neuronal elements  of  verified  or radiatum  reported  here  and S c h w a r t z k r o i n  neurons  (Wong  et a l .  1979) . T h e r e f o r e , t h e i n t r a c e l l u l a r p o t e n t i a l s t o be d e s c r i b e d a r e considered  representative  of  somatic  a c t i v i t y of C A l pyramidal neurons. cell de  bodies w i t h i n the No  1934),  the  impalements w i t h i n proximal  portion  pyramidal  cells.  boundary o f s t r a t u m pyramidale  this layer either  However,  exists  The 165  basal with  results of  somatic  or  these  single  the present study  and  81  and  apical  potentials  microelectrode  dendrite,  dendritic  (Lorente  intracellular  r e c o r d i n g s from t h e  apical  dendrites  limitations will  in  of mind,  be d e s i g n a t e d  as  in origin.  intracellular single  that  may i n c l u d e  r e c o r d i n g s from w i t h i n s t r a t u m pyramidale "somatic"  apical  Considering the d i s p e r s i o n of  possibility  of  and  were  pyramidal  to  be  dendritic described  impalements not  neuron.  were o b t a i n e d f r o m  obtained Membrane  of from  over  recordings. A l l were r e c o r d e d  either  the  soma  from or  d u a l impalements o f a  c h a r a c t e r i s t i c s d e f i n e d as  s a t i s f a c t o r y f o r i n c l u s i o n o f d a t a were r e s t i n g p o t e n t i a l s o f a t  - 78 least  -55mV  amplitudes criteria to 5).  and of  input  evoked  resistance potentials  since this characteristic  the recording p o s i t i o n along The  action  term  "current  potential  spike"  at  measured  p o t e n t i a l baseline preceding width,  and  voltage  spikes  were  taken  potential. discharge  from  Voltage  threshold  was t a k e n  spike The  of  cell  stratum  within  estimated  artefact.  Amplitude, evoked  first  action  for  current  evoked  synaptically  f o r stimulus  from  the  this  intensities  was t a k e n  The  evoked  AP  amplitude  just  threshold  as  lens  was  the ventral  of  measured  subsequently  cell  dividing  the t o t a l  65um. Any i m p a l e m e n t s f a l l i n g intermediate  to  half into  visually  into  dividing i n half,  thirds,  sections  thereby  approximating  b e t w e e n 65pm d i v i s i o n s w e r e  the distance  a  pyramidale  layer to the fissure  either  distance  with  the microscope, or  T h i s was done b y  400um d i s t a n c e f r o m t h e  along  distance of the dendritic  point  objective  t h e hippocampal f i s s u r e .  value  (width  to the resting  as a f r a c t i o n o f t h e d i s t a n c e from s t r a t u m  partitioning  a  and h a l f w i d t h  measurements f o r c u r r e n t  pyramidale.  graticule  and  e f f e r e n t pathways  with respect  a x i s was m e a s u r e d  electrode  the  amplitude  of  a r e those  reference p o i n t from which t h e r e c o r d i n g d i s t a n c e  recording  to  injection  activation.  the pyramidal border  (Chapter  a s e q u i v a l e n t t o t h e a v e r a g e peak  o f t h e u n d e r l y i n g EPSP for  the  axis  evoked s p i k e s "  the stimulus  threshold  according  i s u s e d t o r e f e r t o an  of afferent or  Spike  was  cell  intracellular  in t h e hippocampal s l i c e . amplitude)  n o t used as a c c e p t a n c e  "stimulus  a c t i v a t e d through s t i m u l a t i o n  half  were  was f o u n d t o v a r y  through  depolarizing current, while  18 megohm o r more. The  the pyramidal  evoked  evoked  of  of  adjacent  given  division  p o i n t s . This technique proved of judging the pyramidale,  and  t o be a r e a s o n a b l y a c c u r a t e method  distance of a e s t i m a t e s made  r e c o r d i n g e l e c t r o d e from in this  way w e r e  found  c o m p a r a b l e t o t h o s e o b t a i n e d by measurement w i t h t h e Unless otherwise stated, d e n d r i t i c l e a s t 150um f r o m t h e v e n t r a l b o r d e r  stratum to  graticule.  impalements were o b t a i n e d of stratum  be  pyramidale.  at  - 80 4-3.  Results  Membrane  Characteristics  The and  -  membrane p o t e n t i a l  dendritic  response of p y r a m i d a l c e l l  impalements to  p u l s e i n j e c t i o n s are  a series  of  square  somatic  wave  current  shown i n F i g 4 . 1 A B . C u r r e n t / v o l t a g e  (I/V)  f  p r o f i l e s e x h i b i t a l i n e a r d i s p l a c e m e n t o f membrane p o t e n t i a l both  locations  over  a  range  of  a p p r o x i m a t e l y -80mV t o -55mV. F o r this be  r a n g e , a t i m e - d e p e n d e n t sag observed  in  s l o p e of the  v a l u e . Examples this Fig  form of  rectification pulse  relation  dendritic  was  at p o t e n t i a l s and  as  observed  an  a  (Hotson et a l .  increase  depolarized  dendritic  could  impalements,  also  injections  of somatic  beyond  for in  from  resting  recordings in  p a r t i c u l a r l y e v i d e n t are  the  which  shown i n  4.1C,D.  resting  and  dendritic  potential,  (n=83; mean +/.55mV  resistance calculated +/-  and  from  membrane p o t e n t i a l  "anomalous r e c t i f i c a t i o n "  r e c t i f i c a t i o n was  Somatic  +/-  of the  pulses  t o as  current I/V  hyperpolarizing  somatic  Anomalous  depolarizing  extending  both  response r e f e r r e d 1979).  potentials  at  sem)  (n=27) at the as  with  25.9  an  average  in intrasomatic for  dendritic  s o m a t i c and +/-  respectively  impalements  dendritic  1).  of  recording  intrasomatic  (Table  comparable v a l u e s  value  62.5 sites,  and  +/and  (Table 1).  l e v e l were a l s o  .57megohm (n=92;  2.06megohm (n=14) f o r  locations,  membranes had  mean +/-  sem)  -dendritic  of  ,73mV 64.3 Input  similar, and  24.4  recording  -  FIG. 4.1 membrane  Pyramidal c e l l potential  hyperpolarizing  below.  responses  (Ri) f o r somatic  depolarizing dendritic  range  (D) impalement.  and a l l subsequent response  from  comparison.  shown  a  range  of  (I/V) p l o t  square  wave  and c a l c u l a t e d  and d e n d r i t i c of  membrane  f o r another  Action  potentials  pyramidal  neurons  The input  responses are shown potential somatic truncated.  f i g u r e s , the i n t r a s o m a t i c and  separate  d e n d r i t i c (B)  current pulse i n j e c t i o n s .  rectification is  (A) and a p i c a l  to  and d e p o l a r i z i n g  Anomalous  -  somatic  corresponding c u r r e n t / v o l t a g e resistances  81  are  i n the  (C)  and  In t h i s  intradendritic presented f o r  - 82  Somatic  -  Dendritic  2 0 msec  -  TABLE  1  Average  resistance dendritic stratum  resting  (Ri) of  CAl  pyramidal  (mean +/-  b r a c k e t s ) . The a v e r a g e v a l u e only f o r those had  been  recordings  constructed.  -  membrane  impalements obtained  pyramidale  83  potential cell  at least  (RMP) and i n p u t  somatic  and  apical  150um f r o m t h e b o r d e r o f  sem; number o f i m p a l e m e n t s shown i n o f i n p u t r e s i s t a n c e was  i n which f u l l  calculated  current/voltage  profiles  - 84 -  TABLE 1  SOMATIC  DENDRITIC (> 150 JJM)  (MV)  R  ±  I  (fin)  64.3  62.5  RF1P  ±  .73  ±  .55  (83)  (27)  25.9  24.4  .57 (92)  ± 2.06 (14)  - 85 Current  Evoked  Suprathreshold  Depolarizing discharge neuron.  in In  discharge assigned 1"  reliably  general, each  form  The  current  evoked  location  was  very  of  Type  response  to  somatic  these  cells,  current  four  activation,  potential  similar  and  r e f e r r e d t o as  rapid  (width  arising  was  decline  at  characteristics were d e t e c t e d . the  half  cell  those  of spike  varied  spikes  recorded  amplitude  The in a  and  "burst" of  within the  spike  increase  each s p i k e  in  i n the  was r e c o r d e d a t  differences i n the  s o m a t i c and  following  levels of  of depolarization.  axis,  of greater  of  characteristics of  with  evoked  a  p r e d i c t a b l e manner,  level  cell  evoked  interspike interval  o f Type 1 d i s c h a r g e  were  less  by r e p e t i t i v e  amplitude  greater  evoked  For instance,  but  a l l levels  frequency  in  amplitude),  pyramidal  of current  pyramidal  h a l f w i d t h than recovery  the  high  decrease  in  from a s l i g h t l y  of  for  ( F i g 4.2). Higher  increased.  burst  While t h e b a s i c p a t t e r n levels  spikes  current  p o t e n t i a l s followed  within the  a  depolarizing  progressive  "Type  and d e n d r i t i c i m p a l e m e n t s  e v o k e d an i n i t i a l  injection  evoked s p i k e s  halfwidth  a  c o u l d be  most common f o r m o f s p i k e  (12/16)  fast  (AP)  a p p e a r e d t o be r e s t r i c t e d t o  the  injection  action  with  current  displaying  of  current  depolarizing  was  both  sequence  depolarizing  burst  1  In  repetitive  three  discharge  (Type 3 ) .  in  (21/36).  burst  spike  of the pyramidal  action  basic patterns,  membrane  discharge  of  repetitive  d e n d r i t i c membranes  t o e i t h e r o f two  dendritic  all  evoked  and "Type 2" f o r t h e p u r p o s e o f c o m p a r i s o n . A t h i r d  common  as  currents  s o m a t i c and  in  Responses  d e n d r i t i c spikes  i n the somatic amplitude apical  region  and s h o r t e r  d e n d r i t e . The  the i n i t i a l  burst  o f AP  -  FIG  4.2  The  increasing (A-C)  and  "Type 1"  86  response  l e v e l s of d e p o l a r i z i n g dendritic  impalement  c a l i b r a t i o n b e t w e e n s o m a t i c and  -  of  a CAl  current  p y r a m i d a l neuron applied  to a  (D-F). Note d i f f e r e n c e dendritic  recordings.  to  somatic  in voltage  - 87 -  Dendritic  Somatic D.  E. —  B.  c.  ill  J  J 120  J  I nA 10 mV 20 msec  - 88 discharge  could  also  differ  between  somatic  recording s i t e s .  I n t h e soma, s p i k e a m p l i t u d e  from t h e i n i t i a l  decline  fifth  dendritic  usually  recovered  w i t h i n t h e b u r s t by  approximately  to eighth action p o t e n t i a l of the repetitive spike  In c o n t r a s t ,  the recovery  c o u l d d e p e n d upon t h e less  and  recovery  of spike  amplitude  degree o f c u r r e n t  observed  the  depolarizing current pulse  greater  in  injection the  the  train.  the dendrite (7/12),  amplitude  with  of  ( F i g 4.2 D - F ) . I n a d d i t i o n ,  the  somatic  s p i k e s were e v o k e d f r o m a c o n s i s t e n t v o l t a g e t h r e s h o l d , w i t h t h e first  evoked a c t i o n  potential arising  depolarization  regardless of  current pulse  ( F i g 4.2A-C;  absolute  the amplitude  membrane  discharge,  with  displayed  the  increasing directly  a similar of  voltage threshold  voltage of the breakpoint  dendritic  from  with the  apparent  breakpoint level of  of  the depolarizing measured as  of spike discharge).  no  voltage  level  However,  threshold f o r spike  current  the  f o r AP  activation  injection  (Fig  4.2D-F). Type 2 s p i k e d i s c h a r g e frequency  burst  of  afterhyperpolarization discharge  was c h a r a c t e r i z e d by an i n i t i a l fast  (AHP)  ( F i g 4.3A-G).  This  commonly o b s e r v e d i n b o t h  spikes and  repetitive  pattern  somatic  and  appeared  to  displaying  an  obvious  anomalous  threshold  range  of  current  f o r AP d i s c h a r g e ,  response i n f u l l  be  most  activity varied  activity  prevalent rectification  injection  current  an spike  was l e s s (10/36)  i n membrane in  ( c f 4.1C,D).  the Near  i n j e c t i o n e v o k e d t h e Type  f o r m , i n c l u d i n g an i n i t i a l  AHP, and r e p e t i t i v e s p i k e pattern of  of  by  single  (4/16) and d e n d r i t i c  impalements  depolarizing  followed  high  discharge with the  burst of spikes,  ( F i g 4.3A,D). However,  2 an  the  degree o f d e p o l a r i z a t i o n ,  - 89 with r e p e t i t i v e spikes encroaching of c u r r e n t  injection  Once  again,  upon t h e AHP a t h i g h e r  ( F i g 4.3A-C and  certain  D-G).  characteristics  of t h e evoked s p i k e  d i f f e r e d between somatic  and d e n d r i t i c r e c o r d i n g  the  the i n i t i a l burst  soma, s p i k e s w i t h i n  levels  l o c a t i o n s . In  o f Type 2  discharge  were e v o k e d upon an u n d e r l y i n g d e p o l a r i z a t i o n , e x h i b i t n g a r a p i d decline  i n amplitude  and i n c r e a s e  underlying depolarization during  spike  recover  in  amplitude current  amplitude  somatic  of  impalements  burst  "intermediate  Component  action  inflections  region  were  levels  of current  the  first  dendritic  spike  Several  injection spike),  a  spikes  of the  recorded  ( F i g 4.3G). as s m a l l  intermediate  i n the  somatic  ( F i g 4.3A-C; m e a s u r e m e n t s r e f e r t o while could  the  voltage  threshold  appear t o i n c r e a s e  and  voltage  for  directly  ( F i g 4.3D-F). o f Type 2  Type 1 s p i k e a c t i v a t i o n . F o r  halfwidth  of the  similar voltage threshold f o r a l l  of the evoked c h a r a c t e r i s t i c s  amplitude,  some  underlying  be d e t e c t e d  edge  of the current pulse  were s i m i l a r t o t h a t o f the  or f a l l i n g  In  spike, similar to  then  activation  with the amplitude  large  potentials  from  depression  spike  e t a l . (1982)  evoked  evoked  to f i f t h  a particularly  could  could  l e v e l of  ( F i g 4.3D-F).  s p i k e " of Benardo  Once a g a i n ,  burst  upon t h e  l a r g e r and b r o a d e r  on t h e r i s i n g  ( F i g 4.3G).  initial  the current pulse, the  the t h i r d  merged on t o p o f  characteristics  greater degree of  currents  (3/10),  t o form a  the  during  a  However, t h e  i n d e n d r i t i c impalements.  c o u l d depend  displaying  depolarizing  depolarization  spike  recordings  injection,  dendritic  the  i n spike  following  d e n d r i t i c spikes  higher  initial  and c h a n g e  t h e b u r s t w e r e more a c c e n t u a t e d  Although  with  i n halfwidth.  discharge instance,  threshold of the f i r s t  -  FIG.  4.3  increasing (A-C) was D-F.  The  "Type 2"  90  response of  l e v e l s of depolarizing  and d e n d r i t i c taken H-K.  illustrating  from  impalement  a  separate  "Type  3"  -  CAl pyramidal  neurons t o  current applied to a  (D-F).  The Type 2 r e s p o n s e o f  dendritic  responses  somatic  of  impalement dendritic  from t h a t i n recordings  t h e c u r r e n t e v o k e d and s p o n t a n e o u s d i s c h a r g e o f  separate d e n d r i t i c  i m p a l e m e n t s i n H , l and  >  J,K.  G  two  Somatic  Dendritic  2 0 msec  - 92 current  evoked  discharge.  spike  were  However, two  comparable  in  subsequent  p a t t e r n o f AP  Type 1 and 2  major d i s t i n g u i s h i n g f e a t u r e s  larger d e p o l a r i z a t i o n underlying the i n i t i a l the  both  burst of spikes  a f t e r h y p e r p o l a r i z a t i o n observed discharge.  Type 1  and 2 a c t i v i t y  A third  and i n f r e q u e n t  only encountered i n evoked  and  were t h e r e f o r e  impalements i l l u s t r a t i n g  these  form of s p i k e a c t i v i i t y  discharge t h i s type  of  on a r i s i n g  two  3 a c t i v i t y during amplitude  c u r r e n t evoked response  depolarization with a  from  one  constant  over  the  spike  and J ) .  a p p e a r e d t o be a d i s t i n c t  o f r e c o r d i n g . The e x a c t  fast spikes  dendritic  "slow"  ( F i g 4.3H  impalement  form  time  of  Type  p a t t e r n and  i n Type 3 a c t i v i t y c o u l d to  another,  recording  b u t was  vary  generally  f r o m a. g i v e n d e n d r i t i c  impalement. I n c o n t r a s t , slow s p i k e c h a r a c t e r i s t i c s v a r i e d one d e p o l a r i z i n g p u l s e  to the  next,  in  burst  one d e n d r i t e c h a n g e d f r o m Type 2 t o  the course  of i n i t i a l  current  a low t h r e s h o l d a l l - o r - n o n e  This pattern of spike generation although  The  was  separate d e n d r i t i c  evoked from t h e t h i r d or subsequent f a s t s p i k e  of d i s c h a r g e ,  (5/36)  o f r e s p o n s e a r e shown i n F i g  f o r c o m p a r i s o n . The  dendrites consisted of  of f a s t spikes  activation,  apparent.  dendritic recording locations.  spontaneous  4.3H,I and J,K  was most  and  i n t h e Type 2  most r e a d i l y d i s t i n g u i s h e d n e a r t h r e s h o l d f o r s p i k e where t h e AHP o f Type 2 d i s c h a r g e  were t h e  from  evoked f o r example w i t h  an  amplitude  i n t h e r a n g e o f 50-75mV and h a l f w i d t h b e t w e e n 9-20msec  in  dendritic  one  recording.  a c c o m p a n i e d by g r a d u a l  form  current  of  which  injection  3  activity  was  often  d e p o l a r i z i n g s h i f t s o f membrane p o t e n t i a l  t h a t l e d t o spontaneous the  Type  was  i n the  all-or-none bursts of spike very  similar  discharge,  t o t h a t evoked  same d e n d r i t i c i m p a l e m e n t  through  ( F i g 4.3H,I  -  and  J,K).  feature  In c o n t r a s t ,  -  93  spontaneous a c t i v i t y  o f c e l l s d i s p l a y i n g Type 1 o r  2  S t i m u l u s Evoked S u b t h r e s h o l d S y n a p t i c Graded s y n a p t i c the  pyramidal  alveus,  stratum  stimulation the  oriens  was  pyramidal  cell  of  region alveus  at  differed  (n=8)  the  IPSP  displaying mean +/-  in dendritic  intensities  an  between  average  sem)  i n the  recordings  (p <.  i n h i b i t o r y p o t e n t i a l was  in  cases  However,  appeared  the  dendritic  hyperpolarizing (5-10 of  mV),  and  was  stratum oriens  equally  SO  region,  evoked  .014 often  could  (Fig  latency 25.6  4.4A,D).  (SO)  The  was  of  17.7  an  +/-  1.72msec  and  i n amplitude,  be  (data not  shown).  synaptic  inputs  average  +/-  low  spikes  of  peak  dendritic  potential.  reversed  to  a  depolarization the  discharge  regardless  coursing  of pyramidal c e l l s  (EPSP)  (4.4B,E).  l a r g e s t amplitude i n the peak a m p l i t u d e o f 14  of  through  evoked a graded e x c i t a t o r y p o t e n t i a l  EPSP  displaying  potential  The  dendritic  apical dendrites  for  Students t - t e s t ) .  effective in blocking  afferent  to  of i n h i b i t o r y  slight depolarizing  IPSP  evoked  of  i n b o t h s o m a t a and The  a  resting potential  Stimulation  inputs  s o m a t i c and  soma and  Alvear  sites.  p o t e n t i a l t h r o u g h s l i g h t membrane  orthodromically  p o l a r i t y at  as  peak  the  subthreshold  d e n d r i t i c recording  dendritic many  activation  of  of  ( F i g 4.4).  d i s c h a r g e evoked a graded i n h i b i t o r y  latency  1.35msec (n=6;  stimulation  of s t r a t u m pyramidale  and  locations,  to  i n h i b i t o r y synaptic  recurrent  (IPSP) at b o t h s o m a t i c of  recorded at a l l l e v e l s  response  activate  a common  discharge.  stratum radiatum  through  the  antidromic spike  in  or  used to  i n t e r n e u r o n s i n the Stimulation  axis  not  Potentials  p o t e n t i a l s were  cell  was  +/-  somatic 2.48mV  - 94 FIG.  4.4  recorded  Stimulus  i n pyramidal c e l l  from t h e border intensities stimulation stratum  evoked  subthreshold  of  stimulation. the  radiatum  alveus  (C,F).  potentials  soma (A-C) and d e n d r i t e s (D-F)  of stratum pyramidale  of  synaptic  superimposed  Potentials  (A,D),  stratum  A l l somatic  o b t a i n e d f r o m t h e same i m p a l e m e n t  at  were oriens  recordings  increasing evoked  five single  impalement. sweeps.  by  (B E), or f  (A-C)  were  (membrane s l i g h t l y d e p o l a r i z e d  i n A) , w h i l e .the r e c o r d i n g s o f D and F were t a k e n f r o m a dendritic  200um  A l l potentials  a r e averaged  single  records of  St im: Site  Somatic A.  Alveus _  B.  Stratum Oriens - i  C.  Stratum Radiatum  Dendritic D.  (200p)  - 96 (n=5;  mean +/  -  s e m ) . The EPSP  was o f s m a l l e r  apical dendrite, exhibiting a  peak a m p l i t u d e o f 6.4  200/im f r o m t h e b o r d e r o f s t r a t u m spike  threshold).  negative-going edge  of  "notch"  somatic  corresponding the  Near  or  dendritic  space  Stimulation i n stratum  of  amplitude,  +/-  spike  again,  (SR)  also  evoked  an a v e r a g e  a  of  value  from t h e border o f  an  somatic  average  pyramidal  (n=34; EPSPs  the extracellular  amplitude  of  stratum  graded  21.2 +/-  pyramidale  EPSP was o f s m a l l e r  value  population  peak  o f 12.9 +/- .72mV i n  set to spike threshold).  o b s e r v e d as a r e f l e c t i o n o r "notch"  spike  could  on t h e r i s i n g  Once  often  be  edge o f e v o k e d  ( F i g 4.4C; R i c h a r d s o n e t a l . 1 9 8 4 a ; T u r n e r e t a l . 1 9 8 4 ) .  Stimulus  Evoked S u p r a t h r e s h o l d  Stimulus  evoked  markedly from t h a t  Responses  action  of current  potential injection  discharge  spike  in  intracellular  Suprathreshold both  spikes  alvear stimulation  somatic arose  recordings  and  directly  dendritic from  of  differed  i n that antidromic  o r t h o d r o m i c s t i m u l a t i o n c o n s i s t e n t l y evoked a s i n g l e  in  response i n  collateral/commissural  s o m a t a and d e n d r i t e s  s e m ) . The  exhibiting  somatic impalements  EPSPs  ( F i g 4.4B,E), a response  Schaffer  dendrites, exhibiting  mean  a  o b s e r v e d on t h e r i s i n g  EPSPs  radiatum  1.13mV a t 200jum d i s t a n c e (n=22;  1.55mV  discharge,  ( F i g 4.4C,F). T h i s p o t e n t i a l had t h e g r e a t e s t  in d i s t a l  -  (see C h a p t e r 5 ) .  e x c i t a t o r y p o t e n t i a l i n both neurons  f o r spike  frequently  +/  ( n = l l ; EPSPs s e t t o  to reflections of the population  extracellular  afferents  pyramidale  threshold was  amplitude i n the  all-or-none  the pyramidal  evoked a s h o r t  or  latency  cell. spike  l o c a t i o n s ( F i g 4.5A,D). T h e s e  baseline  w i t h an i n v a r i a n t o n s e t  FIG.  4.5  recorded (D-F) the  Characteristic in  pyramidal c e l l  locations. alveus  ( D F ) . Somatic while  dendritic  single dendritic pyramidale). In suprathreshold v o l t a g e and  stratum  each case, intensities  and  evoked  (200pm  dendritic  by s t i m u l a t i o n  (B,E), or stratum  (D-F)  of  radiatum separate  were o b t a i n e d from  from the  r e c o r d i n g s are of  apical  were t a k e n f r o m t h r e e  recordings  impalement  evoked s p i k e d i s c h a r g e  (A-C)  was  oriens  r e c o r d i n g s (A-C)  f  cells  somatic  Spike discharge  (A D), f  stimulus  stimulation.  border  of  shown f o r Note  time base c a l i b r a t i o n of a l l r e c o r d i n g s .  the  a  stratum sub-  and  common  Stim.Site  Somatic A.  Alveus . J  B Stratum Oriens  C. Stratum Radiatum  Dendritic (200>u) D.  - 99 latency  at  a  antidromic pyramidal radiatum  given  response  stimulus of  the  and a p i c a l d e n d r i t e s o f t h e  ( F i g 4.5C,F) e v o k e d a s i n g l e a l l - o r - n o n e s p i k e f r o m  an  location  EPSP  at  a l l levels  along  in  the  amplitude  of  discharge  the  illustrated  from  of  At stimulus both  somatic  jitter"),  arising  approximately  synaptic  potential  from  in  As  s p i k e s were evoked w i t h  dendritic spike displaying  higher  discharge  the  peak  from  a  latency  i s clearly  intensity  or the  than  those  similar to  initial  evoked  spike  and  threshold  dendritic  onset l a t e n c y  spikes  ("latency  stimulus  intensity  ( F i g 4.6C,D). I n t h e  onset  voltage  was  illustrated  somatic  threshold,  l e v e l of d e p o l a r i z a t i o n ( F i g 4.6A).  from t h a t  voltage threshold  was e v o k e d a t two  stimulus  soma were  a p r o g r e s s i v e l y s h o r t e r and  activation differed  no c l e a r  characteristic discharge  the  given  d e n d r i t i c l o c a t i o n s are  r e g i o n , s p i k e s were evoked from a c o n s i s t e n t  of  any  t h e peak o f t h e e x c i t a t o r y  ( F i g 4.6A,B).  less variable latency to  regardless  at  i n t e n s i t i e s near  variability  arising  the  i n halfwidth  and  displayed considerable  spikes  each o f  at the  synaptically  somatic  generation,  increased,  The  ( c f 4.5A-C and D-F) .  recorded  more n a r r o w  cell.  dendrite.  i n F i g 4.6.  spike  be s i m i l a r axis  4.5, s p i k e s  properties  recorded  pyramidal  evoked from  were f o u n d t o  and  oriens  the  dendro-somatic  i n the apical  Some  of  action potentials  However, as shown i n F i g  with  an  or  three stimulation s i t e s  for  representing  ( F i g 4.5B,E)  c h a r a c t e r i s t i c s of  recorded  soma  neuron. S t i m u l a t i o n of stratum  underlying  greater  intensity,  i n the  However, soma i n  f o r AP g e n e r a t i o n .  i n F i g 4.6B  i n which  This spike  i n t e n s i t i e s of s t i m u l a t i o n . At the  (12V),  s p i k e s were  falling  phase o f  evoked from e i t h e r t h e EPSP.  With  9V  - 100 FIG.  4.6  pyramidal shown i n  Stratum cell  radiatum  somatic  each case  -  evoked s p i k e d i s c h a r g e  (A,C)  and  with several  recorded  dendritic locations  superimposed  i n t e n s i t i e s w e r e s e t f o r t h r e s h o l d (A,B)  sweeps.  (B D), f  Stimulus  or s u p r a t h r e s h o l d  a c t i v a t i o n of s p i k e d i s c h a r g e i n each l o c a t i o n .  Similar  were  potentials.  obtained  r e c o r d i n g s were  for  stratum  taken from  oriens separate  a p i c a l d e n r i t e s 200um f r o m t h e b o r d e r  evoked  impalements of stratum  in  (C,D)  results  o f somata pyramidale.  All or  Somatic  B. Dendritic ( 2 0 0 p )  2 0 mV 5 msec  - 102 s t i m u l a t i o n , a spike could again underlying  EPSP  and  o b s e r v e d a t t h e 12V  rise  voltage  stimulation  t o produce  and  a  t h r e s h o l d w e l l below t h a t  of hippocampal a f f e r e n t  a characteristic  amplitude  of  evoked  potentiation"  1977;  T u r n e r e t a l . 1 9 8 4 ) . The s o m a t i c  four  pulses  radiatum  is  intensities, o f EPSP  a  10Hz  shown  in  stimulus  and  with continued  train  times  t o onset.  frequencies  With  subthreshold  of a  evoked  of  in  p r i o r to the  spikes  layer, a in  the  Schwartzkroin  during  the frequency  stimulus  either  generation result  somatic  region  (  dendritic  of  level  the  was  d i d not appear  repetitive Richardson  found a t  pyramidal  discharge  or d e n d r i t i c impalements.  and P r i n c e 1980) . T h e r e f o r e , discharge  spike  of multiple population  indicating  less  s t i m u l a t i o n or w i t h  multiple  somatic  t r a i n and  to a shorter,  With prolonged  activation,  action potential  frequency  stratum  single all-or-none  However, r e p e t i t i v e d e n d r i t i c s p i k e d i s c h a r g e  the c e l l  (Schwartzkroin  delivered to  stimulation displayed a shift  variable latency  occur  pattern  and d e n d r i t i c l e v e l . A c t i o n p o t e n t i a l s  were evoked a t comparable  be  the rate of  and d e n d r i t i c r e s p o n s e t o  the generation  spike at both the somatic  could  inputs  r e p e t i t i v e s t i m u l a t i o n brought about a p o t e n t i a t i o n  amplitude  greater  4.7.  Fig  increase i n  p o t e n t i a l s , a response  commonly r e f e r r e d t o as " f r e q u e n c y  of  smaller  intensity.  High frequency i s known  at  be e v o k e d , b u t f r o m a  cell  stimulation of afferent synaptic  spikes at  activation et  in  of  a l . 1984a;  a similar pattern both the  to  somatic  of and  response t o high  inputs.  - 103 FIG.  4.7  Response  dendrite  (B) t o 10Hz  afferent  inputs  Illustrated  of  pyramidal  frequency at  l a s t two  pulses  cell  soma  (A)  and  s t i m u l a t i o n of stratum  subthreshold  are four i n d i v i d u a l  t r a i n w i t h the  -  stimulus  pulses of a ten  apical radiatum  intensities. pulse  stimulus  i n e i t h e r case showing a sub-  s u p r a t h r e s h o l d example from s e p a r a t e  stimulus  trains.  and  A. Somatic  B. Dendritic  ,  0  _j20mV 5 msec  - 105 Comparison of S u p r a t h r e s h o l d  -  Stimulus  and  Current  Evoked  Responses A c h a r a c t e r i z a t i o n of pyramidal  cell  a c t i v i t y has  revealed  t h a t d e p o l a r i z i n g c u r r e n t evokes a v a r i e t y of s p i k e responses i n the d e n d r i t i c r e g i o n , i n c l u d i n g the l a r g e and  small  amplitude  r e p e t i t i v e discharge  fast spikes,  l a r g e s l o w a c t i o n p o t e n t i a l ( c f 4.3). evoked s p i k e i s of l a r g e amplitude in  orthodromic  Upon  narrow h a l f w i d t h ,  those  s t i m u l a t i o n , the stimulus  initial  appeared  distinctly  halfwidth were  examination,  (Fig  activated  spikes  4.8A-C). upon  superimposed,  stimulus  and  the  revealing  current  evoked  4.8D-F). S i m i l a r r e s u l t s although  the  initial  a  basic  action  in  and  and  c u r r e n t evoked of  4.8). spikes  amplitude  similarity  somatic  i n amplitude  amplitude  spikes  the l e v e l  demonstrates  (Fig  and spikes  the  between  p o t e n t i a l discharge  r e c o r d i n g l o c a t i o n s . I n T a b l e 2,  further  action  underlying depolarization,a l l  dendritic  h a l f w i d t h at  c u r r e n t evoked  when s t i m u l u s e v o k e d  and  stimulus  evoked  or  terms  were o b t a i n e d  difference  evoked through a n t i -  and  in  evoked the  stimulus  dendrites  current  same  To  d i r e c t l y compared  However,  a  determine  and  stimulus  different  of  However, t h e most commonly  and  p o t e n t i a l o f d e n d r i t i c membrane was  both  or the generation  a r e p e t i t i v e manner i n Type 1 o r 2 a c t i v i t y .  s i m i l a r i t y b e t w e e n t h i s s p i k e and  of  was  and  not  impalements, h a l f w i d t h of  as m a r k e d as  a comparison of  of the  (Fig  soma and  similarity  spike apical  i n waveform  c u r r e n t evoked f a s t s p i k e s of the p y r a m i d a l  in  cell.  of  - 106 FIG.  4.8  Comparison o f  potential  discharge  dendrite.  A-C.  superimposed  s t i m u l u s and in  a  Current  c u r r e n t evoked  pyramidal  evoked  discharge  (C).  e v o k e d s p i k e s were e v o k e d on t h e same u n d e r l y i n g that  current  is  upon s p i k e s a c t i v a t e d by s t i m u l a t i o n o f t h e  ( A ) , s t r a t u m o r i e n s (B) o r s t r a t u m r a d i a t u m  as  cell  necessary evoked  to  spike  elicit then  potentials are  t a k e n from  from t h e border  of stratum  D-F.  action apical shown alveus Stimulus  depolarization  c u r r e n t e v o k e d d i s c h a r g e , and a  superimposed t h e same  pyramidale.  f o r comparison. A l l  dendritic  i m p a l e m e n t 200um  Stim.Site A.  Alveus  Stratum B.  Stratum Radiatum C.  _J  F.  - J  ,J20mV 2 msec  - 108 TABLE  2  Average  amplitude) of cell  amplitude  c u r r e n t and  somata  and  stratum  pyramidale  resting  membrane  apical  dendrites  (mean  +/-  potential).  discharge  halfwidth  s t i m u l u s evoked  s p i k e c h a r a c t e r i s t i c s were t a k e n Type 1 o r 2  and  sem;  (width  spikes  200jum  from  amplitudes  Measurements  e v o k e d by d e p o l a r i z i n g  half  i n pyramidal the border of measured from  of  from the f i r s t  at  current  evoked  spike of  either  current.  Stimulus  e v o k e d s p i k e s w e r e e v o k e d by s t i m u l a t i o n o f t h e a l v e u s ,  stratum  oriens, or stratum  radiatum.  average are given  i n brackets.  the somatic according amplitude  The number o f i m p a l e m e n t s f o r  or d e n d r i t i c l e v e l  t o a one and  Stimulus  evoked s p i k e s a t e i t h e r  were n o t s i g n i f i c a n t l y  way ANOVA t e s t . C u r r e n t  current  evoked  each  dendritic  different  evoked somatic spike  amplitude  h a l f w i d t h w e r e f o u n d t o be s i g n i f i c a n t l y d i f f e r e n t f r o m  spike and  stimulus  e v o k e d s p i k e s . However, t h i s d i f f e r e n c e c a n be a t t r i b u t e d t o t h e l e v e l of  membrane d e p o l a r i z a t i o n  (see F i g 4 . 8 ) .  upon w h i c h  s p i k e s a r e evoked  -  TABLE  109  -  2  SPIKE AMPLITUDE SOMATIC  (MV)  DENDRITIC  (200  STIMULUS  SPIKE  (MSEC)  HALFWIDTH  SOMATIC  DENDRITIC  (200  UM)  UM) I  INJECT.  69.6  90.3  CURRENT  ±  2.04  ±  87.3  +  2.77  ±  ORIENS  ±  4.06 (4)  RADIATUM  ±  ±  2.68  2.36 (9)  .97  (23)  1.34  ±  ±  .055  1.33  ±  1.17  .87  .039 (11)  .066 (12)  (4)  ±  .069 (11)  .84  65.0  ±  .028  .14 (2)  (7)  (12)  90.0  STRATUM  2.42  59.5  ±  ±  .96  (11)  87.2  .051  i.80  (6)  62.9  (9)  STRATUM  +  (9)  (11)  ALVEUS  3.42  1.06  ±  .037 (20)  - 110 Spike  -  Pre-Potentials Somatic  recordings  of  uncovered the  presence of  "fast pre-potentials"  given  small  to  a  pyramidal  amplitude  all-or-none  evoking somatic spike discharge and  1977;  Spencer  characterized  by  a  and fast  amplitude, a fast i n i t i a l decay.  Although  s t u d y , FPPs  The  or  membrane  e i t h e r case, the  peak o f  from the  the  FPP  inputs  the  edge o f  falling  train.  4.9C,D). recorded in  FPPs In  in  the  fast  the  Knowles  relatively  in  i n the  the  are  small of  present  soma w e r e  discharge  also  i n pyramidal  cell Fast  are  illustrated  in Fig  by  stimulation  of e i t h e r  current  4.9.  stratum  all-or-none  t o FPP  activation  and  was  spontaneous  was  pre-potentials  greatly were  FPP  stimulation  a  on  progressive the  stimulus  also effective in locations discharge  impalement,  during  depolarized. also  arising  discharge  during  dendritic  following cellular cell  FPP  p o t e n t i a l , with  injection  somatic cases,  a l s o evoke  In  manner n e a r  pre-potential. Repetitive  synaptic  of  i n j e c t i o n ( F i g 4.9A,B).  action potential discharge  current  both  a  of  Schwartzkroin  examined  e v o k e d i n an  latency  immediately  spontaneous  FPP  (10-20Hz) c o u l d  several  which  by  fast  Depolarizing  evoking  time  i n the  Lomo 1966;  "uncovered" through h y p e r p o l a r i z a t i o n  EPSP, w i t h  of a f f e r e n t  decrease  of  was  peak o f t h e  name  level.  evoked  (5-20mV) the  a  a s e c o n d s l o w e r component  those observed  r a d i a t u m , and  often  These p o t e n t i a l s  rise,  systematically  apical dendrites were  of  (FPPs),  Dudek 1981;  1961).  d e c a y and  dendritic  pre-potentials  the  rate  characteristics  s o m a t a and  oriens  not  s i m i l a r to  recorded at the  Kandel  have  p o t e n t i a l capable  ( A n d e r s e n and  S c h w a r t z k r o i n 1981b; M a c V i c a r and  1975;  neurons  observed  (Fig was the  However, a t more  - Ill FIG.  4.9  discharge  Characteristics i n pyramidal  (B,D,F).  A,B.  uncovered  through  sweeps  shown  E,F.  somata  fast  in  membrane A).  f  injection  r e s t i n g membrane p o t e n t i a l .  and a p i c a l  radiatum  (FPP)  dendrites  evoked  spikes  h y p e r p o l a r i z a t i o n (5-20mV; s e v e r a l  C,D.  S u c c e s s i v e sweeps  pre-potential  (A C,E)  FPPs u n d e r l y i n g s t r a t u m  depolarizing current C).  cell  of  FPP  discharge  (action  evoked  through  potential truncated  of spontaneous  FPP d i s c h a r g e  in at  -  112  -  Stim. Site Stmt. Rad.  Current  Spont. -T5 20  - I nA JlOmV 5 msec  - 113 s t a b l e r e s t i n g p o t e n t i a l s , as shown i n F i g 4.9E,F. The  frequency  o f s p o n t a n e o u s FPP  shift i n  discharge  c o u l d be  membrane p o t e n t i a l , d i s p l a y i n g and  altered with a  an i n c r e a s e w i t h  depolarization  r e d u c t i o n o r b l o c k a d e w i t h membrane h y p e r p o l a r i z a t i o n . The  a given  amplitude  of fast pre-potentials varied s l i g h t l y  impalement depending  on t h e r e s t i n g membrane  within  potential  (see S c h w a r t z k r o i n  1 9 7 7 ) , b u t moreso b e t w e e n d i f f e r e n t  pyramidal  n e u r o n s . However,  of the  no c l e a r  difference  in  between s o m a t i c FPPs e v o k e d average  the  amplitude  of  evoked  thus f a r , FPPs  c o u l d be f o u n d  and d e n d r i t i c r e c o r d i n g s . F o r i n s t a n c e , o f t h o s e  t h r o u g h SR  amplitude  s t i m u l a t i o n , somatic  of  d e n d r i t i c FPPs 14 +/of  c a s e s documented  15  +/-  1.4mV  3.4mV  FPPs d i s p l a y e d an  (n=5;  mean +/- sem) and  (n=4; m e a s u r e d f r o m  the breakpoint  discharge). A  "pre-potential"  action  potential  pyramidal  cell  discharge  axons  previously described (Spencer  was  also  following  ( F i g 4.10).  i n somatic  and K a n d e l  found  recordings  1 9 6 1 b ) , and  terminology  an i n i t i a l  used f o r t h e  s p i n a l motoneuron spike  underlying  uncovered  at the  hyperpolarization antidromic  antidromic  stimulation of efferent  This  potential  has  been  of the pyramidal  cell  was t h o u g h t  activation of a spike i n the i n i t i a l r e f e r r e d t o as  to underly  t o represent the  segment r e g i o n . I t i s  segment o r " I S " s p i k e , f o l l o w i n g t h e components o f s p i k e a c t i v a t i o n  i n the  (Coombs e t a l . 1 9 5 7 a , b ) . I n many c a s e s , the  antidromic  somatic (Fig  stimulus  conditioning antidromic  thus  or  action  dendritic level  4.10A,B).  applied  potential  2.0  s p i k e c o u l d be u s e d  could  be  t h r o u g h membrane  Alternatively, -  the IS  2.4msec  a  following  i n conjunction  membrane h y p e r p o l a r i z a t i o n t o r e v e a l t h e I S s p i k e  test a  with  ( F i g 4.10C,D).  - 114 FIG.  4.10  Initial  evoked a n t i d r o m i c membrane  (B D). r  hyperpolarization conjunction  and  spikes IS  in  (IS) s p i k e s  somatic  spikes  (A,B),  with antidromic  condition-test superimposed  segment  were or  i n each case.  a l l recordings  are taken  (A,C) and a p i c a l uncovered  through  paired pulse  i n t e r v a l s o f 2.0  underlying  dendritic  t h r o u g h membrane  hyperpolarization  sweeps a r e  p o t e n t i a l i s truncated  from separate  in  s t i m u l a t i o n (C,D) a t  - 2.4 msec. S e v e r a l  Action  alvear  impalements.  i n A,  Dendritic  c.  _T 10 2 D.  Paired. Pulse  JlOmV I msec  - 116 The  characteristics  similar  to  those  of of  amplitude  of  decay  IS spikes  pre-potential  including a fast  FPP. in  similar  to  a l l cells  pre-potentials.  and a the  and  those  o f 19 +/- 2.3mV (n=6; mean  3.6mV (n=6) i n t h e d e n d r i t e of  exhibiting  means o f a c t i v a t i o n s e r v i n g  fall  g r e a t d i f f e r e n c e between somatic  the orthodromically  A n t i d r o m i c a l l y evoked "IS s p i k e s " c o u l d almost  initial  rise  cell  l o c a t i o n s , with amplitudes  rather  rate of  between p y r a m i d a l  +/- sem) i n t h e soma and 21 +/values  ( c f 4.9). For  t o r e s t i n g p o t e n t i a l . Although  v a r i e d somewhat  i m p a l e m e n t s , t h e r e was no dendritic  w e r e f o u n d t o be v e r y  IS spike displayed a f a s t  a two component d e c a y ,  s e c o n d more p r o l o n g e d  potential  the fast  instance, the antidromic and  this  evoked  i n f a c t be o b s e r v e d  FPP d i s c h a r g e ,  with only the  t o d i s t i n g u i s h IS spikes  from  fast  - 117 -  4-4.  Discussion  The  present  properties  of  s t u d y has evoked  membranes o f t h e more c o n s i s t e n t  potentials  findings  Given the  noted exceptions  in  ( i e . spike membranes  properties,  input  basic  was  amplitude  were  were  impalements, or  found  linear  to  s o m a t i c and  s i m i l a r membrane and s t i m u l u s  Potentials  over  found with  to a  instance,  a  given  rectification  be c o m p a r a b l e i n s o m a t i c and  similar  evoked  current  pulse  the dendritic  range  similar  to  that  response  injections at  and  described  1983b; H a l l i w e l l  displayed for and  H o t s o n e t a l . 1 9 7 9 ; S e g a l and B a r k e r 1 9 8 4 ) . However, pharmacological  mechanisms o f  not  b e e n e x a m i n e d , i t s h o u l d be n o t e d t h a t  of  membrane  properties  a s p e c t s o f evoked  the dendritic  i s based  at  Adams since  comparison  on t h e q u a l i t a t i v e  activity.  E v o k e d i n h i b i t o r y and e x c i t a t o r y recorded  HPC  r e s p o n s e have  t h e above  largely  to  membrane r e s p o n s e  of potentials,  the  be  sites.  o f waveform  and b o t h c u r r e n t  s o m a t i c membrane (Brown and G r i f f i t h 1982;  recording  halfwidth),  exhibit  the  i n the pattern  absolute value and  depolarizing  l o c a t i o n . For  anomalous  i n the  dendritic  c h a r a c t e r i s t i c s o f r e s t i n g membrane p o t e n t i a l and  hyperpolarizing either  and  ( H P C ) . One o f  dendritic  and E v o k e d S y n a p t i c  resistance  dendritic  somatic  the  discharge.  Membrane P r o p e r t i e s The  s o m a t i c and  synaptic potentials,  evoked s p i k e  in  characterize  was a g e n e r a l s i m i l a r i t y  activity  dendritic  further  hippocampal pyramidal c e l l  of evoked  parameters  served t o  both  the  somatic  synaptic potentials and d e n d r i t i c  could  level of the  - 118 pyramidal  cell.  potentials density  By c o m p a r i n g t h e e v o k e d c h a r a c t e r i s t i c s o f t h e s e  to  the  analysis  results (CSD;  Chapter  possible s i t e of o r i g i n of example,  stimulation  obtained 3),  these  of  through  into  the  p o t e n t i a l s c a n be g a i n e d .  For  pyramidal  some  current-source  cell  insight  axons  i n the alveus  e v o k e d a g r a d e d I P S P i n b o t h t h e soma and a p i c a l d e n d r i t i c t r e e . This  potential  membrane,  has  been  representing  conductance  through  interneurons  (Allen  Dingledine The  well a  GABA  the et  characterized  action  a l .  principle  s i t e Of  1977;  interneuron  electrophysiologically  dendritic  electrotonic  recurrent  Andersen  axonal  ( L e u n g 1979a,b)  membrane  may  conduction  dendro-somatic a x i s .  et  in Clinhibitory  a l .  of  1969; 1981a).  t e r m i n a t i o n has been de No 1934) and  t o somatic  and  proximal  n e u r o n s . The a l v e a r e v o k e d  thus  correspond  inhibitory  In support  of this,  to  a  pyramidale  dendritic  field  following  population  spike  (Chapter  source  is  the  the 3;  currents ~ along current-source  the  density source  and a c u r r e n t s i n k i n t h e  generation  of  Leung 1979a,b).  expected  result  an  antidromic  A long  f o r current  duration movement  a s s o c i a t e d w i t h t h e a c t i v a t i o n of a C l - conductance a t the body  layer  of  the  pyramidal  cell  current sink could thus represent flow  through  pyramidal  inhibitory current intradendritic  IPSP  source  cell in  a p a s s i v e compensatory  stratum  cell  p o p u l a t i o n . The d e n d r i t i c  dendritic  displayed a  IPSP  passive  i n d i c a t e the presence of a long duration current  near t h e r e g i o n o f stratum  current  increase  ( C a j a l 1911; Lorente  d e n d r i t i c membranes o f p y r a m i d a l  profiles  of  somatic  and Langmoen 1 9 8 0 ; K n o w l e s and S c h w a r t z k r o i n  l o c a l i z e d both anatomically  in  mediated  f o r HPC  elements  pyramidale.  l o n g e r peak  current  towards the In fact, the  l a t e n c y than  the  -  somatic  inhibitory  potential,  depolarizing  potential,  movement  current  of  intradendritic passive  in  Such a p r o c e s s might  limit  intracellular  evoked with  comparison  r e g i o n s . The a l v e a r  evoked  of  activity  evoked  the  can  active  t o conduct  inhibitory  through  the a b i l i t y  to the  termination  somatic the  along the  cell. relationships  and  applied to excitatory synaptic  stimulation  of  (SO) s t i m u l a t i o n e v o k e d  t h e s o m a t i c and  cell.  be r e q u i r e d t o d e t e r m i n e  sink-source  be  through a  of the pyramidal  synaptic  of  a  inward  i n f a c t be e x p e c t e d c o n s i d e r i n g  interneuronal  as  an  the somatic region  inputs. Stratum oriens  cell,  conduction  axis of the pyramidal  similar  EPSP a t b o t h  often  consistent  dendritic  f u r t h e r work w i l l  of  dendro-somatic  potentials  was  result  excitatory potentials  r e g i o n . However,  A  and  I P S P c o u l d t h u s be a d e q u a t e l y e x p l a i n e d  p o t e n t i a l evoked  exact  in  electrotonic  of d e n d r i t i c  a  -  119  afferent  synaptic  an  intracellular  d e n d r i t i c l e v e l of  the pyramidal  w i t h t h e g r e a t e s t peak a m p l i t u d e i n t h e r e g i o n o f t h e c e l l  layer.  These  results  current-source  coincide  those  current  sink  i n the basal  f o l l o w e d by t h e c o n d u c t i o n and d e c a y  t h r o u g h t h e somata population. evoked  obtained  through  d e n s i t y a n a l y s i s , i n w h i c h SO s t i m u l a t i o n  a l a r g e a m p l i t u d e EPSP and field  with  In  and a p i c a l d e n d r i t e s  contrast,  the largest  stratum  EPSP i n t h e  dendritic  site  subsequent  c o n d u c t i o n towards  The above  of  origin  data indicate  for  currents  of the pyramidal  radiatum  (SR)  apical dendrite  f r o m CSD  dendritic  of synaptic  s m a l l e r peak a m p l i t u d e i n s o m a t i c r e c o r d i n g s . agree w i t h those d e r i v e d  evoked  cell  stimulation  and  an EPSP o f  These r e s u l t s a l s o  a n a l y s i s , i n d i c a t i n g an a p i c a l the  SR  the somatic that a  evoked  EPSP,  and  a  region.  s y n a p t i c p o t e n t i a l evoked  - 120 within  one  through  a l a r g e extent of the dendro-somatic  serve  to  given  region  emphasize  pyramidal  cell  Schwartzkroin  of  the  (Brown  short  et  also imply  along t h i s  a l . 1981;  cell  a x i s . These length  Turner  and  presence  be t a k e n  can conduct  i n t r i n s i c membrane p r o p e r t y , and a t t e m p t s  the  potentials.  an e v o k e d  by i t s e l f  of  similarity  dendritic  of  results  1 9 8 4 ; T u r n e r and  i n part f o r the  of somatic  that the  a x i s cannot  pyramidal  electrotonic  1 9 8 0 ) , and may a c c o u n t  i n evoked c h a r a c t e r i s t i c s I t would  the  potential  as e v i d e n c e  f o r an  to locate the site  o r i g i n o f a n y p o t e n t i a l must i n c o r p o r a t e a d d i t i o n a l  of  experimental  strategies.  Current  Evoked  Spikes  A n a l y s i s of somatic revealed current for  that  spike  1-3).  However,  imply t h e presence region. Furthermore, absolute  and  in  response  take a v a r i e t y  be  o f forms,  used i n  the present  of d i s t i n c t the some  stressed  pyramidal  grouped  patterns  or  here (Types  that  the grouping of  study  i s n o t meant t o  cell  types  d i s t i n c t i o n between t h e s e transition  potentials  to depolarizing  comparison i n t o three b a s i c  i t should  discharge patterns  not  discharge  injection could  the purpose of  and d e n d r i t i c c u r r e n t e v o k e d  i n the  CAl ,  patterns i s  combination  of  these  c h a r a c t e r i s t i c s c a n o c c u r , a s shown b y t h e o c c a s i o n a l c h a n g e dendritic spike discharge presence  f r o m Type 2  t o Type 3  a c t i v i t y . The  o f t h r e e d i f f e r e n t p a t t e r n s o f c u r r e n t evoked  c o u l d n o t be a t t r i b u t e d as v a l u e s o f  to the quality of  r e s t i n g p o t e n t i a l and  in  cellular  discharge  impalement,  input resistance f o r  cells  d i s p l a y i n g e a c h f o r m o f d i s c h a r g e were c o m p a r a b l e . I n  addition,  orthodromic  EPSP-IPSP  stimulation  would  reliably  evoke  an  - 121 s e q u e n c e and a s i n g l e f a s t  spike i n both somatic  and  dendritic  impalements r e g a r d l e s s o f t h e form o f c u r r e n t evoked Therefore, stimulus  membrane evoked  suggesting  spike  that the  inherent property injury  or  characteristics, discharge  variability  recording. Rather,  s y n a p t i c p o t e n t i a l s , and appeared  pattern of current  of the c e l l in  be  and n o t t h e r e s u l t  of  of  the i n t r a c e l l u l a r  s t u d i e s would i n d i c a t e  the d i f f e r e n t i a l p a t t e r n o f c u r r e n t evoked a c t i v i t y ionic basis of spike generation apical  i n the pyramidal  reflects the  cell  soma  i n v e s t i g a t i o n s have shown t h a t f a s t s p i k e s  t h e soma a n d a p i c a l d e n d r i t e t h e Na+  channel  thought t o  blocker  of pyramidal  1979).  be m e d i a t e d  In  and  contrast,  represent  and a r e  b e e n shown t o  and  by  therefore  Na+ c h a n n e l s  and S l a w s k y 1977;Wong  intermediate  regenerative  i n both  are blocked  through voltage-dependent  d e n d r i t i c membrane h a v e probably  cells  t e t r o d o t o x i n (TTX)  ( H o t s o n and P r i n c e 1 9 8 0 ; S c h w a r t z k r o i n  al.  that  dendrite.  Previous  al.  intact, an  quality  pharmacological  to  e v o k e d a c t i v i t y was  i n question the  responses.  spikes  of  be TTX i n s e n s i t i v e ,  and  Ca+2 c h a n n e l  slow  et  activation  (Wong e t  1 9 7 9 ) . S i m i l a r l y , t h e membrane d e p o l a r i z a t i o n u n d e r l y i n g t h e  initial by Ca+2  burst of fast spikes channel  antagonists  i s i n s e n s i t i v e t o TTX b u t ( Schwartzkroin  reduced  and S l a w s k y 1 9 7 7 ;  Wong and P r i n c e 1 9 7 8 ; Wong e t a l . 1 9 7 9 ) . The current  distinguishing evoked  contribution generation.  activity  of In this  the generation  feature  Na+  might  or  regard,  of fast  between  Ca+2  the  therefore channel  Type 1  Na+ s p i k e s ,  be  three  forms o f  the  relative  conductance  discharge  to  was d o m i n a t e d  w h i l e Type  2 activity  c o m p r i s e d o f Na+ s p i k e a c t i v a t i o n a s w e l l a s a l a r g e  AP by was  underlying  - 122 depolarization spikes.  Type  and 2  the  discharge  afterhyperpolarization discharge, a  occasional  also d i s t i n c t  following  p o t e n t i a l shown  conductance  i n HPC s o m a t i c  Hotson  P r i n c e 1980).  and  was  exhibiting  depolarizing  range  the  i n d i s p l a y i n g an  initial  to represent  membrane (Brown Although not  Type 2 d i s c h a r g e a p p e a r e d impalements  generation of intermediate  burst  of spike  a Ca+2-dependent K+ and G r i f f i t h  1983a;  absolute i n occurrence,  t o c o r r e l a t e with somatic or d e n d r i t i c anomalous  of  current  rectification injection  in  (cf  the  4.1C,D),  a  phenomenon r e l a t e d t o t h e a c t i v a t i o n o f a v o l t a g e - d e p e n d e n t Ca+2 conductance  (Benardo  et a l .  1982; Hotson  et a l .  1979).  Thus,  Type 1 s p i k e d i s c h a r g e .would a p p e a r t o be m e d i a t e d p r i m a r i l y Na+  channel  conductance,  while  a c t i v i t y c o n t r i b u t e d t o Type  both  Na+  and  t h e p r i n c i p l e charge  d i s c h a r g e . Of i n t e r e s t discharge  patterns  dendritic  level,  with  be  Type  recorded i n both somatic to  carrier  made 1  at  and d e n d r i t i c  s o m a t i c and  t h u s p o s s i b l e t h a t one f o r m 2) i s e x p r e s s e d a t given  indicate f o r m o f AP  dendritic  the  much  commonly  as  compared  i n t h e p a t t e r n o f evoked recording  sites,  o f s p i k e d i s c h a r g e (Type 1  both the  between  s o m a t i c and  more  impalements  of  s o m a t i c and  dendritic  i t is  o r Type  level of a  cell. Further  that  f o rthis  both  activity  t h a t o f Type 2. G i v e n t h e s i m i l a r i t y  a c t i v i t y between  further  i s the fact that the d i s t i n c t i o n  could  channel  2 d i s c h a r g e . The p r e p o n d e r a n c e  s l o w s p i k e g e n e r a t i o n i n Type 3 a c t i v i t y w o u l d Ca+2 i o n s a s  Ca+2  by  examination  potentials  channel  thought  conductance  impalements  of  were  the  of  current  to  indicate  most  pyramidal  evoked the  responses  reveals  a c t i v a t i o n o f Ca+2  commonly r e c o r d e d i n d e n d r i t i c cell.  For  instance,  the  - 123 depolarization  underlying  the burst  discharge  of greater  amplitude  was  Ca+2-dependent i n t e r m e d i a t e dendritic  recording  somatic  previous  locations.  membrane.  i n d e n d r i t i c membrane, a n d  These  r e s u l t s would suggest a  i o n i c channels along  Similar  investigations,  but  Ca+2-dependent i n t e r m e d i a t e dendritic  membrane  by  intermediate  and  d e n d r i t e , but these levels  of  dendrites  the  the pyramidal  Instead,  dendrite  1979).  t h a t slow  is  depolarizing  most  current  However,  routinely  separate  being  evoked  i n loss  early  investigations  of of  dendritic cells  of  exhibit  behaviour  are  known  i n response  to  of  over  study  to current  might studies  pyramidal  through  cell  injection  of  (Wong e t a l .  1.5nA  was  as such a c t i o n  the dendritic  b o t h C A l a n d CA3 p y r a m i d a l which  i n the  was  difference  previous  o f 2nA o r g r e a t e r  injection  level  investigators  factors. First,  attempted i n the present  result  and d e n d r i t i c  spike generation  current  often  i n o n l y 8/36  common f o r m o f a c t i o n  of previous  i n the order  study,  observed ata l l  detected  t h e most  commonly  current  present  s p i k e a c t i v a t i o n . The a p p a r e n t  be a t t r i b u t e d t o t h r e e have r e p o r t e d  evoked  be e v o k e d i n t h e a p i c a l  a t both t h e somatic  r e s u l t s and t h o s e  in  readily  depolarizing  were i n f r e q u e n t l y  tree,  drawn  that  e t a l . 1979). I n t h e  potentials  t h a t o f Na+-dependent between these  of  dendritic  reported  s l o w s p i k e s were  slow spike could  examined.  were  studies  injection  dendritic  potential discharge  conclusions these  or  ( B e n a r d o e t a l . 1 9 8 2 ; Wong the  i n Type 2  a x i s , w i t h a g r e a t e r d e n s i t y o f Ca+2 c h a n n e l s i n  than  in  spikes  o r s l o w s p i k e s were o n l y o b s e r v e d i n  non-uniform d i s t r i b u t i o n of cell  of fast  impalement.  not would  Secondly,  a c t i v i t y were p e r f o r m e d on  (Wong e t a l . 1 9 7 9 ) , t h e more  pronounced  injection  latter  burst-like  (Wong e t a l . 1 9 7 9 ;  - 124 Wong  and  Prince  dendritic  activity  guinea-pig, the  were  while the  finding that  stained  a l l previous  performed  on  results of this  CAl  personal  CAl pyramidal  Protein  pyramidal  (CaBP),  cells  pyramidal  by  or  absence  channel a c t i v i t y , guinea-pig  of  the  CaBP  t h e above  pyramidal  from  i s the  of the guinea-pig antibodies  are  specific for  as i s c h a r a c t e r i s t i c a l l y  of  may  on  c e l l s of the  to this point  rat  1982). Although t h e  be i n c o n s e q u e n t i a l  r e s u l t s do  found  ( D r . K.G. B a i m b r i d g e ,  c o m m u n i c a t i o n ; B a i m b r i d e and M i l l e r  presence  studies  work w e r e o b t a i n e d  cells  immunohistochemically  Calcium-Binding for  Finally,  r a t h i p p o c a m p u s . Of p o s s i b l e r e l e v a n c e  recent not  1978).  indicate that  neurons are not s t r i c t l y  t o Ca+2 r a t and  comparable i n  a l l  respects.  Stimulus  Evoked  The from  stimulus  that  giving  Spikes  evoked  cell  dendritic pyramidal  with a  recording cell  p r o f i l e analyses  all-or-none  short onset sites.  has  been  (Gessi e t a l .  response  oriens  spike arising  differed  injection i n  spike at a l l l e v e l s  Antidromic well  from  through  i n the laminar  p o t e n t i a l s i n both  the  a l . 1967)  (Chapter 3 ) , i n d i c a t i n g t h a t t h e a l v e a r  represents  or  field  directly  spike discharge  characterized  of the  b o t h s o m a t i c and  1966; Leung 1979a,b; S p e r t i e t  somata and a p i c a l d e n d r i t e s stratum  current  latency at  of extracellular  i n v i t r o preparation  evoked  depolarizing  cell  a x i s . The a l v e a r e v o k e d s p i k e was e v o k e d  from b a s e l i n e  and  through  rise to a single  pyramidal  in vivo  evoked response o f t h e pyramidal  radiatum  an  antidromic  of pyramidal also  t h e peak o f  spike  cells.  invasion of  Stimulation  evoked a s i n g l e  of  all-or-none  the synaptic depolarization  at  - 125  both  the  somatic  and  displayed similar  -  dendritic  level.  Orthodromic  evoked c h a r a c t e r i s t i c s  at a l l  dendro-somatic a x i s ,  including latency j i t t e r  AP  the  discharge,  and  response  to  spikes  l e v e l s of the  near t h r e s h o l d f o r  stimulus  intensity  or  r e p e t i t i v e s t i m u l a t i o n of the afferent input.  the  S o m a t i c and d e n d r i t i c s p i k e s d i f f e r e d  characteristically in  exact  with  form  exhibiting  a  of  the  evoked  l a r g e r amplitude  spike,  and more  somatic  spikes  narrow h a l f w i d t h  than  d e n d r i t i c s p i k e s . However, a t a n y g i v e n  l e v e l of the c e l l  both  were evoked w i t h  similar  spikes  evoked  through  radiatum  were t h u s  alvear  amplitude  and  and  stimulation  halfwidth.  of  comparable  orthodromic  either  in  spikes Dendritic  stratum  waveform  to  oriens  that  of  or a  spike  antidromically  i n v a d i n g t h e d e n d r i t i c a r b o r i z a t i o n . AP d i s c h a r g e dendritic  recording  locations  voltage threshold f o r spike orthodromic  spikes  were  t h r e s h o l d , as judged by of s p i k e d i s c h a r g e . conspicuous  differed  activation. evoked  at  the absolute  i n somatic  and  i n terms o f t h e  In the somatic  region,  a  voltage  consistent  voltage of  the breakpoint  I n c o n t r a s t , d e n d r i t i c membrane d i s p l a y e d no  voltage  orthodromically  also  axis,  threshold  evoked  spikes  f o r spike arising  generation,  at  various  with  levels of  d e p o l a r i z a t i o n f r o m t h e u n d e r l y i n g EPSP. A s s u m i n g t h a t d e n d r i t i c spikes a r i s e through  a c t i v a t i o n of voltage-dependent  t h e above r e s u l t s would s u g g e s t t h a t t h e g e n e r a t o r dendritic dendritic  spike  is  located  distant  to  s i t e f o r the  p o s i t i o n of the  r e c o r d i n g e l e c t r o d e . This s i t e might correspond  o r more l o c a t i o n s w i t h i n t h e d e n d r i t i c t r e e the  the  channels,  somatic  intracellular  region  of  the  characteristics  pyramidal of  ("hot s p o t s " ) cell.  orthodromic  However, spike  t o one or  to the  discharge  - 126  -  would be c o n s i s t e n t with r e s u l t s obtained density  analysis  p o t e n t i a l s and  (Chapter  3).  current-source  through  Laminar  density  current-source  profiles  of  revealed t h a t  field  SO of  s t i m u l a t i o n evoked a s i n g l e s p i k e response at a l l l e v e l s of pyramidal  cell  a x i s , with  the s p i k e  conducting  from  SR the  the  cell  l a y e r through the d e n d r i t i c f i e l d . The  i n t r a c e l l u l a r s p i k e would  thus be  evoked c h a r a c t e r i s t i c s at  the  expected to  somatic  and  display similar  dendritic  level  (ie  i n t e n s i t y or r e p e t i t i v e s t i m u l a t i o n ) ,  response  and  to  stimulus  to the a l v e a r  evoked  spike a n t i d r o m i c a l l y invading the d e n d r i t i c a r b o r i z a t i o n . the i n t r a c e l l u l a r p r o p e r t i e s of s p i k e d i s c h a r g e accord with  the data  obtained  from  CSD  Thus,  are at l e a s t  analysis  in  indicating a  somatic s i t e of o r i g i n f o r the evoked d e n d r i t i c s p i k e .  Stimulus  Vs. Current  A comparison revealed  a  Evoked  of the  basic  Spikes stimulus  similarity  and  c u r r e n t evoked response  between  the  c h a r a c t e r i s t i c s of  c u r r e n t evoked Na+-dependent s p i k e s and  a c t i o n p o t e n t i a l s evoked  through  efferent  stimulation  instance, activity  of  depolarizing i n both somatic  afferent current  or evoked  similar  pathways.  For  patterns  of  and d e n d r i t i c l o c a t i o n s , with  s p i k e s e x h i b i t i n g a g r e a t e r amplitude and more narrow than  those  in  dendritic  c u r r e n t evoked d e n d r i t i c  recording  sites.  spike d i s p l a y e d no  t h r e s h o l d f o r a c t i v a t i o n , while  In  somatic halfwidth  a d d i t i o n , the  consistent voltage  spike discharge  i n the  somatic  region arose from a s i m i l a r membrane p o t e n t i a l f o r a l l l e v e l s of current i n j e c t i o n . discharge to  those  In f a c t ,  revealed that evoked through  a d i r e c t comparison of  stimulus evoked s p i k e s curent  injection  fast  spike  were comparable  i n terms  of amplitude  - 127 and  halfwidth  when  evoked  -  upon  a  s i m i l a r l e v e l o f membrane  depolarization. The  major d i s t i n c t i o n  spike generation stimulus  giving  current  r e s u l t s can  be  a  rise  to  repetitive  explained  a  r e c u r r e n t a c t i v a t i o n o f an  pyramidal  cells  discharge In the  capable  a c t i v a t i o n can the somatic Wong and  spike  discharge.  and  s i t e of o r i g i n  and  However, of in  inhibitory synaptic potential  in  suppressing Dingledine  appropriate  dendritic level  with  i s known t o r e s u l t  of  P r i n c e 1979) . The  of s t i m u l u s  discharge,  Cl-  repetitive and  channel  1980).  blockers,  (Schwartzkroin  similarity  spike  Langmoen  stimulus  also lead to r e p e t i t i v e spike generation  and  evoked  by t h e f a c t t h a t s t i m u l a t i o n  ( A n d e r s e n e t a l . 1969;  presence of  current  single  spike  a f f e r e n t or e f f e r e n t pathways i n the s l i c e the  and  f o u n d i n t h e p a t t e r n o f AP  activation  depolarizing these  was  between s t i m u l u s  and  i n evoked  at  both  Prince  1980;  characteristics  c u r r e n t evoked s p i k e s might then imply  a  common  f o r a l l evoked f a s t s p i k e s of the p y r a m i d a l  cell.  Spike P r e - P o t e n t i a l s The and  evoked  c h a r a c t e r i s t i c s of  antidromic  initial  segment  of  pyramidal  somatic  region  previous  investigators  1961;  MacVicar  the  and  Dudek  and  present  extends these  of  FPPs  Previous and  and  "IS"  (IS)  ( A n d e r s e n and  Schwartzkroin study  fast pre-potentials  1981;  P r i n c e 1980;  spikes  spikes  cell  recorded  i n the  have been d e s c r i b e d  Lomo 1966; Schwartzkroin  S p e n c e r and  dendritic  1975,1977;  Kandel 1961b).  The  activation  recording locations.  i n v e s t i g a t i o n s h a v e drawn a d i s t i n c t i o n b e t w e e n t h e  I S s p i k e b a s e d upon i n i t i a l  by  Kandel et a l .  f i n d i n g s i n r e p o r t i n g the in  (FPPs)  r e p o r t s t h a t the  I S s p i k e was  FPP of  -  greater  amplitude  than  similar  i n waveform, w i t h  "FPP"  the  FPP  study,  given  location.In  has been  potentials  and  1981b;  p o t e n t i a l s may  this  and  on  a  Kandel  in  1961  and P r i n c e  (Knowles  1 9 8 0 ; T a y l o r and  increasing  t o note  between  of the FPP  i n amplitude  membrane at  during  p r e - p o t e n t i a l s " evoked  between t h e s e  a l l l e v e l s o f membrane  at different  Therefore,  given the  of the FPP  potentials  levels of  similarity in  and I S s p i k e , t h e  i s probably  unwarranted,  be r e f e r r e d t o a s f a s t p r e - p o t e n t i a l s .  spike following a  Spencer and evidence  in  in  antidromic  i n pyramidal  cell  somata  b e e n p r o p o s e d t o r e p r e s e n t an e l e c t r o t o n i c a l l y d e c a y e d  the  these  that IS s p i k e s a r e u s u a l l y  FPPs are observed  Fast p r e - p o t e n t i a l s recorded  within  and  h y p e r p o l a r i z a t i o n (Schwartzkroin 1977). I n  and e v o k e d c h a r a c t e r i s t i c s  both w i l l  d e s c r i p t i o n o f these  I t i s p o s s i b l e t h e r e f o r e t h a t t h e I S s p i k e and F P P  "fast  distinction and  the f i r s t  potential,  r e s t i n g membrane p o t e n t i a l . shape  antidromic a c t i v a t i o n of the  hyperpolarized  stimulation, while  t o be v e r y  i n amplitude at  d i f f e r e n c e i n amplitude  regard, i t i s important  represent  were found  r e l a t e t o the f a c t that the amplitude  t o membrane  polarization.  inability to  1 9 6 1 b ) . However, i n t h e  spike  Schwartzkroin  w i t h membrane  uncovered  IS  addition,  Spencer  an  no g r e a t d i f f e r e n c e  Dudek 1 9 8 2 ) . The r e p o r t e d  response  upon  p r e - p o t e n t i a l (Kandel e t a l . 1961;  reported since  by  Schwartzkroin  can v a r y  and  1 9 7 7 ; S p e n c e r and K a n d e l  present  any  -  FPP,  the  a n t i d r o m i c a l l y evoke a f a s t Schwartzkroin  128  failure  dendritic Kandel  of active  propagation  arborization  (Andersen  1 9 6 1 b ) . One w o u l d  dendritic  recordings  waveforms i n t e r m e d i a t e i n a m p l i t u d e  dendritic  a t some and  fast  point  Lomo 1 9 6 6 ;  t h e r e f o r e expect of  have  to find  pre-potential-like  between t h e s o m a t i c  FPP  and  - 129  -  the much l a r g e r d e n d r i t i c s p i k e . However, somatic and FPPs  fell  within  activated  a  through  therefore that a  similar  antidromic  evoked  of amplitude  stimulation.  fast pre-potential in  not represent the p a s s i v e an  range  dendritic  It  dendritic  and c o u l d be  would  the somatic  appear  region  may  conduction and e l e c t r o t o n i c decay  spike.  Further  evidence t o support  c o n c l u s i o n w i l l be presented i n Chapter  this  6.  Previous s t u d i e s have p r o v i d e d evidence t h a t at l e a s t f a s t p r e - p o t e n t i a l s represent  some  the e l e c t r o t o n i c conduction  of a  s p i k e across a gap j u n c t i o n between neighboring pyramidal (Andrew et a l . 1982; presence of  MacVicar  gap j u n c t i o n s  and Dudek 1981). Support  between pyramidal  from f r e e z e - f r a c t u r e s t u d i e s (Schmalbruch the  ability  to  attain  dye-coupling  neurons f o l l o w i n g i n t r a c e l l u l a r pyramidal c e l l somata or and Dudek  1981). The p o i n t  between  pyramidal  cells  somatic or  dendritic level  Dudek 1981;  Schmalbruch and Jahnsen  p a i r of second  to  occur  electrotonic  neurons.  Such  gap a  contact  at e i t h e r  the  MacVivar  and  a l . 1982;  1981), and dual  neurons i s  impalements  reflected  (MacVicar and Dudek  i n somata  junctions  mechanism  into  a l . 1982;  and a p i c a l  would  between help  the  1981). Fast  d e n d r i t e s of CAl  adjacent to  of a  in  pyramidal c e l l s might thus be adequately e x p l a i n e d on the of  and  t h a t s p i k e d i s c h a r g e i n one  pre-potential  p r e - p o t e n t i a l s recorded  come  1981)  of gap j u n c t i o n a l  (Andrew et  e l e c t r o t o n i c a l l y coupled as a f a s t  and Jahnsen  the  of two or more pyramidal  thought  of pyramidal c e l l s have shown  for  neurons has  a p i c a l d e n d r i t e s (Andrew et  is  cells  i n j e c t i o n of L u c i f e r Yellow  MacVicar  of  basis  pyramidal  account  for  the  s i m i l a r i t y i n the evoked c h a r a c t e r i s t i c s and waveform of somatic and  dendritic  FPPs,  and  antidromic  activation  of  the f a s t  - 130 -  p r e - p o t e n t i a l at both the somatic and d e n d r i t i c l e v e l . the c h a r a c t e r i z a t i o n of FPP only at a p r e l i m i n a r y required  to  pre-potentials  However,  d i s c h a r g e i n d e n d r i t i c membrane  is  stage, and s u b s t a n t i a l l y more work w i l l be  definitively i n the pyramidal  locate cell.  the  origin(s)  of  fast  - 131 5-0. THE  5-1.  -  EVOKED CHARACTERISTICS OF ACTION POTENTIAL DISCHARGE ALONG DENDRO-SOMATIC AXIS OF THE  CAl PYRAMIDAL NEURON  Introduction  Laminar p r o f i l e a n a l y s i s of e x t r a c e l l u l a r f i e l d in the CAl  region have i n d i c a t e d  a retrograde conduction  spike response from  the c e l l body  region,  a  suggesting  somatic  p r o p e r t i e s of s p i k e been  discharge  characterized  pyramidal  cell  and  of  i n the  pyramidal  intracellular apical  the p r o p e r t i e s  the somatic and  of i n t r a c e l l u l a r  dendritic  3 ) . The cell  basic  have  also  recordings  from  dendrites  (Chapter  p r o v i d i n g the b a s i s f o r a comparison of i n t r a - and spike a c t i v i t y at  of a  o r i g i n f o r the evoked  neurons (Chapter  through  somata  l a y e r through the  site  d e n d r i t i c s p i k e i n CAl pyramidal  potentials  4),  extracellular  d e n d r i t i c l e v e l . Several spike d i s c h a r g e  of  c o i n c i d e with  the c h a r a c t e r i s t i c s of e x t r a c e l l u l a r p o p u l a t i o n s p i k e  responses  recorded  instance,  in  stratum  stimulation single  of  pyramidale  afferent  evoked  spike  or  in  and  radiatum.  efferent  somatic  and  For  pathways gave r i s e to a dendritic  impalements,  s i m i l a r to t h a t observed f o r e x t r a c e l l u l a r f i e l d p o t e n t i a l s . a d d i t i o n , the exact waveform of the i n t r a c e l l u l a r spike  differed  between somatic and d e n d r i t i c l o c a t i o n s , with the somatic e x h i b i t i n g a g r e a t e r amplitude i n the d e n d r i t e . response i n negativity  amplitude and voltage  pyramidale  that  in  biphasic in  threshold  of  c o n s i s t e d of  stratum  radiatum  population  that spike  a l a r g e amplitude was  waveform. Furthermore,  orthodromic  spike  s h o r t e r h a l f w i d t h than  S i m i l a r l y , the e x t r a c e l l u l a r  stratum while  and  In  of  smaller  the lack  of  i n t r a d e n d r i t i c s p i k e s would  - 132 indicate  that  t h e s p i k e had o r i g i n a t e d  recording electrode, However,  laminar  indicate  that  of the  properties be  s u g g e s t e d by f i e l d  profiles  of  remote from t h e  potential  extracellular  field  in a  p r o g r e s s i v e manner  potentials  pyramidal c e l l  population.  of spike activation  spike  through the d e n d r i t i c More i n s i g h t  i n the pyramidal c e l l  into  the  might  thus  g a i n e d by comparing v a r i o u s p a r a m e t e r s o f i n t r a c e l l u l a r  spike  discharge a t d i f f e r e n t s i t e s along the dendro-somatic S u c h an a n a l y s i s prevailing dendrite  of  spots of  would a l s o  provide  the  pyramidal  of  membrane,  the  located  tree  initiate  in dendrites physically cut  across  1982;  dendritic the  the proximal-mid  Masukawa  dendritic  and  Prince  the c e l l  stratum radiatum 1984).  of that  At  propagation subsequent  amplitude of  the  passive  of  which  spike  electrotonic  the  activation knife  (Benardo e t a l .  the somatic l e v e l , the  suggests  within  a  pre-potential,  f a i l u r e of active  dendritic  conduction  to  tree  and a  the c e l l  region  ( A n d e r s e n and Lomo 1 9 6 6 ; S p e n c e r and K a n d e l 1 9 6 1 b ) .  model  of  spike  characteristics passive  activation of  electrotonus,  the  would  predict  intradendritic  including  a  can  i n the  body b y a  s p i k e i s observed i n t h e form o f a f a s t  small  o r more  membrane  recordings of spike  i s o l a t e d from  have  spike t o hot  a t one  spike discharge independently  soma h a s come f r o m i n t r a c e l l u l a r  apical  ( A n d e r s e n and Lomo 1 9 6 6 ;  S p e n c e r and K a n d e l 1 9 6 1 b ) . E v i d e n c e t h a t fact  i n the  a dendritic  possibly  dendritic  of the  Previous investigations  s i t e f o r generation of  dendritic  branchpoints  cell.  axis.  a direct test  hypothesis concerning spike generation  assigned the  in  analysis.  t h e c h a r a c t e r i s t i c s o f t h e evoked p o p u l a t i o n  r e s p o n s e change field  as  at a s i t e  a  spike  decline  change  This  i n the  indicative in  body  of  a m p l i t u d e and  133  -  increase cell  i n both width  body  layer.  properties  of  laminar  According  onset l a t e n c y with p r o x i m i t y  However,  the  those observed f o r on  and  this  dendritic  pattern  spike  of  change  profiles  of  field  potentials  spike  distance  intracellular might  somatic or  from  spike then  pyramidale.  c h a r a c t e r i s t i c s along  dendritic site  a  in  the  response  gradual  i n onset l a t e n c y the greater  stratum  provide  the  i n the CAl region.  t o these r e s u l t s , t h e evoked s p i k e d i s p l a y s a  recording  to  are i n d i r e c t contrast to  the e x t r a c e l l u l a r population  c h a n g e i n w a v e f o r m and i n c r e a s e  axis  -  An  the  a n a l y s i s of  the dendro-somatic  means o f d i s t i n g u i s h i n g b e t w e e n a  of o r i g i n  f o r the  evoked d e n d r i t i c  spike. The p r e s e n t of  the  study  properties  dendro-somatic a x i s amplitude, view  of  halfwidth, the  fact  indicate  a  somatic  attempts  were  also  therefore provides  of  action  of the  that  potential  pyramidal  voltage  a comparative a n a l y s i s  cell,  threshold  of  origin  made  in  the  population.  the  latency. In  d e n s i t y a n a l y s i s would  f o r the d e n d r i t i c spike,  present  study  to record the  evoked a c t i v i t y o f i s o l a t e d d e n d r i t i c elements o f t h e cell  along  i n c l u d i n g those of  and o n s e t  current-source  site  discharge  pyramidal  - 134 5-2.  -  Methods  D a t a was  pooled  dendro-somatic a x i s halfwidth,  and  from a l l i n t r a c e l l u l a r r e c o r d i n g s  along  of pyramidal  amplitude,  voltage  n e u r o n s and  threshold  measured  methods d e s c r i b e d p r e v i o u s l y ( C h a p t e r of  single  pyramidal  measurements pyramidal  of  cell  neurons  spike  c o u l d not  spike  obtained  activity  experiments, pyramidale high  low  dendritic  impedance  to record the  impedance  each  of  intradendritic  three  spike  simultaneously  stratum  pyramidale.  withdrawn from the dendritic display  of  Extracellular  the c e l l  region of the same s t i m u l u s extradendritic  as  these stratum  and  the  evoked  of the  by  waveforms  digital  To  spike was  in then the  micrometer PZ-550).  body and  in  the  then c o l l e c t e d  at  the  a v o i d any  over time,  response  Inchworm  a l a t e n c y comparison of  spike potentials.  intensity  to the depth of  at the c e l l  impalement were  potential  electrode  (Burleigh  Upon  threshold for  population  a  a  apical  layer.  to  returned  depth  intensity for  the  set  monitored  electrode  the e x t r a c e l l u l a r  in  was  intradendritic  i m p a l e m e n t and  dendritic  For  impalement, s t i m u l u s  with that  potential  intracellular  placed  that i n  pathways  The  recording,  dendritic  potentials.  within  generation,  recorded  the  estimate  and  electrode  d i r e c t l y below  the  of  e x t r a c e l l u l a r p o p u l a t i o n s p i k e , and  obtaining a stable intradendritic for  field  absolute  regions  at the somatic  the  impalements  indirect  e l e c t r o d e was  intracellular  region  different  be made. However, an  extracellular  to  obtained,  through a comparison of evoked  and  a  not  at  of the l a t e n c y f o r s p i k e discharge l e v e l was  according  4). Since dual  were  latency  spike  the  intra-  and  p o s s i b l e change i n  threshold  intradendritic  - 135 responses the  were u s u a l l y c o l l e c t e d  recording  site.  p a t h w a y had b e e n intensity  was  population  However,  i f  set  spike  to  at  evoke the  an a t t e m p t  the  cell  from  the stimulus i n t e n s i t y of a  changed b e f o r e e l e c t r o d e  c o l l e c t i o n of i n t r a d e n d r i t i c In  just p r i o r to withdrawal  same  layer  withdrawal, stimulus amplitude  and l a t e n c y  as t h a t r e c o r d e d  during  potentials.  to assess d e n d r i t i c a c t i v i t y  independently of  t h a t i n t h e soma, two d i s s e c t i o n t e c h n i q u e s were u s e d t o p l a c e a knife cut i n the to  separate the  neurons.  proximal-mid cell  bodies  apical dendritic  and  region  apical dendrites of  i n order pyramidal  The f i r s t method e m p l o y e d a " m i c r o - k n i f e " c o n s i s t i n g o f  a s m a l l r a z o r b l a d e c h i p g l u e d t o a t h i n wooden d o w e l mounted on a  micro-manipulator.  transferral  Slices  cut  immediately  following  t o t h e r e c o r d i n g chamber u n d e r o b s e r v a t i o n w i t h a 4X  d i s s e c t i n g microscope. slice  were  along  the  The b l a d e was l o w e r e d  intended  line  of  cut  slowly through both  within  stratum  r a d i a t u m and d i r e c t l y  a c r o s s t o t h e a l v e a r t i s s u e on e i t h e r  of  r e g i o n . S m a l l l a t e r a l movements  the cut d e n d r i t i c  axis of the blade  w e r e o f t e n made a t  obtain a  separation  complete  tissue.  In  In  the  immediately placed  second  individually  pre-cooled  plastic  "anchored"  with  microscope  to  Immediately  three  sectioning  i n drops petri  before cutting  to on  f o u r s l i c e s were the  tissue  of c o l d oxygenated dish.  masking tape prevent  body  of the s l i c e .  method,  following  cut to  some c a s e s ,  c u t s were e x t e n s i v e enough t o a l l o w s e p a r a t i o n o f t h e c e l l r e g i o n from t h e remainder  side  along the  the bottom of t h e  of s l i c e  the  The  to the  movement a slice,  petri stage of during  taken  c h o p p e r and  medium w i t h i n dish a 3X  was  a  then  dissectng  microdissection.  the corner of a torn  piece  - 136 of t i s s u e paper  was u s e d t o  drop c o n t a i n i n g the s l i c e , surface of the p e t r i  draw away most o f t h e f l u i d allowing  t h e s l i c e t o r e s t upon  d i s h . The t i p o f  the intended  held at approximately the  progress  compressive "rolled"  of  l i n e of  45 d e g r e e s  the  cut  damage t o t h e  and  immediately  placed  from  on  repeated f o r the next s l i c e . the t i s s u e , only three t o  vertical  so as  r e g i o n . The b l a d e  of f r e s h l y oxygenated the  cut  slice  t o watch  was  cold  in  medium  and t h e p r o c e d u r e  f o u r s l i c e s were c u t p e r  manner  o f 3-5  min) was  not found  h e a l t h , as c o n t r o l s l i c e s m a i n t a i n e d were  then  I n o r d e r t o a v o i d h y p o x i c damage t o  The t i m e r e q u i r e d f o r c u t t i n g  conditions  was  to minimize the p o s s i b i l i t y of  was c o m p l e t e d .  same  blade  blade  dissection,  q u i c k l y t r a n s f e r r i n g them t o t h e r e c o r d i n g chamber o n c e  (total  was  a c r o s s and t h r o u g h t h e s l i c e  one s m o o t h m o t i o n . A few d r o p s was  and t h e  d i s s e c t i o n . The  dendritic  a l o n g t h e c u r v e d edge  the  a #10 s c a l p e l b l a d e  p l a c e d on t h e p e t r i d i s h a t t h e edge o f t h e s l i c e aligned along  i n the  found  to  a c t i v i t y comparable t o s l i c e s  slices  cutting  i n t h e above  t o compromise  slice  i n t h e p e t r i d i s h under t h e  exhibit  electrophysiological  removed d i r e c t l y  fron the  tissue  chopper t o t h e r e c o r d i n g chamber. Slices r e c o v e r y and the  were  was  a  equilibration to  concentration  medium  allowed  of  elevated  period  1.6mM  recovery of cut t i s s u e elements,  a t l e a s t 45 m i n f o r  bath conditions.  extracellular from  of  to  calcium 2.5-3mM  as o u t l i n e d  I n some c a s e s , i n the perfusing to  enhance t h e  i n the procedure  of  Benardo e t a l . (1982). The e v o k e d a c t i v i t y  in  the CAl region of  was e x a m i n e d p r i m a r i l y by e x t r a c e l l u l a r response  dissected  slices  recording techniques  t o s t i m u l a t i o n o f s t r a t u m r a d i a t u m i n p u t s . The  in  maximal  - 137 s o m a t i c and/or d e n d r i t i c response stimulus  i n t e n s i t y ) was r e c o r d e d  extending CA2 t o  to either side of  the subicular  pyramidale  was  dendritic  CA2-subicular  axis  somatic recording the  slice,  t h e c u t from t h e l a t e r a l  attached  was  the cut  to  recorded  (dendritic  site).  the  at  border o f  region of  slice,  and  along  the  a l lpoints  electrode  below  the  I f t h e e x c i s e d p o r t i o n was removed  from  cut region, while  through  the  maximal  stimulation  stimulating  entire  extent of  electrode  electrode t o maintain  directly  on e i t h e r  d e n d r i t i c a c t i v i t y was of  stratum  was  the  c u t . In order  recorded t o achieve  radiatum a f f e r e n t inputs, the  usually  a constant  distance of approximately  moved  with  the recording  stimulating-recording  400pm. I n o t h e r  cases,  electrode  the stimulating  e l e c t r o d e p o s i t i o n i n t h e CA2 r e g i o n was k e p t c o n s t a n t recording The site,  p o s i t i o n o f t h e k n i f e c u t , l o c a t i o n o f each  and t h e maximal evoked s o m a t i c  histological  Slices  were  first  5 hours flat  before  sectioning.  surface of  (Tissue-Tek)  (Tissue-tek;  in a  C a + 2 - a c e t a t e s o l u t i o n f o r a t l e a s t 24  on t h e  Compound  session f o r  placed  t r a n s f e r r e d t o a 20% s u c r o s e - p h o s p h a t e b u f f e r e d  placed  at  l o c a t i o n were n o t e d i n a d e t a i l e d d i a g r a m o f t h e  analysis.  f o r m a l i n / 1%  recording  or d e n d r i t i c response  and t i s s u e p r e p a r e d a t t h e end o f a r e c o r d i n g  (pH 7.4)  while the  e l e c t r o d e was moved.  each r e c o r d i n g slice,  stratum  somatic  s o m a t i c and d e n d r i t i c a c t i v i t y was r e c o r d e d  side of the  t o 60V  at a sequential series of sites  r e g i o n . When  still  activity  t o SR s t i m u l a t i o n (up  on  a pre-cut  frozen  cutting  stage  the  12-14 d e g r e e s c e n t i g r a d e )  c u t a n d mounted on g l a s s  slides.  h o u r s and  saline  S l i c e s were  10%  solution  individually d r o p o f O.C.T.  of  a  a n d 22pm s e r i a l  S l i c e t i s s u e was t h e n  cryostat sections stained  -  with  cresyl  extent of relation  violet the k n i f e  of neuronal  and  138  -  microscopically  cut at  a l l levels  examined of the  elements to p r e v i o u s l y  to assess slice  recorded  and  the the  activity.  - 139 5-3.  Results  S p i k e A m p l i t u d e and  Halfwidth  Representative potentials  examples  recorded at the  evoked s p i k e s  were  amplitude  and  region,  level  in  the  s t i m u l a t i o n was decreasing  the  and  greatest  of  in  evoked  dendro-somatic axis values  are  recording  the  to the  taken  schematic  diagram  values  cell  as of  the  a  rat  In  site  both antidromic progressive body l a y e r  are and  decline  EPSP  apical  calculate  5.2  input  SR  distance  and  a l l the the  average of  stratum pyramidale Figs  and  dendrite,  case, the  anatomical  was  ( F i g 5.1C).  used to  In each  the  e v o k e d by  were c o l l e c t e d a t  the  (somatic  5.4  a  the  scaled average  r e s i s t a n c e at  each  These c a l c u l a t i o n s r e v e a l  that  orthodromic i n t r a c e l l u l a r i n amplitude  from  Conversely,  p y r a m i d a l n e u r o n and  also provided.  in  c h a r a c t e r i s t i c s along  o f r e s t i n g membrane p o t e n t i a l o r  recording  decline  amplitude  5.IB).  of the  and  border of  "0"um).  the  i n F i g 5.1  In  e v o k e d EPSP  body l a y e r  potential  against  from the  recordings  SO  and  body.  distance  (Fig  region  ( F i g s 5.2-5.4).  plotted  site  The  of  apical dendritic tree  value  A l l stimulus  a gradual  with  5.1).  rise  D a t a s u c h as t h a t shown  average  displayed  (Fig  the  the maximal a m p l i t u d e  halfwidth  of  with proximity  l e v e l s of the  i n F i g 5.1.  apical dendrite  rate  orthodromic  l o c a t i o n s along  somatic region, d e c l i n i n g i n  along  amplitude  and  of the pyramidal c e l l  spikes  increase  a l s o l a r g e s t i n the rise  various  found to e x h i b i t  border of stratum pyramidale  rate of  antidromic  illustrated  s h o r t e s t h a l f w i d t h at the dendritic  of  soma and  a p i c a l d e n d r i t i c t r e e are  the  -  spikes  with distance  ( F i g 5 . 2 A - C ) . However, t h e  display a  from the  cell  change i n a c t i o n p o t e n t i a l  - 140 FIG  5.1  O s c i l l o s c o p e t r a c e s of t y p i c a l  potential discharge dendrites  65,  pyramidale. alveus  recorded  165  Spike  and  change i n s p i k e  spike activation.  along  cells,  the c e l l  from  was  s t i m u l u s evoked a c t i o n cell  the  border  e v o k e d by  (B) o r s t r a t u m  amplitude  and  s o m a t a and  for stimulus Somatic  while  of  radiatum  stratum  halfwidth with  intensities  recordings  the  ( C ) . Note  the  recording  several near  r e c o r d i n g s were t a k e n  dendritic  apical  s t i m u l a t i o n of  body l a y e r . I n e a c h c a s e ,  a r e shown s u p e r i m p o s e d  separate  265pm  oriens  d i s t a n c e from the c e l l  for  i n pyramidal  discharge  (A), stratum  gradual  -  sweeps  threshold  from  three  at each l o c a t i o n  a x i s are from a s i n g l e d e n d r i t i c impalement. Note  t h e common v o l t a g e and  time base c a l i b r a t i o n  for a l l recordings.  Stim. Site A.  Recording Site (p)  - 142 FIG  5.2  Plots  spikes recorded  of the  +/-  sem;  from the  measured  recordings  are  average amplitude  i n pyramidal c e l l  varying distances  from  taken  radiatum  D.  fissure  potentials  (RMP)  Ojnm  dark  e a c h p o i n t a r e shown a t were c a l c u l a t e d  a p i c a l dendrites at (mean Somatic  d i s t a n c e . S p i k e s were e v o k e d o r i e n s (B)  or  a l v e u s and  Average  standard error l i e s  w i t h i n the border  are those  (n)  Average  from the l a r g e s t evoked s p i k e at each standard error bars  membrane  impalements i n p l o t s  number o f i m p a l e m e n t s  and v a l u e s w i t h o u t  cell  hippocampal  resting  the base o f the graphs.  in  by  stratum  of the r a t pyramidal  of the  lines.  The  evoked  stratum pyramidale  f o r the pyramidal c e l l  a r e shown i n l o w e r p a r t o f D.  of s t i m u l u s  membrane p o t e n t i a l ) .  diagram  the border  by  of  (A), stratum  Schematic  scale with denoted  border  as  the alveus  drawn t o  s o m a t a and  resting  s t i m u l a t i o n of (C).  -  A-C for  values  location, which  the  of the i l l u s t r a t e d p o i n t .  A. Stim. Site  B. Alveus  * I ^80H f70H  C.  Stratum Oriens  i i  < 60  Stratum Radiatum  i  CD  £ 50 H CL  if)  4 0 nH  9 2 4  0  3 10 10  100  4  200  300  0  3  7 10 12 2  100  200  I -  1  1  300  9  2 4  I  '  0  D.  cr  8 r  2 4  E  ffi o  ° gj  o  4  6  12 3  I  9  0 100 200 300 Apical Dendritic Recording Site (/J)  5  100  23 6 4  200  300  - 144 amplitude  along  manner. A  sharp i n i t i a l  w i t h i n lOOum and  a  the  dendritic drop i n  from stratum  second  rapid  150-200um f r o m variability  the  occurred  in  followed  distal  l a y e r . These  quality  of  i n a non-linear  spike amplitude  pyramidale,  decline  the c e l l  in  tree  was o b s e r v e d by a " p l a t e a u "  d e n d r i t i c impalements  results are  n o t due  d e n d r i t i c impalements, as t h e  average value  of resting  location  w i t h i n t h e r a n g e o f 62-68mV ( F i g 5.2D). R a t h e r ,  fell  comparison  of  pyramidal  spike  cell  configuration branchpoints  suggests correlate  dendrite  distal  values  transition  anatomical  in  The  of the three  i n spike  increase  dendritic dendritic  stimulation  and o r t h o d r o m i c  and  a  l o c a t i o n s . Since  from t h e  halfwidth i s  i n spike width  region  variability  along  p l o t o f t h e average  change i n s p i k e  ( F i g 5.3A)  spikes  through the  more p r o n o u n c e d the majority of  action potentials  in Fig  explained  i n r e s t i n g membrane p o t e n t i a l  of  impalements. characteristics  of  action  injection of depolarizing current recording  of the  spike width with distance  t h e o b s e r v e d c h a n g e i n s p i k e h a l f w i d t h c a n n o t be  cellular  a  l o c a t i o n o f major  evoked from each  were o b t a i n e d  on t h e b a s i s o f a  site  Representative 5.4A)  of  diagram  ina  apical  in  scaled  i s also evident  5.3B,C), w i t h a g r a d u a l  increase  5.2,  the  f o r both antidromic  proximal-mid  these  to  a  points  ( F i g 5 . 3 ) . Once a g a i n , t h e  similar (Fig  that  increase  halfwidth of spikes sites  to  recording  of the apical d e n d r i t i c t r e e .  A progressive the a p i c a l  membrane p o t e n t i a l f o r e a c h  amplitude  to  along  the  demonstrate  cell  axis  t h e c u r r e n t evoked  that the fast  evoked  also varied according  pyramidal  photographs of  potentials  by  to the  ( F i g 5.4). response ( F i g  Na+ s p i k e was e v o k e d w i t h t h e  - 145 FIG.  5.3  amplitude)  Plots  of  the  average  halfwidth  o f s t i m u l u s evoked s p i k e s recorded  (width  at half  i n pyramidal  cell  s o m a t a and a p i c a l d e n d r i t e s a t v a r y i n g d i s t a n c e s f r o m t h e b o r d e r of stratum from  pyramidale  r e s t i n g membrane  (mean  +/-  potential).  sem;  h a l f amplitude  Somatic  recordings  measured are taken  as Oum d i s t a n c e . S p i k e s were e v o k e d b y s t i m u l a t i o n o f t h e a l v e u s ( A ) , s t r a t u m o r i e n s (B) o r impalements  stratum radiatum  ( C ) . The number  (n) f o r e a c h p o i n t a r e shown a t t h e b a s e o f  of  graphs.  Stim. Site  Alveus A.  CD  Stratum Radiatum  Stratum Oriens B.  C.  TD Z5  E < o _C  1.5  H  5J'  u CD CO  ° E 1.0  •  C  I  4  "O  CD  CO  J  5n  J  7  I  0  5  3  10  100  8  3  200  I  4  300  0  1 4  4  100  7  10  12  200  3  I  4  12 2 0 1  300  0  Apical Dendritic Recording Site ( p )  100  200  5  3 ,  1  300  - 147 FIG.  5.4  action  A.  Oscilloscope  p o t e n t i a l discharge  apical  dendrites  pyramidale.  B.  100 Plots  -  t r a c e s of  recorded  and  i n pyramidal  165um  of  the  typical  from  the  average  current cell  somata  border of  amplitude  (B)  (width at h a l f amplitude) of the c u r r e n t evoked  recorded  in  varying distances +/-  sem).  Somatic  cell  from the  somata border of  recordings  are  and  apical  stratum taken  as  pyramidale  The  c u r r e n t e v o k e d s p i k e i n e i t h e r Type 1 o r Type 2  number o f  impalements  base of each graph.  (n)  for  each p o i n t are  spike at  (mean  distance.  A m p l i t u d e s were m e a s u r e d f r o m r e s t i n g membrane p o t e n t i a l first  and  dendrites  Oum  and  stratum  halfwidth  pyramidal  evoked  for  the  discharge.  shown a t  the  Spike Amplitude (mV) =3 I  (ji  CD  ->J  o  o  o  i_  CO  (X)  o  o  J  ^  >  I  I  o-, = CO o  ro  3  o o  Q  ro  o'  cn ro  o o  CD  O  >  CD CL  9. o o  o  1  O  S  CD  O  CL  O  Spike Width at Half Amplitude (msec)  JO CD  O O  b ^• • • i  13 iQ  CO  O n  o ro b  CJI i  i  i  i  i  i  i  i _  o CD CoL  tf>  CD  OS  ro  ro o o  ro  -•—i  OJ  o -  8frT  -  - 149 greatest  a m p l i t u d e and s h o r t e s t h a l f w i d t h i n t h e s o m a t i c  of  pyramidal  the  cell.  l o c a t i o n s then d i s p l a y e d halfwidth with distance similar  response  discharge,  the  calculate the  Spikes  region  evoked i n d e n d r i t i c r e c o r d i n g  a d e c r e a s e i n a m p l i t u d e and i n c r e a s e i n from t h e c e l l  was f o u n d  layer  f o r spikes  f i r s t evoked  spike  average amplitude  ( F i g 5.4A). S i n c e  evoked i n  in  Type 1  e i t h e r case  and h a l f w i d t h  of the  current  the dendro-somatic a x i s  the  i n t h e p l o t s o f F i g 5.4B,C w e r e d e r i v e d  relatively decline  few  cell  impalements,  i n spike amplitude  and i n c r e a s e  apical dendrite, with the greatest in distal  Voltage  ( F i g 5.4B).  results  Voltage  i n halfwidth  along the  configuration  Discharge  threshold f o r orthodromic spike  according  to  the  of the  recording  pyramidal  c a s e o f SR s t i m u l a t i o n , s p i k e  activation also  position  cell  t h r e s h o l d was f o u n d t o be  lowest  increasing with distance  a p i c a l dendrite. In contrast, spike threshold  SO  stimulation  progressive from  greatest  variable quality  pyramidale.  range  variability  of  of c e l l u l a r  input  would again  soma,  the distance  impalement, as input  f o r a l l recording dendritic  the  These r e s u l t s  r e s t i n g membrane p o t e n t i a l and acceptable  at  decrease the greater stratum  the  In the  the  was  along  ( F i g 5.5A).  in the somatic region, gradually  synaptic  from  do i n d i c a t e a  change i n s p i k e  of Orthodromic Spike  dendro-somatic a x i s  site  Although  dendritic locations.  Threshold  varied  the  or 2  was u s e d t o  evoked s p i k e along average values  a  spike  i n response t o exhibiting  of the  due t o a  average values o f  locations  suggest that  a  recording  are not  resistance f e l l  threshold  along  within  an  ( F i g 5.5C). A  f o r two  forms o f  the action p o t e n t i a l  - 150 F I G 5.5  P l o t s of  d i s c h a r g e evoked  the average  by s t i m u l a t i o n  radiatum at varying distances the pyramidal c e l l . as t h e  voltage threshold of stratum  for spike  o r i e n s or  stratum  along the dendro-somatic axis,  V o l t a g e t h r e s h o l d a t e a c h l o c a t i o n was  a v e r a g e peak  amplitude  of  t h e evoked  of  taken  EPSP when s e t t o  j u s t s p i k e t h r e s h o l d ( m e a s u r e d f r o m r e s t i n g membrane p o t e n t i a l ) . Somatic r e c o r d i n g s diagram of plots  in  are taken  the r a t A.  C.  as Oum  pyramidal neuron  Average r e s t i n g  (n) f o r s t r a t u m  the top of A while others Average v a l u e s without the standard error l i e s point.  drawn t o  B.  Schematic  s c a l e below the  membrane p o t e n t i a l  input r e s i s t a n c e f o r the impalements impalements  distance.  (RMP)  shown i n A. The number  r a d i a t u m e v o k e d EPSPs a r e are g i v e n a t t h e base of  standard error bars  the  of  shown a t  each graph.  are those  w i t h i n the boundary of  and  in  which  illustrated  - 151 -  A. Stim. Site  •Stratum Radiatum oStr. Oriens n  ° E  34 2  5  4  10 12 2 2 6 4  I  20  ^  CD  a  TD TD  CL  o to  § * 5  E E 10  i  CL CO  5  CD  0  J  o O  5  1 4  3  7  2  I  I  B.  Radiatum  Pyr  St. Oriens  C. ^  68  n  CL  cr 6 0  I  *  *  4  3  8  13 2  5  2  v  3  4  5  J  19 2 4  CD CJ  .  CI  Q-  ~ £ ^ or  40 20  23  2  2  10  0 100 200 300 Apical Dendritic Recording Site ( / J )  - 152 recorded to the that  w i t h i n an  recording the  synaptic cell  Stimulus  dual  i n the present at d i f f e r e n t  for  stimulus  intensity,  the  intracellular  a given  at  cells  pyramidale  to the both  discharge  in for  determining  the  a  direct  the  (Richardson  as  apical  impalement  (see  in  necessary the  extracellular  dendritic  level.  to the  of  negative-going  s p i k e response  l a t e n c y of the p o p u l a t i o n  spike  l a t e n c y of  compared A  5.2  the  to  intrasomatic  that  of  second estimate  activation  the  Taylor  in  Dudek  dendrite.  waveform  between  comparing  evoked  or  and  r e l a t i o n s h i p between  extracellular  addition,  dramatically  by  a l . 1984;  such,  spike  In  i t was  and  i n time  i n d i c a t i o n of the  and  measured  intrasomatic spike discharge  et  The  be  latency j i t t e r  discharge  somatic  obtained  comparison  extracellular population  dendritic the  vary  l a t e n c y of  correlates  u s e d as an  generation,  dendritic  either  pyramidal  were n o t  neuron.  could  for spike  Turner et a l . 1984). t h u s be  cells  impalements. Therefore,  s t u d i e s have shown t h a t  latency  region of the  pyramidal  discharge  latency  potentials  spike  i n the  preventing  cell  p h a s e o r peak o f t h e  and  of  response  CAl pyramidal  can  noting  spike threshold for  i n f l u e n c e o f s u c h f a c t o r s as  pyramidal  to estimate  1984;  was  i t i s worth  s p i k e onset l a t e n c y c o u l d not  spike  to the  stratum  which  distal  Latency  study,  according  Previous  regard,  impalements of pyramidal  regions  latency  field  this  in  equivalent  Evoked Spike  separate  i s generated at a s i t e  ( F i g 5.5A).  Since  the  location  i n p u t was  body  apical dendrite  e l e c t r o d e . In  only  -  was  spike of  the  obtained  by  intradendritic  spike  immediate v i c i n i t y of Methods).  In  this  the way,  - 153 i n t r a d e n d r i t i c AP  discharge  c o u l d be  e x t r a d e n d r i t i c s p i k e waveforms extracellular The  field  recorded  p o t e n t i a l s (Chapter  field  stratum  records  potentials  o r i e n s or  obtained  hippocampal  from  slice).  on  revealed  peak l a t e n c y spike in  single  than the  by  is  shown i n  of  falling  pyramidale  edge  these  lowest  set  intradendritic  of  records  spikes  were  of found  positive/negative extracellular dendritic pyramidal  region cell  during  population  from the  F i g 5.6). found  potentials  5.6.  the  recorded  In  each  a l i g n with the  suprathreshold  biphasic  stimulation  3). S p e c i f i c a l l y ,  case,  in  the  of  the  the  onset  i n t r a d e n d r i t i c s p i k e corresponded to t h a t of the p o s i t i v e  e x t r a d e n d r i t i c p o t e n t i a l , while the  breakpoint  of decay of  s p i k e a l i g n e d w i t h the t e r m i n a t i o n of the negative  component  the d e n d r i t i c f i e l d  lines  5.6).  to  recording  spike p o t e n t i a l recorded  (Chapter  The  i m p a l e m e n t i s shown i n  Fig to  longer  A comparison of  d e n d r i t i c s p i k e to the e x t r a d e n d r i t i c f i e l d f o l l o w i n g withdrawal  cell  population  r e s p o n s e s was  pyramidale.  spike  a  d i s t a n c e of the d e n d r i t i c  s i t e from the border of stratum  the  and  at the  of the  traces in  two  and  impalement  evoked at  o r peak  ( t o p two  of  (all  intradendritic  s p i k e s were  i n l a t e n c y between  immediately  F i g 5.6  p o t e n t i a l response recorded  increase d i r e c t l y w i t h the  of the  profiles  s t i m u l a t i o n of  dendritic  comparison  that d e n d r i t i c  stratum  difference  the  laminar  of  3).  evoked  radiatum a  A  a c t i v a t i o n to the f i e l d layer  compared t o t h e l a t e n c y  r e l a t i o n s h i p between i n t r a d e n d r i t i c s p i k e d i s c h a r g e  extracellular alveus,  -  While  extracellular  p o t e n t i a l ( d e n o t e d by  the p o s i t i v e / n e g a t i v e EPSP i s n o t  as e v i d e n t  dotted  component o f  the  SR  in this particular  in  the of Fig  evoked example,  t h e o n s e t o f t h e p o s i t i v e - g o i n g d e f l e c t i o n on t h e e x t r a d e n d r i t i c  - 154 FIG.  5.6  A comparison  of  the l a t e n c y of i n t r a d e n d r i t i c  discharge to e x t r a c e l l u l a r f i e l d pyramidale  (population  (extradendritic) dendritic the  Stimulus spike  latency  (A),  stratum  stratum  intensities  over  from  field a  pyramidale.  withdrawal  stratum radiatum  from  stimulation  field  Intradendritic 200um  of  (C).  potentials  lines indicate  intradendritic  impalement  the  for intradendritic  shown) and  sweeps. D o t t e d  potentials.  single  stratum  to threshold  between  in  spike  (B) o r s t r a t u m r a d i a t u m  sweeps  10 s u c c e s s i v e  recorded  were e v o k e d by  oriens  (several  and  following  were s e t  relationship  extradendritic obtained  spike)  immediately  activation  averaged  potentials  impalement. P o t e n t i a l s  alveus  -  the  spikes  and  recordings  were  from the border  of  Stratum Radiatum  5 msec  - 156 EPSP  can  be  seen  to  -  correspond  to  the  rising  edge o f  the  i n t r a d e n d r i t i c s p i k e at t h i s l o c a t i o n ( F i g 5.6C).  I n t r a d e n d r i t i c Spike F r a c t i o n a t i o n Previous  investigators  dendritic spike  have  of pyramidal  reported  that  neurons c o u l d  be  as  evidence  dendritic  for  spike  membrane  activation.  In  (Wong e t a l . 1 9 7 9 ) . T h i s  exhibiting  the  d e n d r i t i c s p i k e was  discharge  present  at  a study,  the  alveus,  generation  stratum  c o u l d be  for  of  spike of  the  oriens,  or  stratum  extracellular  spike  population  pyramidale.  at  radiatum,  the  cell  of  the  body l a y e r ( F i g  s p i k e s evoked w e l l beyond  the  peak  or  edge o f  the  comprised of  two  intradendritic The  spike  all-or-none  amplitude  stimulation  ( F i g 5.7A-C).  intensity,  the  clearly  distinct  second in  appeared  components.  p o t e n t i a l o f 9-25mV a m p l i t u d e s p i k e w i t h an  on t h e  falling  EPSP.  fractionated  principle  spike  greater  the  f r a c t i o n a t i o n observed f o r those population  a  r e l a t e d to  of  the  with  the  degree  of  discharge,  of  with  b e y o n d t h e peak  5 . 7 A - C ) . F r a c t i o n a t i o n o f t h e d e n d r i t i c s p i k e was spike  This  e v o k e d by s t i m u l a t i o n  at a l a t e n c y  to  spots"  o c c a s i o n a l l y observed i n d e n d r i t i c recording  f r a c t i o n a t e d s p i k e evoked  latency  taken  fractionation  l o c a t i o n s 200-300um f r o m t h e b o r d e r o f s t r a t u m p a t t e r n of s p i k e  r e s u l t was  threshold  a  into  "fractionation"  r e g i o n a l "hot  low  evoked  separated  m u l t i p l e c o m p o n e n t s , a r e s p o n s e r e f e r r e d t o as a of the s p i k e waveform  the  The  be first  was  a  small  t h a t gave r i s e t o a second, l a r g e r  that could  vary w i t h the  I f evoked at component  arising  to  was  intensity  of  just-threshold stimulus of  from the f a l l i n g  small amplitude edge o f t h e  and first  - 157 FIG.  5.7  230pm  Fractionation  from  the  border  d i s c h a r g e evoked through oriens  (B)  or  extracellular  of  increasing  intensities  into  spike  each case,  two  (C)  to  stratum  radiatum  of  stimulation. all-or-none  o f t h e second  10Hz r e p e t i t i v e  dendritic  spike  amplitude  to  increased  recorded A-C.  (A),  compared  recorded  stratum to  Note f r a c t i o n a t i o n o f components,  and  train  is  of the  peak  fractionate  evident.  The  o b t a i n e d from a s e p a r a t e d e n d r i t i c C.  the  component a t h i g h e r s t i m u l u s dendritic the alveus  latency into  spike i n (D) o r  amplitude  and d e c r e a s e d i n a  two  component  w a v e f o r m . A s l i g h t h y p e r p o l a r i z a t i o n o f t h e membrane d u r i n g stimulus  the  i n stratum  stimulation, the f u l l  in  Spike  a r e superimposed f o r  stimulation of  ( E ) . During  eventually  is  response  D,E. F r a c t i o n a t i o n  response  spikes  pyramidale.  s e v e r a l sweeps  principle  increase i n amplitude intensities.  stratum  radiatum  population  In  intradendritic  stimulation of the alveus  stratum  pyramidale.  spikes  of  a n t i d r o m i c response  the  i n A was  i m p a l e m e n t f r o m t h a t o f B and  Stim. Site  Alveus  Stratum Oriens  Stratum Radiatum  B.  C.  Single Sweeps  2 msec  - 159 potential.  As  potential  could  increasing  stimulus  in  intensity  exhibit  amplitude,  onset l a t e n c y u n t i l the no and  l o n g e r be d i s c e r n e d  was  the  increased,  peculiar  progressively  t h e second  characteristic  arising with  b r e a k b e t w e e n t h e two (best i l l u s t r a t e d  of  a shorter  components  f o r the case of  could alvear  SO s t i m u l a t i o n i n F i g 5 . 7 A , B ) . Fractionation  dendritic  of  spike  impalements d u r i n g  or a f f e r e n t pathways s p i k e evoked slightly  the  could  o f t e n occur  repetitive stimulation of  ( F i g 5.7D,E). I n e i t h e r c a s e ,  in full  f o r m on  i n peak l a t e n c y  in distal  the f i r s t  during the  efferent  a dendritic  p u l s e would  increase  stimulus trairi,  with the  s e c o n d component g r a d u a l l y d e c l i n i n g  i n amplitude  initial  small all-or-none potential.  As shown i n F i g 5.7D, t h e  second  larger  potential  could  stimulation, while the f i r s t be  evoked  at  a  dendritic spike  fractionation  spike.  discharge  in  level,  o f Wong e t  obtained  could  dendritic  membrane  constant  a l . (1979),  be  In  response  repetitive  amplitude  through the  i n the related  this  fractionation  through h y p e r p o l a r i z a t i o n  s t i m u l a t i o n of stratum  r e s u l t s c o u l d be  during  train.  was a c h i e v e d  membrane d u r i n g  lost  an  component d e c l i n e d o n l y s l i g h t l y t o  comparatively  majority of the stimulus In the study  be  to reveal  present to  case,  radiatum  inputs.  study,  and  of the of the Similar  again the  t h e l a t e n c y o f t h e evoked  the  onset l a t e n c y f o r spike  to  SR  s t i m u l a t i o n would i n c r e a s e  hyperpolarization  at  both  the somatic  with  and d e n d r i t i c  w i t h t h e r e s u l t t h a t t h e d e n d r i t i c s p i k e would e x h i b i t  fractionation  into the separate  components d e s c r i b e d  above.  a  - 160  -  I s o l a t i o n o f A p i c a l D e n d r i t i c E l e m e n t s by K n i f e C u t s  i n the  CAl  Region In order could occur  to determine  whether d e n d r i t i c  independently of  t h a t at  t h e soma,  made t o p h y s i c a l l y s e p a r a t e t h e soma and pyramidal  cells  by a  Benardo e t a l . (1982).  k n i f e cut  activation  attempts  were  a p i c a l dendrites of  according to  Briefly,  spike  t h i s was  the technique  performed  through  of a " m i c r o - k n i f e " c o n s i s t i n g of a s m a l l razor blade c h i p t o a wooden d o w e l was  mounted on a m i c r o - m a n i p u l a t o r .  c u t w h i l e r e s t i n g upon  the  recording  proximal-mid observation slice  chamber stratum  lowering  radiatum  (see Methods f o r  tissue  extracellular  followed  the  blade  under  details). this  procedure,  f i e l d p o t e n t i a l s were r e c o r d e d  within  across  attempts and  to  SR  cut  evoked  f r o m w i t h i n and  dendritic  activity.  However,  encountered  in  of the  s l i c e t i s s u e . The  on  evoked  difficulties  achieve separation  r e g i o n w i t h o u t damaging r e m a i n i n g  the  microscopic  to assess the c h a r a c t e r i s t i c s of  .trying to  glued  net  e i t h e r s i d e of the cut  several  use  tissue  direct  Initial  were  cell  body  majority  p r o b l e m s were r e l a t e d t o t h e f a c t t h a t t h e n y l o n n e t w i t h i n r e c o r d i n g chamber d i d n o t p r o v i d e a d e q u a t e s u p p o r t a complete  cut of the s l i c e .  of the nylon one  For  strands  little  allowed  the  net  surface  pressure of the blade, r e q u i r i n g f u r t h e r k n i f e w i t h the p o s s i b i l i t y of compressive  pliability to  i n only  intended l i n e  o r no b a c k g r o u n d  t o p u s h t h e b l a d e a g a i n s t . The also  the  for obtaining  s l i c e would r e s u l t  o r a few n y l o n s t r a n d s c r o s s i n g b e n e a t h t h e  f o r which  of  i n s t a n c e , the net c o n f i g u r a t i o n  f i b e r s s u p p o r t i n g the  cut i n the CAl region, o f f e r i n g  of  Slice  the surface of the nylon  by  CAl  bend  support  of the  nylon  beneath  f o r w a r d advance of damage t o  of  the the  surrounding  - 161 tissue.  In f a c t ,  evidence  of  including  evoked e x t r a c e l l u l a r p o t e n t i a l s o f t e n  damage  to  the  "intact"  evoked p o t e n t i a l s o f low  discharge  of p o p u l a t i o n  Histological  t o make  analysis revealed  small repeated  the bottom the of  of the s l i c e .  nylon net  net  and  amplitude  or the  the c e l l  body l a y e r u s i n g  cuts along  axis  slice,  repetitive body l a y e r .  of  obtaining  this  technique  of the blade  t h i s procedure often  the s l i c e ,  extensive  the  displayed  of a cut  t h a t t h e o n l y means  However,  supporting  tension  regions  s p i k e responses at  complete s e p a r a t i o n of the c e l l was  -  resulting  jarring  i n a b i l i t y to s u c c e s s f u l l y section s l i c e s  of  severed  i n a sudden l o s s slice  tissue.  i n t h i s manner  f o r c u t t i n g the s l i c e  to  chamber. I n  slice  was  placed  the  recording  i n a small  p l a s t i c p e t r i d i s h and  a  s c a l p e l blade  observation. This technique  was  described  c o m p l e t e and  the c e l l  f o u n d t o be  s e c t i o n (22um) f r o m  t h i s manner and  stained with cresyl v i o l e t the e x c i s i o n  apical dendritic  a cut s l i c e to  microscopic  f a r superior to  r e g i o n . SR  of pyramidal  pyramidale  revealed  t h a t SR  cell  evoked  bodies  cut  i n the  intact  in  5.8B,  from  recorded  A recording  of  neuronal  i s shown i n F i g  evoked p o t e n t i a l s  or radiatum  that  at  the the  electrode region  of  p o t e n t i a l s were c o m p a r a b l e  t h a t o f c o n t r o l h i p p o c a m p a l t i s s u e , w i t h m a x i m a l e v o k e d EPSPs  o f 12mV  and  population spikes  o f up  multiple population spike discharge. of  under  a hippocampal s l i c e  i n d i c a t e d l o c a t i o n s a r e shown i n F i g 5.8C. i n stratum  the  reliable separation  tissue. A thin  placed  t h i s case,  body l a y e r w i t h m i n i m a l damage t o s u r r o u n d i n g  demonstrating  prior  d r o p o f o x y g e n a t e d medium w i t h i n a  c u t by  above, a l l o w i n g  The  required  the development of another technique placement w i t h i n  at  the evoked  EPSP and  population  t o 27mV w i t h no  evidence  However, t h e peak  of  amplitude  spike progressively declined  - 162 FIG.  5.8  Thin  sections  (22um)  of  cresyl  h i p p o c a m p a l t i s s u e f r o m an i n t a c t s l i c e cut across regio superior f o r from  violet  and a s l i c e w i t h a k n i f e  i s o l a t i o n of the apical dendrites  somata o f C A l p y r a m i d a l c e l l s .  The m a j o r s u b f i e l d s o f t h e  h i p p o c a m p a l f o r m a t i o n a r e shown i n A, i n c l u d i n g r e g i o n s o f C A l and CA3, and t h e d e n t a t e somata o f C A l p y r a m i d a l  cells  of  pyramidale.  tissue  in  stratum  staining of c e l l s part to g l i a l somata  pyramidale  in  (dark  circles).  C. S t r a t u m  locations  indicated  placed  in  The  (DG). The s t a i n e d dark  the  by t h e  and s t r a t u m  in  B from  a r e marked i n radiatum  stratum pyramidale  to right.  radiatum  and moved  enlargement  of  the f i m b r i a l  recorded  in  stratum radiatum a t  asterisk  i n B and C.  at  the  and s t r a t u m  side of the  recording electrode to  approximately  radiatum  (open  The s t i m u l a t i n g e l e c t r o d e  with the  distance of stratum  on  cell  loss of stratum  radiatum evoked p o t e n t i a l s recorded  stratum  a constant  i n large  of pyramidal  electrode positions  triangles)  band  d i f f u s e and s c a t t e r e d  removal  i s indicated  from l e f t  recording electrode maintain  gyrus  a r e r e p r e s e n t e d by t h e  B, t h e  C A l . Recording  stratum pyramidale  was  In  knife cut  r a d i a t u m , moving  the hippocampal  i n s t r a t u m r a d i a t u m c a n be a t t r i b u t e d  elements.  by t h e  stained  evoked  the position  400um. fiber  D. An potential  indicated  by t h e  A.  -y  -yv-  -|f  •  5 msec I mV i  5 msec  5 mV  -  as t h e (Fig  r e c o r d i n g e l e c t r o d e was  164  -  moved t o w a r d s t h e edge o f t h e  5.8C). A r a p i d d e c l i n e i n the amplitude  in stratum  radiatum  approximately almost a l l  was  from  examined,  the  border  the maximal  evoked beyond t h i s p o i n t w i t h s t i m u l u s was  i n the  (up t o along  range  4mV)  of the  be  evoked  the e n t i r e extent  synaptic projections  and  intact  be  "EPSPs" to  i n stratum  (Fig  5.8D).  afferent Therefore,  radiatum  a t t r i b u t e d t o c o m p r e s s i v e damage t o t h e d e n d r i t i c  t h a t might have been s u s t a i n e d d u r i n g some s l i c e s ,  a postsynaptic  limited distance within subsequent  histological  region  analysis  s c a t t e r e d or d i s p l a c e d pyramidal  be  evoked  of the s l i c e .  revealed  cell  could region  the c u t t i n g procedure.  p o t e n t i a l could  the cut  60V  radiatum  that  the l a c k of evoked s y n a p t i c p o t e n t i a l s i n s t r a t u m not  the  region. pyramidal the c e l l in  an  It  cell appeared  cell  dendrites  However, of  s o m a t a a t t h e edge o f  the  that  a p i c a l d e n d r i t e s had  body r e g i o n , p r e v e n t i n g  isolated  still  therefore,  dendrite.  a  presence  evoked p o t e n t i a l s i n t h i s case c o u l d thus correspond pyramidal  In  over  i n c i s i o n d i r e c t l y above t h e p o s i t i o n o f the evoked a c t i v i t y .  of  In  fiber potentials  of the cut, demonstrating  were s t i l l  of  i n t e n s i t i e s o f up  recorded  over  incision.  amplitude  o f 0.2-0.4mV. However, l a r g e  could  EPSP  then observed w i t h i n the cut region  100-150pm slices  of the evoked  cut  not  attached the  to  activity  to the c e l l  vast  majority  survived separation  an a n a l y s i s o f s p i k e  The  body of from  activation  - 165 5-4. D i s c u s s i o n Previous cell  have  spike  studies  reported  in  apical  on t h e evidence  of the pyramidal  f o r the i n i t i a l  dendritic  e l e c t r o t o n i c conduction region  evoked a c t i v i t y  membrane,  of the  a c t i v a t i o n of a  with  a  dendritic spike  subsequent  t o the somatic  ( A n d e r s e n e t a l . 1 9 6 0 ; A n d e r s e n and Lomo 1 9 6 6 ; C r a g g  and  Hamlyn 1 9 5 5 ; S c h w a r t z k r o i n  1 9 7 7 ; S p e n c e r and K a n d e l 1 9 6 1 b ) .  One  would thus expect t o f i n d  evidence i n the c o n f i g u r a t i o n of  the  d e n d r i t i c s p i k e o f an e l e c t r o t o n i c c o n d u c t i o n the  cell  body  region.  In  fact,  a comparative  a n a l y s i s of spike c h a r a c t e r i s t i c s along revealed  a  p r o g r e s s i v e change  discharge,  including  towards  intracellular  the dendro-somatic  i n several  spike  and d e c a y  parameters of  amplitude,  halfwidth,  axis spike  voltage  t h r e s h o l d and l a t e n c y t o o n s e t .  Evoked  Characteristics  Dendro-Somatic  of  Spike  o f an  e l e c t r o t o n i c conduction  from t h e d e n d r i t i c r e g i o n would halfwidth with  However, s p i k e s through current increase opposite dendritic The (hot  in  the  of a spike  be a d e c r e a s e i n a m p l i t u d e  proximity to  evoked from  each o f  halfwidth  to that  with  body l a y e r .  the stimulus  pathways o r  d i s t a n c e from stratum  expected f o r a  and  the c e l l  i n j e c t i o n d i s p l a y e d a decrease i n amplitude  and  pyramidale,  somatopetal conduction  of  the  spike. initial  spot)  in  voltage threshold along  Along  Axis  One c o n s e q u e n c e  increase i n  Discharge  the  activation of dendritic f o r spike  dendro-somatic  a spike  membrane  from a  would  activation at axis.  An  also  generator  p r e d i c t a low  one o r  analysis  zone  more p o i n t s  of s y n a p t i c a l l y  - 166 evoked  spike  threshold  voltage  threshold  for spike  apical dendrite. threshold  was  did  reveal  a  discharge  However, t h e distinctly  gradual  along  progression  different  for  synaptic d e p o l a r i z a t i o n of the pyramidal for  spike discharge  in  the  mid-distal  pyramidal  cell  in  the  apical  would  apical  l a y e r . In  dendrite,  dendrite. A that  of  change i n  two  cell.  strict  (1)  an  t h a t a s p i k e a t any g i v e n  Voltage  declining  threshold  decreasing  threshold  was  greatest  towards  complementary  same  dendritic  appear  to  apical  impalement;  with distance  along  of these r e s u l t s spike  is  voltage-dependent membrane.  An  to  activation  alternative  spike threshold  (2)  the a p i c a l dendrite  can  thresholds  of  usual spike  explanation  i s that a d e n d r i t i c spike  conclusion  would  understanding  of  discharge for  (recall  are taken from the  5.1). Neither  the  most  and  measurements cf  in  neuronal  the v a r i a b i l i t y i n  i s generated at a  remote from t h e d e n d r i t i c r e c o r d i n g  l o c a t i o n . In f a c t , the  location  i n which spike discharge  in  the  pyramidal  cell  evoked from a c o n s i s t e n t v o l t a g e the c e l l  b o d y l a y e r . The  by t h e i n i t i a l  t h r e s h o l d was  i n the region  a b o v e r e s u l t s m i g h t t h e n be  action  potential  would  then  site only was of  explained  a c t i v a t i o n of a spike i n the somatic region,  a subsequent retrograde  by  body,  dendritic  l o c a t i o n along  threshold  conform  the  i n the c e l l  interpretation  e n t i r e l y d i f f e r e n t voltage  that  spike  forms o f evoked  e f f e c t i v e l y e v o k e d by an EPSP o f b a s a l d e n d r i t i c o r i g i n ,  be e v o k e d a t two  cell  c o n t r a s t , a c t i o n p o t e n t i a l s evoked  threshold apparently  imply  the pyramidal  r e s p o n s e t o SR s t i m u l a t i o n  SO s t i m u l a t i o n d i s p l a y e d t h e h i g h e s t with voltage  change i n t h e  and  s p i k e i n v a s i o n o f d e n d r i t i c membrane. An  conducting  superimpose  back upon  through the  the d e n d r i t i c tree  coincident  level  of  - 167 depolarization within  the  apparent l a c k of v o l t a g e The  results  spike discharge activation  comparison of  dendrite, giving  threshold  obtained  dendritic  Although s t r i c t  potentials  apical  in  the  spike discharge  the  an  of  of  pyramidal  indirect  expect the d e n d r i t i c  would  s h o r t e r l a t e n c y than the p o p u l a t i o n reflecting  recorded  t h a t i n the  recorded  i n stratum  the  pyramidal  s p i k e at the  extradendritic  axis. somatic  cell  to conduct w i t h with distance  population.  spike  l a y e r , suggesting  the that  in  the  a x i s of the pyramidal  initial  spikes  activation Chapter  density  3,  analysis region  body l a y e r .  c h a r a c t e r i s t i c s of  evoked d e n d r i t i c  onset l a t e n c y along  cell  to  potential  i n c r e a s i n g l a t e n c y through the d e n d r i t i c  h a l f w i d t h , and  the  described  found through c u r r e n t - s o u r c e  from the c e l l  Therefore,  intradendritic  positive/negative  As  a  pyramidale,  f o r somatic  radiatum f o l l o w i n g suprathreshold  cell  t h i s p o t e n t i a l was  result for  for  evoked at  i n stratum  average l a t e n c y  a  field  latency  at the  s p i k e t o be  somatic region. Furthermore,  with  amplitude,  obtained,  i n d e n d r i t i c l o c a t i o n s were e v o k e d s u b s e q u e n t  aligned  of the  the  precede that  spike  spike  However, i n t r a d e n d r i t i c s p i k e s w e r e e v o k e d b e y o n d  peak o f t h e p o p u l a t i o n spikes  of the  for  cell.  at d i f f e r e n t l e v e l s of the dendro-somatic  level,  discharge.  initial  extracellular  estimate  spike a c t i v a t i o n  a potential  the  spike to  Should d e n d r i t i c one  an  latency  concept  s p i k e l a t e n c y were not  intradendritic  provides  a n a l y s i s of the  locations  measures of  r i s e to  in dendritic locations.  an  also contradict  in  -  the  dendro-somatic  are d i r e c t l y opposed t o the  a c t i v a t i o n of  a spike  expected  from w i t h i n  d e n d r i t i c a r b o r i z a t i o n . I n a d d i t i o n , an  a n a l y s i s of the  threshold for spike  dendrite  a c t i v a t i o n i n the  spike  d i d not  the  voltage reveal  - 168 r e g i o n s o f d e n d r i t i c membrane e x h i b i t i n g for  spike discharge  voltage  (hot s p o t s ) . In  threshold  synaptic  or  f o r spike  current  a low v o l t a g e  fact,  discharge  evoked  the only  cell  body l a y e r .  may h a v e b e e n  i s o l a t e d o r remote  Although  was f o u n d i n t h e  d e n d r i t i c "hot  to the d e n d r i t i c  obtained,  a l l data  are consistent with the i n i t i a l  a  in  somatic  spike  subsequent  the  retrograde  region  conduction  the  spots"  impalements  activation of  of the pyramidal of  consistent  i n response t o e i t h e r  depolarizations  region of the  threshold  spike  cell,  and a  through  the  dendritic arborization. Some comes  of  from  discharge  the  the to  potentials  strongest close  the  at  evidence  forthis interpretation  correlation  of  components  of  spike  a l l levels  of  the  intracellular  spike  extracellular  dendro-somatic  field  axis.  For  i n s t a n c e , t h e onset l a t e n c y o f d e n d r i t i c s p i k e s evoked from a l l three  pathways  shown t h r o u g h the  cell  of t r a n s i t i o n At  layer.  The  population  to  i n spike  spike  spike  p o t e n t i a l waveform  analysis to between  100pm  a  from  stratum  changed  from  positive/negative  distal  pyramidale, a  component  stratum  of  radiatum  the  dendritic potential point  amplitude  recorded  i n the  Similarly, the loss of the  extracellular  correlates  the  negative-going  e x h i b i t a decrease i n  cell.  and  pattern  3 ) . This l o c a t i o n corresponds t o the approximate  region of the pyramidal  negative  intra-  from  the dendro-somatic  increase i n halfwidth with respect t o spikes  somatic  conduct  f u r t h e r noted i n the  c o n f i g u r a t i o n along  at which i n t r a d e n d r i t i c spikes and  field  correlation  component  to  the  density  spike potentials i s  approximately  extracellular  (Chapter  current-source  body  extracellular  axis.  corresponded  dendritic  t o t h e second r a p i d  spike i n decline  - 169 in  amplitude  spike.  and i n c r e a s e  Anatomical  indicate  that  approximate  these  points of  structure  configuration  the proximal  conduction dendritic  of  dendritic in  past  change i n s p i k e close  cell  correspond  to the  3).  of  The  distal  result  f o r the case of a  profiles  that  elements.  the c o n c l u s i o n s a  the  between  change i n  spike  indicate  spike  in  retrograde  conducting  ( G o l d s t e i n and  mechanism  Rail  underlying the  dendro-somatic  axis,  i n t r a - and e x t r a c e l l u l a r s p i k e  of  the  field  activity  in  I n t r a c e l l u l a r data would  d e r i v e d form  distal  waveform i s  spike  r e s u l t s obtained from  representative  pyramidal c e l l  indicating  of  c h a r a c t e r i s t i c s along the  indicate are  dendritic  regardless  correspond  branchpoints  structure  spike  of b i f u r c a t i o n of  r a d i a t u m may  dendritic  apical in  then  S i m i l a r l y , the  stratum  the  change  past the point  shaft.  correlation  properties  conduction  individual  thus  current-source density  spike  potential  support  analysis,  through  the apical  border  of stratum  r e g i o n (Chapter 3 ) .  D e n d r i t i c Spike Beyond pyramidale  Fractionation  approximately  200jum  (approximately half  fissure), dendritic  evidence  pyramidal  r e g i o n s . An a l t e r a t i o n i n a c t i o n p o t e n t i a l  However,  waveform.  rat  branchpoints  past the branchpoint of a neuronal  the  the  intradendritic  s t r a t u m r a d i a t u m may  secondary  f a c t the expected  1974).  of the  transition  (Chapter  of the spike  characteristics  of  major  i n proximal  to a conduction  in  measurements  location  dendritic  in halfwidth  Spike for  the  the distance to  spikes could  fractionation  multiple sites  from  exhibit has  of spike  a  the hippocampal  "fractionation" in  previously  been  generation i n  taken  as  dendritic  - 170 membrane  (Wong  et  f r a c t i o n a t i o n was both antidromic of  the  indicate  that  an  for  of  an  antidromic  orthodromic  peak  of  Thus, i t had  been  the would  evoked  conducting  from  spike discharge. spike  does  not  activation  of  low  "reflected"  i n neighboring  spike  (Parnas Close  Thus,  examination  components  spike  necessarily  for spike a c t i v a t i o n  -  a  b a s i c form a  of  small  as  dendritic  this  retrograde  region  be  separated  a  in the  spike  might  be  fractionated  "fractionated"  (cf  4.9  and  revealed  into  potential  second s p i k e  pre-potential  spike  phenomenon  all-or-none  and  of the  fast  second l a r g e r  spots,  d e n d r i t i c b r a n c h e s as a  could  amplitude,  of  threshold  the  1979).  fractionated  constant  a  hot  the  similar  a p i c a l d e n d r i t e . However, t h e s e r e s u l t s c a n n o t r u l e o u t of d e n d r i t i c  case  components  body l a y e r . These r e s u l t s spike  orthodromic  spike  through  a f r a c t i o n a t i o n of waveform  p o s s i b l e presence  The  the  spikes  i n d i c a t e the presence of m u l t i p l e s i t e s the  study, evoked  pyramidale.  at the c e l l  exhibit  observed  fractionation  present  beyond  dendritic  discharge that  evoked  i n stratum  fractionated  s o m a t i c r e g i o n can to  the  for d e n d r i t i c spikes  spike  spike recorded  that  In  or orthodromic s t i m u l a t i o n , w i t h a l l  s u b s e q u e n t t o AP also  1979).  observed  fractionated  population appear  al.  -  that  essentially of  amplitude.  spike thus resembles  4.10). In  the  two  relatively  of v a r i a b l e  underlying  a  the  a c t i v a t i o n of  a  fact, fractionated  spikes displayed  s i m i l a r evoked  discharge,  u n c o v e r e d t h r o u g h membrane h y p e r p o l a r i z a t i o n o r  being  c h a r a c t e r i s t i c s to that of  r e p e t i t i v e s t i m u l a t i o n . However, f r a c t i o n a t i o n o f t h e also s t r o n g l y c o r r e l a t e d to parameter  of  AP  discharge  the  l a t e n c y of s p i k e  directly  spike  FPP  was  activation, a  r e l a t e d to the  recording  - 171 d i s t a n c e from stratum s p i k e was  found at  pyramidale.  A greater  comparatively  f r a c t i o n a t i o n of  long l a t e n c i e s to  onset,  more s e p a r a t i o n o f s p i k e c o m p o n e n t s when e v o k e d upon t h e edge o f t h e  EPSP.  I t i s important  this latency f a l l of the body  close  amplitude  and  Langmoen  ( A l g e r and  t h e e v o k e d s p i k e may  for  thus  Nicoll  correspond  1980)  or  1984). t o an  the  correlation  distance  cell  perhaps  from  between the  dependence o f  Such a  spike  cell  i n h i b i t i o n of  spike  the amplitude  as  of a  well  as  in  movements  mechanism c o u l d  fractionation  layer,  in  F r a c t i o n a t i o n of  as a r e t r o g r a d e l y c o n d u c t e d d e n d r i t i c s p i k e s h i f t s  dendro-somatic a x i s .  at  activation  peak l a t e n c y t o w i t h i n t h e t i m e o f i n h i b i t o r y c u r r e n t  the  falling  t o note t h a t s p i k e s evoked  to the approximate time  (Dingledine  d e n d r i t i c membrane  along  with  i n h i b i t o r y p o s t s y n a p t i c conductance observed at the region  the  explain  and  recording  the  intensity  f r a c t i o n a t e d s p i k e . However,  f u r t h e r work i s r e q u i r e d t o i d e n t i f y t h e f a c t o r s r e s p o n s i b l e f o r spike fractionation,  and  to  o f s p i k e a c t i v a t i o n t o FPP  assess  e v o k e d i n d e n d r i t i c membrane  cell  body l a y e r  accomplish would ensure cell  subsequent t h i n  and  apical  and  the  cell  apical dendrites  from  the  region.  To  d e v e l o p e d by w h i c h t h e  cut  reliable dendrites,  s e c t i o n i n g and  analysis  was  be  of t h a t at  k n i f e cut  technique  a complete  somata  t i s s u e . An  made t o i s o l a t e  a  action p o t e n t i a l could  independently  by p l a c i n g a  this,  form  Dendrites  t o d e t e r m i n e w h e t h e r an  body, a t t e m p t s were  this  discharge.  Evoked A c t i v i t y of I s o l a t e d A p i c a l In order  the s i m i l a r i t y of  in  s e p a r a t i o n of as  histological  of evoked a c t i v i t y  the CAl  confirmed analysis  i n cut s l i c e s  pyramidal through of  slice  revealed  - 172 m i n i m a l damage t o p y r a m i d a l of  the  cut  dendritic  or  to  field  afferent  under  postsynaptic a c t i v i t y elements cut.  the  c o u l d be  of  projecting  In  through  contrast,  recorded from  region the  little  isolated  or  could  3 ) . Evoked a c t i v i t y  then correspond  the c e l l  bodies of  to d e n d r i t i c  pyramidal c e l l s  stratum pyramidale. activity  would i n d i c a t e t h a t  In f a c t ,  within  the  knife  this distance cell  a t t h e edge  of a  structures attached to  i n the  intact portion  i n the  few  slices  region  of  the c u t ,  r e v e a l e d the presence  of pyramidal c e l l  histological  somata  at the  i n d i c a t e t h a t most i f n o t a l l i s o l a t e d  d e n d r i t e s had n o t s u r v i v e d s e p a r a t i o n f r o m t h e c e l l  apical  dendrites  and  recorded action p o t e n t i a l dendrite  (Benardo  However, t h e  et  somata  of pyramidal c e l l s ,  d i s c h a r g e from w i t h i n  al.  1982;  t e c h n i q u e used  Masukawa  i n these  be s u c c e s s f u l l y e m p l o y e d  and p r o v e d  inadequate  complete  s e p a r a t i o n of d e n d r i t i c  region. I t these  and  thus  be p o s s i b l e  studies  were  obtained  that dendritic  from  "intact"  1984).  cutting  in  the  work,  obtaining  s t r u c t u r e s from the c e l l  may  have  "isolated"  i n the present  or at l e a s t u n r e l i a b l e  apical  isolation  Prince  studies for  s l i c e c o u l d not t o be  the  and  The  body r e g i o n .  P r e v i o u s i n v e s t i g a t o r s h a v e r e p o r t e d o b t a i n i n g an of  of  demonstrating  edge o f t h e c u t a b o v e t h e l o c a t i o n o f t h e e v o k e d p o t e n t i a l . r e s u l t s would thus  no  dendritic  l a t e r a l branching of the pyramidal  a p i c a l d e n d r i t e (Chapter  analysis  fibers cut.  A n a t o m i c a l measurements  evoked  s t r u c t u r e s o u t s i d e the  b e y o n d a p p r o x i m a t e l y 150um f r o m t h e b o r d e r o f t h e  i s w i t h i n t h e range  cut  cell  -  body  recordings i n  dendrites  still  a t t a c h e d t o s o m a t a n o t removed by t h e k n i f e c u t . C o n s i d e r i n g t h e difficulties of  evoked  associated with activity  from  t h i s technique, the within  an  isolated  demonstration d e n d r i t e would  - 173 require  additional  anatomical  -  confirmation  through i n j e c t i o n of a s u i t a b l e c e l l techniques presently cell  dendrites  region,  and  available,  cannot  the  survive  i n the  separation of  cell  body l o s s  s t a i n . Therefore,  i t would appear  possibility  independent of that  of c e l l  that  not  the  pyramidal  from the  dendritic  body has  using  cell  body  spike  activation  y e t been  adequately  tested. The  p r e s e n t s t u d y has  further characterized  of a c t i o n p o t e n t i a l discharge of the has  CAl pyramidal c e l l .  demonstrated  i n s o m a t i c and  change i n  amplitude, halfwidth, voltage  of change i n results dendritic  current-source  in  the  discharge the  by  a  density  region  model The  of  analysis  properties  and  onset l a t e n c y  arborization.  of  a  along  the d i r e c t i o n  within  r e s u l t s obtained  spike  the the  through  (Chapter 3 ) , i n d i c a t i n g t h a t  soma-axon  retrograde  spike  c h a r a c t e r i s t i c s of  the  i n the pyramidal neuron i s hillock.  Action  i n d e n d r i t i c membrane t h e n f o l l o w s t h a t  result  of  d i r e c t l y opposed to  intracellular  spike generation the  activity  the  of spike generation  thus c o i n c i d e with the  s i t e f o r Na+  d e n d r i t i c membranes  d e n d r o - s o m a t i c a x i s . However,  arborization.  spike discharge  initial  threshold  these c h a r a c t e r i s t i c s i s  predicted  properties  A d e t a i l e d a n a l y s i s of s p i k e  a gradual  the p y r a m i d a l c e l l  the  potential  i n the  soma  as  i n v a s i o n of the d e n d r i t i c  - 174 6-0.  The  GENERAL SUMMARY AND DISCUSSION  S i t e of O r i g i n of Pyramidal The  present  study  has s e r v e d  of evoked d e n d r i t i c s p i k e s hippocampus.  A  current-source the  initial  site  analysis  of spike discharge or orthodromic  t h e soma-axon h i l l o c k .  through in  dendritic  the  characteristics  Na+ s p i k e s  recorded  These  results  cell.  regarding  According  a  indicate that  i n d e n d r i t i c l o c a t i o n s then o f t h e s p i k e from t h e c e l l  of  Furthermore,  Na+  evoked by  spikes  a n t i - or  s i t e of o r i g i n  directly  cell  orthodromic  f o r a l l evoked  dendrite.  opposed  to  d e n d r i t i c spike activation  tree at regional  low t h r e s h o l d  the  existing  i n the pyramidal  "hot spots" of  f o r spike  the  e v o k e d by  t o t h i s model, d e n d r i t i c s p i k e s a r e evoked  within the dendritic exhibiting  activity  arborization.  i n the pyramidal are  potentials,  region of  conduction  t o those  field  stimulation i s i n the  s t i m u l a t i o n may i n d i c a t e a s o m a t i c  hypothesis  of  cell for  the  depolarizing current  n e u r o n s o f mammalian  i n the pyramidal  Evoked s p i k e s  a r i s e through a retrograde  similarity  i n CAl pyramidal  comprehensive  Spikes  to identify the s i t e of o r i g i n  d e n s i t y , and i n t r a c e l l u l a r  both antidromic  layer  Cell Dendritic  discharge  from  membrane  (Andersen  and  Lomo 1 9 6 6 ; S p e n c e r and K a n d e l 1961b; T r a u b and L l i n a s 1 9 7 9 ; Wong e t a l . 1 9 7 9 ) . As a r e s u l t , s y n a p t i c d e p o l a r i z a t i o n c a n g i v e  rise  to  that  the i n i t i a l  activation of a spike at the d e n d r i t i c l e v e l  e l e c t r o t o n i c a l l y conducts t o the c e l l as a f a s t p r e - p o t e n t i a l 1977;  b o d y t o a p p e a r i n t h e soma  ( A n d e r s e n and Lomo 1 9 6 6 ;  Schwartzkroin  Wong e t a l . 1 9 7 9 ) . A s p i k e e v o k e d w i t h i n t h e d e n d r i t e  t h u s summate w i t h s y n a p t i c c u r r e n t s , i n c r e a s i n g t h e for  AP  discharge  at  t h e axon  hillock.  Several  can  probability  results of the  175  -  present  study  hypothesis. revealed the  argue  For  soma-axon  dendritic  locations.  was  Finally,  of  of  a  the  of  to  that  spike  from  s i n k was cells,  the observed  the  for  analysis evoked  in  and  in  not  the  the d e n d r i t i c  the  dendrite  above  change i n  spike along  predicted  .the  density  pyramidal  intracellular  spike recorded  extradendritic  analysis to  for  electrotonic  to the  cell  cell.  dendritic  spikes  dendrite  by  Rather, arise  a spike  at  the  the  body.  through  initiated  a  in  aligned CSD  Therefore,  the  of a d e n d r i t i c s i t e  of  indicate  retrograde the  of  through  dendritic  results  and  shown  body l a y e r .  contention  recorded  pyramidale,  potential  the c e l l  support the  spikes  pyramidal  i n stratum  field  conduct from  e v i d e n c e does not origin  region  of  i n t r a d e n d r i t i c s p i k e s were e v o k e d f o l l o w i n g t h e peak  the p o p u l a t i o n with  validity  current-source  In a d d i t i o n ,  the  opposite  conduction  the  shortest latency current  hillock  configuration axis  against  instance,  t h a t the  -  region  level  of  that  evoked  i n v a s i o n of of the  the  the  soma-axon  hillock.  I m p l i c a t i o n s of the Present The  findings  understanding  of  f a c t t h a t the cell  body  does not  presented pyramidal  Study in  t h i s work  cell  dendritic spike  implies immediately  physiology.  is  The  retrograde  For  our  generation  conduction  of a s p i k e  body.  In  fact,  in  of a s p i k e from the  the e l e c t r o t o n i c decay of a s p i k e from the d e n d r i t e cell  at  the the  spike a c t i v a t i o n  l a y e r a l s o i n d i c a t e s t h a t a f a s t p r e - p o t e n t i a l does not  pyramidal  current  instance,  evoked f o l l o w i n g t h a t  that dendritic  c o n t r i b u t e d i r e c t l y to the  axon h i l l o c k .  question  FPPs of s i m i l a r  the cell  reflect  towards  the  amplitude  and  - 176  -  w a v e f o r m were r e c o r d e d a t b o t h t h e and  the  change i n c o n f i g u r a t i o n  was  opposite  action  potential  Although  the  determined, previous spike  to that  dendritic level The  junction  results  threshold  d e n d r i t i c membrane  of the  of  origin  interpretation  of  electrotonically 1966;  Spencer  dendritic spike of s p i k e  decayed  M a c V i c a r and critical  the  fast  1961b).  was  t a k e n as  further  dendritic  and  dendro-somatic represent a were  that  the  following  pyramidale  the  and  fractionated  d i s c h a r g e at the at  suggest  spike A  dendritic  cell  hot  by spike  was  not  or  the in  dendritic  cell,  spots  and  the  represent  fractionation  and  an  Lomo the  evidence for multiple  sites  regions  (Wong e t  the the  of  and  originate the  spike  somatic  the  a l . 1979).  FPP  arisen  detected  the  does  fractionated  not  spikes  population spike  in  suggesting  subsequent to  on  at  retrograde along  body l a y e r . F u r t h e r m o r e , s p i k e  spots  of  activation  antidromic stimulation, had  somatic  Dudek 1 9 8 1 ) .  (Andersen  In a d d i t i o n , . peak  a  of  of  that  dendritic spike.  evoked  stratum  configuration axis  in  apparent  pyramidal c e l l ,  conduction  not  hot  the  the  was  Dendritic  s p i k e s were f o u n d t o  of  the  spike  pyramidal  dendritic  Kandel  level  tree.  evaluation  However, as d i s c u s s e d a b o v e , somatic  an  proposed  pre-potentials  and  in  at e i t h e r  s p o t s f o r Na+  in  somatic  generation  been  account f o r the  spikes  that  discharge  pyramidal c e l l .  were o r i g i n a l l y p r o p o s e d t o  decay of  e l e c t r o t o n i c c o n d u c t i o n of  for a  hot  dendrite  dendritic  have  located  also press  level,  along the  the  FPP  (Andrew e t a l . 1982;  e v i d e n c e f o r low  site  within  the  dendritic  a somatopetal  explanations  including  a gap  spike  underlying  alternative  across  for  from  mechanism  studies,  of the  predicted  arising  s o m a t i c and  spike  discharge  current-source  - 177 density profiles, spike discharge regions the  of  along  the  particularly  apical  spike at  and measurement  dendrite. a dendritic  intense  threshold  m i g h t be  hot spot  activation,  discharge  dendro-somatic axis  low  It  such  threshold d i d not  for  reveal  spike a c t i v a t i o n i n  argued t h a t  that  cell  for  only occurs  as  of the pyramidal  generation  during  of a  periods  of  found  during epileptiform  population.  However, p r e l i m i n a r y  analysis of multiple population s i m i l a r pattern of spike  of the voltage  spike discharge  has r e v e a l e d  a c t i v a t i o n as t h a t f o u n d  a  under normal  c o n d i t i o n s , w i t h each s p i k e  i n the burst o r i g i n a t i n g at the c e l l  body l a y e r . I t i s p o s s i b l e  that spike discharge  hot  spot  was  Nevertheless, supporting  simply  existence  i t may  characteristic of aspects  detected  a l t e r n a t i v e explanations  the  Therefore,  not  not  cell  the  dendritic  present  hot spots  in dendritic  be  necessary  to  spots  study.  c a n be o f f e r e d  of  d e n d r i t i c hot  of pyramidal  in  at a  invoke  to  f o r data membrane.  the s p e c i a l  account f o r  physiology.  The p r o p e r t i e s o f t h e e v o k e d d e n d r i t i c s p i k e w o u l d question  t h e p o s s i b l e means o f  dendro-somatic blockade of  axis.  a p p l i c a t i o n of tetrodotoxin  spike  is  Wong e t a l .  actively  conduction  Previous  the pyramidal  e t a l . 1982;  investigations  cell  apical  1979), suggesting through  on t h e  of the spike along  basis of a passive  the  have  injection that  a  through (Benardo  a voltage-dependent study  electrotonic  d e n d r i t i c a x i s . As d i s c u s s e d  be i n t e r p r e t e d as a p a s s i v e  the  the d e n d r i t i c  3, t h e i n v a s i o n o f t h e d e n d r i t i c r e g i o n b y a c u r r e n t could  shown  d e n d r i t i c spike  (TTX) o r QX-314  generated  further  of a spike along  c o n d u c t a n c e . However, t h e r e s u l t s o f t h e p r e s e n t be e x p l a i n e d  certain  d e p o l a r i z a t i o n and  can  Na+ also  conduction  in  Chapter  source/sink subsequent  - 178 active  spike  recording potential  generation  location. and  capacitance  of  gradual  dendritic decay  a  flow  high  change  field  of  the  electrotonus  in  conducted  biphasic  with  be e x p e c t e d the  component  of  the  for  electrotonic  ( i e a spike)  long time constant  waveform  the  positive/negative  would a l s o  associated  towards  along  (Nobles  spike  1966).  through  the  w o u l d t h e n be a f u n c t i o n o f t h e r a t e o f r i s e and intracellular could  dendritic  on  region The  at  laminar  the s p i k e through the proximal  a  for  the  conducting  profiles  of  may however  each l o c a t i o n .  account  potential  presence  radiatum  spike  thus  spike  stratum  a  frequency  postive/negative  potentials.  spike  However,  membrane w i t h a c o m p a r a t i v e l y The  a  current source/sink current  conduction  as  Passive biphasic  through  of e x t r a c e l l u l a r  current  the field  sink i n the proximal  i n d i c a t e an a c t i v e p r o p a g a t i o n  of  dendritic region.  I n t r a c e l l u l a r data would a l s o  support  the contention of  p a s s i v e e l e c t r o t o n i c decay o f t h e s p i k e through t h e m a j o r i t y the  dendritic  amplitude  arborization.  and i n c r e a s e d  Intracellular  i n h a l f w i d t h w i t h d i s t a n c e from t h e c e l l  at the l e v e l of the pyramidal f o r spike  e x p l a i n e d by  cell  axis.  4.8).  The  discharge  activation  b o d y . The l a c k o f a in  the  dendrite  conducted spike  only  voltage  could  be  superimposing  l e v e l o f d e p o l a r i z a t i o n a t any p o i n t a l o n g t h e  In fact, the  be a l t e r e d w i t h t h e no c l e a r  cell  an e l e c t r o t o n i c l l y  upon t h e e x i s t i n g  of  spikes declined i n  l a y e r , and d i s p l a y e d a s i n g l e c o n s i s t e n t v o l t a g e t h r e s h o l d  threshold  a  amplitude  l e v e l o f membrane p o l a r i z a t i o n ,  reversal potential evidence  of the d e n d r i t i c spike  for a  f o r the  suggesting  dendritic spike  Na+-dependence  could  (see F i g  of d e n d r i t i c spike  may r e l a t e t o t h e methods u s e d i n a p p l y i n g Na+  channel  -  blockers. In  the study  179  i n which  membrane, t h e d r u g was p e r f u s e d al.  -  TTX was  across  applied to dendritic  the entire s l i c e  (Wong e t  1 9 7 9 ) , and b l o c k a d e o f t h e d e n d r i t i c s p i k e may h a v e  occurred  secondarily t o i n h i b i t i o n of somatic spike discharge.  Similarly,  injection of the anesthetic  impalement  (Benardo e t a l . for  1982) c a n n o t n e c e s s a r i l y  be t a k e n as  a blockade of the spike at the d e n d r i t i c l e v e l ,  may h a v e proximal  diffused to  the region  of the  channel blockers  evidence  as t h e  drug  soma-axon h i l l o c k ( o r  dendrite). A test f o r the ionic basis of the  s p i k e may t h e r e f o r e  The  QX-314 i n t o a d e n d r i t i c  dendritic  r e q u i r e a more s e l e c t i v e a p p l i c a t i o n o f  t o d e n d r i t i c membrane o f t h e p y r a m i d a l  P o s s i b l e S i g n i f i c a n c e of D e n d r i t i c Spikes  Na+  cell.  to Pyramidal  Cell  Function The  retrograde  conduction  of a spike through the  a r b o r i z a t i o n c o u l d be o f i m p o r t a n t f u n c t i o n . For spike  invading  instance, the the  consequence t o pyramidal  large depolarization  dendrite  dendritic  would  rapidly  cell  i n d u c e d by a  depolarize  the  m a j o r i t y o f the d e n d r i t i c s t r u c t u r e over a short p e r i o d of time. Through  activation  unrelated  of  intrinsic  to spike discharge,  voltage-dependent  such a d e p o l a r i z a t i o n could  to increase the e f f e c t i v e length constant  of the c e l l ,  the t r a n s f e r o f s y n a p t i c c u r r e n t s t o the c e l l Sugimori 1984).  Alternatively, a  region of afferent termination of s y n a p t i c  d e p o l a r i z a t i o n by  inward s y n a p t i c will along  certainly the  cell  c u r r e n t s . The influence the axis,  channels  altering  layer  spike conducting  might a l s o l i m i t reducing  the  retrograde form o f the  serve  enhancing  (Llinas  through the  the time  course  driving force  invasion  and  of a  for spike  extracellular potentials  shape o f  the e x t r a c e l l u l a r  -  voltage The  gradient  i n the CAl region  dendritic spike w i l l  extracellular  potentials dendritic The might  level  pyramidal Under  the  and the  pyramidal  (Richardson  retrograde  also  flow,  i n determining  upon  (Richardson  et a l .  will  be  ephaptic  cell  at  to  conditions,  influence of f i e l d  b o t h t h e s o m a t i c and  of the spike through the dendrite  the  multiple  spike  activation  system  (Andersen  Schwartzkroin  and  pharmacological synaptic  spike  et  cell  of  by  a  recurrent  inhibitory  a l . 1969; D i n g l e d i n e  Prince  1980).  synaptic  i n the presence of  the action of  depolarization  depolarizing  shift  (PDS)  can  1980;  and W y l e r 1 9 7 9 ) . The PDS i s  thought  to  (Schwartzkroin  arise  i n part  from  voltage-dependent  Ca+2  channels  in  (Schwartzkroin  P r i n c e 1980;  and  P r i n c e 1979).  activation of  In f a c t ,  a dendritic  under t h e s e c o n d i t i o n s g i v e s  the inhibitory  may be t o p r e v e n t  previous  evoking  and  activation  Wyler 1979;  h a s shown t h a t  by a n t i d r o m i c  stimulation  r i s e t o Ca+2 s p i k e d i s c h a r g e  atthe  P r i n c e 1980). Therefore,  feedback network  to the  through the dendrite  of  membrane  one  pyramidal  t h e a c t i v a t i o n o f d e n d r i t i c Ca+2  t h e Na+ s p i k e c o n d u c t i n g  Prince  Ca+2-dependent,  and  work  a large  of  dendritic  Schwartzkroin  Na+ s p i k e  d e n d r i t i c l e v e l ( S c h w a r t z k r o i n and function of  the  inhibitory  elicit  capable  spike a c t i v a t i o n  Schwartzkroin  feedback  and Langmoen 1 9 8 0 ;  However,  block  e v o k e d by  i s restricted to  r e p e t i t i v e somatic  by  discharge  discharge  e f f e r e n t pathways  a g e n t s known t o  inputs,  paroxysmal  pyramidal  afferent or  single  cell  important  c e l l s thought c h a r a c t e r i s t i c o f e p i l e p t i f o r m a c t i v i t y .  normal  Wong and  an  e t a l . 1984a,b; T u r n e r e t a l . 1984).  conduction  contribute  stimulation of  and  1984b).  therefore modify the c h a r a c t e r i s t i c s o f  current  consideration  -  180  spikes  from t h e region  - 181 of the c e l l The the  layer.  p r e s e n t s t u d y has  pyramidal c e l l  demonstrated  a r i s e through  d e n d r i t e by  a spike  initiated  hillock,  finding  that  a  present concept of  activity  including  been  the dentate  shown  comparable of  pyramidal c e l l  to the  spikes of  retrograde i n v a s i o n of the region  of the  to  exist  Llinas  and  1979),  Sugimori  (Furshpan  and  i n our pattern  i n o t h e r CNS  (Jefferys  the  soma-axon  physiology. 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