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Action potential discharge in somata and dendrites of CA1 pyramidal neurons of mammalian hippocampus.. 1985

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ACTION POTENTIAL DISCHARGE IN SOMATA AND DENDRITES OF CA1 PYRAMIDAL NEURONS OF MAMMALIAN HIPPOCAMPUS: AN ELECTROPHYSIOLOGICAL ANALYSIS by RAY W. TURNER B . S c , U n i v e r s i t y 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 i n THE FACULTY OF GRADUATE STUDIES (Department of Physiology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1985 © Raymond W i l l i a m Turner, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (.3/81) ABSTRACT The e l e c t r o p h y s i o l o g i c a l p r o p e r t i e s o f s o m a t i c and d e n d r i t i c membranes o f CA1 p y r a m i d a l neurons were i n v e s t i g a t e d u s i n g t h e r a t in v i t r o hippocampal s l i c e p r e p a r a t i o n . A comprehensive a n a l y s i s o f 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 , c u r r e n t - s o u r c e d e n s i t y (CSD) and i n t r a c e l l u l a r a c t i v i t y has s e r v e d t o i d e n t i f y the s i t e o f o r i g i n o f a c t i o n p o t e n t i a l (AP) d i s c h a r g e i n C A l p y r a m i d a l neurons. 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 o f C A l p y r a m i d a l c e l l s was evoked by s u p r a t h r e s h o l d s t i m u l a t i o n o f the a l v e u s ( a n t i d r o m i c ) or a f f e r e n t s y n a p t i c i n p u t s i n s t r a t u m o r i e n s (SO) or s t r a t u m r a d i a t u m (SR). Laminar p r o f i l e s o f t h e " s t i m u l u s evoked" 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 were r e c o r d e d a t 25jum i n t e r v a l s a l o n g t h e dendro-somatic a x i s o f the p y r a m i d a l c e l l 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 l a t e n c y p o p u l a t i o n s p i k e response and c u r r e n t s i n k was r e c o r d e d i n s t r a t u m p y r a m i d a l e or t h e p r o x i m a l s t r a t u m o r i e n s , a r e g i o n c o r r e s p o n d i n g t o somata and axon h i l l o c k s o f C A l p y r a m i d a l neurons. A b i p h a s i c p o s i t i v e / n e g a t i v e s p i k e p o t e n t i a l ( c u r r e n t s o u r c e / s i n k ) was r e c o r d e d i n d e n d r i t i c r e g i o n s , w i t h b o t h components i n c r e a s i n g i n peak l a t e n c y t h r o u g h t h e d e n d r i t i c f i e l d w i t h d i s t a n c e from t h e b o r d e r o f s t r a t u m p y r a m i d a l e . 3) A c o m p a r a t i v e i n t r a c e l l u l a r a n a l y s i s o f evoked a c t i v i t y i n so m a t i c and d e n d r i t i c membranes r e v e a l e d a b a s i c s i m i l a r i t y i n the p a t t e r n o f AP d i s c h a r g e a t a l l l e v e l s o f t h e dendro-somatic a x i s . S t i m u l a t i o n o f t h e a l v e u s , SO, or SR evoked a s i n g l e s p i k e w h i l e i n j e c t i o n o f d e p o l a r i z i n g c u r r e n t evoked a r e p e t i t i v e i i i t r a i n o f s p i k e s grouped f o r c o m p a r a t i v e purposes i n t o t h r e e b a s i c p a t t e r n s o f AP d i s c h a r g e . 4) Both c u r r e n t and s t i m u l u s evoked i n t r a c e l l u l a r s p i k e s d i s p l a y e d a p r o g r e s s i v e d e c l i n e i n a m p l i t u d e and i n c r e a s e i n h a l f w i d t h w i t h d i s t a n c e from the b o r d e r of s t r a t u m p y r a m i d a l e . 5) The o n l y 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 f o r i n t r a c e l l u l a r s p i k e d i s c h a r g e was found i n t h e r e g i o n o f t h e c e l l body, w i t h no a pparent t h r e s h o l d f o r s p i k e a c t i v a t i o n i n d e n d r i t i c l o c a t i o n s . 6 ) S t i m u l u s evoked i n t r a d e n d r i t i c s p i k e s were evoked beyond the peak of t h e p o p u l a t i o n s p i k e r e c o r d e d i n s t r a t u m p y r a m i d a l e , and a l i g n e d w i t h t h e b i p h a s i c e x t r a d e n d r i t i c f i e l d p o t e n t i a l shown t h r o u g h l a m i n a r p r o f i l e a n a l y s i s t o conduct w i t h i n c r e a s i n g l a t e n c y from th e c e l l body l a y e r . The evoked c h a r a c t e r i s t i c s o f 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 CAl p y r a m i d a l c e l l s are i n t e r p r e t e d t o i n d i c a t e t h e i n i t i a l g e n e r a t i o n o f a s p i k e i n t h e r e g i o n o f t h e soma-axon h i l l o c k and a subsequent r e t r o g r a d e s p i k e i n v a s i o n o f d e n d r i t i c a r b o r i z a t i o n s . i v ACKNOWLEDGEMENTS For s u p p o r t and guidance over t h e c o u r s e o f my graduate program I am i n d e b t e d t o Dr. James J . M i l l e r . I would a l s o l i k e t o e x p r e s s my s i n c e r e thanks t o Thomas L. R i c h a r d s o n f o r d a t a 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 a d v i c e , and f o r t h e e n d l e s s hours o f d i s c u s s i o n , s u p p o r t and c o l l a b o r a t i v e e f f o r t t h a t made t h i s work p o s s i b l e . Thanks a l s o t o o t h e r members o f the l a b o r a t o r y and department whose c o n t r i b u t i o n s and comments were g r e a t l y a p p r e c i a t e d . I g r a t e f u l l y acknowledge t h e e x p e r t a s s i s t a n c e o f K u r t Henze and Rob Andersen i n photography and p r e p a r a t i o n of f i g u r e s . I a l s o w i s h t o thank the members o f t h e a d v i s o r y committee and Dr. P. C a r l e n , t h e e x t e r n a l examiner f o r r e v i e w i n g t h i s t h e s i s . F i n a l l y , I would l i k e t o acknowledge 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 E x a m i n a t i o n i A b s t r a c t i i Acknowledgements i v T a b l e o f C o n t e n t s v L i s t o f F i g u r e s v i i i L i s t o f T a b l e s x i 1-0. INTRODUCTION 1 1-1. Anatomy 1 - The Hippocampal F o r m a t i o n 1 - Laminae and Neuronal Elements o f Regio S u p e r i o r (CAl) 2 - A f f e r e n t S y n a p t i c I n p u t s t o Regio S u p e r i o r 6 1) S c h a f f e r C o l l a t e r a l s 7 2) Commissural P r o j e c t i o n s 7 1-2. The Hippocampal S l i c e P r e p a r a t i o n 8 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 CA l P y r a m i d a l Neurons 9 1) Evoked 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) 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 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. R e c o r d i n g Chamber 21 2- 3. S t i m u l a t i n g and R e c o r d i n g Techniques 22 3- 0. CURRENT-SOURCE DENSITY ANALYSIS OF ACTION POTENTIAL DISCHARGE IN THE CAl REGION OF THE HIPPOCAMPUS 27 3- 1. I n t r o d u c t i o n 27 v i 3-2. Methods 31 - 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 i o n s 31 - S t i m u l a t i n g and R e c o r d i n g P r o c e d u r e s 35 3-3. R e s u l t s 40 - 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 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 C A l 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 - S u b t h r e s h o l d E x c i t a t o r y S y n a p t i c P o t e n t i a l s 47 - Orthodromic P o p u l a t i o n S p i k e Responses 52 3- 4. D i s c u s s i o n 65 4- 0. COMPARATIVE INTRACELLULAR ANALYSIS OF SOMATIC AND DENDRITIC ELECTROPHYSIOLOGY OF THE C A l PYRAMIDAL NEURON 73 4-1. I n t r o d u c t i o n 73 4-2. Methods 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 S y n a p t i c P o t e n t i a l s . . 93 - S t i m u l u s Evoked S u p r a t h r e s h o l d Responses 96 - Comparison o f S u p r a t h r e s h o l d S t i m u l u s and C u r r e n t Evoked S p i k e s 105 - S p i k e 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 117 - Membrane P r o p e r t i e s and Evoked S y n a p t i c P o t e n t i a l s 117 - C u r r e n t Evoked S p i k e s 120 v i i - 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 - S p i k e P r e - P o t e n t i a l s 127 5-0. EVOKED CHARACTERISTICS OF ACTION POTENTIAL DISCHARGE ALONG THE DENDRO-SOMATIC AXIS OF THE CA l PYRAMIDAL NEURON 131 5-1. I n t r o d u c t i o n 131 5-2. Methods 134 5-3. R e s u l t s 139 - 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 - V o l t a g e T h r e s h o l d o f Orthodromic S p i k e D i s c h a r g e . . 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 S p i k e F r a c t i o n a t i o n 156 - 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 Elements by K n i f e 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 A l o n g the Dendro-Somatic A x i s 165 - D e n d r i t i c S p i k e 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 171 6- 0. GENERAL SUMMARY AND DISCUSSION 174 - The S i t e of O r i g i n o f P y r a m i d a l C e l l D e n d r i t i c S p i k e s 174 - I m p l i c a t i o n s o f t h e P r e s e n t Study 175 - The 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 S p i k e s t o P y r a m i d a l C e l l F u n c t i o n 179 REFERENCES 182 v i i i LIST OF FIGURES FIG. 1.1: Schematic i l l u s t r a t i o n o f t h e i n t r i n s i c s y n a p t i c o r g a n i z a t i o n o f t h e hippocampal s l i c e . . . 3 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 f i e l d p o t e n t i a l s i n the CA1 r e g i o n 10 FIG. 3.1: Schematic diagram o f the r a t p y r a m i d a l c e l l and placement 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 the CA1 r e g i o n 36 FIG. 3.2: Laminar p r o f i l e s o f a l v e a r a n t i d r o m i c evoked 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 and c u r r e n t - s o u r c e d e n s i t y a l o n g t h e p y r a m i d a l c e l l a x i s 44 FIG. 3.3: Laminar p r o f i l e s o f 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 and c u r r e n t - s o u r c e d e n s i t y evoked t h r o u g h s u b t h r e s h o l d s t i m u l a t i o n o f s t r a t u m o r i e n s o r s t r a t u m r a d i a t u m 49 FIG. 3.4: Laminar p r o f i l e s o f 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 and c u r r e n t - s o u r c e d e n s i t y evoked t h r o u g h s u p r a t h r e s h o l d s t i m u l a t i o n o f s t r a t u m o r i e n s 53 FIG. 3.5: Laminar p r o f i l e s o f 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 and c u r r e n t - s o u r c e d e n s i t y evoked t h r o u g h s u p r a t h r e s h o l d 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 57 FIG. 3.6: A comparison o f t h e t i m i n g r e l a t i o n s h i p between evoked 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 and c u r r e n t - s o u r c e d e n s i t y a t the s o m a t i c and a p i c a l d e n d r i t i c l e v e l o f the p y r a m i d a l c e l l 60 FIG. 3.7: Peak l a t e n c y o f the n e g a t i v e v o l t a g e p o t e n t i a l i x and c u r r e n t s i n k a l o n g t h e p y r a m i d a l c e l l a x i s f o l l o w i n g s u p r a t h r e s h o l d s t i m u l a t i o n o f t h e a l v e u s or s t r a t u m r a d i a t u m 62 FIG. 4.1: Somatic and a p i c a l d e n d r i t i c membrane p o t e n t i a l response t o a s e r i e s o f square wave i n t r a c e l l u l a r c u r r e n t p u l s e i n j e c t i o n s 81 FIG. 4.2: "Type 1" c u r r e n t 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 s o m a t i c and d e n d r i t i c membranes 86 FIG. 4.3: "Type 2" and "Type 3" c u r r e n t 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 s o m a t i c and d e n d r i t i c membranes 90 FIG. 4.4: 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 p o t e n t i a l s i n p y r a m i d a l c e l l soma and a p i c a l d e n d r i t e 94 FIG. 4.5: 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 p y r a m i d a l c e l l soma and a p i c a l d e n d r i t e 97 FIG. 4.6: C h a r a c t e r i s t i c s o f s t r a t u m r a d i a t u m 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 p y r a m i d a l c e l l soma and a p i c a l d e n d r i t e 100 FIG. 4.7: Somatic and a p i c a l d e n d r i t i c response t o 10Hz r e p e t i t i v e s t i m u l a t i o n of s t r a t u m r a d i a t u m a f f e r e n t s y n a p t i c i n p u t s 103 FIG. 4.8: Comparison o f s t i m u l u s and c u r r e n t evoked a c t i o n p o t e n t i a l s i n t h e 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 106 FIG. 4.9: C h a r a c t e r i s t i c s o f f a s t p r e - p o t e n t i a l d i s c h a r g e i n p y r a m i d a l c e l l soma and a p i c a l d e n d r i t e I l l FIG. 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 " i n i t i a l segment" s p i k e s i n p y r a m i d a l c e l l soma X and a p i c a l d e n d r i t e 114 FIG. 5.1: R e p r e s e n t a t i v e photographs o f s t i m u l u s evoked s p i k e s a t v a r i o u s l o c a t i o n s a l o n g t h e dendro-somatic a x i s o f t h e p y r a m i d a l c e l l 140 FIG. 5.2: P l o t s o f s t i m u l u s evoked s p i k e a m p l i t u d e a l o n g the d e n d ro-somatic a x i s o f t h e p y r a m i d a l c e l l . . . . 1 4 2 FIG. 5.3: P l o t s o f s t i m u l u s evoked s p i k e h a l f w i d t h a l o n g t h e dendro-somatic a x i s o f the p y r a m i d a l c e l l 145 FIG. 5.4: P l o t s o f c u r r e n t evoked s p i k e a m p l i t u d e and h a l f w i d t h a l o n g the dendro-somatic a x i s o f the p y r a m i d a l c e l l 147 FIG. 5.5 P l o t o f v o l t a g e t h r e s h o l d f o r o r t h o d r o m i c s p i k e d i s c h a r g e a l o n g the dendro-somatic a x i s o f t h e p y r a m i d a l c e l l 150 FIG. 5.6: Comparison of s t i m u l u s evoked 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 t o 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 i n s t r a t u m p y r a m i d a l e and radiatum....154 FIG. 5.7: F r a c t i o n a t i o n o f s t i m u l u s evoked i n t r a d e n d r i t i c s p i k e s 157 FIG. 5.8: S t i m u l u s evoked a c t i v i t y of d e n d r i t e s i s o l a t e d from t h e c e l l body by a k n i f e c u t i n t h e C A l r e g i o n 162 x i LIST OF TABLES TABLE 1: Average r e s t i n g membrane p o t e n t i a l and i n p u t r e s i s t a n c e o f s o m a t i c and a p i c a l d e n d r i t i c impalements o f t h e p y r a m i d a l c e l l 83 TABLE 2: Average a m p l i t u d e and h a l f w i d t h o f c u r r e n t and s t i m u l u s evoked i n t r a c e l l u l a r s p i k e s i n t h e p y r a m i d a l c e l l soma and a p i c a l d e n d r i t e 108 1-0. INTRODUCTION - 1 - 1-1. Anatomy The Hippocampal F o r m a t i o n The hippocampus i s a f o r e b r a i n c o r t i c a l s t r u c t u r e found as a s m a l l gyrus beneath t h e i n f e r i o r horn o f t h e l a t e r a l v e n t r i c l e . P r i m i t i v e homologues o f t h e hippocampus can be t r a c e d as f a r back as t h e c y c l o s t o m e s (Angevine 1975), w i t h a p r o g r e s s i v e development o f the s t r u c t u r e t h r o u g h v e r t e b r a t e phylogeny. A l t h o u g h c o m p r i s i n g a major p o r t i o n o f lower mammalian c o r t i c a l t i s s u e , t h e e x p a n s i o n o f n e o c o r t e x and 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 s i z e of t he hippocampus i n more advanced v e r t e b r a t e forms. For i n s t a n c e , i n t h e r a t , t h e hippocampus forms a l o n g t u b e - l i k e 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 t e m p o r a l p o l e beneath t h e f l o o r o f t h e l a t e r a l v e n t r i c l e . I n man, t h e hippocampus 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 , f o r m i n g the f l o o r o f the i n f e r i o r horn o f t h e l a t e r a l v e n t r i c l e and e x t e n d i n g from w i t h i n a few c e n t i m e t e r s o f t h e t e m p o r a l p o l e t o t h e s p l e n i u m o f t h e corpus c a l l o s u m . In c o r o n a l s e c t i o n , t h e hippocampal f o r m a t i o n appears t o a r i s e as an e x t e n s i o n o f the e n t o r h i n a l c o r t e x f o l d e d back on i t s e l f t o form two i n t e r l o c k i n g g y r i , t h e hippocampus p r o p e r ( h e r e a f t e r r e f e r r e d t o as hippocampus), and d e n t a t e g y r u s ( F i g 1.1). Ramon y C a j a l (1911) d i v i d e d t h e hippocampus i n t o r e g i o s u p e r i o r and r e g i o i n f e r i o r , i n r e f e r e n c e t o t h e o r g a n i z a t i o n of the p r i n c i p l e c e l l t y p e o f t h e s t r u c t u r e , t h e p y r a m i d a l neuron ( F i g 1.1). L o r e n t e de No (1934) f u r t h e r s u b d i v i d e d t h e - 2 - hippocampus i n t o f o u r f i e l d s a b b r e v i a t e d by t h e l e t t e r s CA (Cornu Ammonis). Of t h e s e t h e CA1 and CA3 a r e a s were d i v i d e d i n t o r e g i o n s d e s i g n a t e d a, b, and c a c c o r d i n g t o c y t o l o g i c a l f e a t u r e s or e f f e r e n t p r o j e c t i o n s o f p y r a m i d a l neurons i n each f i e l d . The CA1 r e g i o n d e f i n e s t h e d o r s a l a s p e c t o f p y r a m i d a l c e l l s , CA2 and CA3 t h e g i a n t p y r a m i d a l c e l l s i n t h e v e n t r a l a s p e c t o f the hippocampus, and CA4 t h e " m o d i f i e d " p y r a m i d a l c e l l s l o c a t e d w i t h i n t h e h i l a r r e g i o n . The boundary between CA2 and CA1 i s t a k e n as t h e l i m i t between r e g i o s u p e r i o r and r e g i o i n f e r i o r . As t h e p r e s e n t s t u d y was c o n f i n e d t o p y r a m i d a l c e l l s i n t h e CA1 r e g i o n o f t h e hippocampus, f u r t h e r a n a t o m i c a l d e s c r i p t i o n w i l l be r e s t r i c t e d t o r e g i o s u p e r i o r . Laminae and Neuronal Elements o f Regio S u p e r i o r (CAD Regio s u p e r i o r can be d i v i d e d i n t o s i x s t r a t a r u n n i n g p a r a l l e l t o t h e c e l l l a y e r e x t e n d i n g from t h e v e n t r i c u l a r e p i t h e l i a l s u r f a c e t o t h e hippocampal f i s s u r e ( C a j a l 1911). The most o b v i o u s s t r a t u m i s t h e s t r a t u m p y r a m i d a l e , a l a y e r o f packed p y r a m i d a l c e l l somata t h r e e t o f o u r c e l l s deep w i t h t h e b a s a l and a p i c a l d e n d r i t i c s h a f t s o f p y r a m i d a l c e l l s e x t e n d i n g p e r p e n d i c u l a r t o t h e c e l l l a y e r ( F i g 1.1). One or more d e n d r i t e s a r i s e from t h e b a s a l p o r t i o n o f t h e p y r a m i d a l c e l l and one l a r g e d e n d r i t e from t h e a p i c a l r e g i o n , g i v i n g the c e l l a t r i a n g u l a r or " p y r a m i d a l " appearance. The b a s a l d e n d r i t e s undergo p r o f u s e b r a n c h i n g 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 extend i n t o t h e s t r a t u m o r i e n s and o c c a s i o n a l l y i n t o t h e a l v e a r r e g i o n . The a p i c a l d e n d r i t e p r o j e c t s r a d i a l l y t h r o u g h t h e s t r a t u m r a d i a t u m , lacunosum and m o l e c u l a r e , t h e l a t t e r two o f t e n r e f e r r e d t o as s t r a t u m lacunosum-moleculare i n t h e r a t ( F i g 1.1; C a j a l 1911). - 3 - FIG. 1.1 U l t r a s t r u c t u r a l anatomy of the t r a n s v e r s e hippocampal s l i c e . Recordings were r e s t r i c t e d to the CAl f i e l d of Regio S u p e r i o r . N o t a t i o n s : S . O r i . = stratum o r i e n s S.Pyr. = stratum pyramidale S.Rad. = stratum radiatum S.Lac. = stratum lacunosum S.Mol. = stratum moleculare S.C. = S c h a f f e r c o l l a t e r a l s B.C. = basket c e l l CA I (Regio Superior) Area Dentata - 5 - The apical dendrite i s characterized by a thick proximal shaft in stratum radiatum with a greater degree of arborization occurring in the mid-distal stratum radiatum. In the rat hippocampus, many apical dendrites exhibit profuse l a t e r a l branching at the border of stratum lacunosum-moleculare, although the maximal l i m i t of dendritic extension i s found at the hippocampal f i s s u r e . Dendritic spines are r e l a t i v e l y sparse on the proximal apical dendritic shaft but are profusely dis t r i b u t e d on dendritic branches, increasing in number toward stratum lacunosum-moleculare (Westrum and Blackstad 1962). The axons of CA1 pyramidal neurons course r a d i a l l y through stratum oriens and consolidate within the alvear white matter to project to the subicular pyramidal c e l l layer or to jo i n efferent f i b e r s of the fimbria (Knowles and Schwartzkroin 1981b; Lorente de No 1934). Ramon y Cajal (1911) and Lorente de No (1934) described at least thirteen neuronal c e l l types in regio superior d i s t i n c t from the pyramidal neuron. However, the most common of these are the basket c e l l s , a set of interneurons believed to mediate recurrent i n h i b i t i o n of pyramidal c e l l s (Allen et a l . 1977; Andersen et a l . 1964,1969; Dingledine and Langmoen 1980; Knowles and Schwartzkroin 1981a; Tombol et a l . 1979). Although c y t o l o g i c a l features can be used to distinguish between several subtypes of basket c e l l s (Lorente de No 1934), certain c h a r a c t e r i s t i c s can be considered common to each c l a s s . The c e l l bodies of basket c e l l s are generally larger than adjacent pyramidal neurons, and are located within stratum pyramidale or stratum oriens. Most have a basal and apical dendritic tree extending p a r a l l e l to pyramidal c e l l dendrites within stratum - 6 - oriens and stratum radiatum, respectively. The axon extends into stratum radiatum and lacunosum-moleculare, sending several c o l l a t e r a l s back to form horizontal projections in the proximal stratum radiatum, with f i n a l termination in the form of a basket-like plexus around pyramidal c e l l somata and proximal dendrites (Lorente de No 1934). A major source of excitatory synaptic input to these c e l l s i s derived from c o l l a t e r a l projections of CAl pyramidal c e l l axons. Basket c e l l interneurons are thought to be i n h i b i t o r y in nature, each providing a potent recurrent i n h i b i t i o n of somata, proximal dendrites and axon h i l l o c k s of as many as 500 pyramidal neurons (Andersen et a l . 1969; Knowles and Schwartzkroin 1981a; Somogyi et a l . 1983). In addition, interneurons with anatomical and physiological c h a r a c t e r i s t i c s similar to that of basket c e l l s have also been proposed to bring about a feed-forward i n h i b i t i o n of pyramidal neurons in the CAl region (Alger and N i c o l l 1982; Andersen et a l . 1969; Ashwood et a l . 1984; Knowles and Schwartzkroin 1981a; Schwartzkroin and Mathers 1978). Thus, through formation of extensive synaptic contact with pyramidal neurons, basket c e l l s are believed to be responsible for the major balance of i n h i b i t o r y influence in the region. Afferent Synaptic Inputs to Regio Superior A c h a r a c t e r i s t i c feature of the hippocampus i s the lamellar organization of afferent synaptic inputs and the laminar d i s t r i b u t i o n of terminal projections onto basal and apical dendritic structures (Andersen et a l . 1971a; Raisman et a l . 1965). For instance, the majority of afferent synaptic inputs enter the hippocampus approximately perpendicular to the - 7 - septo-hippocampal p o l e , and p r o j e c t i n a l a m i n a r f a s h i o n t o form s y n a p t i c c o n t a c t a l o n g d i s c r e t e r e g i o n s o f t h e dendro-somatic a x i s (Raisman e t a l . 1965). A l t h o u g h a wide v a r i e t y o f 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 systems i n n e r v a t e t h e CA1 r e g i o n , t h e p r e s e n t s t u d y was c o n f i n e d t o two o f t h e major a f f e r e n t s , t h e S c h a f f e r c o l l a t e r a l and t h e com m i s s u r a l i n p u t s t o r e g i o s u p e r i o r . 1) S c h a f f e r C o l l a t e r a l s A major i n p u t from r e g i o i n f e r i o r o f t h e hippocampus t o CA1 was f i r s t d e s c r i b e d by S c h a f f e r i n 1892. T h i s system o r i g i n a t e s as an axon 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 c e l l s s h o r t l y b e f o r e t h e e f f e r e n t f i b e r e n t e r s t h e f i m b r i a . These c o u r s e back t h r o u g h s t r a t u m o r i e n s and p y r a m i d a l e o f CA2 and the n t u r n down t o p r o j e c t t h r o u g h s t r a t u m r a d i a t u m o f r e g i o s u p e r i o r ( L o r e n t e de No 1934). There, en-passage 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 a re formed on t h e a p i c a l d e n d r i t e s o f CA1 p y r a m i d a l neurons (Andersen and Lomo 1966; Westrum and B l a c k s t a d 1962). 2) Commissural P r o j e c t i o n s Commissural i n p u t s t o t h e CA1 r e g i o n o f the d o r s a l hippocampus a r i s e i n l a r g e p a r t from t h e CA3 f i e l d o f t h e c o n t r a l a t e r a l hippocampus (Raisman e t a l . 1965). E f f e r e n t CA3 f i b e r s e n t e r t h e f i m b r i a and proceed i n a r o s t r a l d i r e c t i o n t o c r o s s t h e m i d l i n e i n t h e v e n t r a l p s a l t e r i u m . There t h e f i b e r s t u r n and p r o g r e s s caudad t h r o u g h t h e c o n t r a l a t e r a l f i m b r i a and a l v e u s t o e n t e r r e g i o s u p e r i o r and form 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 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 p y r a m i d a l neuron ( B l a c k s t a d 1956; Raisman e t a l . 1965). - 8 - 1-2. The Hippocampal S l i c e P r e p a r a t i o n The l a m i n a r o r g a n i z a t i o n o f t h e hippocampus and the a n a t o m i c a l s p e c i f i c i t y o f s y n a p t i c i n p u t s onto p y r a m i d a l neurons make t h i s system an i d e a l model f o r t h e s t u d y o f t h e 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 c o r t i c a l neurons i n t h e mammalian CNS. O r i g i n a l i n v e s t i g a t i o n s i n t o t h e evoked e l e c t r i c a l a c t i v i t y o f t h e hippocampus were performed t h r o u g h s t e r e o t a x i c 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 e l e c t r o d e s i n t h e a c u t e , a n e s t h e t i z e d a n i m a l . A l t h o u g h a g r e a t d e a l o f i n f o r m a t i o n has been g a i n e d i n t h i s way, t h e r e a re s e v e r a l drawbacks t o t h i s approach, i n c l u d i n g the e f f e c t s o f a n e s t h e t i c s on evoked p o t e n t i a l s , and d i f f i c u l t i e s i n d e t e r m i n i n g t h e e x a c t s i t e o f r e c o r d i n g and s t i m u l a t i n g e l e c t r o d e s . Many o f t h e s e problems have been overcome t h r o u g h development o f t h e i n v i t r o s l i c e p r e p a r a t i o n (Skrede and Westgaard 1971). W i t h t h i s t e c h n i q u e , s m a l l s e c t i o n s o f n e u r o n a l t i s s u e can be m a i n t a i n e d i n v i t r o i n t h e absence o f a n e s t h e t i c , and are found t o e x h i b i t 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 comparable t o t h o s e o b s e r v e d i n t h e i n t a c t a n i m a l ( S c h w a r t z k r o i n 1975,1977). The l a m i n a r o r g a n i z a t i o n o f t h e hippocampus makes t h i s s t r u c t u r e p a r t i c u l a r l y w e l l s u i t e d t o t h e a p p l i c a t i o n o f t h e s l i c e t e c h n i q u e . For example, a s l i c e t a k e n p e r p e n d i c u l a r t o t h e l o n g i t u d i n a l a x i s o f t h e hippocampus p r e s e r v e s t h e m a j o r i t y o f a f f e r e n t s y n a p t i c i n p u t s , and s t i m u l a t i n g and r e c o r d i n g e l e c t r o d e s can be a c c u r a t e l y p l a c e d w i t h i n t h e major s t r a t a o f 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 o b s e r v a t i o n . T h e r e f o r e , t h r o u g h use o f t h e s l i c e p r e p a r a t i o n a l l s t r a t a o f t h e C A l r e g i o n become a c c e s s i b l e t o e l e c t r o p h y s i o l o g i c a l a n a l y s i s , - 9 - p e r m i t t i n g a c h a r a c t e r i z a t i o n o f evoked a c t i v i t y a l o n g t h e e n t i r e d e n d ro-somatic a x i s o f the p y r a m i d a l neuron. For t h e s e r e a s o n s , t h e hippocampal s l i c e t e c h n i q u e was used i n t h e p r e s e n t s t u d y . 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 C A l P y r a m i d a l Neurons 1)Evoked 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 The 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 f i e l d p o t e n t i a l s o f CA l p y r a m i d a l neurons evoked by s t i m u l a t i o n o f a f f e r e n t or e f f e r e n t pathways have been d e s c r i b e d i n d e t a i l i n p r e v i o u s i n v e s t i g a t i o n s (Andersen 1960; Andersen e t a l . 1966a,b; Andersen and Lomo 1966; Cragg and Hamlyn 1955; G l o o r e t a l . 1963; Leung 1979a,b,c; S p e r t i e t a l . 1967). E l e c t r i c a l s t i m u l a t i o n o f p y r a m i d a l c e l l axons w i t h i n t h e a l v e a r r e g i o n evokes an a n t i d r o m i c response w i t h i n p y r a m i d a l neurons, 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 n e g a t i v e - g o i n g p o t e n t i a l i n s t r a t u m p y r a m i d a l e ( F i g 1.2A). T h i s waveform r e p r e s e n t s t h e summed synchronous d i s c h a r g e o f a p o p u l a t i o n o f p y r a m i d a l neurons, and i s r e f e r r e d t o as an 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 " (Andersen e t a l . 1971b). A c t i v a t i o n o f S c h a f f e r c o l l a t e r a l / c o m m i s s u r a l (Sch/Comm) a f f e r e n t i n p u t s i n s t r a t u m r a d i a t u m 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 on t h e r i s i n g edge of a l a r g e r , l o n g e r d u r a t i o n n e g a t i v i t y i n s t r a t u m r a d i a t u m ( F i g 1.2B). The i n i t i a l component i s graded i n n a t u r e and independent of e x t r a c e l l u l a r c a l c i u m (arrows i n F i g 1.2B,C), e v i d e n t as a t r i p h a s i c p o t e n t i a l i n low c a l c i u m medium (expanded i n F i g - 10 - FIG.1.2 C h a r a c t e r i s t i c s 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 i n the CA1 r e g i o n . A. An 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 r e c o r d e d i n s t r a t u m p y r a m i d a l e evoked t h r o u g h s t i m u l a t i o n o f e f f e r e n t p y r a m i d a l c e l l axons i n t h e a l v e a r r e g i o n , shown f o r i n c r e a s i n g i n t e n s i t i e s o f s t i m u l a t i o n . B. The e x c i t a t o r y p o s t s y n a p t i c p o t e n t i a l (EPSP) and p o p u l a t i o n s p i k e evoked t h r o u g h s t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s i n s t r a t u m r a d i a t u m . Both t h e s o m a t i c ( S t . Pyr.) and a p i c a l d e n d r i t i c response ( S t . Rad.) are shown f o r i n c r e a s i n g s t i m u l u s i n t e n s i t i e s . The arrow denotes t h e f i b e r p o t e n t i a l a s s o c i a t e d w i t h a c t i v i t y o f a f f e r e n t a x o n a l p r o j e c t i o n s i n s t r a t u m r a d i a t u m . C. The e f f e c t o f l o w e r i n g the e x t r a c e l l u l a r Ca+2 c o n c e n t r a t i o n from 1.6mM t o .ImM on s t r a t u m r a d i a t u m evoked s o m a t i c ( S t . Pyr.) and a p i c a l d e n d r i t i c p o t e n t i a l s ( S t . Rad.). Note t h e g r a d u a l d e c l i n e i n a m p l i t u d e o f the EPSP and p o p u l a t i o n s p i k e , r e v e a l i n g a Ca+2-independent f i b e r p o t e n t i a l 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 ( a r r o w ) . D. An expanded view o f t h e f i b e r p o t e n t i a l i n C shown a t a h i g h e r s t i m u l u s i n t e n s i t y , i l l u s t r a t i n g t he t r i p h a s i c n a t u r e o f the f i b e r p o t e n t i a l i n t h e absence o f an e x t r a c e l l u l a r EPSP.  1.2D). T h i s waveform i s t h e compound a c t i o n p o t e n t i a l a s s o c i a t e d w i t h t h e a c t i v i t y o f Sch/Comm axons i n s t r a t u m r a d i a t u m and i s r e f e r r e d t o as a " f i b e r p o t e n t i a l " . The second component i s a smooth graded n e g a t i v e waveform w i t h a maximal n e g a t i v i t y i n t h e r e g i o n o f Sch/Comm a f f e r e n t i n p u t s (Andersen 1960; Andersen e t a l . 1966a; Andersen and Lomo 1966; Leung 1979c). T h i s p o t e n t i a l i s c alcium-dependent ( F i g 1.2C) and r e p r e s e n t s t h e e x t r a c e l l u l a r r e f l e c t i o n o f the e x c i t a t o r y p o s t s y n a p t i c p o t e n t i a l (EPSP) a s s o c i a t e d w i t h s y n a p t i c d e p o l a r i z a t i o n 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 (Andersen 1960; Andersen and Lomo 1966; Leung 1979c). The EPSP conducts 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 f i e l d , d e c l i n i n g i n a m p l i t u d e and r e v e r s i n g i n p o l a r i t y j u s t p r o x i m a l t o t h e c e l l l a y e r t o appear as a p o s i t i v e - g o i n g p o t e n t i a l i n s t r a t u m p y r a m i d a l e ( F i g 1.2B). W i t h h i g h e r s t i m u l u s i n t e n s i t i e s , a s harp n e g a t i v e - g o i n g " p o p u l a t i o n s p i k e " i s evoked upon t h e p o s i t i v e - g o i n g s y n a p t i c waveform i n s t r a t u m p y r a m i d a l e , r e f l e c t i n g t h e synchronous d i s c h a r g e o f p y r a m i d a l neurons f o l l o w i n g s y n a p t i c d e p o l a r i z a t i o n o f t h e c e l l p o p u l a t i o n (Andersen e t a l . 1971b; F i g 1.2B). A c t i v a t i o n o f commissural a f f e r e n t s i n s t r a t u m o r i e n s evokes a s i m i l a r sequence o f p o t e n t i a l s a l o n g t h e b a s a l d e n d r i t i c a x i s o f t h e p y r a m i d a l neuron (not shown). S t i m u l a t i o n o f s t r a t u m o r i e n s evokes a f i b e r p o t e n t i a l and a n e g a t i v e - g o i n g e x t r a c e l l u l a r EPSP i n t h e b a s a l d e n d r i t i c r e g i o n ( G e s s i e t a l . 1966; Leung 1979c). The EPSP n e g a t i v i t y i s maximal i n t h e r e g i o n o f a f f e r e n t s y n a p t i c i n p u t s , d e c l i n e s i n a m p l i t u d e t h r o u g h t h e 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 s t r a t u m p y r a m i d a l e (Leung 1979c). Higher i n t e n s i t i e s o f s t i m u l a t i o n can then b r i n g about n e u r o n a l d i s c h a r g e and t h e g e n e r a t i o n o f a - 1 3 - population spike at the c e l l layer. 2) Evoked Action Potential Discharge According to the t r a d i t i o n a l model of neuronal function, all-or-none action potential (AP) discharge could occur only in the region of the soma-axon h i l l o c k . The d e n d r i t i c arborization of a c e l l served as the p r i n c i p l e s i t e for termination of afferent excitatory synaptic inputs, and for the passive electrotonic conduction of synaptic currents to the soma-axon h i l l o c k region. However, t h i s view has been su b s t a n t i a l l y modified in recent years with the knowledge that dendritic membrane can also exhibit electroresponsive properties, and that regional variations in the d i s t r i b u t i o n of ionic channels can lead to regenerative action potential discharge in the dendritic tree (Llinas 1975; Llinas and Sugimori 1980b; Wong et a l . 1979). The evoked a c t i v i t y of somatic and dendritic membranes of a neuron has been most thoroughly examined in the Purkinje c e l l of the cerebellum (Ekerot and Oscarrson 1981; Llinas and Nicholson 1971; Llinas and Sugimori 1980a,b, 1984; Nicholson and Llinas 1971). In these neurons, voltage-dependent Na+ channels in the region of the axon h i l l o c k give r i s e to fast Na+-dependent spikes in response to stimulation of climbing f i b e r afferent inputs. However, synaptic depolarization of the dendritic tree can also evoke prolonged all-or-none Ca+2-dependent spikes within the dendritic arborization through the action of i n t r i n s i c voltage-dependent Ca+2 channels (Llinas and Sugimori 1980a,b). Ca+2 spikes may arise at one or more locations within the dendritic tree at presumed "hot spots" of low threshold dendritic membrane, and can determine the 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 soma ( L l i n a s 1975; L l i n a s and N i c h o l s o n 1971). For i n s t a n c e , Ca+2 s p i k e s can pro 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 d e n d r i t i c t r e e t o form a p r o l o n g e d d e p o l a r i z a t i o n c a p a b l e of e v o k i n g 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 a t t h e s o m a t i c l e v e l . I n t h i s way, t h e d e n d r i t i c s p i k e can s e r v e t o "boost t h e w e i g h t " o f a f f e r e n t s y n a p t i c i n p u t s by e n s u r i n g a r e l i a b l e d e n d r o-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 h i l l o c k ( L l i n a s 1975). A d i f f e r e n t i a l d i s t r i b u t i o n o f i o n i c c h a n n e l s can thus c o n f e r a n o n - u n i f o r m i t y t o t h e e x c i t a b i l i t y o f membrane a l o n g the d e n d r o -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 c e l l , 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 r e g e n e r a t i v e Ca+2 s p i k e s i n t h e d e n d r i t e s , w h i l e v o l t a g e - d e p e n d e n t Na+ ch a n n e l s and Na+-dependent s p i k e a c t i v a t i o n a r e r e s t r i c t e d t o the s o m a t i c r e g i o n . The d i s t r i b u t i o n o f Na+ and Ca+2 c h a n n e l s are i n f a c t so pronounced i n t h i s neuron t h a t s o m a t i c Na+ s p i k e s cannot a c t i v e l y r e i n v a d e t h e d e n d r i t i c a r b o r i z a t i o n , but e l e c t r o t o n i c a l l y decay t h r o u g h t h e p r o x i m a l p o r t i o n o f t h e d e n d r i t i c t r e e ( L l i n a s 1975; L l i n a s and S u g i m o r i 1980b). A c t i v e , a l l - o r - n o n e responses can thus be evoked a t b o t h t h e s o m a t i c and d e n d r i t i c l e v e l o f a c e l l , but t h e e x a c t form and c h a r a c t e r i s t i c s o f t h e evoked p o t e n t i a l depend upon t h e s p e c i f i c d i s t r i b u t i o n 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 i o n i c c h a n n e l s . A second c o r t i c a l neuron thought t o e x h i b i t a non-uniform membrane e x c i t a b i l i t y i s t h e p y r a m i d a l neuron of mammalian hippocampus. The p r o p e r t i e s o f s p i k e d i s c h a r g e a t t h e l e v e l o f the p y r a m i d a l c e l l soma were f i r s t c h a r a c t e r i z e d i n v i v o t h r o u g h - 15 - the 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 p o t e n t i a l s (Andersen 1960; Andersen et a l . 1966a fb; Andersen and Lomo 1966; Leung 1979a,b; S p e r t i et a l . 1977), e x t r a c e l l u l a r s i n g l e u n i t d i s c h a r g e (Andersen et a l . 1971b; Green et a l . 1961; Kandel et a l . 1961; Eu l e r and Green 1960) and i n t r a c e l l u l a r r e c o r d i n g s i n the v i c i n i t y of the pyramidal c e l l soma (Andersen et a l . 1966a,b; Andersen and Lomo 1966; Kandel et a l . 1961; Kandel and Spencer 1961; Spencer and Kandel 1961a,b). The evoked c h a r a c t e r i s t i c s of sp i k e d i s c h a r g e i n pyramidal c e l l somata have a l s o been examined i n the i n v i t r o p r e p a r a t i o n through both e x t r a - and i n t r a c e l l u l a r r e c o r d i n g techniques (Richardson et a l . 1984a; Schwartzkroin 1975,1977; Schwartzkroin and P r i n c e 1980; Schwartzkroin and Slawsky 1977). S t i m u l a t i o n of e f f e r e n t pyramidal c e l l axons i n the alveus evokes an ant i d r o m i c s p i k e i n v a s i o n of pyramidal c e l l somata, as i n d i c a t e d by a s h o r t l a t e n c y i n t r a c e l l u l a r a c t i o n p o t e n t i a l corresponding i n time to the 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 i n stratum pyramidale. 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 inputs impinging upon e i t h e r b a s a l or a p i c a l d e n d r i t i c s t r u c t u r e s a l s o evokes a s i n g l e s p i k e superimposed upon the u n d e r l y i n g EPSP i n pyramidal c e l l somata (Schwartzkroin 1975,1977). As with antidromic responses, the l a t e n c y of the orthodromic i n t r a c e l l u l a r s p i k e i s found to c o i n c i d e with the nega t i v e - g o i n g 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 i n stratum pyramidale, r e v e a l i n g a c l o s e correspondence between i n t r a c e l l u l a r s p i k e d i s c h a r g e and the e x t r a c e l l u l a r waveform (Richardson et a l . 1984a; Schwartzkroin and P r i n c e 1980; Turner et a l . 1984). Given the laminar o r g a n i z a t i o n of the hippocampus, an e l e c t r o p h y s i o l o g i c a l a n a l y s i s of evoked a c t i v i t y can a l s o be - 16 - c a r r i e d out a t t h e d e n d r i t i c l e v e l o f t h e p y r a m i d a l c e l l . The c h a r a c t e r i s t i c s o f d e n d r i t i c a c t i v i t y were f i r s t examined i n v i v o t h r o u g h an a n a l y s i s o f l a m i n a r p r o f i l e s o f evoked 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 l o n g t h e den d r o - s o m a t i c a x i s o f the C A l p y r a m i d a l c e l l (Andersen 1959; Andersen 1960; Cragg and Hamlyn 1955; F u j i t a and Sak a t a 1962). These e a r l y s t u d i e s r e p o r t e d t h e p o s s i b l e o c c u r r e n c e of s p i k e g e n e r a t i o n i n t h e 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 f o l l o w i n g s t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s . D e n d r i t i c s p i k e s were d e t e c t e d as a sha r p n e g a t i v e - g o 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 i n t h e d e n d r i t i c r e g i o n (presumably Na+-dependent) and were thought t o a c t i v e l y p r o p a g a t e from a d e n d r i t i c s i t e o f o r i g i n a l o n g t h e p y r a m i d a l c e l l a x i s (Andersen 1960; Cragg and Hamlyn 1955; F u j i t a and Saka t a 1962). I n agreement w i t h t h i s , i n t r a c e l l u l a r r e c o r d i n g s i n t h e r e g i o n o f p y r a m i d a l c e l l somata r e v e a l e d t h e pr e s e n c e o f a l l - o r - n o n e Na+-dependent " f a s t p r e - p o t e n t i a l s " ( F P P s ) , thought t o be t h e s o m a t i c r e p r e s e n t a t i o n o f d e n d r i t i c s p i k e d i s c h a r g e (Andersen and Lomo 1966; S c h w a r t z k r o i n 1977; Spencer and Kan d e l 1961b). W i t h t h e r e c o r d i n g s t a b i l i t y a f f o r d e d by t h e s l i c e p r e p a r a t i o n , a l i m i t e d number o f i n v e s t i g a t o r s have now succeeded i n o b t a i n i n g m i c r o e l e c t r o d e impalements o f p y r a m i d a l c e l l d e n d r i t e s , and have r e p o r t e d t h e pr e s e n c e o f evoked Na+ and Ca+2-dependent a c t i o n p o t e n t i a l s i n t h e d e n d r i t i c r e g i o n (Benardo e t a l . 1982; Masukawa and P r i n c e 1984; Wong and P r i n c e 1979; Wong e t a l . 1979). The g e n e r a t i o n o f d e n d r i t i c s p i k e s i n p y r a m i d a l neurons i s thought t o e x e r t c o n s i d e r a b l e i n f l u e n c e on t h e p a t t e r n o f c e l l d i s c h a r g e i n b o t h normal 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 t o th e p r e v a i l i n g h y p o t h e s i s , s y n a p t i c a c t i v a t i o n o f t h e p y r a m i d a l - 17 - c e l l under normal c o n d i t i o n s g i v e s r i s e t o a s i n g l e f a s t (Na+-dependent) d e n d r i t i c s p i k e t h a t c o nducts t o t h e c e l l body 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 t h e s o m a t i c r e g i o n . Through summation w i t h u n d e r l y i n g d e n d r i t i c p o s t s y n a p t i c p o t e n t i a l s , t h e d e n d r i t i c Na+ s p i k e may s e r v e t o "boost t h e we i g h t " o f s y n a p t i c i n p u t s by i n c r e a s i n g the p r o b a b i l i t y f o r AP d i s c h a r g e a t t h e axon h i l l o c k (Andersen and Lomo 1966; S c h w a r t z k r o i n 1977; Spencer and Kandel 1961). I n c o n t r a s t , t h e a c t i v a t i o n o f e x c i t a t o r y s y n a p t i c i n p u t s i n t h e pr e s e n c e o f p h a r m a c o l o g i c a l agents known t o reduce t h e e f f i c a c y o f i n h i b i t o r y feedback networks can evoke a b u r s t d i s c h a r g e o f b o t h Na+ and Ca+2-dependent 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 d e n d r i t i c d e p o l a r i z a t i o n evoked i n t h i s way can e l i c i t 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 i n t h e c e l l body, l e a d i n g t o t h e p r o p o s a l t h a t 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 u n d e r l y t h e m u l t i p l e s p i k e d i s c h a r g e 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 i n t h e hippocampus ( S c h w a r t z k r o i n and P r i n c e 1980; S c h w a r t z k r o i n and Wyler 1979; Wong and P r i n c e 1979; Wong e t a l . 1979). - 18 - 1-4. THE PRESENT STUDY P r e v i o u s i n v e s t i g a t i o n s o f t h e c h a r a c t e r i s t i c s o f evoked a c t i v i t y i n t h e hippocampus have r e p o r t e d e v i d e n c e f o r a c t i o n p o t e n t i a l d i s c h a r g e a t b o t h t h e s o m a t i c and d e n d r i t i c l e v e l o f t h e C A l p y r a m i d a l neuron. 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 p l a y an i m p o r t a n t r o l e i n d e t e r m i n i n g t h e f i n a l o u t p u t o f t h e p y r a m i d a l c e l l , and a b e r r a n t d e n d r i t i c d i s c h a r g e may even c o n t r i b u t e t o t h e p a t h o l o g y o f e p i l e p s y i n mammalian c o r t i c a l s t r u c t u r e s . I d e n t i f i c a t i o n o f t h e f a c t o r s r e s p o n s i b l e f o r g e n e r a t i o n o f d e n d r i t i c s p i k e s would thus be an i m p o r t a n t s t e p i n u n d e r s t a n d i n g t h e d i s c h a r g e p a t t e r n s o f a c o r t i c a l neuron i n t h e mammalian CNS. Much i n f o r m a t i o n has been g a i n e d about th e p r o p e r t i e s o f t h e f a s t Na+-dependent d e n d r i t i c s p i k e , as t h e s e s p i k e s are r e l i a b l y evoked under the more p h y s i o l o g i c a l c o n d i t i o n s o f s y n a p t i c d e p o l a r i z a t i o n (Andersen 1960; Andersen and Lomo 1966; Andersen et a l . 1966a,b; Cragg and Hamlyn 1955; F u j i t a and S a k a t a 1962; Masukawa and P r i n c e 1984; S c h w a r t z k r o i n 1977; Spencer and Kandel 1961b). Based on i n t r a c e l l u l a r r e c o r d i n g s a t t h e s o m a t i c l e v e l , t h e s i t e f o r g e n e r a t i o n o f t h e Na+ s p i k e was t e n t a t i v e l y proposed t o e x i s t a t b r a n c h p o i n t s o f t h e d e n d r i t i c t r e e a t "hot s p o t s " o f d e n d r i t i c membrane (Spencer and Kandel 1961b), analagous t o t h a t proposed f o r Ca+2 s p i k e g e n e r a t i o n i n t h e c e r e b e l l a r P u r k i n j e neuron ( L l i n a s 1975). However, d e s p i t e e x t e n s i v e a n a l y s i s o f 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 t h e p y r a m i d a l c e l l , t h e e x a c t s i t e of g e n e r a t i o n of a d e n d r i t i c s p i k e has not y e t been i d e n t i f i e d . The p r e s e n t s t u d y was t h e r e f o r e undertaken t o c h a r a c t e r i z e - 19 - and compare t h e e l e c t r o p h y s i o l o g i c a l p r o p e r t i e s o f s o m a t i c and d e n d r i t i c membranes o f CA1 p y r a m i d a l neurons i n o r d e r t o 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 p o t e n t i a l s i n 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 . The r e s u l t s t o be d e s c r i b e d are d i v i d e d i n t o t h r e e s t a g e s of e x p e r i m e n t a t i o n . The f i r s t i s a l a m i n a r p r o f i l e a n a l y s i s o f evoked 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 i n t h e CAT r e g i o n , and d e t e r m i n a t i o n o f c u r r e n t - s o u r c e d e n s i t y r e l a t i o n s h i p s a l o n g t h e dendro-somatic a x i s o f t h e p y r a m i d a l c e l l (Chapter 3 ) . The second i s an i n t r a c e l l u l a r i n v e s t i g a t i o n o f the membrane p r o p e r t i e s , s y n a p t i c p o t e n t i a l s , and s p i k e c h a r a c t e r i s t i c s of the 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 neurons (Chapter 4 ) . The f i n a l s e t o f e x p e r i m e n t s examine v a r i o u s parameters o f i n t r a c e l l u l a r s p i k e d i s c h a r g e a t a l l l e v e l s o f t h e p y r a m i d a l c e l l a x i s , and a s s e s s 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 a c t i v i t y and 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 (Chapter 5 ) . A g e n e r a l summary and d i s c u s s i o n o f the d a t a and c o n c l u s i o n s d e r i v e d from t h e s e e x p e r i m e n t s are p r e s e n t e d i n Chapter 6. 2-0. Methods - 20 - 2-1. S u r g i c a l Procedure Experiments were performed on t h e hippocampal f o r m a t i o n o f male W i s t a r r a t s (150-250g; C h a r l e s R i v e r , M o n t r e a l ) u s i n g t h e i n v i t r o s l i c e p r e p a r a t i o n . The a n i m a l s were d e c a p i t a t e d and t h e s k i n and c o n n e c t i v e t i s s u e o v e r l y i n g the s k u l l removed by s c a l p e l . C r a n i a l bones were removed w i t h rongeurs and t h e d u r a mater 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 removed w i t h f o r c e p s . A c o l d (1-4 degrees c e n t i g r a d e ) m o d i f i e d R i n g e r ' s s o l u t i o n c o n s i s t i n g o f 124mM NaCl, 3mM KC1, 0.75mM KH2P04, 1.6mM CaC12, 1.2mM MgS04, 24mM NaHC03, lOmM D-glucose (Sigma) preoxygenated w i t h 95% 02 / 5% C02 was poured over t h e exposed c o r t e x t o c o o l and oxygenate th e t i s s u e . Complete c o r o n a l c u t s were made between t h e o c c i p i t a l c o r t e x and c e r e b e l l u m and a c r o s s t h e f r o n t a l c o r t e x . The c e r e b e l l u m was d i s c a r d e d and t h e r e m a i n i n g b r a i n l i f t e d out w i t h a s p a t u l a and p l a c e d i n a p e t r i d i s h f i l l e d w i t h c o l d oxygenated medium. The r i g h t and l e f t hemispheres were s e p a r a t e d by a s a g i t t a l s c a l p e l c u t and one hemisphere immersed i n a beaker o f c o l d oxygenated medium f o r l a t e r d i s s e c t i o n . The r e m a i n i n g hemisphere was t i p p e d up onto the c o r o n a l s u r f a c e o f the f r o n t a l c o r t e x and t h e m i d b r a i n s e p a r a t e d from th e c o r t i c a l t i s s u e , l e a v i n g t h e v e n t r o - m e d i a l s u r f a c e o f t h e hippocampus exposed. A s p a t u l a was p l a c e d beneath the c u r v a t u r e o f t h e f i m b r i a and f o r n i x t o c a r e f u l l y " r o l l " t h e hippocampus away from the s u b i c u l a r c o r t e x , and the r e m a i n i n g c o r t e x c u t away. The hippocampus was then p l a c e d on t h e c u t t i n g s t a g e o f a S o r v a l l t i s s u e chopper and s l i c e s o f 400um t h i c k n e s s - 21 - were c u t 10 degrees o b l i q u e t o t h e l o n g i t u d i n a l a x i s o f t h e hippocampus. Each s l i c e was l i f t e d from the b l a d e w i t h a OOO b r u s h , p l a c e d i n a s m a l l p e t r i d i s h o f medium, and t r a n s f e r r e d t o a n y l o n net w i t h i n t h e r e c o r d i n g chamber. S l i c e s were t h e n a l l o w e d a t l e a s t 45 min f o r r e c o v e r y and e q u i l i b r a t i o n t o t h e r e c o r d i n g b a t h environment. Once t h e t i s s u e had been p l a c e d i n c o l d oxygenated medium, i t was found t h a t the time r e q u i r e d f o r d i s s e c t i o n was not as c r i t i c a l t o t h e h e a l t h o f a s l i c e as t h e c a r e t a k e n d u r i n g d i s s e c t i o n . T h e r e f o r e , f o l l o w i n g a d i s s e c t i o n t ime of 5-7 min, s l i c e s c o u l d be c u t from the second hemisphere w i t h no apparent d e t r i m e n t t o s l i c e v i a b i l i t y (as d e t e r m i n e d by 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 ) . 2-2. R e c o r d i n g Chamber The r e c o r d i n g chamber i s d e s i g n e d t o c r e a t e an a r t i f i c i a l environment r e s e m b l i n g t h e i n v i v o c o n d i t i o n so as t o m a i n t a i n n e u r o n a l t i s s u e i n a v i a b l e s t a t e f o r extended p e r i o d s o f time f o l l o w i n g removal from the a n i m a l . B r i e f l y , t h i s i s a c c o m p l i s h e d by p r o v i d i n g s l i c e s w i t h an oxygenated a r t i f i c i a l c e r e b r o s p i n a l f l u i d warmed t o body tem p e r a t u r e and an atmosphere o f warmed, h u m i d i f i e d oxygen/carbon d i o x i d e gas. The chamber i s c o n s t r u c t e d from p l e x i g l a s s and c o n s i s t s o f an o u t e r c i r c u l a r water j a c k e t and a s e p a r a t e i n n e r c i r c u l a r r e c o r d i n g w e l l . The o u t e r water j a c k e t i s p a r t i a l l y f i l l e d w i t h d i s t i l l e d water and warmed by a h e a t i n g c o i l t o m a i n t a i n an i n n e r r e c o r d i n g chamber tem p e r a t u r e o f 35 +/- 0.5 degrees c e n t i g r a d e . P l a s t i c t u b i n g c onnected t o t h e o u s i d e of the b a t h - 22 - runs t h r o u g h t h e o u t s i d e water j a c k e t and d e l i v e r s warmed, preoxygenated 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 a t a r a t e o f 2-3 ml/min ( D i a l - a - F l o , Sorenson R e s e a r c h Co., Utah ). Excess medium i s then drawn o f f by s u c t i o n t h r o u g h a p i e c e of p l a s t i c t u b i n g i n a w e l l connected t o t h e r e c o r d i n g chamber. Media o f a l t e r e d i o n i c c o n s t i t u e n c y can be i n t r o d u c e d w i t h o u t i n t e r r u p t i o n o f f l o w t h r o u g h a three-way v a l v e o u t s i d e o f t h e chamber. S l i c e s a re p l a c e d on a n y l o n net w i t h i n t h e r e c o r d i n g chamber and i l l u m i n a t e d by r e f l e c t e d l i g h t from below t h e chamber. A 95% oxygen / 5% carbon d i o x i d e gas m i x t u r e i s th e n passed t h r o u g h t h e o u t e r chamber t o s u p e r f u s e s l i c e s w i t h a warm, h u m i d i f i e d gas 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 Techniques B i p o l a r s t i m u l a t i n g e l e c t r o d e s c o n s t r u c t e d from t w i s t e d 62um nichrome w i r e were mounted on a m i c r o - m a n i p u l a t o r ( N a r i s h i g e ) and p o s i t i o n e d i n major s t r a t a of t h e hippocampal s l i c e under d i r e c t o b s e r v a t i o n w i t h t h e a i d o f a 4X d i s s e c t i n g m i c r o s c o p e ( C a r l Z e i s s ) . Square wave p u l s e s o f 0.1 msec d u r a t i o n and 1-70V i n t e n s i t y were d e l i v e r e d from i s o l a t i o n u n i t s ( M e d i c a l Systems Corp., Model DS2) c o n t r o l l e d by a f o u r c h a n n e l p u l s e g e n e r a t o r ( D i g i t i m e r , M e d i c a l Systems C o r p . ) . S t i m u l i were d e l i v e r e d a t a b a s e l i n e r a t e o f s t i m u l a t i o n o f 1/7 sees and mi n i m a l s t i m u l u s 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 l o n g - t e r m a l t e r a t i o n s i n t h e c h a r a c t e r i s t i c s o f evoked p o t e n t i a l s (Turner e t a l . 1982). A l l r e c o r d i n g s i n t h e p r e s e n t work were c a r r i e d out i n t h e - 23 - CAlb r e g i o n o f t h e m i d - d o r s a l r a t hippocampus. E x t r a c e l l u l a r r e c o r d i n g s were o b t a i n e d a l o n g t h e e n t i r e e x t e n t of t h e p y r a m i d a l c e l l a x i s , w h i l e i n t r a c e l l u l a r impalements o f n e u r o n a l elements were r e s t r i c t e d t o s t r a t u m p y r a m i d a l e and s t r a t u m r a d i a t u m . R e c o r d i n g e l e c t r o d e s were c o n s t r u c t e d u s i n g e i t h e r a N a r i s h i g e or F r e d e r i c k Haer m i c r o e l e c t r o d e p u l l e r . E x t r a c e l l u l a r e l e c t r o d e s o f 2-6 megohm impedance were p u l l e d from e i t h e r g l a s s c a p i l l a r y t u b i n g ( F r e d e r i c k Haer, Omega Dot Tubing , 1.5mm O.D.) or from 2.0mm g l a s s t u b i n g and f i l l e d w i t h 2M Na C l . F i b e r f i l l e d e l e c t r o d e s were b a c k f i l l e d w i t h a 31 gauge hypodermic n e e d l e w h i l e t h e t i p s o f n o n - c a p i l l a r y e l e c t r o d e s were broken back under m i c r o s c o p i c o b s e r v a t i o n ( L e i t z W e t z l e r , Germany) t o a dia m e t e r o f l-2um b e f o r e b a c k f i l l i n g w i t h e l e c t r o l y t e . I n t r a c e l l u l a r e l e c t r o d e s o f 30-80 megohm impedance were c o n s t r u c t e d from g l a s s c a p i l l a r y t u b i n g ( F r e d e r i c k Haer, 1.5mm O.D.) and b a c k f i l l e d w i t h 1M K+-acetate. E x t r a c e l l u l a r e l e c t r o d e s were mounted on a m i c r o - m a n i p u l a t o r ( N a r i s h i g e ) and i n t r a c e l l u l a r e l e c t r o d e s t o a m a n i p u l a t o r equipped w i t h a v a r i a b l e speed p i e z o e l e c t r i c advance ( B u r l e i g h Inchworm PZ-550) . R e c o r d i n g e l e c t r o d e s were connected t o t h e p r e a m p l i f i e r headstage o f a d u a l m i c r o p r o b e (WPI, Model KS700) and p o t e n t i a l s r e c o r d e d i n r e f e r e n c e t o a s i l v e r - s i l v e r c h l o r i d e b a t h ground. 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 were f i l t e r e d u s i n g a O.lHz-lOkHz bandpass and i n t r a c e l l u l a r responses a DC-lOkHz bandpass. Recorded e l e c t r i c a l a c t i v i t y was d i s p l a y e d on a d u a l beam s t o r a g e o s c i l l o s c o p e ( T e k t r o n i x ) and photographed o r l e d t o a PDP 11/23 computer f o r s t o r a g e and subsequent a n a l y s i s . M i c r o e l e c t r o d e s o f 30-80 megohm impedance were found t o be - 24 - s a t i s f a c t o r y f o r o b t a i n i n g impalements o f e i t h e r somata o r a p i c a l d e n d r i t e s o f p y r a m i d a l c e l l s . The c u r r e n t p a s s i n g c a p a b i l i t y o f an e l e c t r o d e was f i r s t examined by immersing t h e t i p o f t h e e l e c t r o d e i n t o t h e a r t i f i c i a l CSF o f t h e r e c o r d i n g chamber. Command p u l s e s t o t h e probe were p r o v i d e d by a custom-made " p r o b e - d r i v e r " , and e l e c t r o d e s b a l a n c e d t h r o u g h use of a c o n v e n t i o n a l Wheatstone b r i d g e c i r c u i t w h i l e m o n i t o r i n g t h e e l e c t r o d e v o l t a g e response t o a square wave c u r r e n t p u l s e o f up to 1.5 nA. E l e c t r o d e s were c o n s i d e r e d a c c e p t a b l e f o r i n t r a c e l l u l a r work i f c a p a b l e o f p a s s i n g .75 nA o f inward or outward c u r r e n t w i t h m i n i m a l v o l t a g e d e f l e c t i o n ( < 2.0mV ). I n t r a c e l l u l a r e l e c t r o d e s were p l a c e d w i t h i n s t r a t u m p y r a m i d a l e or s t r a t u m r a d i a t u m under d i r e c t m i c r o s c o p i c o b s e r v a t i o n , and advanced s l o w l y t h r o u g h t h e s l i c e . Impalements of t h e p y r a m i d a l c e l l were o b t a i n e d by a p p l y i n g b r i e f (0.5 sec) b u r s t s o f h i g h - f r e q u e n c y c a p a c i t a n c e feedback t h r o u g h t h e e l e c t r o d e . P e n e t r a t i o n o f t h e c e l l membrane was s i g n i f i e d by a sharp drop o f t h e v o l t a g e response i n t h e o r d e r o f -30 t o -60mV, and a h y p e r p o l a r i z i n g c u r r e n t o f 0.25-0.75nA was i m m e d i a t e l y a p p l i e d t o a i d r e c o v e r y o f t h e c e l l r e s t i n g membrane p o t e n t i a l . The p e n e t r a t i o n was c o n s i d e r e d s t a b l e i f t h e r e s t i n g p o t e n t i a l was m a i n t a i n e d f o l l o w i n g g r a d u a l removal o f h y p e r p o l a r i z i n g c u r r e n t over l - 2 m i n f o l l o w i n g i n i t i a l c e l l p e n e t r a t i o n . The c e l l was t h e n a l l o w e d 5-15min t o r e c o v e r from t h e pr o c e d u r e o f m i c r o e l e c t r o d e impalement. P y r a m i d a l c e l l impalements o b t a i n e d i n t h i s manner c o u l d be m a i n t a i n e d i n a s t a b l e s t a t e f o r up t o 4 h r s , w i t h m i n i m a l change i n membrane c h a r a c t e r i s t i c s ( i e . 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 r e c o r d i n g p e r i o d . - 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 o f membrane r e c o v e r y was e s t i m a t e d by c a l c u l a t i n g t h e i n p u t r e s i s t a n c e o f the membrane, as dete r m i n e d by t h e membrane response t o a s e r i e s o f square wave c u r r e n t p u l s e 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 b a l a n c e d , and s i x t o t w e l v e inward and outward square wave c u r r e n t p u l s e i n j e c t i o n s o f up t o l.OnA (100 msec d u r a t i o n ) a p p l i e d a c r o s s t h e membrane. The membrane response was r e c o r d e d , and a c u r r e n t / v o l t a g e (I/V) graph l a t e r c o n s t r u c t e d by p l o t t i n g t h e a m p l i t u d e o f t h e membrane v o l t a g e a g a i n s t t h a t o f t h e c o r r e s p o n d i n g c u r r e n t p u l s e . A b e s t - f i t s t r a i g h t l i n e was drawn t h r o u g h t h e l i n e a r range o f membrane v o l t a g e d e f l e c t i o n s , and the s l o p e o f t h e l i n e t a k e n as t h e v a l u e of t h e c e l l i n p u t r e s i s t a n c e ( R i ) . A l t e r n a t i v e l y , t h e i n p u t r e s i s t a n c e c o u l d be e s t i m a t e d by t h e a m p l i t u d e o f t h e membrane v o l t a g e s h i f t i n response t o a s i n g l e 0.5 or l.OnA inward c u r r e n t p u l s e . A l t h o u g h t h e R i v a l u e c a l c u l a t e d i n t h i s manner was not as a c c u r a t e as t h a t o b t a i n e d 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 c u r r e n t / v o l t a g e p l o t , t h i s method was commonly used i n d i s t a l d e n d r i t i c impalements as t h e a p p l i c a t i o n o f a f u l l range o f c u r r e n t p u l s e i n j e c t i o n s ( p a r t i c u l a r l y over 1.5nA) c o u l d l e a d t o a d e c l i n e i n the q u a l i t y o r sudden l o s s o f t h e d e n d r i t i c impalement. Data was c o l l e c t e d from c e l l s e x h i b i t i n g an i n p u t r e s i s t a n c e o f a t l e a s t 18 megohm or more, w i t h t h e R i v a l u e o f impalements used i n t h e p r e s e n t s t u d y r a n g i n g from 18-37 megohm. The 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 m o n i t o r e d u s i n g a d i g i t a l v o l t a g e d i s p l a y , and t h e a b s o l u t e v a l u e o f t h e r e s t i n g p o t e n t i a l t a k e n as t h e b a s e l i n e v o l t a g e s h i f t o b s e r v e d upon w i t h d r a w a l o f t h e 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 t o t h e e x t r a c e l l u l a r compartment. - 26 - Further information on recording arrangements for s p e c i f i c experiments are provided in the METHODS section of relevant chapters. - 27 - 3-0. CURRENT-SOURCE DENSITY ANALYSIS OF ACTION POTENTIAL DISCHARGE IN THE CAl REGION OF THE HIPPOCAMPUS 3-1. Introduction Of paramount importance to the understanding of neuronal function i s the i d e n t i f i c a t i o n of the actual s i t e for action potential (AP) generation, the f i n a l output of a neuron following integration of excitatory and i n h i b i t o r y synaptic potentials. In most c e l l s , the conductance mechanism responsible for spike generation i s thought to exist in the axon h i l l o c k , a region under the influence of currents passively conducting from the point of synaptic termination (Edwards and Ottoson 1958; Kado 1973; Ringham 1971; Smith et a l . 1982; Smith 1983). However, i t i s now thought that neuronal membrane can display a non-uniform e x c i t a b i l i t y along the dendro-somatic axis, giving r i s e to action potential discharge at both the somatic and dendritic l e v e l . This c h a r a c t e r i s t i c could be a s i g n i f i c a n t factor in the integration of synaptic inputs i f the dendritic spike should summate with synaptic currents and increase the p r o b a b i l i t y for AP discharge at the axon h i l l o c k (Andersen and Lomo 1966; Kandel and Spencer 1961; Llinas 1975; Llinas and Sugimori 1980b; Wong et a l . 1979) One neuron thought to exhibit Na+-dependent spike discharge in both somatic and dendritic locations i s the pyramidal neuron of mammalian hippocampus. Evidence for spike generation in the pyramidal c e l l apical dendrite came from early investigations into 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 f i e l d potentials evoked through stimulation of afferent excitatory synaptic - 28 - i n p u t s (Andersen 1959; Andersen 1960; Cragg and Hamlyn 1955; F u j i t a and S a k a t a 1962). These s t u d i e s r e p o r t e d an evoked n e g a t i v e s p i k e p o t e n t i a l a t a l l l e v e l s o f t h e p y r a m i d a l c e l l a x i s , s u g g e s t i n g 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 t h a t s u b s e q u e n t l y p r o p a g a t e d i n b o t h d i r e c t i o n s a l o n g t h e p y r a m i d a l c e l l s t r u c t u r e (Andersen 1960; Andersen and Lomo 1966; Andersen e t a l . 1966; Cragg and Hamlyn 1955; F u j i t a and Sak a t a 1962). F u r t h e r 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 from i n t r a c e l l u a r r e c o r d i n g s i n p y r a m i d a l c e l l somata o f s m a l l Na+-dependent " f a s t p r e - p o t e n t i a l s " i n f e r r 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 decayed s p i k e a r i s i n g from w i t h i n t h e d e n d r i t i c t r e e (Andersen and Lomo 1966; S c h w a r t z k r o i n 1977; Spencer and Kand e l 1961b). M i c r o e l e c t r o d e impalements o f t h e 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 f i n a l l y c o n f i r m e d t h a t s t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s gave 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 p y r a m i d a l c e l l d e n d r i t i c membrane (Wong e t a l . 1979). However, t h e a c t u a l s i t e f o r g e n e r a t i o n o f t h e Na+-dependent d e n d r i t i c s p i k e i s p r e s e n t l y unknown. S i n c e e x t r a c e l l u l a r p o t e n t i a l s a re g e n e r a t e d t h r o u g h t h e a c t i v a t i o n o f i o n i c c h a n n e l s i n n e u r o n a l membrane, t h e s i t e f o r s p i k e g e n e r a t i o n i n t h e d e n d r i t e might be d e t e c t e d i n t h e 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 f i e l d p o t e n t i a l s a l o n g t h e p y r a m i d a l c e l l a x i s . S e v e r a l l a m i n a r p r o f i l e a n a l y s e s o f evoked f i e l d p o t e n t i a l s have been c a r r i e d out i n t h e C A l r e g i o n . However, t h e s i t e o f o r i g i n o f s p i k e s evoked t h r o u g h s t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s has been p l a c e d a t v a r i o u s p o i n t s a l o n g t h e p y r a m i d a l c e l l s t r u c t u r e . For i n s t a n c e , some i n v e s t i g a t o r s have a s s i g n e d t h e l o w e s t t h r e s h o l d f o r s p i k e g e n e r a t i o n t o p r o x i m a l d e n d r i t i c membrane (Andersen 1959; - 29 - Andersen 1960; F u j i t a and Sa k a t a 1962), w h i l e o t h e r s t u d i e s have r e p o r t e d i n i t i a l s p i k e a c t i v a t i o n i n e i t h e r s o m a t i c (Andersen e t a l . 1961) or d i s t a l d e n d r i t i c r e g i o n s o f t h e p y r a m i d a l c e l l (Andersen 1960; Andersen e t a l . 1966a,b; Andersen and Lomo 1966; Cragg and Hamlyn 1955). The v a r i a b i l t y i n t h e s e r e s u l t s may r e l a t e t o the f a c t t h a t the m a j o r i t y o f e x t r a c e l l u l a r i n v e s t i g a t i o n s i n t o t h e 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 i n t h e p y r a m i d a l c e l l have r e l i e d s o l e l y upon the i n t e r p r e t a t i o n o f e x t r a c e l l u l a r p o t e n t i a l waveforms. A l t h o u g h much i n f o r m a t i o n has been g a i n e d i n t h i s way, t h e low r e s i s t i v i t y o f the e x t r a c e l l u l a r medium r e s u l t s i n a d i s s i p a t i o n o f v o l t a g e over space t h r o u g h volume c o n d u c t i o n , r e d u c i n g the a c c u r a c y by which f i e l d p o t e n t i a l a n a l y s i s can l o c a t e the t r u e s i t e o f i o n i c c h a n n e l s u n d e r l y i n g t h e evoked p o t e n t i a l ( G l o o r e t a l . 1963; Leung 1979b; M i t z d o r f 1985; N i c h o l s o n 1973). A more a c c u r a t e r e s o l u t i o n o f n e u r o n a l a c t i v i t y i s o b t a i n e d t h r o u g h c u r r e n t - s o u r c e d e n s i t y (CSD) a n a l y s i s , i n whi c h t h e i o n i c c h a n n e l s r e s p o n s i b l e f o r g e n e r a t i o n o f an evoked p o t e n t i a l can be s p a t i a l l y l o c a l i z e d i n terms of a net l o s s ( s i n k ) or a c c u m u l a t i o n (source) o f c u r r e n t w i t h r e s p e c t t o t h e e x t r a c e l l u l a r medium ( P i t t s 1952). U n l i k e f i e l d p o t e n t i a l s , t h e d e n s i t y o f c u r r e n t i n the e x t r a c e l l u l a r space can be w e l l l o c a l i z e d , w i t h the r e s u l t t h a t CSD p r o f i l e s p r o v i d e a f a r s u p e r i o r e s t i m a t e o f t h e l o c a t i o n , magnitude and time c o u r s e of transmembrane c u r r e n t s as compared t o t h a t o b t a i n e d t h r o u g h f i e l d p o t e n t i a l a n a l y s i s (Freeman and Stone 1969; M i t z d o r f 1980; N i c h o l s o n 1973; N i c h o l s o n and Freeman 1975). At t h e p r e s e n t t i m e , a c u r r e n t - s o u r c e d e n s i t y a n a l y s i s of s p i k e d i s c h a r g e i n the C A l p y r a m i d a l c e l l has o n l y been - 30 - performed f o r p o t e n t i a l s evoked by a n t i d r o m i c s t i m u l a t i o n o f p y r a m i d a l c e l l axons (Leung 1979a,b). In t h e s e s t u d i e s , t h e s i t e f o r i n i t i a l s p i k e a c t i v a t i o n i n p y r a m i d a l c e l l s was found i n t h e r e g i o n o f t h e soma-axon h i l l o c k , 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 s i n k i n p r o x i m a l s t r a t u m o r i e n s or t h e s t r a t u m p y r a m i d a l e . Both t h e b a s a l and a p i c a l d e n d r i t i c r e g i o n s were s u b s e q u e n t l y invaded by a s h o r t d u r a t i o n s o u r c e / s i n k , p r o v i d i n g s t r o n g e v i d e n c e t h a t a s p i k e evoked at t h e c e l l l a y e r can conduct i n a r e t r o g r a d e manner t h r o u g h t h e d e n d r i t i c a r b o r i z a t i o n o f p y r a m i d a l neurons. Comparable s t u d i e s have not y e t been performed f o r s p i k e d i s c h a r g e i n t h e p y r a m i d a l c e l l f o l l o w i n g s t i m u l a t i o n o f a f f e r e n t e x c i t a t o r y s y n a p t i c i n p u t s . T h e r e f o r e , a c u r r e n t - s o u r c e d e n s i t y a n a l y s i s o f evoked 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 was c a r r i e d out i n t h e C A l r e g i o n f o r t h e purpose of l o c a t i n g the o r i g i n o f t h e evoked d e n d r i t i c s p i k e o f p y r a m i d a l neurons. 3-2. Methods - 31 - 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 i o n s C u r r e n t - s o u r c e d e n s i t y a n a l y s i s i s a means by which i o n i c 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 can be l o c a l i z e d i n terms o f c u r r e n t movement w i t h r e s p e c t t o t h e e x t r a c e l l u l a r space ( P i t t s 1952). A movement o f c u r r e n t from t h e e x t r a c e l l u l a r t o t h e i n t r a c e l l u l a r compartment i s d e t e c t e d as a net l o s s o r s i n k o f c u r r e n t , a r e s u l t most o f t e n s i g n i f y i n g a d e p o l a r i z a t i o n o f n e u r o n a l membrane. In c o n t r a s t , t h e f l o w o f c u r r e n t from t h e i n t r a c e l l u l a r t o t h e e x t r a c e l l u l a r compartment i s o b s e r v e d as a net a c c u m u l a t i o n o r s o u r c e o f c u r r e n t i n t h e e x t r a c e l l u l a r space. C u r r e n t - s o u r c e d e n s i t y can be e s t i m a t e d from t h e s p a t i a l d i s t r i b u t i o n o f e x t r a c e l l u l a r v o l t a g e d e f l e c t i o n s and t h e r e s i s t i v i t y o f t h e e x t r a c e l l u l a r medium. The e x t r a c e l l u l a r r e s i s t a n c e t o c u r r e n t f l o w ( c o n d u c t i v i t y ) i s i n l a r g e p a r t d e termined by t h e a n a t o m i c a l s t r u c t u r e o f n e u r o n a l t i s s u e (Freeman and Stone 1969; M i t z d o r f 1985; N i c h o l s o n and Freeman 1975). Most t i s s u e s a r e found t o e x h i b i t d i f f e r e n t v a l u e s o f c o n d u c t i v i t y i n a t l e a s t two p l a n e s o f a r e c t a n g u l a r c o o r d i n a t e system. T h i s can be a t t r i b u t e d t o t h e pr e s e n c e o f n e u r o n a l elements r u n n i n g t r a n s v e r s e t o t h e d i r e c t i o n o f c u r r e n t f l o w ( i e . m y e l i n a t e d f i b e r s ; Freeman and Stone 1969; H a b e r l y and Shepherd 1973) or t o an a l i g n m e n t o f c o r e c o n d u c t o r s ( i e . d e n d r i t e s ) p a r a l l e l t o t h e a x i s o f c u r r e n t f l o w (Freeman and Stone 1969; M i t z d o r f 1985; N i c h o l s o n and Freeman 1975). V a r i a t i o n s i n t h e s t r u c t u r e o f a c e l l u l a r a g g r e g a t e can a l s o 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 r e s i s t a n c e a l o n g - 32 - a p r i n c i p l e c o o r d i n a t e a x i s , w i t h a change i n c o n d u c t i v i t y observed at the border of c e l l l a y e r s or f i b e r bundles ( H a b e r l y and Shepherd 1973; M i t z d o r f 1985). E x p e r i m e n t a l l y , c u r r e n t - s o u r c e d e n s i t y i s approximated as t h e second s p a t i a l d e r i v a t i v e of evoked p o t e n t i a l s r e c o r d e d at e q u i d i s t a n t l o c a t i o n s i n n e u r o n a l t i s s u e . Under i d e a l c o n d i t i o n s , a r e a l - t i m e a n a l y s i s of CSD i s o b t a i n e d by s i m u l t a n e o u s l y r e c o r d i n g p o t e n t i a l s from a 3 - d i m e n s i o n a l a r r a y o f e l e c t r o d e s , t a k i n g i n t o account the c o n d u c t i v i t y o f the e x t r a c e l l u l a r space i n a l l t h r e e d i m e n s i o n s (Freeman and N i c h o l s o n 1975; Freeman and Stone 1969). In t h i s c a s e , the c u r r e n t d e n s i t y a t time t f o r p o i n t x,y,z o f a r e c t a n g u l a r c o o r d i n a t e system i s g i v e n by: S(t,x,y,z) = where ox, ay, a z are t h e c o n d u c t i v i t y v a l u e s f o r t h e x,y,z d i m e n s i o n s , and V ( t ) the e x t r a c e l l u l a r v o l t a g e f o r time t a t p o i n t x,y,z ( N i c h o l s o n and Freeman 1975). However, i n n e u r o n a l t i s s u e d i s p l a y i n g a h i g h degree of l a m i n a r o r g a n i z a t i o n , the c a l c u l a t i o n of c u r r e n t - s o u r c e d e n s i t y can be s i m p l i f i e d t o a 1 - d i m e n s i o n a l a n a l y s i s ( H a b e r l y and Shepherd 1973). Such a c o n d i t i o n i s commonly found i n c o r t i c a l s t r u c t u r e s of the mammalian CNS, where n e u r o n a l t i s s u e i s c o m p r ised of one or a few p r i n c i p l e c e l l t y p e s a r r a n g e d i n a s e r i a l l a m i n a r f a s h i o n . In t h i s c a s e , the l a m i n a r o r g a n i z a t i o n o f n e u r o n a l elements r e s u l t s i n a r e l a t i v e l y h i g h c o n d u c t i v i t y a l o n g the - 33 - d e ndro-somatic a x i s of the p r i n c i p l e c e l l t y p e , and d u r i n g synchronous a c t i v a t i o n of the c e l l p o p u l a t i o n , t h e m a j o r i t y of e x t r a c e l l u l a r c u r r e n t f l o w i s r e s t r i c t e d t o the l a m i n a r p l a n e of t h e t i s s u e ( H a b e r l y and Shepherd 1973). R e l a t i v e l y l i t t l e c u r r e n t f l o w i s found normal t o t h i s a x i s , and e r r o r s i n t r o d u c e d i n t o the c a l c u l a t i o n o f c u r r e n t - s o u r c e d e n s i t y by e x c l u d i n g e x t r a c e l l u l a r c o n d u c t i v i t y e s t i m a t e s i n t h e s e d i m e n s i o n s are found t o be of a q u a n t i t a t i v e r a t h e r than q u a l i t a t i v e n a t u r e ( H a b e r l y and Shepherd 1973). In o t h e r words, the use o f a 1 - d i m e n s i o n a l CSD a n a l y s i s may a f f e c t the a b s o l u t e v a l u e of the c u r r e n t - s o u r c e d e n s i t y measurement, but the p o l a r i t y and time c o u r s e of c u r r e n t s i n k s and s o u r c e s are p r e s e r v e d . The measured v a l u e s of v a r i a t i o n i n the c o n d u c t i v i t y a l o n g th e p r i n c i p l e a x i s o f a l a m i n a t e d s t r u c t u r e have a l s o been found t o e x e r t a minor i n f l u e n c e on the q u a l i t a t i v e a s p e c t s of CSD p r o f i l e s , and the e x t r a c e l l u l a r c o n d u c t i v i t y i s u s u a l l y t r e a t e d as a c o n s t a n t (Freeman and Stone 1969; H a b e r l y and Shepherd 1973; M i t z d o r f 1980,1985; N i c h o l s o n and Freeman 1975). T h e r e f o r e , g i v e n t h e c o n d i t i o n s o f : 1) l a r g e s c a l e synchronous a c t i v a t i o n of a l a m i n a t e d s t r u c t u r e 2) e q u i d i s t a n t r e c o r d i n g o f evoked p o t e n t i a l s p a r a l l e l t o t h e d e n d r o - s o m a t i c a x i s of t h e p r i n c i p l e c e l l t y p e 3) a homogeneous c o n d u c t i v i t y t e n s o r , where ax = ay = a z t h e c a l c u l a t i o n of c u r r e n t - s o u r c e d e n s i t y can be reduced t o a 1 - d i m e n s i o n a l a n a l y s i s g i v e n by: , r t - V a ^ ( V l - V - ( V2 - V S(tyx) = a r = dx (AX) - 34 - V l + V3 ~ 2 V 2 ( A X ) ' where V I , V2, V3 r e p r e s e n t the f i e l d p o t e n t i a l s r e c o r d e d a t t h r e e c o n s e c u t i v e l o c a t i o n s a l o n g the d e n d ro-somatic a x i s s e p a r a t e d by a d i s t a n c e of A _x. A n a t o m i c a l l y , t h e C A l r e g i o n of t h e hippocampus s a t i s f i e s t h e r e q u i r e m e n t s of a 1 - d i m e n s i o n a l CSD a n a l y s i s , w i t h symmetric o r g a n i z a t i o n o f d e n d r i t i c s t r u c t u r e s p e r p e n d i c u l a r t o the p y r a m i d a l c e l l body l a y e r . Through use of t h e s l i c e p r e p a r a t i o n the placement of s t i m u l a t i n g and r e c o r d i n g e l e c t r o d e s can be v i s u a l l y c o n f i r m e d , and t h e p y r a m i d a l c e l l p o p u l a t i o n a c t i v a t e d by s t i m u l a t i o n of a f f e r e n t or e f f e r e n t f i b e r p r o j e c t i o n s . Synchronous d i s c h a r g e o f the c e l l p o p u l a t i o n a l s o g i v e s r i s e t o l a r g e v o l t a g e g r a d i e n t s a l o n g the dendro-somatic a x i s of t h e p y r a m i d a l c e l l (up t o 120 mV/mm; R i c h a r d s o n e t a l . 1984b), r e d u c i n g the degree of c u r r e n t f l o w normal t o t h e l a m i n a r p l a n e of t h e t i s s u e . A l t h o u g h a s l i g h t i n c r e a s e i n r e s i s t i v i t y has been found i n the r e g i o n o f s t r a t u m p y r a m i d a l e (1.5 - 3X d e n d r i t i c r e s i s t i v i t y ; J e f f e r y s 1984), t h i s s l i g h t v a r i a t i o n i n c o n d u c t i v i t y w i l l o n l y a f f e c t the a b s o l u t e v a l u e of c u r r e n t - s o u r c e d e n s i t y measurements, and a homogeneous e x t r a c e l l u l a r c o n d u c t i v i t y can be assumed. T h e r e f o r e , g i v e n a synchronous a c t i v a t i o n o f the p y r a m i d a l c e l l p o p u l a t i o n , a 1 - d i m e n s i o n a l c u r r e n t - s o u r c e d e n s i t y a n a l y s i s can be a p p r o p r i a t e l y a p p l i e d i n the C A l r e g i o n of t h e hippocampus. - 35 - S t i m u l a t i n g and R e c o r d i n g P r o c e d u r e s 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 p o p u l a t i o n was a c h i e v e d t h r o u g h s t i m u l a t i o n o f t h r e e d i s t i n c t pathways i n the hippocampal s l i c e . The f i r s t was s u p r a t h r e s h o l d a c t i v a t i o n o f p y r a m i d a l c e l l e f f e r e n t f i b e r s i n t h e a l v e u s ( S I ; F i g 3.1), known t o r e s u l t 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 r e c u r r e n t a c t i v a t i o n o f i n h i b i t o r y i n t e r n e u r o n s i n t h e r e g i o n o f s t r a t u m p y r a m i d a l e . The second employed s t i m u l a t i o n o f a f f e r e n t e x c i t a t o r y i n p u t s t o t h e b a s a l d e n d r i t i c a r b o r i z a t i o n o f p y r a m i d a l c e l l s t h r o u g h s t i m u l a t i o n o f s t r a t u m o r i e n s (S2; F i g 3.1). A t h i r d s t i m u l a t i n g e l e c t r o d e was p l a c e d i n s t r a t u m r a d i a t u m t o a c t i v a t e S c h a f f e r c o l l a t e r a l / c o m m i s s u r a l e x c i t a t o r y i n p u t s s y n a p s i n g d i r e c t l y upon 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 (S3; F i g 3.1). Laminar p r o f i l e s o f 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 evoked from each o f t h e above pathways ( u s u a l l y one p r o f i l e f o r each s l i c e ) 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 25um i n t e r v a l s p a r a l l e l t o t h e dendro-somatic a x i s o f t h e p y r a m i d a l c e l l . To ensure t h a t r e c o r d i n g p o s i t i o n s were l o c a t e d a l o n g a s t r a i g h t l i n e p a r a l l e l t o t h e d e n d r i t i c s t r u c t u r e , t h e r e c o r d i n g e l e c t r o d e m a n i p u l a t o r was s e c u r e d i n a p o s i t i o n w i t h t h e h o r i z o n t a l t r a v e l o f 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 s t r a t u m p y r a m i d a l e . C o n s e c u t i v e movements o f t h e e l e c t r o d e c o u l d t h e n be made w i t h o u t v i s u a l e s t i m a t i o n of t h e r e c o r d i n g a x i s , and t h e d i s t a n c e o f t h e r e c o r d i n g l o c a t i o n a l o n g t h e p y r a m i d a l c e l l measured d i r e c t l y from t h e micrometer s c a l e o f t h e m a n i p u l a t o r . Evoked p o t e n t i a l s were always r e c o r d e d d u r i n g w i t h d r a w a l o f t h e e l e c t r o d e from the maximum depth of t h e e l e c t r o d e t r a c k i n o r d e r t o a v o i d any a l t e r a t i o n o f t h e FIG. 3.1 Schematic diagram of the r a t p y r a m i d a l c e l l and s t r a t a o f t h e C A l r e g i o n o f the hippocampus, i l l u s t r a t i n g the placement o f s t i m u l a t i n g e l e c t r o d e s f o r 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 p o p u l a t i o n . S t i m u l a t i o n o f the a l v e u s (SI) was used t o evoke an a n t i d r o m i c response t h r o u g h s t i m u l a t i o n o f p y r a m i d a l c e l l axons. A second e l e c t r o d e i n s t r a t u m o r i e n s (S2) was used t o s t i m u l a t e a f f e r e n t s y n a p t i c i n p u t s t o t h e b a s a l d e n d r i t i c t r e e , and a t h i r d i n s t r a t u m r a d i a t u m (S3) f o r s y n a p t i c a c t i v a t i o n o f t h e 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 . S t i m u l u s evoked p o t e n t i a l s were r e c o r d e d p a r a l l e l t o t h e d e ndro-somatic a x i s a t a l l l e v e l s o f t h e p y r a m i d a l neuron. - 37 - S| — Alveus St. Oriens St. Pyramidale St. Radiatum - 38 - e x t r a c e l l u l a r waveform t h a t might accompany compression o f t i s s u e d u r i n g f o r w a r d e l e c t r o d e advancement. P r o f i l e s o f f i e l d p o t e n t i a l s w i t h i n a s i n g l e r e c o r d i n g e l e c t r o d e t r a c k r e v e a l e d t h a t t h e peak a m p l i t u d e o f an evoked p o t e n t i a l c o u l d v a r y w i t h t h e r e c o r d i n g d e p t h , and t h a t t h e depth a t which peak a m p l i t u d e was a t t a i n e d c o u l d d i f f e r a t v a r i o u s p o i n t s a l o n g t h e c e l l a x i s . S i n c e minor v a r i a t i o n s i n waveform 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 consequence t o t h e a c c u r a c y o f c u r r e n t - s o u r c e d e n s i t y measurements, evoked p o t e n t i a l s were c o l l e c t e d at 20jum i n t e r v a l s o f depth up t o 100/am above and below t h e approximate depth o f maximal waveform (determined p r i o r t o s e r i a l p r o f i l e measurements). P o t e n t i a l s a l o n g each e l e c t r o d e t r a c t were th e n averaged by computer ( f o u r t o e l e v e n sweeps), a v o i d i n g the problem o f p o t e n t i a l v a r i a t i o n w i t h r e c o r d i n g d e pth and p r o v i d i n g the e q u i v a l e n t o f an average v a l u e o f c u r r e n t - s o u r c e d e n s i t y over a "column" o f t i s s u e p e r p e n d i c u l a r t o t h e dendro-somatic a x i s o f t h e p y r a m i d a l c e l l . The s t a b i l i t y o f f i e l d p o t e n t i a l s were c o n t i n u o u s l y m o n i t o r e d 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 i n s t r a t u m p y r a m i d a l e , and l a m i n a r p r o f i l e s o m i t t e d from d a t a a n a l y s i s i n t h e event o f any change i n t h e c o n t r o l p o p u l a t i o n s p i k e d u r i n g t h e c o u r s e o f the e x p e r i m e n t . C u r r e n t - s o u r c e d e n s i t y a n a l y s i s was performed i n one dimensio n as a c o n t i n u o u s f u n c t i o n o f time f o r each 25(um i n t e r v a l a l o n g t h e p y r a m i d a l c e l l a x i s . The r e s o l u t i o n of c u r r e n t - s o u r c e d e n s i t y was o p t i m i z e d by v a r y i n g the v a l u e of d e l t a X used i n each c a l c u l a t i o n a c c o r d i n g t o t h e s l o p e o f t h e v o l t a g e g r a d i e n t a l o n g d i f f e r e n t r e g i o n s o f t h e p y r a m i d a l c e l l . I n g e n e r a l , a d e l t a X o f 75um was used i n d e n d r i t i c r e g i o n s , but was s h o r t e n e d t o 25rtm i n a r e a s w i t h a s t e e p v o l t a g e g r a d i e n t , such as i n t h e v i c i n i t y o f s t r a t u m p y r a m i d a l e . I n o r d e r t o i n c r e a s e t h e s i g n a l - n o i s e r a t i o , p o t e n t i a l s were c o l l e c t e d o n - l i n e a t a s a m p l i n g f r e q u e n c y o f up t o 25kHz, and c o u l d be averaged and d i g i t a l l y f i l t e r e d b e f o r e a p p l y i n g 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 i o n s . S i n c e c u r r e n t - s o u r c e d e n s i t y was c a l c u l a t e d i n one d i m e n s i o n , and t h e v a l u e o f t h e e x t r a c e l l u l a r c o n d u c t i v i t y was not d e t e r m i n e d i n t h e p r e s e n t s t u d y , CSD waveforms cannot be c o n s i d e r e d a b s o l u t e q u a n t i t a t i v e e s t i m a t e s o f c u r r e n t d e n s i t y , and u n i t s are not p r o v i d e d on CSD p r o f i l e s . R a t h e r , 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 are used t o compare the 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 o f s i n k - s o u r c e r e l a t i o n s h i p s i n t h e CAl r e g i o n f o l l o w i n g s t i m u l a t i o n o f major e f f e r e n t or a f f e r e n t pathways i n t h e hippocampal s l i c e . - 40 - 3-3. R e s u l t s 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 Neuron In o r d e r t o c o r r e l a t e c u r r e n t - s o u r c e d e n s i t y r e l a t i o n s h i p s t o s p e c i f i c a n a t o m i c a l l o c a t i o n s o f t h e p y r a m i d a l c e l l , a d e t a i l e d knowledge of b o t h t h e s t r u c t u r e and r e l a t i o n s h i p o f t h e c e l l t o v a r i o u s s t r a t a w i t h i n t h e C A l r e g i o n i s r e q u i r e d . Almost a l l measurements o f the l e n g t h and b r a n c h i n g p a t t e r n o f p y r a m i d a l c e l l s t r u c t u r e s have been c a r r i e d out i n t h e mouse, g u i n e a - p i g or r a b b i t ( C a j a l 1911, Cragg and Hamlyn 1955; Green and Maxwell 1961; L o r e n t e de No 1934; Turner and S c h w a r t z k r o i n 1980). An e x c e p t i o n i s Westrum and B l a c k s t a d ' s (1962) e l e c t r o n m i c r o s c o p i c s t u d y o f C A l p y r a m i d a l neurons i n t h e r a t . A l t h o u g h t h e g e n e r a l morphology o f t h e p y r a m i d a l c e l l i s s i m i l a r between s p e c i e s , t h e e x a c t d i m e n s i o n o f c e l l p r o c e s s e s can v a r y c o n s i d e r a b l y . For i n s t a n c e , t h e l e n g t h o f t h e p r o x i m a l s h a f t and d i m e n s i o n o f n e u r o n a l elements i n t h e r a t a r e s a i d t o be " c o n s i d e r a b l y s m a l l e r " than t h o s e i n t h e r a b b i t (Green and Maxwell 1961). The s i z e o f t h e p y r a m i d a l c e l l can a l s o v a r y a c c o r d i n g t o t h e hippocampal s u b f i e l d and a l o n g t h e s e p t o - t e m p o r a l p o l e , w i t h t h e d o r s a l CAlb r e g i o n c o n t a i n i n g some of t h e s m a l l e s t p y r a m i d a l c e l l s o f t h e hippocampal f o r m a t i o n ( L o r e n t e de No 1934). Attempts were thus 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 o f t h e w i d t h o f s t r a t a and o f t h e l e n g t h and b r a n c h i n g p a t t e r n of p y r a m i d a l c e l l d e n d r i t e s i n t h e C A l r e g i o n . T h i s was a c c o m p l i s h e d t h r o u g h l i g h t m i c r o s c o p i c e x a m i n a t i o n o f c r e s y l v i o l e t s t a i n e d hippocampal s l i c e s o r o f p y r a m i d a l c e l l s i n t h i n s e c t i o n s o f hippocampal t i s s u e s t a i n e d i m m u n o h i s t o c h e m i c a l l y f o r - 41 - c a l c i u m - b i n d i n g p r o t e i n (CaBP; Baimbridge and M i l l e r 1 9 8 2 ) . S i n c e subsequent i n t r a d e n d r i t i c r e c o r d i n g s were c o n f i n e d t o t h e a p i c a l d e n d r i t e , a t t e n t i o n was f o c u s e d p r i m a r i l y upon a p i c a l d e n d r i t i c elements i n s t r a t u m r a d i a t u m , w i t h measurements o f d e n d r i t i c d i a m e t e r t a k e n from t h e s t u d y o f Westrum and B l a c k s t a d (1962). Measurements of t h e w i d t h o f s t r a t a i n r a t hippocampus gave v a l u e s f o r t h e a l v e u s o f 70 +/- 17.8 pm (n=5; mean +/- sem), s t r a t u m o r i e n s 160 +/- 18.1 jam (n=5) , s t r a t u m p y r a m i d a l e 44 +/- 2.9jum (n=34) , s t r a t u m r a d i a t u m 303 +/- 7. 8jum (n=18) , and s t r a t u m l a c u n o s u n - m o l e c u l a r e 102 +/- 4.4pm (n=18). P y r a m i d a l c e l l s s p anning t h e d i s t a n c e from t h e deep edge of s t r a t u m o r i e n s t o s t r a t u m lacunosum-moleculare would t h e r e f o r e e x h i b i t a maximal o v e r a l l l e n g t h o f 600jum. T h i s c o n t r a s t s w i t h a l e n g t h o f up t o 1mm i n g o l g i - s t a i n e d p y r a m i d a l c e l l s o f t h e g u i n e a - p i g (Turner and S c h w a r t z k r o i n 1980). D e l i n e a t i o n o f t h e boundary between s o m a t i c and d e n d r i t i c membrane i s o f t e n d i f f i c u l t as t h e c e l l body t a p e r s g r a d u a l l y i n t o t h e a p i c a l d e n d r i t i c s h a f t , but t h e w i d t h and depth o f p y r a m i d a l c e l l somata f a l l w i t h i n t h e range of 10-15pm and 15 - 2 0 r i m , r e s p e c t i v e l y . A l t h o u g h t h e l e n g t h o f t h e p r o x i m a l a p i c a l d e n d r i t i c s h a f t can 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 a range of 25-100pm. The d i a m e t e r o f t h e r a t p y r a m i d a l c e l l d e n d r i t e i s g r e a t e s t i n t h e p r o x i m a l s h a f t (3-4pm) g r a d u a l l y 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 p o i n t (Westrum and B l a c k s t a d 1962). Secondary branches p r o g r e s s r a d i a l l y a t a s l i g h t a n g l e towards t h e hippocampal f i s s u r e , t a p e r i n g t o a d i a m e t e r o f 0.5-lpm (Westrum and B l a c k s t a d 1962). The degree of t r a n s v e r s e s p r e a d o f d e n d r i t i c b r a n c h i n g from t h e r e g i o n o f the - 42 - soma can be as g r e a t as 225um, but i s more u s u a l l y w i t h i n a range 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 i n C A l P y r a m i d a l Neurons The c h a r a c t e r i s t i c s o f evoked 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 and s i n k - s o u r c e r e l a t i o n s h i p s i n t h e C A l r e g i o n were examined over t h e e n t i r e a x i s o f t h e p y r a m i d a l neuron. However, most a t t e n t i o n was f o c u s s e d upon evoked e l e c t r i c a l a c t i v i t y and c u r r e n t - s o u r c e d e n s i t y i n s t r a t u m p y r a m i d a l e and s t r a t u m r a d i a t u m , r e g i o n s c o r r e s p o n d i n g t o t h e somata and a p i c a l d e n d r i t e s o f t h e p y r a m i d a l c e l l p o p u l a t i o n , r e s p e c t i v e l y . 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 The l a m i n a r p r o f i l e s o f 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 ( v o l t a g e ) and a s s o c i a t e d c u r r e n t - s o u r c e d e n s i t y (CSD) evoked t h r o u g h a n t i d r o m i c a c t i v a t i o n o f C A l p y r a m i d a l c e l l s a r e shown i n F i g 3.2. I n t h i s and a l l subsequent f i g u r e s , t h e r e f e r e n c e p o i n t f o r t h e d i s t a n c e o f t h e r e c o r d i n g e l e c t r o d e a l o n g the n e u r o n a l a x i s i s t a k e n as t h e v e n t r a l b o r d e r of s t r a t u m p y r a m i d a l e (Gjum) . The c h a r a c t e r i s t i c s o f t h e a n t i d r o m i c f i e l d p o t e n t i a l were found t o v a r y a c c o r d i n g t o t h e r e c o r d i n g p o s i t i o n a l o n g t h e p y r a m i d a l c e l l s t r u c t u r e ( F i g 3.2A). At t h e l e v e l of s t r a t u m p y r a m i d a l e , a l v e a r s t i m u l a t i o n evoked a s h o r t l a t e n c y n e g a t i v e - g o i n g p o t e n t i a l c h a r a c t e r i s t i c of an 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 ( F i g 3.2A; Leung 1979a,b; S p e r t i e t a l . 1967). A s i m i l a r response was r e c o r d e d a p p r o x i m a t e l y lOGjum on e i t h e r s i d e o f s t r a t u m p y r a m i d a l e , but t h e n e g a t i v i t y o f the p o t e n t i a l was 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 or - 43 - t h e p r o x i m a l s t r a t u m o r i e n s . The p o p u l a t i o n s p i k e t h e n p r o g r e s s i v e l y d e c l i n e d i n a m p l i t u d e and i n c r e a s e d i n l a t e n c y a l o n g t h e a p i c a l d e n d r i t e w i t h d i s t a n c e from th e b o r d e r of s t r a t u m p y r a m i d a l e . I n s t r a t u m r a d i a t u m , t h e n e g a t i v i t y o f t h e s p i k e was c o n t i n u o u s w i t h t h e n e g a t i v e component of a p o s i t i v e / n e g a t i v e d e n d r i t i c p o t e n t i a l ( P i g 3.2A, 150pm). The 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 n e g a t i v e p o t e n t i a l s o f t h e d e n d r i t i c waveform c o n t i n u e d t o i n c r e a s e w i t h d i s t a n c e a l o n g the d e n d r i t i c t r e e , g i v i n g r i s e i n d i s t a l d e n d r i t i c l o c a t i o n s t o a monophasic p o s i t i v e f i e l d p o t e n t i a l . C a l c u l a t i o n o f c u r r e n t - s o u r c e d e n s i t y from l a m i n a r p r o f i l e s o f a n t i d r o m i c f i e l d p o t e n t i a l s a l s o r e v e a l e d a t r a n s i t i o n i n t h e c h a r a c t e r i s t i c s o f c u r r e n t s o u r c e s and s i n k s a l o n g t h e p y r a m i d a l c e l l a x i s ( F i g 3.2B). A l v e a r s t i m u l a t i o n evoked a l a r g e a m p l i t u d e 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 s t r a t u m p y r a m i d a l e or t h e p r o x i m a l r e g i o n o f s t r a t u m o r i e n s , i n d i c a t i n g t h e movement of c u r r e n t from th e e x t r a - t o t h e i n t r a c e l l u l a r 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 c o u l d be o b s e r v e d i n t h e p r o x i m a l - m i d s t r a t u m r a d i a t u m , r e v e a l i n g an outward f o l l o w e d by an inward f l o w o f c u r r e n t i n t h e r e g i o n 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 . Both th e s o u r c e and s i n k o f t h e d e n d r i t i c response d i s p l a y e d a g r a d u a l i n c r e a s e i n peak l a t e n c y w i t h d i s t a n c e from the b o r d e r o f s t r a t u m p y r a m i d a l e . In d i s t a l s t r a t u m r a d i a t u m , t h e s o u r c e component of t h e d e n d r i t i c waveform p r o g r e s s i v e l y i n c r e a s e d 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 t h e c u r r e n t s i n k , e v e n t u a l l y f o r m i n g a monophasic c u r r e n t s o u r c e i n d i s t a l a p i c a l d e n d r i t i c l o c a t i o n s . The s h i f t i n peak l a t e n c y o f the f i e l d p o t e n t i a l n e g a t i v i t y and c u r r e n t s i n k a l o n g t h e p y r a m i d a l c e l l a x i s i s - 44 - FIG. 3.2 A,B. Laminar p r o f i l e s o f 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 ( v o l t a g e ) and c u r r e n t - s o u r c e d e n s i t y (CSD) evoked t h r o u g h 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. Evoked p o t e n t i a l s were c o l l e c t e d a t 25um s t e p s p a r a l l e l t o t h e dendro-somatic a x i s o f t h e p y r a m i d a l c e l l , and B, 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 i n 1 dim e n s i o n f o r each 25pm i n t e r v a l . CSD measurements are d i s p l a y e d w i t h a zer o l i n e t o f a c i l i t a t e c omparison o f s i n k - s o u r c e r e l a t i o n s h i p s . A s c a l e d s c h e m a t i c d i a g r a m o f t h e r a t p y r a m i d a l c e l l i s i n t e r p o s e d 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 denoted 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 a x i s i s t a k e n from t h e v e n t r a l b o r d e r o f s t r a t u m p y r a m i d a l e (Oum). C,D. A comparison o f 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 (C) and a s s o c i a t e d CSD measurements (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 somata ( -20pm ) and a p i c a l d e n d r i t e s (160jum ) i n response 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 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). V o l t a g e p o l a r i t y n e g a t i v e down i n t h i s and a l l subsequent f i g u r e s . - 45 - A. Voltage B. CSD v_ -— O CD 2 S CO >̂ QCL o o 2 0 0 - - 1 0 0 - 0 - + 100 - 2 0 0 3 0 0 - 1 10 C. Soma Apical Dend. D. I Sink 1 Source 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 comparing the evoked a n t i d r o m i c response a t the l e v e l o f s t r a t u m p y r a m i d a l e and r a d i a t u m ( F i g 3.2C,D). The n e g a t i v i t y of t h e a n t i d r o m i c f i e l d p o t e n t i a l was evoked a t a peak l a t e n c y of 1.1msec i n t h e c e l l body l a y e r ( F i g 3.2C) and 1.96msec i n the a p i c a l d e n d r i t i c r e g i o n 160um from the bo r d e r o f s t r a t u m p y r a m i d a l e ( F i g 3.2C). A s h i f t i n peak l a t e n c y o f t h e evoked c u r r e n t s i n k i s a l s o a p p a r e n t , w i t h peak l a t e n c y measurements of .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 the 160^um 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 ( F i g 3.2D). T h e r e f o r e , b o t h v o l t a g e and 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 i n d i c a t e t h a t a l v e a r s t i m u l a t i o n r e s u l t s i n t h e i n i t i a l a c t i v a t i o n o f an a n t i d r o m i c s p i k e i n t h e r e g i o n of p y r a m i d a l c e l l somata, as shown by t h e pr e s e n c e of a s h o r t - l a t e n c y p o p u l a t i o n s p i k e and c u r r e n t s i n k i n the v i c i n i t y o f t h e c e l l body l a y e r . The c o n d u c t i o n o f a b i p h a s i c p o t e n t i a l and c u r r e n t s o u r c e / s i n k t h r o u g h s t r a t u m r a d i a t u m f u r t h e r s u g g e s t s a subsequent a n t i d r o m i c s p i k e i n v a s i o n o f t h e 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 . I n f a c t , a s i m i l a r sequence of p o t e n t i a l s c o u l d o f t e n be obser v e d i n s t r a t u m o r i e n s f o l l o w i n g a l v e a r s t i m u l a t i o n , 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 evoked s p i k e can conduct t h r o u g h b o t h the b a s a l 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 p y r a m i d a l c e l l . S i m i l a r r e s u l t s were o b t a i n e d i n p r e v i o u s a n a l y s e s o f a l v e a r evoked p o t e n t i a l s i n t h e i n v i v o hippocampus ( G e s s i e t a l . 1966; Leung 1979a,b; S p e r t i e t a l . 1967), a l l e v i d e n c e s u p p o r t i n g the i n t e r p r e t a t i o n t h a t an a n t i d r o m i c a l l y evoked a c t i o n p o t e n t i a l can s e q u e n t i a l l y i n v a d e t h e soma and d e n d r i t i c a r b o r i z a t i o n s of t h e C A l p y r a m i d a l neuron. A n t i d r o m i c a c t i v a t i o n a l s o evoked a l o n g d u r a t i o n (7-15msec) p o s i t i v e - g o i n g p o t e n t i a l i m m e d i a t e l y f o l l o w i n g the - 47 - p o p u l a t i o n s p i k e i n s t r a t u m p y r a m i d a l e and t h e p r o x i m a l r e g i o n of s t r a t u m o r i e n s and r a d i a t u m ( F i g 3.2B; -90 - 60pm). T h i s p o t e n t i a l was a s s o c i a t e d w i t h a c u r r e n t s o u r c e i n t h e r e g i o n o f t h e c e l l l a y e r ( F i g 3.2B, -25 - 25jum) and a c u r r e n t s i n k i n m i d - d e n d r i t i c l o c a t i o n s . The s i n k component o f t h i s p o t e n t i a l c o u l d t h u s o v e r l a p w i t h t h a t o f t h e d e n d r i t i c a n t i d r o m i c s p i k e , p r e v e n t i n g a comparison of t h e a m p l i t u d e o f a c t i o n p o t e n t i a l c u r r e n t s i n k s a l o n g the a p i c a l d e n d r i t i c a x i s ( F i g 3.2B, 50 - 150jum) . T h i s p o t e n t i a l was not e x t e n s i v e l y examined i n the p r e s e n t s t u d y , but most l i k e l y c o r r e s p o n d s t o t h e r e c u r r e n t a c t i v a t i o n o f i n h i b i t o r y s y n a p t i c i n p u t s i n t h e r e g i o n o f s t r a t u m p y r a m i d a l e (Andersen e t a l . 1969: D i n g l e d i n e and Langmoen 1980; Leung 1979a,b,c; S p e r t i e t a l . 1967). S u b t h r e s h o l d E x c i t a t o r y S y n a p t i c P o t e n t i a l s F i e l d p o t e n t i a l s evoked t h r o u g h s u b t h r e s h o l d s t i m u l a t i o n o f e i t h e r s t r a t u m o r i e n s or s t r a t u m r a d i a t u m d i s p l a y e d c h a r a c t e r i s t i c s r e f l e c t i n g t h e a c t i v a t i o n o f e x c i t a t o r y p o s t s y n a p t i c p o t e n t i a l s (EPSPs) i n d e n d r i t i c l o c a t i o n s o f t h e p y r a m i d a l c e l l p o p u l a t i o n ( F i g 3.3). S t r a t u m o r i e n s (SO) s t i m u l a t i o n evoked a graded n e g a t i v e - g o i n g p o t e n t i a l o f a p p r o x i m a t e l y 20msec d u r a t i o n i n t h e r e g i o n o f p y r a m i d a l c e l l d e n d r i t e s and a p o s i t i v e - g o i n g p o t e n t i a l a t the l e v e l o f t h e c e l l body l a y e r and a p i c a l d e n d r i t e s ( F i g 3.3A). The n e g a t i v i t y of t h e SO evoked p o t e n t i a l was maximal i n s t r a t u m o r i e n s (3.5mV at -100/jm, F i g 3.3A), d e c r e a s i n g i n a m p l i t u d e towards t h e p y r a m i d a l c e l l l a y e r t o i n v e r t t o a p o s i t i v e p o t e n t i a l j u s t p r o x i m a l t o or w i t h i n s t r a t u m p y r a m i d a l e . The p o s i t i v i t y was o f t e n l a r g e s t i n a m p l i t u d e i n t h e p r o x i m a l a p i c a l d e n d r i t i c - 48 - r e g i o n , p r o g r e s s i v e l y d e c l i n i n g i n a m p l i t u d e but r e t a i n i n g a p o s i t i v e p o l a r i t y t h r o u g h o u t the a p i c a l d e n d r i t i c f i e l d ( F i g 3.3A). In some s l i c e s , t h e SO evoked d e n d r i t i c n e g a t i v i t y was f o l l o w e d by a second p o t e n t i a l o f p o s i t i v e p o l a r i t y t h a t i n c r e a s e d i n a m p l i t u d e w i t h p r o x i m i t y t o s t r a t u m p y r a m i d a l e ( F i g 3.3A). T h i s p o t e n t i a l was observed as a p o s i t i v e - g o i n g d e f l e c t i o n j u s t beyond the peak o f t h e n e g a t i v e d e n d r i t i c waveform i n p r o x i m a l s t r a t u m o r i e n s ( -75pm - -125pm, F i g 3.3A), w i t h b o t h p o t e n t i a l s e v i d e n t i n the form o f a b i p h a s i c 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 o f s t r a t u m p y r a m i d a l e (-50pm, F i g 3.3A). In t h i s c a s e , t h e peak l a t e n c y o f t h e b a s a l 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 t o the p y r a m i d a l c e l l l a y e r as the l a t e p o s i t i v e p o t e n t i a l i n c r e a s e d i n a m p l i t u d e towards s t r a t u m p y r a m i d a l e . C u r r e n t - s o u r c e d e n s i t y a n a l y s i s i n d i c a t e d t h a t t h e n e g a t i v e - g o i n g wave i n s t r a t u m o r i e n s was a s s o c i a t e d w i t h a graded l o n g l a s t i n g c u r r e n t s i n k , i n d i c a t i n g an inward movement o f c u r r e n t i n t h e r e g i o n of p y r a m i d a l c e l l b a s a l d e n d r i t e s ( F i g 3.3B). The c u r r e n t s i n k was maximal i n t h e s t r a t u m o r i e n s and de c r e a s e d i n a m p l i t u d e w i t h p r o x i m i t y t o the c e l l l a y e r , i n v e r t i n g t o a c u r r e n t s o u r c e i n s t r a t u m p y r a m i d a l e and t h e p r o x i m a l 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 found t o d e c l i n e i n t h e p r o x i m a l r e g i o n of t h e b a s a l d e n d r i t e c o i n c i d e n t w i t h t h e i n c r e a s e i n a m p l i t u d e of the l a t 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 3.3B), s u g g e s t i n g t h a t the l a t t e r was a s s o c i a t e d w i t h the development of a l a t e c u r r e n t s o u r c e i n t h e r e g i o n o f the p y r a m i d a l c e l l l a y e r . S u b t h r e s h o l d s t i m u l a t i o n of s t r a t u m r a d i a t u m evoked a l o n g d u r a t i o n n e g a t i v e - g o i n g p o t e n t i a l w i t h maximal a m p l i t u d e i n FIG. 3.3 Laminar p r o f i l e s o f 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 ( v o l t a g e ) and c u r r e n t - s o u r c e d e n s i t y (CSD) evoked t h r o u g h s u b t h r e s h o l d s t i m u l a t i o n o f s t r a t u m o r i e n s (A,B) or s t r a t u m r a d i a t u m (C,D) . A,C. Evoked p o t e n t i a l s were c o l l e c t e d a t 25/im s t e p s p a r a l l e l t o t h e dendro-somatic a x i s o f the p y r a m i d a l c e l l , and 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 i n 1 dimensi o n f o r each 25/am i n t e r v a l . CSD measurements are d i s p l a y e d w i t h a z e r o l i n e t o f a c i l i t a t e comparison o f s i n k - s o u r c e r e l a t i o n s h i p s . A s c a l e d s c h e m a t i c diagram o f t h e r a t p y r a m i d a l c e l l i s i n t e r p o s e d 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 denoted 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 a x i s i s t a k e n from t h e v e n t r a l b o r d e r o f s t r a t u m p y r a m i d a l e ( Ojum ) . Stim.Site Stratum Oriens A. Voltage B. CSD 200 -, Stratum Radiatum C. Voltage D. CSD I Sink Source 5mV 10 msec - 5 1 - s t r a t u m r a d i a t u m 150 - 250jum from t h e b o r d e r of s t r a t u m p y r a m i d a l e (3.8mV i n a m p l i t u d e and 22msec i n d u r a t i o n a t 200jum, F i g 3.3C). T h i s p o t e n t i a l d e c l i n e d p r o g r e s s i v e l y i n a m p l i t u d e and i n c r e a s e d i n peak l a t e n c y w i t h p r o x i m i t y t o s t r a t u m p y r a m i d a l e , i n v e r t i n g t o a p o s i t i v e p o l a r i t y i n t h e r e g i o n of t h e c e l l l a y e r . The p o s i t i v e - g o i n g p o t e n t i a l was o f s i m i l a r d u r a t i o n as t h e d e n d r i t i c n e g a t i v i t y and a t t a i n e d a maximal a m p l i t u d e i n t h e p r o x i m a l - m i d s t r a t u m o r i e n s . C u r r e n t - s o u r c e d e n s i t y a n a l y s i s r e v e a l e d t h a t t h e n e g a t i v e p o t e n t i a l evoked i n s t r a t u m r a d i a t u m was a s s o c i a t e d w i t h a graded, l o n g d u r a t i o n c u r r e n t s i n k i n t h e r e g i o n of t h e m i d - d i s t a l a p i c a l d e n d r i t e ( F i g 3.3D). The m a j o r i t y o f c u r r e n t s o u r c i n g was found i n s t r a t u m p y r a m i d a l e or p r o x i m a l s t r a t u m o r i e n s , a r e g i o n c o r r e s p o n d i n g t o 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 at t h e l e v e l o f somata and p r o x i m a l b a s a l d e n d r i t i c e x t e n s i o n s of p y r a m i d a l neurons ( F i g 3.3D). T h e r e f o r e , a c t i v a t i o n o f s y n a p t i c a f f e r e n t s i n s t r a t u m o r i e n s o r r a d i a t u m gave 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 and c u r r e n t s i n k r e s t r i c t e d t o t h e zone of s t i m u l a t e d a f f e r e n t i n p u t s , i n d i c a t i n g a s y n a p t i c d e p o l a r i z a t i o n o f p y r a m i d a l c e l l b a s a l 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 . The d e c l i n e i n a m p l i t u d e o f t h e EPSP and c u r r e n t s i n k and t h e p r e s e n c e o f a c u r r e n t s o u r c e i n s t r a t u m p y r a m i d a l e suggest t h a t evoked EPSPs were conducted e l e c t r o t o n i c a l l y towards t h e p y r a m i d a l c e l l l a y e r . The f a c t t h a t s y n a p t i c c u r r e n t s were found t o s o u r c e beyond s t r a t u m p y r a m i d a l e f u r t h e r i n d i c a t e a c o n d u c t i o n o f t h e EPSP p a s t t h e s o m a t i c r e g i o n and t h r o u g h a t l e a s t t h e p r o x i m a l p o r t i o n o f t h e o p p o s i n g d e n d r i t i c t r e e . The l a t e p o s i t i v e - g o i n g p o t e n t i a l and c u r r e n t s o u r c e - 52 - o b s e r v e d 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 p y r a m i d a l e f o l l o w i n g SO s t i m u l a t i o n l i k e l y r e p r e s e n t an i n h i b i t o r y s y n a p t i c p o t e n t i a l evoked i n t h e p y r a m i d a l c e l l t h r o u g h t h e a c t i o n of i n h i b i t o r y i n t e r n e u r o n s near t h e c e l l body l a y e r (Andersen e t a l . 1969; D i n g l e d i n e and Langmoen 1980; Leung 1 9 7 9 a , b f c ) . T h i s i s s u p p o r t e d by t h e f i n d i n g t h a t the e x t r a c e l l u l a r p o s i t i v i t y c o r r e l a t e s i n time t o an i n h i b i t o r y p o t e n t i a l r e c o r d e d from i n t r a s o m a t i c impalements of t h e p y r a m i d a l neuron ( d a t a not shown). The means by w h i c h i n t e r n e u r o n s were a c t i v a t e d was not d e t e r m i n e d , and the l a t e i n h i b i t o r y p o t e n t i a l was not f u r t h e r examined i n t h e p r e s e n t s t u d y . Orthodromic P o p u l a t i o n S p i k e Responses The l a m i n a r p r o f i l e s of 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 and c u r r e n t - s o u r c e d e n s i t y evoked t h r o u g h s u p r a t h r e s h o l d SO s t i m u l a t i o n are i l l u s t r a t e d i n F i g 3.4A,B. To a i d comparison o f s o m a t i c and a p i c a l d e n d r i t i c a c t i v i t y o f t h e p y r a m i d a l c e l l p o p u l a t i o n , f i e l d p o t e n t i a l s and c o r r e s p o n d i n g CSD measurements r e c o r d e d i n s t r a t u m p y r a m i d a l e and r a d i a t u m (125pm) are shown i n F i g 3.4C,D. The p r o p e r t i e s of t h e SO evoked p o p u l a t i o n s p i k e response were found t o v a r y 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 t o t h e r e c o r d i n g p o s i t i o n a l o n g the a p i c a l d e n d r i t i c a x i s . At t h e l e v e l of s t r a t u m p y r a m i d a l e , SO s t i m u l a t i o n evoked a sharp n e g a t i v e p o p u l a t i o n s p i k e upon the u n d e r l y i n g p o s i t i v i t y o f t h e s y n a p t i c waveform ( F i g 3.4A,C). The s h o r t e s t l a t e n c y p o p u l a t i o n s p i k e was most o f t e n found i n s t r a t u m p y r a m i d a l e or t h e p r o x i m a l s t r a t u m o r i e n s , d i s p l a y i n g a g r a d u a l i n c r e a s e i n 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 a p i c a l d e n d r i t e . A p p r o x i m a t e l y 150pm from t h e b o r d e r o f FIG. 3.4 Laminar p r o f i l e s o f 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 ( v o l t a g e ) and c u r r e n t - s o u r c e d e n s i t y (CSD) evoked t h r o u g h s u p r a t h r e s h o l d s t i m u l a t i o n o f s t r a t u m o r i e n s . A. Evoked p o t e n t i a l s were c o l l e c t e d a t 25pm s t e p s p a r a l l e l t o t h e dendro-somatic a x i s o f the p y r a m i d a l c e l l , and B, 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 i n 1 di m e n s i o n f o r each 25pm i n t e r v a l . CSD measurements are d i s p l a y e d w i t h a z e r o l i n e t o f a c i l i t a t e comparison of s i n k - s o u r c e r e l a t i o n s h i p s . A s c a l e d s c h e m a t i c diagram o f t h e r a t p y r a m i d a l c e l l i s i n t e r p o s e d 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 denoted 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 a x i s i s t a k e n from the v e n t r a l b o r d e r o f s t r a t u m p y r a m i d a l e ( Opm ). C,D. A comparison o f 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 (C) and a s s o c i a t e d CSD measurements (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 somata ( -25pm ) and a p i c a l d e n d r i t e s ( 125pm ) i n response t o s t r a t u m o r i e n s s t i m u l a t i o n , (waveforms t a k e n from p r o f i l e s i n A,B). Note t h a t the onset o f t h e d e n d r i t i c s p i k e response can be d e t e c t e d as a break on t h e r i s i n g edge o f t h e p o s i t i v e - g o i n g d e n d r i t i c p o t e n t i a l r e c o r d e d 125pm from s t r a t u m p y r a m i d a l e . Recording Distance from Border of St. Pyramidale (pm) - $g - - 55 - s t r a t u m p y r a m i d a l e , t h e p o p u l a t i o n s p i k e response c o n s i s t e d o f a b i p h a s i c 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 , d i s t i n g u i s h e d from t h e u n d e r l y i n g waveform by a break on t h e r i s i n g edge of t h e p o s i t i v e - g o i n g s y n a p t i c p o t e n t i a l (see F i g 3.4C). The p o s i t i v e and n e g a 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 c o n t i n u e d t o i n c r e a s e i n l a t e n c y t h r o u g h t h e m i d - d e n d r i t i c r e g i o n , w i t h t h e n e g a t i v i t y " d r o p p i n g o u t " i n d i s t a l d e n d r i t i c l o c a t i o n s t o 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 . C a l c u l a t i o n o f c u r r e n t - s o u r c e d e n s i t y uncovered a sharp c u r r e n t s i n k o f s h o r t e s t peak l a t e n c y i n t h e r e g i o n o f s t r a t u m p y r a m i d a l e superimposed upon t h e so u r c e o f c u r r e n t a s s o c i a t e d w i t h s y n a p t i c a c t i v a t i o n o f t h e b a s a l d e n d r i t e ( F i g 3.4B, -25pm; F i g 3.4D). The c u r r e n t s i n k g r a d u a l l y i n c r e a s e d i n 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 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 w i t h t h e s i n k component o f a s o u r c e / s i n k i n t h e m i d - d e n d r i t i c r e g i o n ( F i g 3.4B, 125jum; F i g 3.4D). Both t h e s o u r c e and s i n k o f t h e d e n d r i t i c response i n c r e a s e d i n l a t e n c y w i t h d i s t a n c e from s t r a t u m p y r a m i d a l e , w i t h t h e c u r r e n t s i n k g r a d u a l l y d e c l i n i n g t o g i v e r i s e t o a monophasic c u r r e n t s o u r c e i n d i s t a l d e n d r i t i c membrane. The p o s s i b l e " c o n t a m i n a t i o n " o f t h e d e n d r i t i c s i n k by c u r r e n t f l o w 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 s o u r c e f o l l o w i n g the p o p u l a t i o n s p i k e response a t t h e c e l l l a y e r i s p a r t i c u l a r l y e v i d e n t i n F i g 3.4B (50 - 125pm). A p o p u l a t i o n s p i k e response c o u l d a l s o be d e t e c t e d a t a l l l e v e l s o f t h e p y r a m i d a l c e l l a x i s f o l l o w i n g s u p r a t h r e s h o l d s t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s i n s t r a t u m r a d i a t u m ( F i g 3.5). I n t h e r e g i o n o f t h e p y r a m i d a l c e l l l a y e r , SR s t i m u l a t i o n evoked a l a r g e p o p u l a t i o n s p i k e upon t h e p o s i t i v e - g o i n g a p i c a l d e n d r i t i c s y n a p t i c p o t e n t i a l ( F i g 3.5A, -50pm; F i g 3.5C). The - 56 - l a r g e s t a m p l i t u d e and s h o r t e s t l a t e n c y p o p u l a t i o n s p i k e evoked by SR s t i m u l a t i o n was most o f t e n found i n s t r a t u m p y r a m i d a l e or p r o x i m a l s t r a t u m o r i e n s . The peak n e g a t i v i t y o f t h e p o p u l a t i o n s p i k e t h e n i n c r e a s e d i n l a t e n c y and d e c r e a s e d i n a m p l i t u d e t h r o u g h the p r o x i m a l a p i c a l d e n d r i t i c f i e l d . Beyond 50 - 100pm from t h e v e n t r a l b o r d e r o f s t r a t u m p y r a m i d a l e , t h e s p i k e response c o n t i n u e d t h r o u g h t h e mid s t r a t u m r a d i a t u m as a p o s i t i v e / n e g a t i v e waveform superimposed upon t h e e x t r a c e l l u l a r n e g a t i v i t y o f t h e a p i c a l d e n d r i t i c EPSP ( F i g 3.5A fC). The 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 n e g a t i v e components o f t h e d e n d r i t i c s p i k e response i n c r e a s e d t h r o u g h s t r a t u m r a d i a t u m , c o n v e r t i n g i n d i s t a l d e n d r i t i c l o c a t i o n s t o a monophasic p o s i t i v e - g o i n g d e f l e c t i o n a t a l a t e n c y beyond t h e peak o f t h e e x t r a c e l l u l a r EPSP. 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 r e v e a l e d t h a t SR s t i m u l a t i o n evoked a l a r g e a m p l i t u d e s h o r t l a t e n c y c u r r e n t s i n k i n t h e r e g i o n o f s t r a t u m p y r a m i d a l e ( F i g 3.5A, -50pm) t h a t g r a d u a l l y i n c r e a s e d 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 s t r a t u m r a d i a t u m . T h i s waveform c o n t i n u e d as t h e s i n k component o f a d e n d r i t i c s o u r c e / s i n k , w i t h b o t h t h e so u r c e and s i n k p r o g r e s s i v e l y i n c r e a s i n g i n l a t e n c y t h r o u g h t h e d e n d r i t i c t r e e t o superimpose upon t h e l o n g l a s t i n g c u r r e n t s i n k i n t h e r e g i o n o f s y n a p t i c t e r m i n a t i o n ( F i g 3.5B, 200pm; F i g 3.5D). A s i m i l a r sequence o f p o t e n t i a l s and c u r r e n t s o u r c e / s i n k ( s ) were a l s o found t o conduct t h r o u g h 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 p y r a m i d a l c e l l s f o l l o w i n g 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 a f f e r e n t i n p u t s ( F i g 3.5). Thus, t h e c h a r a c t e r i s t i c s o f t h e SR evoked p o p u l a t i o n s p i k e response a l o n g t h e p y r a m i d a l c e l l a x i s a re not u n l i k e t h o s e - 57 - FIG. 3.5 Laminar p r o f i l e s o f 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 ( v o l t a g e ) and c u r r e n t - s o u r c e d e n s i t y (CSD) evoked t h r o u g h s u p r a t h r e s h o l d s t r a t u m r a d i a t u m s t i m u l a t i o n . A. Evoked p o t e n t i a l s were c o l l e c t e d a t 25pm s t e p s p a r a l l e l t o t h e dendro-somatic a x i s o f t h e p y r a m i d a l c e l l , and B, 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 i n 1 dimensi o n f o r each 25pm i n t e r v a l . CSD measurements a r e d i s p l a y e d w i t h a z e r o l i n e t o f a c i l i t a t e comparison o f s i n k - s o u r c e r e l a t i o n s h i p s . A s c a l e d s c h e m a t i c diagram o f t h e r a t p y r a m i d a l c e l l i s i n t e r p o s e d 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 denoted 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 the c e l l a x i s i s t a k e n from the v e n t r a l b o r d e r o f s t r a t u m p y r a m i d a l e ( Opm ). C,D. A comparison o f 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 (C) and CSD measurements (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 somata ( -50pm ) and a p i c a l d e n d r i t e s ( 200pm ) i n response t o s t r a t u m r a d i a t u m s t i m u l a t i o n (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. Voltage B. CSD 2 0 0 - i — &> "D Q Q E - E Q 0 0 - _ <1> O H o 2 t o >> + 1 0 0 O>C0 2 0 0 - 1 o o a> ^ 3 0 0 J c. Soma 20 D. Apical Dend. ~i Sink 1 Source J 5 m V 5 msec evoked t h r o u g h a n t i d r o m i c a c t i v a t i o n o r s y n a p t i c d e p o l a r i z a t i o n o f t h e b a s a l d e n d r i t i c t r e e ( c f 3.2 and 3.4). T h i s p o i n t i s more c l e a r l y i l l u s t r a t e d i n F i g 3.6, where evoked f i e l d p o t e n t i a l s i n s t r a t u m p y r a m i d a l e and r a d i a t u m are a l i g n e d w i t h a s s o c i a t e d c u r r e n t - s o u r c e d e n s i t y measurements t o p r o v i d e a d i r e c t comparison o f responses evoked by SR and a l v e a r s t i m u l a t i o n ( r e c o r d s t a k e n from 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 the- f a l l i n g edge and peak o f b o t h t h e a l v e a r and SR evoked p o p u l a t i o n s p i k e a l i g n w i t h t h e r i s i n g edge and peak o f t h e evoked c u r r e n t s i n k ( F i g 3.6A,C). S i m i l a r l y , i n s t r a t u m 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 components o f t h e d e n d r i t i c s p i k e p o t e n t i a l evoked t h r o u g h s t i m u l a t i o n o f e i t h e r pathway c o r r e s p o n d i n onset and d u r a t i o n t o t h e d e n d r i t i c c u r r e n t s o u r c e and 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 between t h e a l v e a r and SR evoked p o p u l a t i o n s p i k e response i s f u r t h e r emphasized by comparing t h e peak l a t e n c y o f t h e n e g a t i v e f i e l d p o t e n t i a l and c u r r e n t s i n k a t v a r i o u s p o i n t s a l o n g t h e p y r a m i d a l c e l l a x i s ( F i g 3.7; measurements t a k e n from F i g s 3.2 and 3.5). Both t h e n e g a t i v i t y o f t h e f i e l d p o t e n t i a l and t h e c u r r e n t s i n k evoked by a l v e a r o r SR s t i m u l a t i o n were 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 , p r o g r e s s i v e l y i n c r e a s i n g i n l a t e n c y t h r o u g h b o t h t h e b a s a l and a p i c a l d e n d r i t i c r e g i o n s . A l t h o u g h t h e l a t e n c y o f t h e evoked p o p u l a t i o n s p i k e and c u r r e n t s i n k were s i m i l a r a l o n g most of t h e p y r a m i d a l c e l l a x i s , some d e v i a t i o n i n t h e peak l a t e n c y o f t h e c u r r e n t s i n k was found i n p r o x i m a l d e n d r i t i c r e g i o n s as a r e s u l t o f c u r r e n t f l o w towards t h e l a t e s o u r c e o f c u r r e n t f o l l o w i n g p o p u l a t i o n s p i k e d i s c h a r g e i n s t r a t u m p y r a m i d a l e ( F i g 3.7). - 60 - FIG. 3_,_6_ A comparison o f t h e t i m i n g r e l a t i o n s h i p between evoked 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 and a s s o c i a t e d c u r r e n t - s o u r c e d e n s i t y (CSD) measurements 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 somata (pop s p i k e ) and a p i c a l d e n d r i t e s ( e x t r a - dend). A fB. Somatic (A) and d e n d r i t i c response (B, 160pm ) t o s u p r a t h r e s h o l d s t i m u l a t i o n o f p y r a m i d a l c e l l axons i n t h e a l v e u s . C,D. Somatic (C) and d e n d r i t i c response (D) t o s u p r a t h r e s h o l d s t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s i n s t r a t u m r a d i a t u m . Zero l i n e s a r e i n c l u d e d on CSD measurements t o f a c i l i t a t e comparison o f s i n k - s o u r c e r e l a t i o n s h i p s . Stim.Site Alveus A Pop Spike i — i 2 msec Stratum Radiatum -• Sink 1 Source 5 m V 5 msec FIG. 3.7 Peak l a t e n c y measurements o f the 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 n e g a t i v i t y ( v o l t a g e ) and c u r r e n t s i n k (CSD) a t 25pm i n t e r v a l s a l o n g t h e dendro-somatic a x i s o f t h e p y r a m i d a l c e l l i n response 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 (A) or s t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s i n s t r a t u m r a d i a t u m ( B ) . L a t e n c y measurements were c a l c u l a t e d by computer from t h e l a m i n a r p r o f i l e s o f F i g s 3.2 and 3.5, w i t h d i s t a n c e a l o n g the p y r a m i d a l c e l l a x i s t a k e n from the v e n t r a l b o r d e r o f s t r a t u m p y r a m i d a l e ( Opm ) . A s c h e m a t i c diagram o f t h e r a t p y r a m i d a l c e l l drawn t o s c a l e i s shown f o r comparison and 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 f o r each s l i c e a re denoted by d o t t e d l i n e s . Stim. Site A. 2 0 0 - l o o A o + 100 A 2 0 0 A 3 0 0 J Alveus Voltage CSD o 4 - Stratum Radiatum Voltage CSD • o • o • o m • o • o • o B. 4 ^ o J • o • o c* • 0» • o • o • o • o • o • o T" I I 1 1 2 3 4 7 8 9 10 Latency (msec) - 6 4 - Both the alvear and orthodromic evoked response were also found to display a non-uniform increase in the peak latency of the spike waveform through the dendritic f i e l d of the pyramidal c e l l population. In proximal stratum radiatum, the negativity of the population spike displayed l i t t l e s h i f t in peak latency over the i n i t i a l 100pm distance from stratum pyramidale. However, the peak latency of the negativity increased in the approximate region of t r a n s i t i o n of the spike waveform to a biphasic positive/negative p o t e n t i a l , with both components exhibiting a greater degree of latency s h i f t through mid stratum radiatum. The greatest s h i f t in peak latency of the negative potential was found in d i s t a l dendritic regions, just p r i o r to loss of the negative component of the dendritic spike response. - 65 - 3-4. D i s c u s s i o n C u r r e n t - s o u r c e d e n s i t y a n a l y s i s o f evoked 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 l o n g t h e p y r a m i d a l c e l l a x i s has s e r v e d t o 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 e x c i t a t o r y s y n a p t i c p o t e n t i a l s and s p i k e d i s c h a r g e i n C A l p y r a m i d a l neurons. S t i m u l a t i o n o f s t r a t u m o r i e n s (SO) or r a d i a t u m (SR) evoked 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 c u r r e n t s i n k s p a t i a l l y l o c a l i z e d t o r e g i o n s o f t h e p y r a m i d a l c e l l s t r u c t u r e c o n s i s t e n t w i t h t h e known d i s t r i b u t i o n o f a f f e r e n t s y n a p t i c p r o j e c t i o n s t o r e g i o s u p e r i o r . In t h e case of SO s t i m u l a t i o n , t h e n e g a t i v i t y and c u r r e n t s i n k was maximal i n t h e s t r a t u m o r i e n s , a r e g i o n c o r r e s p o n d i n g t o t h e p o i n t o f t e r m i n a t i o n o f c o m m i s s u r a l i n p u t s upon t h e p y r a m i d a l c e l l b a s a l d e n d r i t i c t r e e ( B l a c k s t a d 1956; Raisman e t a l . 1965). SR s t i m u l a t i o n evoked a maximal n e g a t i v i t y and c u r r e n t s i n k i n t h e m i d - d i s t a l s t r a t u m r a d i a t u m , an a r e a encompassing the t e r m i n a l p r o j e c t i o n s o f S c h a f f e r c o l l a t e r a l and c o m missural a f f e r e n t i n p u t s ( L o r e n t e de No 1934; Raisman e t a l . 1965; Westrum and B l a c k s t a d 1962). In e i t h e r c a s e , t h e s y n a p t i c a l l y evoked d e n d r i t i c n e g a t i v i t y and c u r r e n t s i n k d e c l i n e d i n a m p l i t u d e w i t h p r o x i m i t y t o s t r a t u m p y r a m i d a l e , and was a s s o c i a t e d w i t h a s o u r c e of c u r r e n t w i t h i n and beyond the r e g i o n o f t h e c e l l l a y e r . The a m p l i t u d e , s p a t i a l d i s t r i b u t i o n and p o l a r i t y o f evoked s y n a p t i c p o t e n t i a l s a l o n g the p y r a m i d a l c e l l a x i s i n d i c a t e an a c t i v e s y n a p t i c 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 and e l e c t r o t o n i c c o n d u c t i o n o f EPSPs from th e p o i n t o f s y n a p t i c t e r m i n a t i o n towards t h e p y r a m i d a l c e l l l a y e r . In a d d i t i o n , s y n a p t i c p o t e n t i a l s were found t o conduct beyond t h e s o m a t i c - 6 6 - l e v e l and t h r o u g h some p o r t i o n o f t h e o p p o s i n g d e n d r i t i c a r b o r i z a t i o n , as i n d i c a t e d by t h e p r e s ence of an e x t r a c e l l u l a r p o s i t i v i t y and c u r r e n t s o u r c e beyond s t r a t u m p y r a m i d a l e . I n f a c t , t h e p r e s e n c e and g r a d u a l d e c l i n e i n a m p l i t u d e o f t h e SO evoked EPSP a l o n g t h e a p i c a l d e n d r i t e has now been c o n f i r m e d t h r o u g h i n t r a d e n d r i t i c r e c o r d i n g p r o c e d u r e s (Chapter 5 ) . T h e r e f o r e , s y n a p t i c c u r r e n t s evoked w i t h i n one d e n d r i t i c t r e e o f t h e p y r a m i d a l neuron (as w i t h SO s t i m u l a t i o n ) are found t o conduct 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 m a j o r i t y o f t h e p y r a m i d a l c e l l s t r u c t u r e , a r e s u l t e m p h a s i z i n g t h e s h o r t e l e c t r o t o n i c l e n g t h o f t h e C A l p y r a m i d a l neuron (Brown e t a l . 1981; Turner 1984; Turner and S c h w a r t z k r o i n 1980). A synchronous 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 was evoked by s u p r a t h r e s h o l d s t i m u l a t i o n o f t h e a l v e u s , as shown by t h e g e n e r a t i o n o f a s h o r t l a t e n c y p o p u l a t i o n s p i k e i n t h e r e g i o n o f t h e p y r a m i d a l c e l l l a y e r . T h i s response was a s s o c i a t e d w i t h t h e immediate onset o f a c u r r e n t s i n k , i n d i c a t i n g an inward f l o w o f c u r r e n t and t h e g e n e r a t i o n o f a c t i o n p o t e n t i a l s i n t h e presumed r e g i o n o f t h e soma and axon h i l l o c k o f C A l p y r a m i d a l neurons. A n t i d r o m i c s t i m u l a t i o n a l s o evoked an e x t r a c e l l u l a r s p i k e response i n the a p i c a l d e n d r i t i c r e g i o n , o b s e r v e d as a b i p h a s i c 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 i n t h e mid s t r a t u m r a d i a t u m . T h i s waveform was found t h r o u g h c u r r e n t - s o u r c e d e n s i t y a n a l y s i s t o c o r r e s p o n d t o a c u r r e n t s o u r c e / s i n k c o n d u c t i n g from the c e l l l a y e r t h r o u g h t h e a p i c a l d e n d r i t i c f i e l d . Such a response would be e x p e c t e d f o r t h e case of an a c t i o n p o t e n t i a l c o n d u c t i n g towards a r e c o r d i n g l o c a t i o n , where a l o c a l c i r c u i t d e p o l a r i z a t i o n p r e c e d i n g 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 an i n i t i a l s o u r c e o f c u r r e n t f o l l o w e d by a c u r r e n t s i n k a t t h e t i m e - 6 7 - of spike generation at the recording s i t e . Thus, the evoked c h a r a c t e r i s t i c s of the antidromic response indicate the i n i t i a l generation of an action potential in the region of the pyramidal c e l l body and a sequential depolarization of dendritic membrane, resulting in a retrograde invasion of the spike through the apical dendritic arborization. As mentioned, a similar sequence of current source/sink relationships was observed in stratum oriens following alvear stimulation, suggesting an antidromic spike invasion of both the basal and apical dendritic tree. The above results agree with those of previous investigators, in which alvear evoked potentials in the in vivo hippocampus were interpreted to indicate a sequential spike invasion of axon h i l l o c k , soma, and basal and apical dendritic structures (Gessi et a l . 1966; Leung 1979a,b; Sperti et a l . 1967). Activation of afferent synaptic inputs terminating upon either the basal or apical dendritic tree also evoked a population spike response at a l l l e v e l s of the pyramidal c e l l axis. Suprathreshold stimulation of stratum oriens or radiatum evoked a population spike and current sink of shortest peak latency in stratum pyramidale or the proximal stratum oriens. These waveforms were continuous with a biphasic positive/negative potential or current source/sink that progressively increased in latency through both basal and apical dendritic f i e l d s . The pattern of spike generation following stimulation of afferent synaptic inputs was thus very similar to that found for antidromically evoked spike discharge. In each case, the shortest latency population spike was found in the region of the pyramidal c e l l layer, with a n t i - and orthodromic - 68 - spike responses exhibiting a s h i f t in peak latency with distance from stratum pyramidale. In addition, a d i r e c t comparison of alvear and SR evoked potentials revealed a similar relationship between e x t r a c e l l u l a r f i e l d potentials and current-source density waveforms at both the somatic and apical dendritic l e v e l of pyramidal neurons. Therefore, the sequence of spike generation in the pyramidal c e l l following activation of afferent synaptic inputs would appear indistinguishable from that found for an action potential evoked through antidromic stimulation. S p e c i f i c a l l y , synaptic depolarization of either d e n d r i t i c tree results in the i n i t i a l generation of a spike in the region of the pyramidal c e l l layer, with a subsequent retrograde spike invasion of both the basal and apical dendritic arborizations. Occasionally, short latency current sinks could be found in both the proximal stratum oriens and stratum pyramidale following suprathreshold activation of any of the three stimulus pathways. These current sinks might represent sequential AP discharge in the axon h i l l o c k and somatic region of the pyramidal c e l l , corresponding to the i n i t i a l segment (IS) and soma-dendritic (SD) components of spike discharge proposed for the spinal motoneuron (Coombs et a l . 1957a,b). However, the r e l a t i v e size and t i g h t packing of pyramidal neurons within stratum pyramidale does not permit a r e l i a b l e d i s s o c i a t i o n between the a c t i v i t y of i n i t i a l segment and somatic membranes of pyramidal c e l l s through current-source density analysis. The s i t e for i n i t i a l spike generation in the pyramidal c e l l can thus only be referred to the region of the soma - axon h i l l o c k . Regardless of the form of ac t i v a t i o n , conduction of the - 69 - s p i k e t h r o u g h t h e a p i c a l d e n d r i t i c t r e e was found t o o c c u r i n a non-uniform 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 lOOum of t h e d e n d r i t i c f i e l d , t h e e x t r a c e l l u l a r s p i k e response d i s p l a y e d a m i n i m a l s h i f t i n l a t e n c y and a c o n d u c t i o n v e l o c i t y o f 0.33 m/sec (peak l a t e n c y measurements t a k e n from t h e a n t i d r o m i c f i e l d p o t e n t i a l n e g a t i v i t y ) . In t h e m i d - d i s t a l d e n d r i t e , t h e s p i k e p o t e n t i a l i n c r e a s e d markedly i n l a t e n c y and slowed i n c o n d u c t i o n v e l o c i t y (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 l e v e l i n t h e d i s t a l d e n d r i t i c r e g i o n as 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 . These 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 o f .36m/sec f o r t h e f i b e r s o f t h e S c h a f f e r c o l l a t e r a l / c o m m i s s u r a l p r o j e c t i o n and 1.2m/sec f o r m y e l i n a t e d p y r a m i d a l c e l l axons i n t h e a l v e a r r e g i o n ( T i e l e n e t a l . 1981). There would thus appear t o be two t r a n s i t i o n p o i n t s o f s p i k e c o n d u c t i o n t h r o u g h t h e a p i c a l d e n d r i t e , as judged by t h e change i n shape and c o n d u c t i o n v e l o c i t y o f t h e e x t r a c e l l u l a r s p i k e waveform. Of i n t e r e s t i s t h e f a c t t h a t t h e s e p o i n t s c o r r e l a t e t o t h e approximate l o c a t i o n o f major b r a n c h p o i n t s o f t h e r a t 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 . As d e s c r i b e d e a r l i e r , t h e p r o x i m a l s h a f t o f t h e a p i c a l d e n d r i t e b i f u r c a t e s i n t h e range o f 25-100um from t h e p y r a m i d a l c e l l soma, w i t h more e x t e n s i v e secondary b r a n c h i n g i n d i s t a l s t r a t u m r a d i a t u m or l a c u n o s u m - m o l e c u l a r e . The f a s t i n i t i a l c o n d u c t i o n v e l o c i t y i n t h e p r o x i m a l s t r a t u m r a d i a t u m might t h e n c o r r e s p o n d t o s p i k e c o n d u c t i o n t h r o u g h t h e t h i c k p r o x i m a l d e n d r i t i c s h a f t , w i t h a s l o w i n g o f c o n d u c t i o n v e l o c i t y i n t h e r e l a t i v e l y t h i n secondary d e n d r i t i c b r a n c h e s . The presence o f a monophasic p o s i t i v e - g o i n g p o t e n t i a l i n d i s t a l s t r a t u m r a d i a t u m may f u r t h e r i n d i c a t e a l o s s o f s p i k e c o n d u c t i o n i n t h e r e g i o n o f secondary d e n d r i t i c - 7 0 - b r a n c h i n g . P r e v i o u s i n v e s t i g a t i o n s have r e p o r t e d t h a t s t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s i n s t r a t u m r a d i a t u m r e s u l t s i n t h e i n i t i a l a c t i v a t i o n o f a s p i k e from w i t h i n t h e 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 (Andersen 1 9 6 0 ; Andersen and Lomo 1 9 6 6 ; Cragg and Hamlyn 1 9 5 5 ; F u j i t a and Sakata 1 9 6 2 ) . The f i r s t e v i d e n c e f o r d e n d r i t i c s p i k e d i s c h a r g e came from l a m i n a r p r o f i l e a n a l y s e s o f evoked 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 , and c o n s i d e r a t i o n o f t h e l a t e n c y o f f i e l d p o t e n t i a l components a l o n g t h e p y r a m i d a l c e l l a x i s . Some of t h e s e s t u d i e s r e p o r t e d an evoked n e g a t i v e s p i k e p o t e n t i a l o f s h o r t e s t peak l a t e n c y i n t h e p r o x i m a l s t r a t u m r a d i a t u m , s u g g e s t i n g a p r o x i m a l d e n d r i t i c o r i g i n f o r t h e evoked s p i k e (Andersen 1 9 6 0 ; F u j i t a and S a k a t a 1 9 6 2 ) . I n t h e p r e s e n t s t u d y , t h e p o p u l a t i o n 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 l o c a t e d i n some s l i c e s i n t h e p r o x i m a l r e g i o n o f s t r a t u m r a d i a t u m . However, subsequent c u r r e n t - s o u r c e d e n s i t y a n a l y s i s r e v e a l e d 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 found i n s t r a t u m p y r a m i d a l e or p r o x i m a l s t r a t u m o r i e n s . S i n c e a more a c c u r a t e s p a t i a l 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 can be a c h i e v e d t h r o u g h c u r r e n t - s o u r c e d e n s i t y a n a l y s i s , t h e s i t e f o r AP g e n e r a t i o n can a g a i n be t a k e n as w i t h i n t h e soma-axon h i l l o c k r e g i o n o f p y r a m i d a l neurons. The above r e s u l t s e r v e s t o i l l u s t r a t e t h e d i f f i c u l t i e s t h a t can a r i s e when a t t e m p t i n g t o i d e n t i f y t h e s i t e o f o r i g i n of an evoked response from the s p a t i a l d i s t r i b u t i o n o f 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 . Indeed, t h e a m b i g u i t y o f f i e l d p o t e n t i a l i n t e r p r e t a t i o n o f c u r r e n t d e n s i t y was r e v e a l e d i n one s l i c e , where peak l a t e n c y measurements o f the p o p u l a t i o n s p i k e appeared t o i n d i c a t e a d i f f e r e n t s i t e o f o r i g i n f o r s p i k e s evoked from - 7 1 - each o f t h e t h r e e s t i m u l u s pathways. However, e x a m i n a t i o n o f 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 demonstrated a common s i t e o f o r i g i n f o r a l l evoked s p i k e s i n t h e r e g i o n o f s t r a t u m p y r a m i d a l e , 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 s i n k . O thers have r e p o r t e d a d i s t a l d e n d r i t i c s i t e o f o r i g i n f o r the p y r a m i d a l c e l l d e n d r i t i c s p i k e f o l l o w i n g s t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s (Andersen e t a l . 1966a,b; Andersen and Lomo 1966; Cragg and Hamlyn 1955). In t h e s e s t u d i e s , t h e e x t r a c e l l u l a r r e p r e s e n t a t i o n of a d e n d r i t i c s p i k e was o f t e n t a k e n as t h e peak n e g a t i v i t y o f t h e evoked response a t a l l l e v e l s o f t h e p y r a m i d a l c e l l a x i s . However, i n t h e p r e s e n t s t u d y , t h e n e g a t i v i t y o f t h e SR evoked d e n d r i t i c s p i k e response was found t o be preceded 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 s t r a t u m r a d i a t u m , 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 components c o n d u c t i n g from t h e c e l l body l a y e r . F u r t h e r m o r e , t h e p o s i t i v e / n e g a t i v e s p i k e p o t e n t i a l would o f t e n f a l l beyond t h e peak o f t h e EPSP i n s t r a t u m r a d i a t u m , w i t h t h e r e s u l t t h a t t h e n e g a t i v e s y n a p t i c waveform was i n many cases g r e a t e r i n peak a m p l i t u d e t h a n t h e n e g a t i v e component o f the d e n d r i t i c s p i k e (see F i g 3.5A, 225 and 250um). Measurement o f a s h o r t l a t e n c y n e g a t i v i t y on evoked d e n d r i t i c p o t e n t i a l s would t h e r e f o r e r e p r e s e n t 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 i m p r e s s i o n o f i n i t i a l s p i k e g e n e r a t i o n i n d i s t a l d e n d r i t i c r e g i o n s . One f a c t o r c o n t r i b u t i n g t o t h e d i f f i c u l t y i n i d e n t i f y i n g 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 component of the s p i k e p o t e n t i a l c o u l d a t tim e s be d i f f i c u l t t o d i s t i n g u i s h from t h e u n d e r l y i n g n e g a t i v i t y o f t h e SR evoked EPSP. In t h i s c a s e , t h e d e n d r i t i c s p i k e 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 o f t h e a l v e u s o r s t r a t u m o r i e n s i n t h e same s l i c e would evoke a d i s t i n c t p o s i t i v e / n e g a t i v e s p i k e p o t e n t i a l a t t h e same d e n d r i t i c r e c o r d i n g s i t e . T h i s r e s u l t i s not c o m p l e t e l y u n d e r s t o o d a t t h e p r e s e n t t i m e , but may r e l a t e t o the f a c t t h a t SR s t i m u l a t i o n r e s u l t s i n an a c t i v e s y n a p t i c d e p o l a r i z a t i o n o f t h e a p i c a l d e n d r i t e , a form of membrane a c t i v a t i o n n o t brought about t h r o u g h s t i m u l a t i o n o f t h e a l v e u s o r s t r a t u m o r i e n s . A s p i k e i n v a d i n g t h e a p i c a l d e n d r i t e f o l l o w i n g SR s t i m u l a t i o n may thus encounter membrane o f r e l a t i v e l y h i g h conductance i n t h e r e g i o n o f s y n a p t i c t e r m i n a t i o n , d i s t o r t i n g the e x t r a c e l l u l a r waveform of t h e d e n d r i t i c s p i k e . 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 t h e f a c t t h a t t h e n e g a t i v e component o f the b a s a l d e n d r i t i c s p i k e c o u l d a l s o be d i f f i c u l t t o d i s t i n g u i s h on t h e e x t r a d e n d r i t i c p o t e n t i a l d u r i n g SO s t i m u l a t i o n and s y n a p t i c d e p o l a r i z a t i o n o f the b a s a l d e n d r i t i c t r e e . However, r e g a r d l e s s o f t h e e x a c t form of t h e e x t r a d e n d r i t i c s p i k e p o t e n t i a l , c u r r e n t - s o u r c e d e n s i t y a n a l y s i s would c o n f i r m t h a t t h e p o s i t i v e - g o i n g s p i k e response had conducted from the c e l l body l a y e r t h r o u g h t h e d e n d r i t i c f i e l d . 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 not s u p p o r t the c o n t e n t i o n o f a d e n d r i t i c o r i g i n f o r evoked s p i k e s w i t h i n t h e a p i c a l d e n d r i t i c f i e l d o f t h e C A l p y r a m i d a l neuron. R a t h e r , the i n i t i a l s i t e f o r g e n e r a t i o n o f a s p i k e was l o c a l i z e d t h r o u g h c u r r e n t - s o u r c e d e n s i t y a n a l y s i s t o t h e c e l l body l a y e r o f t h e p y r a m i d a l c e l l p o p u l a t i o n . The p y r a m i d a l c e l l d e n d r i t i c s p i k e would t h u s 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 o f the 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 i n i t i a t e d i n t h e r e g i o n o f the soma-axon h i l l o c k . - 7 3 - 4 - 0 . COMPARATIVE INTRACELLULAR ANALYSIS OF SOMATIC AND DENDRITIC ELECTROPHYSIOLOGY OF C A l PYRAMIDAL NEURONS 4 - 1 . I n t r o d u c t i o n R e g i o n a l v a r i a t i o n s i n t h e d e n s i t y and s p a t i a l d i s t r i b u t i o n of i o n i c c h a n n e l s i n n e u r o n a l membrane can r e s u l t i n a n on-uniform e x c i t a b i l i t y a l o n g the dendro-somatic a x i s o f a c e l l ( L l i n a s 1 9 7 5 ; Traub and L l i n a s 1 9 7 9 ) . The hippocampal p y r a m i d a l c e l l i s one neuron o f the mammalian CNS thought t o e x h i b i t a r e g i o n a l v a r i a t i o n i n 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 p a t t e r n o f evoked a c t i v i t y v a r y i n g a c c o r d i n g t o the form o f a c t i v a t i o n and between s o m a t i c and d e n d r i t i c r e c o r d i n g s i t e s . For i n s t a n c e , s t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s i s found t o evoke a s i n g l e f a s t (Na+-dependent) a c t i o n p o t e n t i a l (AP) a t b o t h the s o m a t i c and a p i c a l d e n d r i t i c l e v e l o f t h e p y r a m i d a l c e l l (Benardo e t a l . 1 9 8 2 ; Kandel e t a l . 1 9 6 1 ; S c h w a r t z k r o i n 1 9 7 5 , 1 9 7 7 ; Wong e t a l . 1 9 7 9 ) . In c o n t r a s t , a c o m b i n a t i o n o f b o t h Na+ and Ca+ 2-dependent p o t e n t i a l s can be evoked i n t h e p y r a m i d a l c e l l by i n t r a c e l l u l a r i n j e c t i o n o f d e p o l a r i z i n g c u r r e n t (Benardo et a l . 1 9 8 2 ; S c h w a r t z k r o i n and Slawsky 1 9 7 7 ; Wong e t a l . 1 9 7 9 ) . However, Ca+ 2-dependent p o t e n t i a l s are more predominant i n d e n d r i t i c than s o m a t i c membrane, w i t h t h e g e n e r a t i o n o f l a r g e Ca+ 2 s p i k e s a t the d e n d r i t i c l e v e l (Benardo e t a l . 1 9 8 2 ; Wong e t a l . 1 9 7 9 ) . The d i f f e r e n t i a l 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 o f s o m a t i c and d e n d r i t i c membranes are thought t o be of i m p o r t a n t consequence t o t h e f i n a l o u t p u t o f t h e p y r a m i d a l c e l l . A c c o r d i n g t o t h e r e s u l t s of p r e v i o u s s t u d i e s , s e l e c t i v e 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 a low t h r e s h o l d f o r Na+ s p i k e - 74 - a c t i v a t i o n (hot s p o t s ) , and s p i k e g e n e r a t i o n i n t h e d e n d r i t e can precede t h a t a t t h e axon h i l l o c k f o l l o w i n g s t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s (Spencer and Kan d e l 1961b; Traub and L l i n a s 1979). The d e n d r i t i c s p i k e t h e n c o n d u c t s t o t h e c e l l body, a p p e a r i n g at t h e s o m a t i c l e v e l as a s m a l l f a s t p r e - p o t e n t i a l superimposed upon t h e s y n a p t i c d e p o l a r i z a t i o n (Andersen and Lomo 1966; S c h w a r t z k r o i n 1977; Spencer and Kandel 1961b). Thus, a s p i k e o r i g i n a t i n g from w i t h i n t h e d e n d r i t e can summate w i t h s y n a p t i c c u r r e n t s , and i n t h i s way i n c r e a s e t h e p r o b a b i l i t y f o r g e n e r a t i o n o f a s p i k e a t t h e soma-axon h i l l o c k . However, a major q u e s t i o n t h a t remains unanswered i s t h e a c t u a l s i t e f o r g e n e r a t i o n o f t h e Na+-dependent d e n d r i t i c s p i k e . Most s t u d i e s have r e p o r t e d e v i d e n c e t o suggest a d e n d r i t i c s i t e of o r i g i n f o r t h e s p i k e r e c o r d e d 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 o f p y r a m i d a l neurons (Andersen 1960; Andersen and Lomo 1966; Cragg and Hamlyn 1955; F u j i t a and S a k a t a 1962; S c h w a r t z k r o i n 1977; Spencer and Kandel 1961b; Traub and L l i n a s 1979; Wong e t a l . 1979). However, r e s u l t s o b t a i n e d t h r o u g h c u r r e n t - s o u r c e d e n s i t y a n a l y s i s o f evoked 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 would not appear t o s u p p o r t t h i s h y p o t h e s i s (Chapter 3 ) . A c c o r d i n g t o t h i s s t u d y , t h e i n i t i a l s i t e f o r s p i k e g e n e r a t i o n i n t h e p y r a m i d a l c e l l i s i n t h e r e g i o n o f t h e soma-axon h i l l o c k , w i t h d e n d r i t i c s p i k e d i s c h a r g e o c c u r r i n g t h r o u g h a r e t r o g r a d e s p i k e i n v a s i o n o f the d e n d r i t i c a r b o r i z a t i o n . G i v e n t h e c o n f l i c t i n g n a t u r e o f t h e above h y p o t h e s e s , i t would seem n e c e s s a r y t o f u r t h e r c h a r a c t e r i z e t h e 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 o f t h e p y r a m i d a l c e l l i n o r d e r t o und e r s t a n d t h e f a c t o r s r e s p o n s i b l e f o r s p i k e g e n e r a t i o n a t t h e so m a t i c and d e n d r i t i c l e v e l . The p r e s e n t s t u d y t h e r e f o r e - 75 - r e p r e s e n t s a c o m p a r a t i v e i n t r a c e l l u l a r a n a l y s i s o f membrane p r o p e r t i e s , s y n a p t i c p o t e n t i a l s , and 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 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 neurons. - 76 - 4-2. Methods I n t r a c e l l u l a r impalements o f n e u r o n a l elements were r e s t r i c t e d t o s t r a t u m p y r a m i d a l e and s t r a t u m r a d i a t u m of t h e CAlb s u b f i e l d o f r e g i o s u p e r i o r . I n t r a c e l l u l a r p o t e n t i a l s r e c o r d e d i n t h e s e s t r a t a c o u l d c o r r e s p o n d t o a c t i v i t y o f t h e somata o r d e n d r i t i c e x t e n s i o n s o f e i t h e r p y r a m i d a l or non- p y r a m i d a l neurons ( C a j a l 1911; L o r e n t e de No 1934). However, s e v e r a l l i n e s o f e v i d e n c e would suggest t h a t t h e r e s u l t s t o be p r e s e n t e d were o b t a i n e d from t h e somata and a p i c a l d e n d r i t e s of p y r a m i d a l neurons. F i r s t l y , t h e p y r a m i d a l neuron i s by f a r t h e most common c e l l t y p e i n t h e C A l r e g i o n , w i t h s t r a t u m p y r a m i d a l e and ra d i a t u m comprised l a r g e l y o f t i g h t l y packed p y r a m i d a l c e l l 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 a r e m a i n l y l o c a l i z e d above s t r a t u m p y r a m i d a l e , a l t h o u g h some are found s c a t t e r e d t h r o u g h t h e p y r a m i d a l c e l l l a y e r . Even fewer numbers o f non - p y r a m i d a l neurons a re found i n s t r a t u m r a d i a t u m ( C a j a l 1911; L o r e n t e de No 1934). T h e r e f o r e , t h e r e l a t i v e 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 s t r u c t u r e s suggest t h a t i n t r a c e l l u l a r r e c o r d i n g s i n C A l would i n h i g h p r o b a b i l i t y c o r r e s p o n d t o impalements o f p y r a m i d a l c e l l s . S e c o n d l y , p r e v i o u s i n v e s t i g a t o r s have compared t h e membrane p r o p e r t i e s and d i s c h a r g e p a t t e r n s o f p y r a m i d a l somata (Knowles and S c h w a r t z k r o i n 1981a; Turner and 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 Mathers 197 8; Wong e t a l . 197 9) t o t h a t o f non - p y r a m i d a l neurons (Ashwood e t a l . 1984; Knowles and S c h w a r t z k r o i n 1981a; S c h w a r t z k r o i n and Mathers 1978; Turner and S c h w a r t z k r o i n 1980). These s t u d i e s i n d i c a t e t h a t p y r a m i d a l and non - p y r a m i d a l c e l l - 77 - elements can be d i s t i n g u i s h e d on t h e b a s i s o f 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 . I n t h e p r e s e n t s t u d y , impaled n e u r o n a l s t r u c t u r e s d i s p l a y e d evoked p a t t e r n s o f a c t i v i t y c h a r a c t e r i s t i c o f p y r a m i d a l c e l l somata and a p i c a l d e n d r i t e s , w h i l e c e l l u l a r elements w i t h p r o p e r t i e s a s c r i b e d t o no n - p y r a m i d a l neurons were v e r y i n f r e q u e n t l y e n c o u n t e r e d . 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 v e r i f i e d t h a t n e u r o n a l elements impaled i n s t r a t u m p y r a m i d a l e or r a d i a t u m 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 t o t h o s e r e p o r t e d here c o r r e s p o n d t o impalements o f somata (Turner and 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 o f p y r a m i d a l neurons (Wong e t 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 re c o n s i d e r e d r e p r e s e n t a t i v e o f s o m a t i c and a p i c a l d e n d r i t i c a c t i v i t y o f C A l p y r a m i d a l neurons. C o n s i d e r i n g the d i s p e r s i o n o f c e l l b o d i e s w i t h i n t h e boundary o f s t r a t u m p y r a m i d a l e ( L o r e n t e de No 1934), t h e p o s s i b i l i t y e x i s t s t h a t i n t r a c e l l u l a r impalements w i t h i n t h i s l a y e r may i n c l u d e r e c o r d i n g s from t h e p r o x i m a l p o r t i o n o f e i t h e r b a s a l or a p i c a l d e n d r i t e s o f p y r a m i d a l c e l l s . However, w i t h t h e s e l i m i t a t i o n s i n mind, r e c o r d i n g s from w i t h i n s t r a t u m p y r a m i d a l e w i l l be d e s i g n a t e d as " s o m a t i c " i n o r i g i n . The r e s u l t s o f the p r e s e n t s t u d y were o b t a i n e d from over 165 s o m a t i c and 81 a p i c a l d e n d r i t i c r e c o r d i n g s . A l l 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 were r e c o r d e d from s i n g l e m i c r o e l e c t r o d e impalements o f e i t h e r the soma or d e n d r i t e , and were not o b t a i n e d from d u a l impalements o f a s i n g l e p y r a m i d a l neuron. Membrane 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 - 7 8 - l e a s t -55mV and i n p u t r e s i s t a n c e of 18 megohm or more. The am p l i t u d e s o f evoked p o t e n t i a l s were not used as acceptance c r i t e r i a s i n c e t h i s c h a r a c t e r i s t i c was found t o v a r y a c c o r d i n g t o t h e r e c o r d i n g p o s i t i o n a l o n g t h e p y r a m i d a l c e l l a x i s (Chapter 5 ) . The term " c u r r e n t evoked s p i k e " i s used t o r e f e r t o an a c t i o n p o t e n t i a l evoked t h r o u g h i n t r a c e l l u l a r i n j e c t i o n o f d e p o l a r i z i n g c u r r e n t , w h i l e " s t i m u l u s evoked s p i k e s " a re th o s e a c t i v a t e d t h r o u g h s t i m u l a t i o n o f a f f e r e n t o r e f f e r e n t pathways i n t h e hippocampal s l i c e . S p i k e a m p l i t u d e and h a l f w i d t h ( w i d t h a t h a l f a m p l i t u d e ) was measured w i t h r e s p e c t t o t h e r e s t i n g p o t e n t i a l b a s e l i n e p r e c e d i n g t h e s t i m u l u s a r t e f a c t . A m p l i t u d e , w i d t h , and v o l t a g e t h r e s h o l d measurements f o r c u r r e n t evoked s p i k e s were t a k e n from t h e f i r s t c u r r e n t evoked a c t i o n p o t e n t i a l . V o l t a g e t h r e s h o l d f o r s y n a p t i c a l l y evoked AP d i s c h a r g e was t a k e n as e q u i v a l e n t t o t h e average peak a m p l i t u d e o f t h e u n d e r l y i n g EPSP f o r s t i m u l u s i n t e n s i t i e s j u s t t h r e s h o l d f o r s p i k e a c t i v a t i o n . The r e f e r e n c e 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 a l o n g t h e p y r a m i d a l c e l l a x i s was measured was t a k e n as t h e v e n t r a l b o r d e r o f s t r a t u m p y r a m i d a l e . The d i s t a n c e o f t h e d e n d r i t i c r e c o r d i n g e l e c t r o d e from t h i s p o i n t was measured w i t h a g r a t i c u l e w i t h i n t h e o b j e c t i v e l e n s o f t h e m i c r o s c o p e , o r e s t i m a t e d as a f r a c t i o n o f the d i s t a n c e from s t r a t u m p y r a m i d a l e t o t h e hippocampal f i s s u r e . T h i s was done by v i s u a l l y d i v i d i n g the 400um d i s t a n c e from the c e l l l a y e r t o t h e f i s s u r e i n h a l f , and s u b s e q u e n t l y d i v i d i n g e i t h e r h a l f i n t o t h i r d s , t h e r e b y p a r t i t i o n i n g t h e t o t a l d i s t a n c e i n t o s e c t i o n s a p p r o x i m a t i n g 65um. Any impalements f a l l i n g between 65pm d i v i s i o n s were g i v e n a v a l u e i n t e r m e d i a t e t o t h e d i s t a n c e o f a d j a c e n t d i v i s i o n p o i n t s . T h i s t e c h n i q u e proved t o be a r e a s o n a b l y a c c u r a t e method o f j u d g i n g t h e d i s t a n c e o f a r e c o r d i n g e l e c t r o d e from s t r a t u m p y r a m i d a l e , and e s t i m a t e s made i n t h i s way were found t o be comparable t o t h o s e o b t a i n e d by measurement w i t h t h e g r a t i c u l e . U n l e s s o t h e r w i s e s t a t e d , d e n d r i t i c impalements were o b t a i n e d a t l e a s t 150um from the v e n t r a l b o r d e r o f s t r a t u m p y r a m i d a l e . - 80 - 4-3. R e s u l t s Membrane C h a r a c t e r i s t i c s The membrane p o t e n t i a l response of p y r a m i d a l c e l l s o m a t i c and d e n d r i t i c impalements t o a s e r i e s o f square wave c u r r e n t p u l s e i n j e c t i o n s a r e shown i n F i g 4.1A fB. C u r r e n t / v o l t a g e (I/V) 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 of membrane p o t e n t i a l a t b o t h l o c a t i o n s over a range of p o t e n t i a l s e x t e n d i n g from a p p r o x i m a t e l y -80mV to -55mV. For h y p e r p o l a r i z i n g p u l s e s beyond t h i s range, a time-dependent sag of t h e membrane p o t e n t i a l c o u l d be o bserved i n b o t h s o m a t i c and d e n d r i t i c impalements, a response r e f e r r e d t o as "anomalous r e c t i f i c a t i o n " (Hotson e t a l . 1979). Anomalous r e c t i f i c a t i o n was a l s o o b s e r v e d f o r d e p o l a r i z i n g c u r r e n t p u l s e i n j e c t i o n s as an i n c r e a s e i n t h e s l o p e o f t h e I/V r e l a t i o n a t p o t e n t i a l s d e p o l a r i z e d from r e s t i n g v a l u e . Examples o f s o m a t i c and d e n d r i t i c r e c o r d i n g s i n w h i c h t h i s form of r e c t i f i c a t i o n was p a r t i c u l a r l y e v i d e n t a r e shown i n F i g 4.1C,D. Somatic and d e n d r i t i c membranes had comparable v a l u e s of r e s t i n g p o t e n t i a l , w i t h an average v a l u e of 62.5 +/- ,73mV (n=83; mean +/- sem) i n i n t r a s o m a t i c r e c o r d i n g s i t e s , and 64.3 +/- .55mV (n=27) f o r d e n d r i t i c impalements (Table 1 ) . I nput r e s i s t a n c e a t t h e s o m a t i c and d e n d r i t i c l e v e l were a l s o s i m i l a r , c a l c u l a t e d as 25.9 +/- .57megohm (n=92; mean +/- sem) and 24.4 +/- 2.06megohm (n=14) f o r i n t r a s o m a t i c and - 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 s , r e s p e c t i v e l y ( T a ble 1 ) . - 8 1 - FIG. 4.1 Pyramidal c e l l somatic (A) and apical dendritic (B) membrane potential responses to a range of square wave hyperpolarizing and depolarizing current pulse i n j e c t i o n s . The corresponding current/voltage (I/V) plot and calculated input resistances (Ri) for somatic and dendritic responses are shown below. Anomalous r e c t i f i c a t i o n of membrane potential in the depolarizing range i s shown for another somatic (C) and dendritic (D) impalement. Action potentials truncated. In t h i s and a l l subsequent figures, the intrasomatic and int r a d e n d r i t i c response from separate pyramidal neurons are presented for comparison. - 82 - Somatic Dendritic 2 0 msec - 8 3 - TABLE 1 Average r e s t i n g membrane p o t e n t i a l (RMP) and i n p u t r e s i s t a n c e (Ri) o f C A l p y r a m i d a l c e l l s o m a t i c and a p i c a l d e n d r i t i c impalements o b t a i n e d a t l e a s t 150um from t h e b o r d e r o f s t r a t u m p y r a m i d a l e (mean +/- sem; number o f impalements shown i n b r a c k e t s ) . The average v a l u e o f i n p u t r e s i s t a n c e was c a l c u l a t e d o n l y f o r t h o s e r e c o r d i n g s i n which f u l l c u r r e n t / v o l t a g e p r o f i l e s had been c o n s t r u c t e d . - 84 - TABLE 1 SOMATIC DENDRITIC (> 150 JJM) RF1P 6 2 . 5 6 4 . 3 (MV) ± .73 ± .55 (83) (27) R I ( f i n ) 2 5 . 9 ± .57 (92) 2 4 . 4 ± 2 . 0 6 (14) - 85 - Current Evoked Suprathreshold Responses D e p o l a r i z i n g c u r r e n t s r e l i a b l y evoked r e p e t i t i v e s p i k e d i s c h a r g e i n somatic and d e n d r i t i c membranes of the pyramidal neuron. In g e n e r a l , c u r r e n t evoked a c t i o n p o t e n t i a l (AP) dis c h a r g e i n each l o c a t i o n was very s i m i l a r and c o u l d be assigned to e i t h e r of two b a s i c p a t t e r n s , r e f e r r e d to as "Type 1" and "Type 2" f o r the purpose of comparison. A t h i r d but l e s s common form of b u r s t d i s c h a r g e appeared to be r e s t r i c t e d to d e n d r i t i c membrane (Type 3 ) . The Type 1 response was the most common form of s p i k e d i s c h a r g e i n both somatic (12/16) and d e n d r i t i c impalements (21/36). In these c e l l s , d e p o l a r i z i n g c u r r e n t evoked a r e p e t i t i v e sequence of f a s t s p i k e s f o r a l l l e v e l s of d e p o l a r i z i n g c u r r e n t i n j e c t i o n ( F i g 4.2). Higher l e v e l s of d e p o l a r i z i n g c u r r e n t evoked an i n i t i a l h i g h frequency " b u r s t " of three to fou r a c t i o n p o t e n t i a l s f o l l o w e d by r e p e t i t i v e s p i k e a c t i v a t i o n , with a p r o g r e s s i v e decrease i n i n t e r s p i k e i n t e r v a l as c u r r e n t i n j e c t i o n was i n c r e a s e d . The c h a r a c t e r i s t i c s of evoked s p i k e s w i t h i n the b u r s t v a r i e d i n a p r e d i c t a b l e manner, d i s p l a y i n g a r a p i d d e c l i n e i n amplitude and i n c r e a s e i n h a l f w i d t h (width at h a l f amplitude), with each s p i k e i n the bu r s t a r i s i n g from a s l i g h t l y g r e a t e r l e v e l of d e p o l a r i z a t i o n . While the b a s i c p a t t e r n of Type 1 di s c h a r g e was recorded at a l l l e v e l s of the pyramidal c e l l a x i s , d i f f e r e n c e s i n 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 somatic and d e n d r i t i c s p i k e s were d e t e c t e d . For i n s t a n c e , s p i k e s evoked i n the somatic region of the pyramidal c e l l were of g r e a t e r amplitude and s h o r t e r h a l f w i d t h than those recorded w i t h i n the a p i c a l d e n d r i t e . The recovery of s p i k e amplitude f o l l o w i n g the i n i t i a l b u r s t of AP - 86 - FIG 4.2 The "Type 1" response of a C A l p y r a m i d a l neuron t o i n c r e a s i n g l e v e l s o f d e p o l a r i z i n g c u r r e n t a p p l i e d t o a s o m a t i c (A-C) and d e n d r i t i c impalement (D-F). Note d i f f e r e n c e i n v o l t a g e c a l i b r a t i o n between s o m a t i c and d e n d r i t i c r e c o r d i n g s . - 87 - Somatic Dendritic D. B. E. — c. ill J J 120 J I nA 10 mV 20 msec - 88 - d i s c h a r g e c o u l d a l s o d i f f e r between s o m a t i c and d e n d r i t i c r e c o r d i n g 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 u s u a l l y r e c o v e r e d from t h e i n i t i a l d e c l i n e w i t h i n t h e b u r s t by a p p r o x i m a t e l y t h e f i f t h t o e i g h t h a c t i o n p o t e n t i a l o f t h e r e p e t i t i v e s p i k e t r a i n . I n c o n t r a s t , t h e r e c o v e r y o f s p i k e a m p l i t u d e i n t h e d e n d r i t e c o u l d depend upon t h e degree o f c u r r e n t i n j e c t i o n ( 7 / 1 2 ) , w i t h l e s s r e c o v e r y o b s e r v e d t h e g r e a t e r t h e a m p l i t u d e o f the d e p o l a r i z i n g c u r r e n t p u l s e ( F i g 4.2 D-F). I n a d d i t i o n , s o m a t i c s p i k e s were evoked from 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 f i r s t evoked a c t i o n p o t e n t i a l a r i s i n g from a s i m i l a r l e v e l o f d e p o l a r i z a t i o n r e g a r d l e s s o f t h e a m p l i t u d e o f t h e d e p o l a r i z i n g c u r r e n t p u l s e ( F i g 4.2A-C; v o l t a g e t h r e s h o l d measured as t h e a b s o l u t e v o l t a g e o f t h e b r e a k p o i n t o f s p i k e d i s c h a r g e ) . However, d e n d r i t i c membrane d i s p l a y e d no apparent t h r e s h o l d f o r AP d i s c h a r g e , w i t h t h e v o l t a g e b r e a k p o i n t f o r s p i k e a c t i v a t i o n i n c r e a s i n g d i r e c t l y w i t h t h e l e v e l o f c u r r e n t i n j e c t i o n ( F i g 4.2D-F). Type 2 s p i k e d i s c h a r g e was c h a r a c t e r i z e d by an i n i t i a l h i g h f r e q u e n c y b u r s t o f f a s t s p i k e s f o l l o w e d by an a f t e r h y p e r p o l a r i z a t i o n (AHP) and r e p e t i t i v e s i n g l e s p i k e d i s c h a r g e ( F i g 4.3A-G). T h i s p a t t e r n of a c t i v i t y was l e s s commonly o b s e r v e d i n b o t h s o m a t i c (4/16) and d e n d r i t i c (10/36) impalements and appeared t o be most p r e v a l e n t i n membrane d i s p l a y i n g an o b v i o u s anomalous r e c t i f i c a t i o n i n t h e d e p o l a r i z i n g range o f c u r r e n t i n j e c t i o n ( c f 4.1C,D). Near t h r e s h o l d f o r AP d i s c h a r g e , c u r r e n t i n j e c t i o n evoked t h e Type 2 response i n f u l l form, i n c l u d i n g an i n i t i a l b u r s t o f s p i k e s , an AHP, and r e p e t i t i v e s p i k e d i s c h a r g e ( F i g 4.3A,D). However, t h e p a t t e r n o f a c t i v i t y v a r i e d w i t h t h e degree o f d e p o l a r i z a t i o n , - 89 - w i t h r e p e t i t i v e s p i k e s e n c r o a c h i n g upon t h e AHP a t h i g h e r l e v e l s of c u r r e n t i n j e c t i o n ( F i g 4.3A-C and D-G). Once a g a i n , c e r t a i n c h a r a c t e r i s t i c s of t h e evoked s p i k e d i f f e r e d between s o m a t i c and 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 s . In the soma, s p i k e s w i t h i n the i n i t i a l b u r s t o f Type 2 d i s c h a r g e were evoked 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 d e c l i n e i n a m p l i t u d e and i n c r e a s e i n h a l f w i d t h . However, t h e u n d e r l y i n g d e p o l a r i z a t i o n and change i n s p i k e c h a r a c t e r i s t i c s d u r i n g the b u r s t were more a c c e n t u a t e d i n d e n d r i t i c impalements. A l t h o u g h s p i k e a m p l i t u d e f o l l o w i n g the i n i t i a l b u r s t c o u l d r e c o v e r i n s o m a t i c r e c o r d i n g s d u r i n g the c u r r e n t p u l s e , the a m p l i t u d e o f d e n d r i t i c s p i k e s c o u l d depend upon the l e v e l o f c u r r e n t i n j e c t i o n , d i s p l a y i n g a g r e a t e r degree o f d e p r e s s i o n w i t h h i g h e r d e p o l a r i z i n g c u r r e n t s ( F i g 4.3D-F). In some d e n d r i t i c impalements (3/10), t h e t h i r d t o f i f t h s p i k e o f t h e i n i t i a l b u r s t merged on t o p o f a p a r t i c u l a r l y l a r g e u n d e r l y i n g d e p o l a r i z a t i o n t o form a l a r g e r and broader s p i k e , s i m i l a r t o the " i n t e r m e d i a t e s p i k e " of Benardo e t a l . (1982) ( F i g 4.3G). Component a c t i o n p o t e n t i a l s c o u l d then be d e t e c t e d as s m a l l i n f l e c t i o n s on t h e r i s i n g or f a l l i n g edge of t h e i n t e r m e d i a t e s p i k e ( F i g 4.3G). Once a g a i n , s p i k e s r e c o r d e d i n the s o m a t i c r e g i o n were evoked from a s i m i l a r v o l t a g e t h r e s h o l d f o r a l l l e v e l s o f c u r r e n t i n j e c t i o n ( F i g 4.3A-C; measurements r e f e r t o the f i r s t evoked s p i k e ) , w h i l e the v o l t a g e t h r e s h o l d 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 c o u l d appear t o i n c r e a s e d i r e c t l y w i t h t h e a m p l i t u d e o f t h e c u r r e n t p u l s e ( F i g 4.3D-F). S e v e r a l o f the evoked c h a r a c t e r i s t i c s o f Type 2 d i s c h a r g e were s i m i l a r t o t h a t o f Type 1 s p i k e a c t i v a t i o n . For i n s t a n c e , t h e a m p l i t u d e , h a l f w i d t h and v o l t a g e t h r e s h o l d of t h e f i r s t - 90 - FIG. 4.3 The "Type 2" response o f C A l p y r a m i d a l neurons t o i n c r e a s i n g l e v e l s o f d e p o l a r i z i n g c u r r e n t a p p l i e d t o a s o m a t i c (A-C) and d e n d r i t i c impalement (D-F). The Type 2 response o f G was t a k e n from a s e p a r a t e d e n d r i t i c impalement from t h a t i n D-F. H-K. "Type 3" responses o f d e n d r i t i c r e c o r d i n g s i l l u s t r a t i n g t h e c u r r e n t evoked and spontaneous d i s c h a r g e o f two s e p a r a t e d e n d r i t i c impalements i n H , l and J,K. > Somatic Dendritic 20 msec - 92 - c u r r e n t evoked s p i k e were comparable i n b o t h Type 1 and 2 d i s c h a r g e . However, two major d i s t i n g u i s h i n g f e a t u r e s were t h e l a r g e r 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 i n i t i a l b u r s t o f s p i k e s and the subsequent a f t e r h y p e r p o l a r i z a t i o n observed i n t h e Type 2 p a t t e r n o f AP d i s c h a r g e . Type 1 and 2 a c t i v i t y were t h e r e f o r e most r e a d i l y d i s t i n g u i s h e d near t h r e s h o l d f o r s p i k e a c t i v a t i o n , where t h e AHP o f Type 2 d i s c h a r g e was most a p p a r e n t . A t h i r d and i n f r e q u e n t form o f s p i k e a c t i v i i t y (5/36) was o n l y e n c o u n t e r e d i n 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 s . The c u r r e n t evoked and spontaneous d i s c h a r g e o f two s e p a r a t e d e n d r i t i c impalements i l l u s t r a t i n g t h i s t y p e o f response are shown i n F i g 4.3H,I and J,K f o r comparison. The c u r r e n t evoked response i n t h e s e d e n d r i t e s c o n s i s t e d o f a low t h r e s h o l d a l l - o r - n o n e b u r s t o f f a s t s p i k e s on a r i s i n g d e p o l a r i z a t i o n w i t h a "slow" s p i k e evoked from t h e t h i r d or subsequent f a s t s p i k e ( F i g 4.3H and J ) . T h i s p a t t e r n o f s p i k e g e n e r a t i o n appeared t o be a d i s t i n c t form of d i s c h a r g e , a l t h o u g h one d e n d r i t e changed from Type 2 t o Type 3 a c t i v i t y d u r i n g the c o u r s e o f r e c o r d i n g . The e x a c t p a t t e r n and a m p l i t u d e o f i n i t i a l f a s t s p i k e s i n Type 3 a c t i v i t y c o u l d v a r y from one d e n d r i t i c impalement t o a n o t h e r , but was g e n e r a l l y c o n s t a n t over t h e time of r e c o r d i n g from 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 from one d e p o l a r i z i n g p u l s e t o the n e x t , evoked f o r example w i t h an a m p l i t u d e i n t h e range o f 50-75mV and h a l f w i d t h between 9-20msec i n one d e n d r i t i c r e c o r d i n g . Type 3 a c t i v i t y was o f t e n accompanied by g r a d u a l 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 a l l - o r - n o n e b u r s t s o f s p i k e d i s c h a r g e , t h e form o f which was v e r y s i m i l a r t o t h a t evoked t h r o u g h c u r r e n t i n j e c t i o n i n t h e same d e n d r i t i c impalement ( F i g 4.3H,I - 93 - and J , K ) . In c o n t r a s t , spontaneous a c t i v i t y was not a common f e a t u r e o f c e l l s d i s p l a y i n g Type 1 or 2 d i s c h a r g e . 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 P o t e n t i a l s Graded s y n a p t i c p o t e n t i a l s were r e c o r d e d a t a l l l e v e l s o f t h e p y r a m i d a l c e l l a x i s i n response t o s t i m u l a t i o n o f t h e a l v e u s , s t r a t u m o r i e n s or s t r a t u m r a d i a t u m ( F i g 4.4). A l v e a r s t i m u l a t i o n was used t o a c t i v a t e i n h i b i t o r y s y n a p t i c i n p u t s t o t h e p y r a m i d a l c e l l t h r o u g h r e c u r r e n t a c t i v a t i o n o f i n h i b i t o r y i n t e r n e u r o n s i n t h e r e g i o n o f s t r a t u m p y r a m i d a l e ( F i g 4.4A,D). S t i m u l a t i o n o f t h e a l v e u s a t i n t e n s i t i e s s u b t h r e s h o l d f o r a n t i d r o m i c s p i k e d i s c h a r g e evoked a graded i n h i b i t o r y p o t e n t i a l (IPSP) at b o t h s o m a t i c and d e n d r i t i c r e c o r d i n g s i t e s . The peak l a t e n c y o f t h e IPSP d i f f e r e d between s o m a t i c and d e n d r i t i c l o c a t i o n s , d i s p l a y i n g an average peak l a t e n c y o f 17.7 +/- 1.35msec (n=6; mean +/- sem) i n t h e soma and 25.6 +/- 1.72msec (n=8) i n d e n d r i t i c r e c o r d i n g s (p <. .014 S t u d e n t s t - t e s t ) . The d e n d r i t i c i n h i b i t o r y p o t e n t i a l was o f t e n low i n a m p l i t u d e , and i n many cases appeared as a s l i g h t d e p o l a r i z i n g p o t e n t i a l . However, t h e d e n d r i t i c IPSP c o u l d be r e v e r s e d t o a h y p e r p o l a r i z i n g p o t e n t i a l t h r o u g h s l i g h t membrane d e p o l a r i z a t i o n (5-10 mV), and was e q u a l l y e f f e c t i v e i n b l o c k i n g t h e d i s c h a r g e of o r t h o d r o m i c a l l y evoked d e n d r i t i c s p i k e s r e g a r d l e s s o f p o l a r i t y a t r e s t i n g p o t e n t i a l ( d a t a not shown). S t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s c o u r s i n g t h r o u g h s t r a t u m o r i e n s (SO) evoked a graded e x c i t a t o r y p o t e n t i a l (EPSP) i n b o t h somata and a p i c a l d e n d r i t e s o f p y r a m i d a l c e l l s (4.4B,E). The SO evoked EPSP was of l a r g e s t a m p l i t u d e i n t h e s o m a t i c r e g i o n , d i s p l a y i n g an average peak a m p l i t u d e of 14 +/- 2.48mV - 94 - FIG. 4.4 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 p o t e n t i a l s r e c o r d e d i n p y r a m i d a l c e l l soma (A-C) and d e n d r i t e s (D-F) 200um from the b o r d e r o f s t r a t u m p y r a m i d a l e superimposed a t i n c r e a s i n g i n t e n s i t i e s o f s t i m u l a t i o n . P o t e n t i a l s were evoked by s t i m u l a t i o n o f the a l v e u s (A,D), s t r a t u m o r i e n s ( B f E ) , o r s t r a t u m r a d i a t u m ( C , F ) . A l l s o m a t i c r e c o r d i n g s (A-C) were o b t a i n e d from t h e same impalement (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 from a s i n g l e d e n d r i t i c impalement. A l l p o t e n t i a l s a re averaged r e c o r d s o f f i v e s i n g l e sweeps. St im: Site Somatic A. Alveus _ B. Stratum Oriens - i C. Stratum Radiatum Dendritic (200p) D. - 96 - (n=5; mean +/- sem). The EPSP was o f s m a l l e r a m p l i t u d e i n t h e a p i c a l d e n d r i t e , e x h i b i t i n g a peak a m p l i t u d e o f 6.4 +/- 1.55mV 200/im from the bord e r o f s t r a t u m p y r a m i d a l e ( n = l l ; EPSPs s e t t o s p i k e t h r e s h o l d ) . Near t h r e s h o l d f o r s p i k e d i s c h a r g e , a n e g a t i v e - g o i n g " n o t c h " was f r e q u e n t l y o b s e r v e d on t h e r i s i n g edge o f s o m a t i c or d e n d r i t i c EPSPs ( F i g 4.4B,E), a response c o r r e s p o n d i n g t o r e f l e c t i o n s o f t h e p o p u l a t i o n s p i k e response i n the e x t r a c e l l u l a r space (see Chapter 5 ) . S t i m u l a t i o n o f S c h a f f e r c o l l a t e r a l / c o m m i s s u r a l a f f e r e n t s i n s t r a t u m r a d i a t u m (SR) a l s o evoked a graded e x c i t a t o r y p o t e n t i a l i n b o t h somata and d e n d r i t e s o f p y r a m i d a l neurons ( 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 a m p l i t u d e i n d i s t a l d e n d r i t e s , e x h i b i t i n g an average v a l u e o f 21.2 +/- 1.13mV a t 200jum d i s t a n c e from t h e b o r d e r o f s t r a t u m p y r a m i d a l e (n=22; mean +/- sem). The s o m a t i c EPSP was o f s m a l l e r peak a m p l i t u d e , e x h i b i t i n g an average v a l u e o f 12.9 +/- .72mV i n so m a t i c impalements (n=34; EPSPs s e t t o s p i k e t h r e s h o l d ) . Once a g a i n , t h e 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 c o u l d o f t e n be observe d as a r e f l e c t i o n or " n o t c h " on t h e r i s i n g edge o f evoked EPSPs ( F i g 4.4C; R i c h a r d s o n e t a l . 1984a; Turner e t a l . 1984). S t i m u l u s Evoked S u p r a t h r e s h o l d Responses 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 d i f f e r e d markedly from t h a t o f c u r r e n t i n j e c t i o n i n t h a t a n t i d r o m i c o r 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 a l l - o r - n o n e s p i k e i n i n t r a c e l l u l a r r e c o r d i n g s o f t h e p y r a m i d a l c e l l . S u p r a t h r e s h o l d a l v e a r s t i m u l a t i o n evoked a s h o r t l a t e n c y s p i k e i n b o t h s o m a t i c and d e n d r i t i c l o c a t i o n s ( F i g 4.5A,D). These s p i k e s a r o s e d i r e c t l y from b a s e l i n e w i t h an i n v a r i a n t o n set FIG. 4.5 C h a r a c t e r i s t i c s t i m u l u s evoked s p i k e d i s c h a r g e r e c o r d e d i n p y r a m i d a l c e l l s o m a t i c (A-C) and a p i c a l d e n d r i t i c (D-F) l o c a t i o n s . S p i k e d i s c h a r g e was evoked by s t i m u l a t i o n o f t h e a l v e u s ( A f D ) , s t r a t u m o r i e n s (B,E), or s t r a t u m r a d i a t u m ( D f F ) . Somatic r e c o r d i n g s (A-C) were t a k e n from t h r e e s e p a r a t e c e l l s w h i l e d e n d r i t i c r e c o r d i n g s (D-F) were o b t a i n e d from a s i n g l e d e n d r i t i c impalement (200pm from t h e b o r d e r o f s t r a t u m p y r a m i d a l e ) . I n each c a s e , r e c o r d i n g s are shown f o r sub- and s u p r a t h r e s h o l d i n t e n s i t i e s o f s t i m u l a t i o n . Note th e common v o l t a g e and time base c a l i b r a t i o n o f a l l r e c o r d i n g s . Stim.Site Somatic A. Alveus . J B Stratum Oriens C. Stratum Radiatum Dendritic (200>u) D. - 99 - l a t e n c y a t a g i v e n s t i m u l u s i n t e n s i t y , r e p r e s e n t i n g an a n t i d r o m i c response o f t h e soma and a p i c a l d e n d r i t e s o f t h e p y r a m i d a l neuron. S t i m u l a t i o n o f s t r a t u m o r i e n s ( F i g 4.5B,E) or r a d i a t u m ( F i g 4.5C,F) evoked a s i n g l e a l l - o r - n o n e s p i k e from an u n d e r l y i n g EPSP a t a l l l e v e l s o f the p y r a m i d a l c e l l . The c h a r a c t e r i s t i c s o f a c t i o n p o t e n t i a l s evoked from each o f t h e t h r e e s t i m u l a t i o n s i t e s were found t o be s i m i l a r a t any g i v e n l o c a t i o n a l o n g the dendro-somatic a x i s ( c f 4.5A-C and D-F) . However, as shown i n F i g 4.5, s p i k e s r e c o r d e d a t t h e soma were g r e a t e r i n a m p l i t u d e and more narrow i n h a l f w i d t h t h a n t h o s e r e c o r d e d i n t h e a p i c a l d e n d r i t e . Some o f t h e p r o p e r t i e s o f s y n a p t i c a l l y evoked s p i k e d i s c h a r g e r e c o r d e d from s o m a t i c and d e n d r i t i c l o c a t i o n s a re i l l u s t r a t e d i n F i g 4.6. At s t i m u l u s i n t e n s i t i e s near t h r e s h o l d f o r s p i k e g e n e r a t i o n , b o t h 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 s p l a y e d c o n s i d e r a b l e v a r i a b i l i t y i n onset l a t e n c y ( " l a t e n c y j i t t e r " ) , a r i s i n g from a p p r o x i m a t e l y t h e peak o f t h e e x c i t a t o r y s y n a p t i c p o t e n t i a l ( F i g 4.6A,B). As s t i m u l u s i n t e n s i t y was i n c r e a s e d , s p i k e s were evoked w i t h a p r o g r e s s i v e l y s h o r t e r and l e s s v a r i a b l e l a t e n c y t o d i s c h a r g e ( F i g 4.6C,D). I n t h e so m a t i c 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 v o l t a g e t h r e s h o l d , w i t h s p i k e s a r i s i n g from a s i m i l a r l e v e l o f d e p o l a r i z a t i o n r e g a r d l e s s o f t h e l a t e n c y t o onset ( F i g 4.6A). However, d e n d r i t i c s p i k e a c t i v a t i o n d i f f e r e d from t h a t i n t h e soma i n d i s p l a y i n g no c l e a r v o l t a g e t h r e s h o l d f o r AP g e n e r a t i o n . T h i s c h a r a c t e r i s t i c i s c l e a r l y i l l u s t r a t e d i n F i g 4.6B i n which s p i k e d i s c h a r g e was evoked a t two i n t e n s i t i e s o f s t i m u l a t i o n . At t h e h i g h e r s t i m u l u s i n t e n s i t y (12V), s p i k e s were evoked from e i t h e r t h e peak or t h e i n i t i a l f a l l i n g phase o f t h e EPSP. W i t h 9V - 100 - FIG. 4.6 S t r a t u m r a d i a t u m evoked s p i k e d i s c h a r g e r e c o r d e d i n p y r a m i d a l c e l l s o m a t i c (A,C) and d e n d r i t i c l o c a t i o n s ( B f D ) , shown i n each case w i t h s e v e r a l superimposed sweeps. S t i m u l u s i n t e n s i t i e s were s e t f o r t h r e s h o l d (A,B) or s u p r a t h r e s h o l d (C,D) a c t i v a t i o n o f 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 . S i m i l a r r e s u l t s were o b t a i n e d f o r s t r a t u m o r i e n s evoked p o t e n t i a l s . A l l r e c o r d i n g s were t a k e n from s e p a r a t e impalements of somata or a p i c a l d e n r i t e s 200um from the b o r d e r o f s t r a t u m p y r a m i d a l e . Somatic B. Dendritic (200p) 20 mV 5 msec - 102 - s t i m u l a t i o n , a s p i k e c o u l d a g a i n be evoked, but from a s m a l l e r u n d e r l y i n g EPSP and a t a v o l t a g e t h r e s h o l d w e l l below t h a t o b s e r v e d a t the 12V i n t e n s i t y . High f r e q u e n c y s t i m u l a t i o n o f hippocampal a f f e r e n t i n p u t s i s known t o produce a c h a r a c t e r i s t i c i n c r e a s e i n t h e r a t e o f r i s e and a m p l i t u d e o f evoked p o t e n t i a l s , a response p a t t e r n commonly r e f e r r e d t o as " f r e q u e n c y p o t e n t i a t i o n " ( S c h w a r t z k r o i n 1 9 7 7 ; Turner e t a l . 1984). The s o m a t i c and d e n d r i t i c response t o f o u r p u l s e s of a 10Hz s t i m u l u s t r a i n d e l i v e r e d t o s t r a t u m r a d i a t u m i s shown i n F i g 4 . 7 . W i t h s u b t h r e s h o l d s t i m u l u s i n t e n s i t i e s , 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 of EPSP a m p l i t u d e and the g e n e r a t i o n of a s i n g l e a l l - o r - n o n e s p i k e a t b o t h t h e s o m a t i c 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 ti m e s d u r i n g the f r e q u e n c y t r a i n and w i t h c o n t i n u e d s t i m u l a t i o n d i s p l a y e d a s h i f t t o a s h o r t e r , l e s s v a r i a b l e l a t e n c y t o o n s e t . W i t h p r o l o n g e d s t i m u l a t i o n or w i t h g r e a t e r f r e q u e n c i e s o f a c t i v a t i o n , m u l t i p l e s p i k e d i s c h a r g e c o u l d be evoked i n e i t h e r s o m a t i c or d e n d r i t i c impalements. 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 d i d not appear t o oc c u r p r i o r t o the g e n e r a t i o n o f m u l t i p l e p o p u l a t i o n s p i k e s a t the c e l l l a y e r , a r e s u l t i n d i c a t i n g r e p e t i t i v e a c t i v a t i o n of s p i k e s i n t h e s o m a t i c r e g i o n ( 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 and P r i n c e 1980) . T h e r e f o r e , a s i m i l a r p a t t e r n o f a c t i o n p o t e n t i a l d i s c h a r g e was found a t b o t h t h e so m a t i c and d e n d r i t i c l e v e l o f the p y r a m i d a l c e l l i n response t o h i g h f r e q u e n c y s t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s . - 103 - FIG. 4.7 Response of p y r a m i d a l c e l l soma (A) and a p i c a l d e n d r i t e (B) t o 10Hz f r e q u e n c y s t i m u l a t i o n of s t r a t u m r a d i a t u m a f f e r e n t i n p u t s a t s u b t h r e s h o l d s t i m u l u s i n t e n s i t i e s . I l l u s t r a t e d are f o u r i n d i v i d u a l p u l s e s o f a t e n p u l s e s t i m u l u s t r a i n w i t h t h e l a s t two p u l s e s i n e i t h e r case showing a sub- and s u p r a t h r e s h o l d example from s e p a r a t e s t i m u l u s t r a i n s . A. Somatic B. Dendritic , 0 _ j 2 0 m V 5 msec - 105 - Comparison of S u p r a t h r e s h o l d S t i m u l u s and C u r r e n t Evoked Responses A c h a r a c t e r i z a t i o n o f p y r a m i d a l c e l l a c t i v i t y has r e v e a l e d 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 o f s p i k e responses i n t h e 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 r e p e t i t i v e d i s c h a r g e o f b o t h l a r g e and s m a l l a m p l i t u d e f a s t s p i k e s , or t h e g e n e r a t i o n o f a 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). However, t h e most commonly evoked s p i k e i s o f l a r g e a m p l i t u d e and narrow h a l f w i d t h , evoked i n a r e p e t i t i v e manner i n Type 1 or 2 a c t i v i t y . To d e t e r m i n e th e s i m i l a r i t y between t h i s s p i k e and t h o s e evoked t h r o u g h a n t i - or o r t h o d r o m i c s t i m u l a t i o n , t h e s t i m u l u s and c u r r e n t evoked a c t i o n p o t e n t i a l o f d e n d r i t i c membrane was d i r e c t l y compared ( F i g 4.8). Upon i n i t i a l e x a m i n a t i o n , s t i m u l u s and c u r r e n t evoked s p i k e s appeared d i s t i n c t l y d i f f e r e n t i n terms o f a m p l i t u d e and h a l f w i d t h ( F i g 4.8A-C). However, when s t i m u l u s evoked s p i k e s were a c t i v a t e d upon t h e same u n d e r l y i n g d e p o l a r i z a t i o n , a l l s p i k e s superimposed, r e v e a l i n g a b a s i c s i m i l a r i t y between s t i m u l u s and c u r r e n t 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 ( F i g 4.8D-F). S i m i l a r r e s u l t s were o b t a i n e d i n s o m a t i c impalements, a l t h o u g h t h e i n i t i a l d i f f e r e n c e i n a m p l i t u d e and h a l f w i d t h o f s t i m u l u s and c u r r e n t evoked s p i k e s was not as marked as i n 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 s . I n T a b l e 2, a comparison of s p i k e a m p l i t u d e and h a l f w i d t h a t t h e l e v e l o f t h e soma and a p i c a l d e n d r i t e s f u r t h e r demonstrates th e s i m i l a r i t y i n waveform of s t i m u l u s and c u r r e n t evoked f a s t s p i k e s o f t h e p y r a m i d a l c e l l . - 106 - FIG. 4.8 Comparison o f s t i m u l u s and c u r r e n t 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 a 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 . A-C. C u r r e n t evoked d i s c h a r g e i s shown superimposed 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 l v e u s ( A ) , s t r a t u m o r i e n s (B) or s t r a t u m r a d i a t u m ( C ) . D-F. S t i m u l u s evoked s p i k e s were evoked on t h e same u n d e r l y i n g d e p o l a r i z a t i o n as t h a t n e c e s s a r y t o e l i c i t c u r r e n t evoked d i s c h a r g e , and a c u r r e n t evoked s p i k e then superimposed f o r compa r i s o n . A l l p o t e n t i a l s a re t a k e n from the same d e n d r i t i c impalement 200um from the b o r d e r o f s t r a t u m p y r a m i d a l e . Stim.Site Alveus Stratum A. B. Stratum Radiatum C. _ J F. - J , J20mV 2 msec - 108 - TABLE 2 Average a m p l i t u d e and h a l f w i d t h ( w i d t h a t h a l f a m p l i t u d e ) o f c u r r e n t and s t i m u l u s evoked s p i k e s i n p y r a m i d a l c e l l somata and a p i c a l d e n d r i t e s 200jum from t h e b o r d e r o f s t r a t u m p y r a m i d a l e (mean +/- sem; a m p l i t u d e s measured from r e s t i n g membrane p o t e n t i a l ) . Measurements o f c u r r e n t 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 from the f i r s t s p i k e of e i t h e r Type 1 or 2 d i s c h a r g e evoked by d e p o l a r i z i n g c u r r e n t . S t i m u l u s evoked s p i k e s were evoked by s t i m u l a t i o n o f t h e a l v e u s , s t r a t u m o r i e n s , o r s t r a t u m r a d i a t u m . The number of impalements f o r each average a r e g i v e n i n b r a c k e t s . S t i m u l u s evoked s p i k e s a t e i t h e r t h e s o m a t i c or d e n d r i t i c l e v e l were not s i g n i f i c a n t l y d i f f e r e n t a c c o r d i n g t o a one way ANOVA t e s t . C u r r e n t evoked s o m a t i c s p i k e a m p l i t u d e and c u r r e n t evoked d e n d r i t i c s p i k e a m p l i t u d e and h a l f w i d t h were found 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 from s t i m u l u s evoked s p i k e s . However, t h i s d i f f e r e n c e can be a t t r i b u t e d t o t h e l e v e l o f membrane d e p o l a r i z a t i o n upon which s p i k e s a re evoked (see F i g 4.8). - 109 - T A B L E 2 S P I K E AMPLITUDE (MV) STIMULUS SOMATIC D E N D R I T I C (200 UM) CURRENT I N J E C T . 90.3 ± 2.04 (11) 69.6 ± 3.42 (9) A L V E U S 87.3 + 2.77 (9) 62.9 ± 2.42 (11) STRATUM ORIENS 87.2 ± 4.06 (4) 59.5 ± 2.68 (12) STRATUM RADIATUM 90.0 ± 2.36 (9) 65.0 ± .97 (23) S P I K E HALFWIDTH (MSEC) SOMATIC D E N D R I T I C (200 UM) 1.06 + .051 (6) I i.80 ± .14 (2) .96 ± .028 (7) 1.34 ± .069 (11) .84 ± .055 (4) 1.33 ± .066 (12) .87 ± .039 (11) 1.17 ± .037 (20) - 110 - S p i k e P r e - P o t e n t i a l s Somatic r e c o r d i n g s o f p y r a m i d a l neurons have o f t e n uncovered th e p r e s ence of " f a s t p r e - p o t e n t i a l s " ( F P P s ) , a name g i v e n t o a s m a l l a m p l i t u d e a l l - o r - n o n e p o t e n t i a l c a p a b l e of e v o k i n g s o m a t i c s p i k e d i s c h a r g e (Andersen and Lomo 1966; Knowles and S c h w a r t z k r o i n 1981b; MacVicar and Dudek 1981; S c h w a r t z k r o i n 1975; 1977; Spencer and K andel 1961). These p o t e n t i a l s are c h a r a c t e r i z e d by a f a s t r a t e o f r i s e , a r e l a t i v e l y s m a l l a m p l i t u d e , a f a s t i n i t i a l decay and a second s l o w e r component of decay. A l t h o u g h not s y s t e m a t i c a l l y examined i n t h e p r e s e n t s t u d y , FPPs s i m i l a r t o t h o s e o b s e r v e d i n t h e soma were a l s o r e c o r d e d at t h e 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 s t i c s o f FPP d i s c h a r g e i n p y r a m i d a l c e l l somata and a p i c a l d e n d r i t e s a r e i l l u s t r a t e d i n F i g 4.9. F a s t p r e - p o t e n t i a l s were evoked by s t i m u l a t i o n o f e i t h e r s t r a t u m o r i e n s or r a d i a t u m , and "uncovered" t h r o u g h h y p e r p o l a r i z a t i o n o f t h e membrane (5-20mV) by c u r r e n t i n j e c t i o n ( F i g 4.9A,B). In e i t h e r c a s e , t h e FPP was evoked i n an a l l - o r - n o n e manner near t h e peak of t h e EPSP, w i t h a c t i o n p o t e n t i a l d i s c h a r g e a r i s i n g from th e peak of t h e f a s t p r e - p o t e n t i a l . R e p e t i t i v e s t i m u l a t i o n o f a f f e r e n t i n p u t s (10-20Hz) c o u l d a l s o evoke FPP d i s c h a r g e on t h e f a l l i n g edge of t h e s y n a p t i c p o t e n t i a l , w i t h a p r o g r e s s i v e d e c r e a s e i n t h e l a t e n c y t o FPP a c t i v a t i o n d u r i n g the s t i m u l u s t r a i n . D e p o l a r i z i n g c u r r e n t i n j e c t i o n was a l s o e f f e c t i v e i n e v o k i n g FPPs i n b o t h s o m a t i c and d e n d r i t i c l o c a t i o n s ( F i g 4.9C,D). In s e v e r a l c a s e s , spontaneous FPP d i s c h a r g e was r e c o r d e d i m m e d i a t e l y f o l l o w i n g c e l l u l a r impalement, d u r i n g the t i m e i n which th e c e l l was g r e a t l y d e p o l a r i z e d . However, spontaneous f a s t p r e - p o t e n t i a l s were a l s o o b s e r v e d at more - I l l - FIG. 4.9 C h a r a c t e r i s t i c s o f f a s t p r e - p o t e n t i a l (FPP) d i s c h a r g e i n p y r a m i d a l c e l l somata (A fC,E) and a p i c a l d e n d r i t e s (B,D,F). A,B. FPPs u n d e r l y i n g s t r a t u m r a d i a t u m evoked s p i k e s uncovered 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 (5-20mV; s e v e r a l sweeps shown i n A ) . C,D. FPP d i s c h a r g e evoked t h r o u g h d e p o l a r i z i n g c u r r e n t i n j e c t i o n ( a c t i o n p o t e n t i a l t r u n c a t e d i n C ) . E,F. S u c c e s s i v e sweeps o f spontaneous FPP d i s c h a r g e a t r e s t i n g membrane p o t e n t i a l . - 112 - Stim. Site Stmt. Rad. Current Spont. -T5 20 - I nA J l O m V 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 f r e q u e n c y o f spontaneous FPP d i s c h a r g e c o u l d be a l t e r e d w i t h a s h i f t i n membrane p o t e n t i a l , d i s p l a y i n g an i n c r e a s e w i t h d e p o l a r i z a t i o n and 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 m p l i t u d e o f f a s t p r e - p o t e n t i a l s v a r i e d s l i g h t l y w i t h i n a g i v e n impalement depending on t h e r e s t i n g membrane p o t e n t i a l (see S c h w a r t z k r o i n 1977), but moreso between d i f f e r e n t p y r a m i d a l neurons. However, o f t h e cases documented t h u s f a r , no c l e a r d i f f e r e n c e i n t h e a m p l i t u d e o f evoked FPPs c o u l d be found between s o m a t i c and d e n d r i t i c r e c o r d i n g s . For i n s t a n c e , o f t h o s e FPPs evoked t h r o u g h SR s t i m u l a t i o n , s o m a t i c FPPs d i s p l a y e d an average a m p l i t u d e o f 15 +/- 3.4mV (n=5; mean +/- sem) and d e n d r i t i c FPPs 14 +/- 1.4mV (n=4; measured from t h e b r e a k p o i n t o f d i s c h a r g e ) . A " p r e - p o t e n t i a l " was a l s o found t o u n d e r l y a n t i d r o m i c a c t i o n p o t e n t i a l d i s c h a r g e f o l l o w i n g s t i m u l a t i o n o f e f f e r e n t p y r a m i d a l c e l l axons ( F i g 4.10). T h i s p o t e n t i a l has been p r e v i o u s l y d e s c r i b e d i n s o m a t i c r e c o r d i n g s o f t h e p y r a m i d a l c e l l (Spencer and Kandel 1961b), and was thought t o r e p r e s e n t t h e a c t i v a t i o n o f a s p i k e i n t h e i n i t i a l segment r e g i o n . I t i s thus r e f e r r e d t o as an i n i t i a l segment o r " I S " s p i k e , f o l l o w i n g t h e t e r m i n o l o g y used f o r t h e components o f s p i k e a c t i v a t i o n i n t h e s p i n a l motoneuron (Coombs e t a l . 1957a,b). I n many c a s e s , t h e IS s p i k e u n d e r l y i n g the a n t i d r o m i c a c t i o n p o t e n t i a l c o u l d be uncovered a t t h e s o m a t i c or d e n d r i t i c l e v e l 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 ( F i g 4.10A,B). A l t e r n a t i v e l y , a t e s t a n t i d r o m i c s t i m u l u s a p p l i e d 2.0 - 2.4msec f o l l o w i n g a c o n d i t i o n i n g a n t i d r o m i c s p i k e c o u l d be used i n c o n j u n c t i o n w i t h 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 IS s p i k e ( F i g 4.10C,D). - 114 - FIG. 4.10 I n i t i a l segment (IS) s p i k e s u n d e r l y i n g a l v e a r evoked a n t i d r o m i c s p i k e s i n s o m a t i c (A,C) and a p i c a l d e n d r i t i c membrane ( B r D ) . IS s p i k e s were uncovered 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 (A,B), or t h r o u g h h y p e r p o l a r i z a t i o n i n c o n j u n c t i o n w i t h a n t i d r o m i c p a i r e d p u l s e s t i m u l a t i o n (C,D) a t c o n d i t i o n - t e s t i n t e r v a l s o f 2.0 - 2.4 msec. S e v e r a l sweeps are superimposed i n each c a s e . A c t i o n p o t e n t i a l i s t r u n c a t e d i n A, and a l l r e c o r d i n g s a re t a k e n from s e p a r a t e impalements. c. P a i r e d . P u l s e Dendritic _T 10 2 D. J l O m V I m s e c - 116 - The c h a r a c t e r i s t i c s o f t h i s p o t e n t i a l were found t o be v e r y s i m i l a r t o th o s e o f t h e f a s t p r e - p o t e n t i a l ( c f 4.9). For i n s t a n c e , t h e a n t i d r o m i c IS s p i k e d i s p l a y e d a f a s t r a t e o f r i s e and a two component decay, i n c l u d i n g a f a s t i n i t i a l f a l l and a second more p r o l o n g e d decay t o r e s t i n g p o t e n t i a l . A l t h o u g h t h e am p l i t u d e o f IS s p i k e s v a r i e d somewhat between p y r a m i d a l c e l l impalements, t h e r e was no g r e a t d i f f e r e n c e between s o m a t i c and d e n d r i t i c l o c a t i o n s , w i t h a m p l i t u d e s o f 19 +/- 2.3mV (n=6; mean +/- sem) i n t h e soma and 21 +/- 3.6mV (n=6) i n t h e d e n d r i t e - v a l u e s r a t h e r s i m i l a r t o thos e o f t h e o r t h o d r o m i c a l l y evoked FPP. 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 i n f a c t be observed i n almost a l l c e l l s e x h i b i t i n g FPP d i s c h a r g e , w i t h o n l y t h e means o f a c t i v a t i o n s e r v i n g t o d i s t i n g u i s h IS s p i k e s from f a s t p r e - p o t e n t i a l s . - 117 - 4-4. D i s c u s s i o n The p r e s e n t s t u d y has s e r v e d t o f u r t h e r c h a r a c t e r i z e t h e p r o p e r t i e s o f evoked p o t e n t i a l s i n s o m a t i c and d e n d r i t i c membranes o f t h e hippocampal p y r a m i d a l c e l l (HPC). One of t h e more c o n s i s t e n t f i n d i n g s was a g e n e r a l s i m i l a r i t y i n t h e p a t t e r n o f evoked a c t i v i t y i n s o m a t i c and d e n d r i t i c r e c o r d i n g s i t e s . G i ven t h e noted e x c e p t i o n s i n t h e a b s o l u t e v a l u e o f waveform parameters ( i e . s p i k e a m p l i t u d e and h a l f w i d t h ) , s o m a t i c and d e n d r i t i c membranes were found t o e x h i b i t s i m i l a r membrane p r o p e r t i e s , s y n a p t i c p o t e n t i a l s , and b o t h c u r r e n t and s t i m u l u s evoked s p i k e d i s c h a r g e . Membrane P r o p e r t i e s and Evoked S y n a p t i c P o t e n t i a l s The b a s i c 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 i n p u t r e s i s t a n c e were found t o be comparable i n s o m a t i c and d e n d r i t i c impalements, w i t h a s i m i l a r evoked response t o h y p e r p o l a r i z i n g o r d e p o l a r i z i n g c u r r e n t p u l s e i n j e c t i o n s a t e i t h e r l o c a t i o n . For i n s t a n c e , t h e d e n d r i t i c membrane response was l i n e a r over a g i v e n range o f p o t e n t i a l s , and d i s p l a y e d anomalous r e c t i f i c a t i o n s i m i l a r t o t h a t d e s c r i b e d f o r HPC so m a t i c membrane (Brown and G r i f f i t h 1983b; H a l l i w e l l and Adams 1982; Hotson e t a l . 1979; S e g a l and B a r k e r 1984). However, s i n c e t h e p h a r m a c o l o g i c a l mechanisms o f t h e d e n d r i t i c response have not been examined, i t s h o u l d be noted t h a t t h e above comparison of membrane p r o p e r t i e s i s based l a r g e l y on t h e q u a l i t a t i v e a s p e c t s o f evoked a c t i v i t y . Evoked i n h i b i t o r y and e x c i t a t o r y s y n a p t i c p o t e n t i a l s c o u l d be r e c o r d e d a t b o t h t h e s o m a t i c and d e n d r i t i c l e v e l o f t h e - 118 - p y r a m i d a l c e l l . By comparing t h e evoked c h a r a c t e r i s t i c s o f t h e s e p o t e n t i a l s t o t h e r e s u l t s o b t a i n e d t h r o u g h c u r r e n t - s o u r c e d e n s i t y a n a l y s i s (CSD; Chapter 3 ) , some i n s i g h t i n t o t h e p o s s i b l e s i t e o f o r i g i n o f t h e s e p o t e n t i a l s can be g a i n e d . For example, s t i m u l a t i o n o f p y r a m i d a l c e l l axons i n t h e a l v e u s evoked a graded IPSP 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 . T h i s p o t e n t i a l has been w e l l c h a r a c t e r i z e d f o r HPC s o m a t i c membrane, r e p r e s e n t i n g a GABA mediated i n c r e a s e i n C l - conductance t h r o u g h t h e a c t i o n o f r e c u r r e n t i n h i b i t o r y i n t e r n e u r o n s ( A l l e n e t a l . 1977; Andersen e t a l . 1969; D i n g l e d i n e and Langmoen 1980; Knowles and S c h w a r t z k r o i n 1981a). The p r i n c i p l e s i t e Of i n t e r n e u r o n a x o n a l t e r m i n a t i o n has been l o c a l i z e d b o t h a n a t o m i c a l l y ( C a j a l 1911; L o r e n t e de No 1934) and e l e c t r o p h y s i o l o g i c a l l y (Leung 1979a,b) t o s o m a t i c and p r o x i m a l d e n d r i t i c membranes o f p y r a m i d a l neurons. The a l v e a r evoked IPSP i n d e n d r i t i c membrane may t h u s c o r r e s p o n d t o a p a s s i v e e l e c t r o t o n i c c o n d u c t i o n o f i n h i b i t o r y c u r r e n t s ~ a l o n g t h e dendro-somatic a x i s . I n s u p p o r t o f t h i s , 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 i n d i c a t e t h e pr e s e n c e o f a l o n g d u r a t i o n c u r r e n t s o u r c e near t h e r e g i o n o f s t r a t u m p y r a m i d a l e and a c u r r e n t s i n k i n t h e d e n d r i t i c f i e l d f o l l o w i n g t h e g e n e r a t i o n o f an 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 (Chapter 3; Leung 1979a,b). A l o n g d u r a t i o n c u r r e n t s o u r c e i s t h e ex p e c t e d r e s u l t f o r c u r r e n t 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 o f a C l - conductance a t t h e c e l l body l a y e r o f t h e p y r a m i d a l c e l l p o p u l a t i o n . The d e n d r i t i c c u r r e n t s i n k c o u l d t h u s r e p r e s e n t a p a s s i v e compensatory c u r r e n t f l o w t h r o u g h p y r a m i d a l c e l l d e n d r i t i c elements towards t h e i n h i b i t o r y c u r r e n t s o u r c e i n s t r a t u m p y r a m i d a l e . I n f a c t , t h e i n t r a d e n d r i t i c IPSP d i s p l a y e d a l o n g e r peak l a t e n c y t h a n t h e - 1 1 9 - s o m a t i c i n h i b i t o r y p o t e n t i a l , and was o f t e n evoked as a d e p o l a r i z i n g p o t e n t i a l , a r e s u l t c o n s i s t e n t w i t h an inward movement o f c u r r e n t i n d e n d r i t i c r e g i o n s . The a l v e a r evoked i n t r a d e n d r i t i c IPSP 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 t h r o u g h a p a s s i v e e l e c t r o t o n i c c o n d u c t i o n o f t h e a c t i v e i n h i b i t o r y p o t e n t i a l evoked i n t h e s o m a t i c r e g i o n o f t h e p y r a m i d a l c e l l . Such a p r o c e s s might i n f a c t be expected c o n s i d e r i n g t h e a b i l i t y o f d e n d r i t i c e x c i t a t o r y p o t e n t i a l s t o conduct t o t h e s o m a t i c r e g i o n . However, f u r t h e r work w i l l be r e q u i r e d t o de t e r m i n e t h e e x a c t l i m i t o f i n t e r n e u r o n a l s y n a p t i c t e r m i n a t i o n a l o n g t h e dendro-somatic a x i s o f t h e p y r a m i d a l c e l l . A s i m i l a r comparison o f s i n k - s o u r c e r e l a t i o n s h i p s and i n t r a c e l l u l a r a c t i v i t y can be a p p l i e d t o e x c i t a t o r y s y n a p t i c p o t e n t i a l s evoked t h r o u g h s t i m u l a t i o n o f a f f e r e n t s y n a p t i c i n p u t s . S t r a t u m o r i e n s (SO) s t i m u l a t i o n evoked an i n t r a c e l l u l a r EPSP a t b o t h t h e s o m a t i c and d e n d r i t i c l e v e l o f t h e p y r a m i d a l c e l l , 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 l a y e r . These r e s u l t s c o i n c i d e w i t h t h o s e o b t a i n e d t h r o u g h c u r r e n t - s o u r c e d e n s i t y a n a l y s i s , i n which SO s t i m u l a t i o n evoked a l a r g e a m p l i t u d e EPSP and c u r r e n t s i n k i n t h e b a s a l d e n d r i t i c f i e l d f o l l o w e d by t h e c o n d u c t i o n and decay o f s y n a p t i c c u r r e n t s t h r o u g h t h e somata and a p i c a l d e n d r i t e s o f t h e p y r a m i d a l c e l l p o p u l a t i o n . I n c o n t r a s t , s t r a t u m r a d i a t u m (SR) s t i m u l a t i o n evoked t h e l a r g e s t EPSP i n t h e a p i c a l d e n d r i t e and an EPSP o f 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 . These r e s u l t s a l s o agree w i t h t h o s e d e r i v e d from CSD 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 d e n d r i t i c s i t e o f o r i g i n f o r t h e SR evoked EPSP, and a subsequent c o n d u c t i o n towards t h e s o m a t i c r e g i o n . The above d a t a i n d i c a t e t h a t a s y n a p t i c p o t e n t i a l evoked - 120 - w i t h i n one g i v e n r e g i o n of t h e p y r a m i d a l c e l l can conduct t h r o u g h a l a r g e e x t e n t o f t h e dendro-somatic a x i s . These r e s u l t s s e r v e t o emphasize t h e s h o r t e l e c t r o t o n i c l e n g t h of t h e p y r a m i d a l c e l l (Brown e t a l . 1981; Turner 1984; Turner and S c h w a r t z k r o i n 1980), and may account i n p a r t f o r t h e s i m i l a r i t y i n evoked c h a r a c t e r i s t i c s o f s o m a t i c and d e n d r i t i c p o t e n t i a l s . I t would a l s o i m p l y t h a t t h e presence o f an evoked p o t e n t i a l a l o n g t h i s a x i s cannot be t a k e n by i t s e l f as e v i d e n c e f o r an i n t r i n s i c membrane p r o p e r t y , and attemp t s t o l o c a t e t h e s i t e o f o r i g i n o f any 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 e x p e r i m e n t a l s t r a t e g i e s . C u r r e n t Evoked S p i k e s A n a l y s i s o f s o m a t i c and d e n d r i t i c c u r r e n t evoked p o t e n t i a l s r e v e a l e d t h a t s p i k e d i s c h a r g e i n response t o d e p o l a r i z i n g c u r r e n t i n j e c t i o n c o u l d t a k e a v a r i e t y o f forms, grouped here f o r t h e purpose o f comparison i n t o t h r e e b a s i c p a t t e r n s (Types 1-3). However, i t s h o u l d be s t r e s s e d t h a t t h e g r o u p i n g o f d i s c h a r g e p a t t e r n s used i n t h e p r e s e n t s t u d y i s not meant t o im p l y t h e pr e s e n c e o f d i s t i n c t p y r a m i d a l c e l l t y p e s i n t h e C A l , r e g i o n . F u r t h e r m o r e , t h e d i s t i n c t i o n between t h e s e p a t t e r n s i s not a b s o l u t e and some t r a n s i t i o n o r c o m b i n a t i o n o f t h e s e c h a r a c t e r i s t i c s can o c c u r , as shown by t h e o c c a s i o n a l change i n d e n d r i t i c s p i k e d i s c h a r g e from Type 2 t o Type 3 a c t i v i t y . The presence 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 d i s c h a r g e c o u l d not be a t t r i b u t e d t o t h e q u a l i t y o f c e l l u l a r impalement, as v a l u e s o f r e s t i n g p o t e n t i a l and i n p u t r e s i s t a n c e f o r c e l l s d i s p l a y i n g each form o f d i s c h a r g e were comparable. I n a d d i t i o n , o r t h o d r o m i c s t i m u l a t i o n would r e l i a b l y evoke an EPSP-IPSP - 121 - sequence and a s i n g l e f a s t s p i k e i n b o t h s o m a t i c and d e n d r i t i c 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 r e s p o n s e s . T h e r e f o r e , membrane c h a r a c t e r i s t i c s , s y n a p t i c p o t e n t i a l s , and s t i m u l u s evoked s p i k e d i s c h a r g e appeared t o be i n t a c t , s u g g e s t i n g t h a t the 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 was an i n h e r e n t p r o p e r t y o f t h e c e l l i n q u e s t i o n and not t h e r e s u l t o f i n j u r y o r v a r i a b i l i t y i n t h e q u a l i t y o f t h e i n t r a c e l l u l a r r e c o r d i n g . R a t h e r , p h a r m a c o l o g i c a l s t u d i e s would i n d i c a t e t h a t 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 r e f l e c t s t h e i o n i c b a s i s o f s p i k e g e n e r a t i o n i n t h e p y r a m i d a l c e l l soma and a p i c a l d e n d r i t e . P r e v i o u s 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 i n b o t h th e soma and a p i c a l d e n d r i t e o f p y r a m i d a l c e l l s a r e b l o c k e d by the Na+ c h a n n e l b l o c k e r t e t r o d o t o x i n (TTX) and are t h e r e f o r e t hought t o be mediated t h r o u g h v o l t a g e - d e p e n d e n t Na+ c h a n n e l s (Hotson and P r i n c e 1980; S c h w a r t z k r o i n and Slawsky 1977;Wong e t a l . 1979). In c o n t r a s t , i n t e r m e d i a t e and slow s p i k e s o f d e n d r i t i c membrane have been shown t o be TTX i n s e n s i t i v e , and p r o b a b l y r e p r e s e n t r e g e n e r a t i v e Ca+2 ch a n n e l a c t i v a t i o n (Wong e t a l . 1979). 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 i n i t i a l b u r s t o f f a s t s p i k e s i s i n s e n s i t i v e t o TTX but reduced by Ca+2 c h a n n e l a n t a g o n i s t s ( S c h w a r t z k r o i n and Slawsky 1977; Wong and P r i n c e 1978; Wong e t a l . 1979). The d i s t i n g u i s h i n g f e a t u r e between t h e t h r e e forms o f c u r r e n t evoked a c t i v i t y might t h e r e f o r e be t h e r e l a t i v e c o n t r i b u t i o n o f Na+ or Ca+2 ch a n n e l conductance t o AP g e n e r a t i o n . I n t h i s r e g a r d , Type 1 d i s c h a r g e was dominated by th e g e n e r a t i o n o f f a s t Na+ s p i k e s , w h i l e Type 2 a c t i v i t y was com p r i s e d o f Na+ s p i k e a c t i v a t i o n as w e l l as a l a r g e u n d e r l y i n g - 122 - d e p o l a r i z a t i o n and t h e o c c a s i o n a l g e n e r a t i o n o f i n t e r m e d i a t e s p i k e s . Type 2 d i s c h a r g e was a l s o d i s t i n c t i n d i s p l a y i n g an a f t e r h y p e r p o l a r i z a t i o n f o l l o w i n g the i n i t i a l b u r s t o f s p i k e d i s c h a r g e , a p o t e n t i a l shown t o r e p r e s e n t a Ca+2-dependent K+ conductance i n HPC s o m a t i c membrane (Brown and G r i f f i t h 1983a; Hotson and P r i n c e 1980). A l t h o u g h not a b s o l u t e i n o c c u r r e n c e , Type 2 d i s c h a r g e appeared t o c o r r e l a t e w i t h s o m a t i c o r d e n d r i t i c impalements e x h i b i t i n g anomalous r e c t i f i c a t i o n i n t h e d e p o l a r i z i n g range o f c u r r e n t i n j e c t i o n ( c f 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 e t a l . 1982; Hotson e t a l . 1979). Thus, Type 1 s p i k e d i s c h a r g e .would appear t o be mediated p r i m a r i l y by Na+ c h a n n e l conductance, w h i l e b o t h Na+ and Ca+2 c h a n n e l a c t i v i t y c o n t r i b u t e d t o Type 2 d i s c h a r g e . The preponderance of slow 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 would f u r t h e r i n d i c a t e Ca+2 i o n s as t h e p r i n c i p l e charge c a r r i e r f o r t h i s form o f AP d i s c h a r g e . Of i n t e r e s t i s t h e f a c t t h a t t h e d i s t i n c t i o n between d i s c h a r g e p a t t e r n s c o u l d be made a t b o t h t h e s o m a t i c and d e n d r i t i c l e v e l , w i t h Type 1 a c t i v i t y much more commonly r e c o r d e d i n b o t h s o m a t i c and d e n d r i t i c impalements as compared to t h a t o f Type 2. Giv e n t h e s i m i l a r i t y i n t h e p a t t e r n o f evoked a c t i v i t y between s o m a t i c and d e n d r i t i c r e c o r d i n g s i t e s , i t i s thus p o s s i b l e t h a t one form o f s p i k e d i s c h a r g e (Type 1 or Type 2) i s e x p r e s s e d a t b o t h t h e s o m a t i c and d e n d r i t i c l e v e l o f a g i v e n c e l l . F u r t h e r e x a m i n a t i o n o f c u r r e n t evoked responses r e v e a l s t h a t p o t e n t i a l s thought t o i n d i c a t e t h e a c t i v a t i o n o f Ca+2 c h a n n e l conductance were most commonly r e c o r d e d i n d e n d r i t i c impalements o f t h e p y r a m i d a l c e l l . For i n s t a n c e , t h e - 123 - d e p o l a r i z a t i o n u n d e r l y i n g the b u r s t o f f a s t s p i k e s i n Type 2 d i s c h a r g e was o f g r e a t e r a m p l i t u d e i n d e n d r i t i c membrane, and Ca+2-dependent i n t e r m e d i a t e or slow s p i k e s were o n l y o b s e r v e d i n 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 s . These r e s u l t s would suggest a non- u n i f o r m d i s t r i b u t i o n o f i o n i c c h a n n e l s a l o n g the p y r a m i d a l c e l l 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 d e n d r i t i c t h a n s o m a t i c membrane. S i m i l a r c o n c l u s i o n s were drawn i n p r e v i o u s i n v e s t i g a t i o n s , but t h e s e s t u d i e s r e p o r t e d t h a t Ca+2-dependent i n t e r m e d i a t e or slow s p i k e s were r e a d i l y evoked i n d e n d r i t i c membrane by i n j e c t i o n o f d e p o l a r i z i n g c u r r e n t (Benardo e t a l . 1982; Wong e t a l . 1979). I n t h e p r e s e n t s t u d y , t h e i n t e r m e d i a t e and slow s p i k e c o u l d be evoked i n t h e a p i c a l d e n d r i t e , but t h e s e p o t e n t i a l s were i n f r e q u e n t l y o b s e r v e d a t a l l l e v e l s o f the d e n d r i t i c t r e e , b e i n g d e t e c t e d i n o n l y 8/36 d e n d r i t e s examined. I n s t e a d , the most common form o f a c t i o n p o t e n t i a l d i s c h a r g e a t b o t h t h e so m a t i c and d e n d r i t i c l e v e l was t h a t o f Na+-dependent s p i k e a c t i v a t i o n . The apparent d i f f e r e n c e between t h e s e r e s u l t s and th o s e of p r e v i o u s i n v e s t i g a t o r s might be a t t r i b u t e d t o t h r e e s e p a r a t e f a c t o r s . F i r s t , p r e v i o u s s t u d i e s have r e p o r t e d t h a t slow s p i k e g e n e r a t i o n i n t h e p y r a m i d a l c e l l d e n d r i t e i s most commonly evoked t h r o u g h i n j e c t i o n o f d e p o l a r i z i n g c u r r e n t i n t h e o r d e r o f 2nA or g r e a t e r (Wong e t a l . 1979). However, c u r r e n t i n j e c t i o n o f over 1.5nA was not r o u t i n e l y a ttempted i n t h e p r e s e n t s t u d y as such a c t i o n would o f t e n r e s u l t i n l o s s o f t h e d e n d r i t i c impalement. S e c o n d l y , e a r l y i n v e s t i g a t i o n s o f d e n d r i t i c a c t i v i t y were performed on bot h C A l and CA3 p y r a m i d a l c e l l s (Wong e t a l . 1979), t h e l a t t e r o f w h i c h a re known t o e x h i b i t more pronounced b u r s t - l i k e b e h a v i o u r i n response t o c u r r e n t i n j e c t i o n (Wong e t a l . 1979; - 124 - Wong and P r i n c e 1978). F i n a l l y , a l l p r e v i o u s s t u d i e s on d e n d r i t i c a c t i v i t y were performed on p y r a m i d a l c e l l s o f t h e g u i n e a - p i g , w h i l e t h e r e s u l t s o f t h i s work were o b t a i n e d from the r a t hippocampus. Of p o s s i b l e r e l e v a n c e t o t h i s p o i n t i s t h e r e c e n t f i n d i n g t h a t C A l p y r a m i d a l c e l l s o f t h e g u i n e a - p i g are not s t a i n e d i m m u n o h i s t o c h e m i c a l l y by a n t i b o d i e s s p e c i f i c f o r C a l c i u m - B i n d i n g P r o t e i n (CaBP), as i s c h a r a c t e r i s t i c a l l y found f o r C A l p y r a m i d a l c e l l s o f the r a t (Dr. K.G. B a i m b r i d g e , p e r s o n a l communication; B a i m b r i d e and M i l l e r 1982). A l t h o u g h t h e pre s e n c e or absence o f CaBP may be i n c o n s e q u e n t i a l t o Ca+2 ch a n n e l a c t i v i t y , t h e above r e s u l t s do i n d i c a t e t h a t r a t and g u i n e a - p i g p y r a m i d a l neurons are not s t r i c t l y comparable i n a l l r e s p e c t s . S t i m u l u s Evoked S p i k e s The s t i m u l u s evoked response o f t h e p y r a m i d a l c e l l d i f f e r e d from t h a t evoked t h r o u g h d e p o l a r i z i n g c u r r e n t i n j e c t i o n i n g i v i n g r i s e t o a s i n g l e a l l - o r - n o n e s p i k e a t a l l l e v e l s o f t h e p y r a m i d a l c e l l a x i s . The a l v e a r evoked s p i k e was evoked d i r e c t l y from b a s e l i n e w i t h a s h o r t onset l a t e n c y a t b o t h s o m a t i c and d e n d r i t i c r e c o r d i n g s i t e s . A n t i d r o m i c s p i k e d i s c h a r g e i n t h e p y r a m i d a l c e l l has been w e l l c h a r a c t e r i z e d t h r o u g h l a m i n a r p r o f i l e a n a l y s e s o f 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 i n b o t h t h e i n v i v o ( G e s s i e t a l . 1966; Leung 1979a,b; S p e r t i e t a l . 1967) and i n v i t r o p r e p a r a t i o n (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 evoked response r e p r e s e n t s an a n t i d r o m i c s p i k e i n v a s i o n o f somata and a p i c a l d e n d r i t e s o f p y r a m i d a l c e l l s . S t i m u l a t i o n o f s t r a t u m o r i e n s or r a d i a t u m a l s o evoked a s i n g l e a l l - o r - n o n e s p i k e a r i s i n g from t h e peak of t h e s y n a p t i c d e p o l a r i z a t i o n a t - 125 - both t h e s o m a t i c and d e n d r i t i c l e v e l . O rthodromic s p i k e s d i s p l a y e d s i m i l a r evoked c h a r a c t e r i s t i c s a t a l l l e v e l s of t h e dendro-somatic a x i s , i n c l u d i n g l a t e n c y j i t t e r near t h r e s h o l d f o r AP d i s c h a r g e , and t h e response t o s t i m u l u s 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 o f the a f f e r e n t i n p u t . Somatic and d e n d r i t i c s p i k e s d i f f e r e d c h a r a c t e r i s t i c a l l y i n the e x a c t form o f t h e evoked s p i k e , w i t h s o m a t i c s p i k e s e x h i b i t i n g a l a r g e r a m p l i t u d e and more 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 any g i v e n l e v e l o f t h e c e l l a x i s , b o t h a l v e a r and o r t h o d r o m i c s p i k e s were evoked w i t h s i m i l a r a m p l i t u d e and h a l f w i d t h . D e n d r i t i c s p i k e s evoked t h r o u g h s t i m u l a t i o n o f e i t h e r s t r a t u m o r i e n s o r r a d i a t u m were thus comparable i n waveform t o t h a t o f a s p i k e a n t i d r o m i c a l l y 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 i n s o m a t i c and 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 s a l s o d i f f e r e d i n terms o f t h e v o l t a g e t h r e s h o l d f o r s p i k e a c t i v a t i o n . I n t h e s o m a t i c r e g i o n , o r t h o d r o m i c s p i k e s were evoked a t 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 , as judged by t h e a b s o l u t e v o l t a g e of t h e b r e a k p o i n t o f s p i k e d i s c h a r g e . 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 c o n s p i c u o u s v o l t a g e t h r e s h o l d f o r s p i k e g e n e r a t i o n , w i t h o r t h o d r o m i c a l l y evoked s p i k e s a r i s i n g a t v a r i o u s l e v e l s o f d e p o l a r i z a t i o n from t h e u n d e r l y i n g EPSP. Assuming t h a t d e n d r i t i c s p i k e s a r i s e t h r o u g h a c t i v a t i o n o f v o l t a g e - d e p e n d e n t c h a n n e l s , t h e above r e s u l t s would suggest t h a t t h e g e n e r a t o r s i t e f o r t h e d e n d r i t i c s p i k e i s l o c a t e d d i s t a n t t o t h e p o s i t i o n o f t h e d e n d r i t i c r e c o r d i n g e l e c t r o d e . T h i s s i t e might c o r r e s p o n d t o one or 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 ("hot s p o t s " ) or t o the s o m a t i c r e g i o n o f t h e p y r a m i d a l c e l l . However, t h e i n t r a c e l l u l a r c h a r a c t e r i s t i c s o f o r t h o d r o m i c s p i k e d i s c h a r g e - 126 - would be consistent with results obtained through current-source density analysis (Chapter 3). Laminar p r o f i l e s of f i e l d potentials and current-source density revealed that SO of SR stimulation evoked a single spike response at a l l l e v e l s of the pyramidal c e l l axis, with the spike conducting from the c e l l layer through the dendritic f i e l d . The i n t r a c e l l u l a r spike would thus be expected to display similar evoked c h a r a c t e r i s t i c s at the somatic and dendritic l e v e l (ie response to stimulus in t e n s i t y or r e p e t i t i v e stimulation), and to the alvear evoked spike antidromically invading the dendritic arborization. Thus, the i n t r a c e l l u l a r properties of spike discharge are at least in accord with the data obtained from CSD analysis indicating a somatic s i t e of o r i g i n for the evoked dendritic spike. Stimulus Vs. Current Evoked Spikes A comparison of the stimulus and current evoked response revealed a basic s i m i l a r i t y between the c h a r a c t e r i s t i c s of current evoked Na+-dependent spikes and action potentials evoked through stimulation of afferent or efferent pathways. For instance, depolarizing current evoked similar patterns of a c t i v i t y in both somatic and dendritic locations, with somatic spikes exhibiting a greater amplitude and more narrow halfwidth than those in dendritic recording s i t e s . In addition, the current evoked dendritic spike displayed no consistent voltage threshold for activation, while spike discharge in the somatic region arose from a similar membrane potential for a l l l e v e l s of current i n j e c t i o n . In fact, a d i r e c t comparison of fast spike discharge revealed that stimulus evoked spikes were comparable to those evoked through curent i n j e c t i o n in terms of amplitude - 127 - and h a l f w i d t h when evoked upon a s i m i l a r l e v e l o f membrane d e p o l a r i z a t i o n . The major d i s t i n c t i o n between s t i m u l u s and c u r r e n t evoked s p i k e g e n e r a t i o n was found i n t h e p a t t e r n of AP d i s c h a r g e , w i t h s t i m u l u s a c t i v a t i o n g i v i n g r i s e t o a s i n g l e s p i k e and d e p o l a r i z i n g c u r r e n t a r e p e t i t i v e s p i k e d i s c h a r g e . However, t h e s e r e s u l t s can be e x p l a i n e d by t h e f a c t t h a t s t i m u l a t i o n o f a f f e r e n t or e f f e r e n t pathways i n t h e s l i c e i s known t o r e s u l t i n t h e r e c u r r e n t a c t i v a t i o n o f an i n h i b i t o r y s y n a p t i c p o t e n t i a l i n p y r a m i d a l c e l l s c a p a b l e o f s u p p r e s s i n g r e p e t i t i v e s p i k e d i s c h a r g e (Andersen e t a l . 1969; D i n g l e d i n e and Langmoen 1980). In t h e p r e s e n c e of a p p r o p r i a t e C l - c h a n n e l b l o c k e r s , s t i m u l u s a c t i v a t i o n can a l s o l e a d t o r e p e t i t i v e s p i k e g e n e r a t i o n a t b o t h th e s o m a t i c and 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 P r i n c e 1980; Wong and P r i n c e 1979) . The s i m i l a r i t y i n evoked c h a r a c t e r i s t i c s o f s t i m u l u s and c u r r e n t evoked s p i k e s might then i m p l y a common s i t e o f o r i g i n f o r a l l evoked f a s t s p i k e s o f t h e p y r a m i d a l c e l l . S p i k e P r e - P o t e n t i a l s The evoked c h a r a c t e r i s t i c s o f f a s t p r e - p o t e n t i a l s (FPPs) and a n t i d r o m i c i n i t i a l segment (IS) s p i k e s r e c o r d e d i n t h e s o m a t i c r e g i o n o f t h e p y r a m i d a l c e l l have been d e s c r i b e d by p r e v i o u s i n v e s t i g a t o r s (Andersen and Lomo 1966; Kandel e t a l . 1961; M a c V i c a r and Dudek 1981; S c h w a r t z k r o i n 1975,1977; S c h w a r t z k r o i n and P r i n c e 1980; Spencer and Kandel 1961b). The p r e s e n t s t u d y e x t e n d s t h e s e f i n d i n g s i n r e p o r t i n g t h e a c t i v a t i o n o f FPPs and " I S " s p i k e s i n 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 s . P r e v i o u s i n v e s t i g a t i o n s have drawn a d i s t i n c t i o n between t h e FPP and IS s p i k e based upon i n i t i a l r e p o r t s t h a t the IS s p i k e was o f - 128 - g r e a t e r a m p l i t u d e than the F P P , and upon an i n a b i l i t y t o a n t i d r o m i c a l l y evoke a f a s t p r e - p o t e n t i a l (Kandel e t a l . 1961; S c h w a r t z k r o i n 1977; Spencer and Kandel 1961b). However, i n the p r e s e n t s t u d y , t h e F P P and IS s p i k e were found t o be v e r y s i m i l a r i n waveform, w i t h no g r e a t d i f f e r e n c e i n a m p l i t u d e a t any g i v e n l o c a t i o n . I n a d d i t i o n , a n t i d r o m i c a c t i v a t i o n of t h e " F P P " has been r e p o r t e d s i n c e t h e f i r s t d e s c r i p t i o n o f t h e s e p o t e n t i a l s by Spencer and Kandel i n 1961 (Knowles and S c h w a r t z k r o i n 1981b; S c h w a r t z k r o i n and P r i n c e 1980; T a y l o r and Dudek 1982). The r e p o r t e d d i f f e r e n c e i n a m p l i t u d e between t h e s e p o t e n t i a l s may r e l a t e t o t h e f a c t t h a t t h e a m p l i t u d e o f the F P P can v a r y w i t h membrane p o t e n t i a l , i n c r e a s i n g i n a m p l i t u d e i n response t o membrane h y p e r p o l a r i z a t i o n ( S c h w a r t z k r o i n 1977). In t h i s r e g a r d , i t i s i m p o r t a n t t o note t h a t IS s p i k e s a r e u s u a l l y uncovered on a h y p e r p o l a r i z e d membrane d u r i n g a n t i d r o m i c s t i m u l a t i o n , w h i l e F P P s are observ e d at a l l l e v e l s o f membrane p o l a r i z a t i o n . 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 the IS s p i k e and F P P r e p r e s e n t " f a s t p r e - p o t e n t i a l s " evoked a t d i f f e r e n t l e v e l s o f r e s t i n g membrane p o t e n t i a l . T h e r e f o r e , g i v e n t h e s i m i l a r i t y i n shape and evoked c h a r a c t e r i s t i c s of the F P P and IS s p i k e , t h e d i s t i n c t i o n between t h e s e p o t e n t i a l s i s p r o b a b l y unwarranted, and b o t h w i l l be r e f e r r e d t o as f a s t p r e - p o t e n t i a l s . F a s t p r e - p o t e n t i a l s r e c o r d e d i n p y r a m i d a l c e l l somata have been proposed 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 decayed d e n d r i t i c s p i k e f o l l o w i n g a f a i l u r e o f a c t i v e p r o p a g a t i o n a t some p o i n t w i t h i n t h e d e n d r i t i c a r b o r i z a t i o n (Andersen and Lomo 1966; Spencer and Kandel 1961b). One would t h e r e f o r e expect t o f i n d e v i d e n c e i n d e n d r i t i c r e c o r d i n g s o f f a s t p r e - p o t e n t i a l - l i k e waveforms i n t e r m e d i a t e i n a m p l i t u d e between the s o m a t i c FPP and - 129 - the much larger dendritic spike. However, somatic and dendritic FPPs f e l l within a similar range of amplitude and could be activated through antidromic stimulation. It would appear therefore that a fast pre-potential in the somatic region may not represent the passive conduction and electrotonic decay of an evoked dendritic spike. Further evidence to support t h i s conclusion w i l l be presented in Chapter 6. Previous studies have provided evidence that at least some fast pre-potentials represent the electrotonic conduction of a spike across a gap junction between neighboring pyramidal c e l l s (Andrew et a l . 1982; MacVicar and Dudek 1981). Support for the presence of gap junctions between pyramidal neurons has come from freeze-fracture studies (Schmalbruch and Jahnsen 1981) and the a b i l i t y to a t t a i n dye-coupling of two or more pyramidal neurons following i n t r a c e l l u l a r i n j e c t i o n of Lucifer Yellow into pyramidal c e l l somata or apical dendrites (Andrew et a l . 1982; MacVicar and Dudek 1981). The point of gap junctional contact between pyramidal c e l l s i s thought to occur at either the somatic or dendritic l e v e l (Andrew et a l . 1982; MacVivar and Dudek 1981; Schmalbruch and Jahnsen 1981), and dual impalements of pyramidal c e l l s have shown that spike discharge in one of a pair of e l e c t r o t o n i c a l l y coupled neurons i s reflected in the second as a fast pre-potential (MacVicar and Dudek 1981). Fast pre-potentials recorded in somata and apical dendrites of CAl pyramidal c e l l s might thus be adequately explained on the basis of electrotonic gap junctions between adjacent pyramidal neurons. Such a mechanism would help to account for the s i m i l a r i t y in 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 fast - 130 - pre-potential at both the somatic and dendritic l e v e l . However, the characterization of FPP discharge in dendritic membrane i s only at a preliminary stage, and substantially more work w i l l be required to d e f i n i t i v e l y locate the origin(s) of fast pre-potentials in the pyramidal c e l l . - 131 - 5-0. EVOKED CHARACTERISTICS OF ACTION POTENTIAL DISCHARGE ALONG THE DENDRO-SOMATIC AXIS OF THE CAl PYRAMIDAL NEURON 5-1. Introduction Laminar p r o f i l e analysis of e x t r a c e l l u l a r f i e l d potentials in the CAl region have indicated a retrograde conduction of a spike response from the c e l l body layer through the dendritic region, suggesting a somatic s i t e of o r i g i n for the evoked dendritic spike in CAl pyramidal neurons (Chapter 3). The basic properties of spike discharge in the pyramidal c e l l have also been characterized through i n t r a c e l l u l a r recordings from pyramidal c e l l somata and apical dendrites (Chapter 4), providing the basis for a comparison of i n t r a - and e x t r a c e l l u l a r spike a c t i v i t y at the somatic and dendritic l e v e l . Several of the properties of i n t r a c e l l u l a r spike discharge coincide 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 population spike responses recorded in stratum pyramidale and radiatum. For instance, stimulation of afferent or efferent pathways gave r i s e to a single evoked spike in somatic and dendritic impalements, similar to that observed for e x t r a c e l l u l a r f i e l d p otentials. In addition, the exact waveform of the i n t r a c e l l u l a r spike d i f f e r e d between somatic and dendritic locations, with the somatic spike exhibiting a greater amplitude and shorter halfwidth than that in the dendrite. S i m i l a r l y , the e x t r a c e l l u l a r population spike response in stratum pyramidale consisted of a large amplitude negativity while that in stratum radiatum was of smaller amplitude and biphasic in waveform. Furthermore, the lack of voltage threshold of orthodromic in t r a d e n d r i t i c spikes would - 132 - i n d i c a t e t h a t the s p i k e had o r i g i n a t e d a t a s i t e remote from t h e r e c o r d i n g e l e c t r o d e , as suggested by f i e l d p o t e n t i a l a n a l y s i s . However, l a m i n a r p r o f i l e s o f 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 i n d i c a t e t h a t 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 s p i k e response change i n a p r o g r e s s i v e manner t h r o u g h t h e d e n d r i t i c f i e l d o f t h e p y r a m i d a l c e l l p o p u l a t i o n . More i n s i g h t i n t o t h e p r o p e r t i e s o f s p i k e a c t i v a t i o n i n t h e p y r a m i d a l c e l l might thus be g a i n e d by comparing v a r i o u s parameters o f i n t r a c e l l u l a r s p i k e d i s c h a r g e a t d i f f e r e n t s i t e s a l o n g t h e de n d r o - s o m a t i c a x i s . Such an a n a l y s i s would a l s o p r o v i d e a d i r e c t t e s t o f t h e p r e v a i l i n g h y p o t h e s i s c o n c e r n i n g s p i k e g e n e r a t i o n i n t h e a p i c a l d e n d r i t e o f t h e p y r a m i d a l c e l l . P r e v i o u s i n v e s t i g a t i o n s have a s s i g n e d t h e s i t e f o r g e n e r a t i o n o f a d e n d r i t i c s p i k e t o hot s p o t s o f d e n d r i t i c membrane, p o s s i b l y l o c a t e d a t one or more b r a n c h p o i n t s o f t h e d e n d r i t i c t r e e (Andersen and Lomo 1966; Spencer and Ka n d e l 1961b). E v i d e n c e t h a t d e n d r i t i c membrane can i n f a c t i n i t i a t e s p i k e d i s c h a r g e i n d e p e n d e n t l y o f t h a t i n t h e soma has come from i n t r a c e l l u l a r r e c o r d i n g s o f s p i k e a c t i v a t i o n i n d e n d r i t e s p h y s i c a l l y i s o l a t e d from the c e l l body by a k n i f e c u t a c r o s s t h e p r o x i m a l - m i d s t r a t u m r a d i a t u m (Benardo e t a l . 1982; Masukawa and P r i n c e 1984). At t h e s o m a t i c l e v e l , t h e d e n d r i t i c s p i k e i s obser v e d i n t h e form o f a f a s t p r e - p o t e n t i a l , t h e s m a l l a m p l i t u d e o f which s u g g e s t s a f a i l u r e o f a c t i v e p r o p a g a t i o n o f t h e s p i k e w i t h i n t h e d e n d r i t i c t r e e and a subsequent p a s s i v e e l e c t r o t o n i c c o n d u c t i o n t o t h e c e l l body r e g i o n (Andersen and Lomo 1966; Spencer and Ka n d e l 1961b). T h i s model o f s p i k e a c t i v a t i o n would p r e d i c t a change i n t h e c h a r a c t e r i s t i c s o f t h e i n t r a d e n d r i t i c s p i k e i n d i c a t i v e o f p a s s i v e e l e c t r o t o n u s , i n c l u d i n g a d e c l i n e i n a m p l i t u d e and - 1 3 3 - i n c r e a s e i n b o t h w i d t h and onset l a t e n c y w i t h p r o x i m i t y t o t h e c e l l body l a y e r . However, t h i s p a t t e r n o f change i n t h e p r o p e r t i e s o f t h e d e n d r i t i c s p i k e a re i n d i r e c t c o n t r a s t t o t h o s e o b s e r v e d f o r t h e 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 response on l a m i n a r p r o f i l e s o f f i e l d p o t e n t i a l s i n t h e C A l r e g i o n . A c c o r d i n g t o t h e s e 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 g r a d u a l change i n waveform and i n c r e a s e i n onset l a t e n c y t h e g r e a t e r t h e r e c o r d i n g d i s t a n c e from s t r a t u m p y r a m i d a l e . An a n a l y s i s o f i n t r a c e l l u l a r s p i k e c h a r a c t e r i s t i c s a l o n g t h e dendro-somatic a x i s might then p r o v i d e a means o f d i s t i n g u i s h i n g between a so m a t i c or d e n d r i t i c s i t e o f o r i g i n f o r t h e evoked d e n d r i t i c s p i k e . The p r e s e n t s t u d y t h e r e f o r e p r o v i d e s a c o m p a r a t i v e a n a l y s i s o f t h e p r o p e r t i e s o f a c t i o n p o t e n t i a l d i s c h a r g e a l o n g t h e dendro-somatic a x i s o f t h e p y r a m i d a l c e l l , i n c l u d i n g t h o s e o f a m p l i t u d e , h a l f w i d t h , v o l t a g e t h r e s h o l d and onset l a t e n c y . In view o f t h e f a c t t h a t c u r r e n t - s o u r c e d e n s i t y a n a l y s i s would i n d i c a t e a s o m a t i c s i t e o f o r i g i n f o r t h e d e n d r i t i c s p i k e , a t t e m p t s were a l s o made i n t h e p r e s e n t s t u d y t o r e c o r d t h e 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 p y r a m i d a l c e l l p o p u l a t i o n . 5-2. Methods - 134 - Data was p o o l e d 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 a l o n g t h e d e n d r o-somatic a x i s o f p y r a m i d a l neurons and s p i k e a m p l i t u d e , h a l f w i d t h , and v o l t a g e t h r e s h o l d measured a c c o r d i n g t o t h e methods d e s c r i b e d p r e v i o u s l y (Chapter 4 ) . S i n c e d u a l impalements of s i n g l e p y r a m i d a l neurons were not o b t a i n e d , a b s o l u t e measurements o f s p i k e l a t e n c y a t d i f f e r e n t r e g i o n s o f t h e p y r a m i d a l c e l l c o u l d not be made. However, an i n d i r e c t e s t i m a t e o f t h e l a t e n c y f o r s p i k e d i s c h a r g e a t t h e s o m a t i c and d e n d r i t i c l e v e l was o b t a i n e d t h r o u g h a comparison of evoked i n t r a c e l l u l a r s p i k e a c t i v i t y and 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 . F or t h e s e e x p e r i m e n t s , a low impedance e l e c t r o d e was p l a c e d i n s t r a t u m p y r a m i d a l e t o r e c o r d t h e 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 a h i g h impedance i n t r a c e l l u l a r e l e c t r o d e w i t h 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 d i r e c t l y below t h a t i n t h e c e l l l a y e r . Upon o b t a i n i n g a s t a b l e i n t r a d e n d r i t i c impalement, s t i m u l u s i n t e n s i t y f o r each of t h e t h r e e pathways was s e t t o t h r e s h o l d f o r i n t r a d e n d r i t i c s p i k e g e n e r a t i o n , and t h e evoked response r e c o r d e d s i m u l t a n e o u s l y w i t h t h a t o f t h e p o p u l a t i o n s p i k e i n s t r a t u m p y r a m i d a l e . The i n t r a d e n d r i t i c e l e c t r o d e was t h e n withdrawn from t h e impalement and r e t u r n e d t o t h e d e pth of the d e n d r i t i c r e c o r d i n g , as m o n i t o r e d by a d i g i t a l micrometer d i s p l a y o f e l e c t r o d e d e pth ( B u r l e i g h Inchworm PZ-550). E x t r a c e l l u l a r p o t e n t i a l waveforms at t h e c e l l body and i n t h e r e g i o n o f t h e d e n d r i t i c impalement were th e n c o l l e c t e d a t the same s t i m u l u s i n t e n s i t y f o r a l a t e n c y comparison of i n t r a - and e x t r a d e n d r i t i c s p i k e p o t e n t i a l s . To a v o i d any p o s s i b l e change i n t h e e x t r a c e l l u l a r p o t e n t i a l over t i m e , t h r e s h o l d i n t r a d e n d r i t i c - 135 - responses were u s u a l l y c o l l e c t e d j u s t p r i o r t o w i t h d r a w a l from t h e r e c o r d i n g s i t e . However, i f the s t i m u l u s i n t e n s i t y o f a pathway had been changed b e f o r e e l e c t r o d e w i t h d r a w a l , s t i m u l u s i n t e n s i t y was s e t t o evoke t h e same a m p l i t u d e and l a t e n c y p o p u l a t i o n s p i k e a t the c e l l l a y e r as t h a t r e c o r d e d d u r i n g c o l l e c t i o n o f i n t r a d e n d r i t i c p o t e n t i a l s . In an attempt t o a s s e s s d e n d r i t i c a c t i v i t y i n d e p e n d e n t l y o f 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 used t o p l a c e a k n i f e c u t i n t h e p r o x i m a l - m i d a p i c a l d e n d r i t i c r e g i o n i n o r d e r t o s e p a r a t e t h e c e l l b o d i e s and a p i c a l d e n d r i t e s o f p y r a m i d a l neurons. The f i r s t method employed 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 dowel mounted on a m i c r o - m a n i p u l a t o r . S l i c e s were c u t i m m e d i a t e l y f o l l o w i n g t r a n s f e r r a l t o t h e r e c o r d i n g chamber under 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 m i c r o s c o p e . The b l a d e was lo w e r e d s l o w l y t h r o u g h t h e s l i c e a l o n g t h e i n t e n d e d l i n e o f c u t b o t h w i t h i n s t r a t u m 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 s i d e of t h e c u t d e n d r i t i c r e g i o n . S m a l l l a t e r a l movements a l o n g t h e a x i s o f t h e b l a d e were o f t e n made at the bottom o f t h e c u t t o o b t a i n a complete s e p a r a t i o n o f s l i c e t i s s u e . I n 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 body r e g i o n from t h e remainder o f t h e s l i c e . In t h e second method, t h r e e t o f o u r s l i c e s were t a k e n i m m e d i a t e l y f o l l o w i n g s e c t i o n i n g on t h e t i s s u e chopper and p l a c e d i n d i v i d u a l l y i n drops o f c o l d oxygenated medium w i t h i n a p r e - c o o l e d p l a s t i c p e t r i d i s h . The p e t r i d i s h was th e n "anchored" w i t h masking tape t o t h e s t a g e o f a 3X d i s s e c t n g microscope t o p r e v e n t movement d u r i n g m i c r o d i s s e c t i o n . Immediately b e f o r e c u t t i n g a s l i c e , t h e c o r n e r o f a t o r n p i e c e - 136 - of t i s s u e paper was used t o draw away most o f the f l u i d i n t h e drop c o n t a i n i n g t h e s l i c e , a l l o w i n g t h e s l i c e t o r e s t upon t h e s u r f a c e o f t h e p e t r i d i s h . The t i p o f a #10 s c a l p e l b l a d e was 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 and t h e b l a d e a l i g n e d a l o n g the i n t e n d e d l i n e o f d i s s e c t i o n . The b l a d e was h e l d a t a p p r o x i m a t e l y 45 degrees from v e r t i c a l so as t o watch t h e p r o g r e s s o f t h e c u t and t o m i n i m i z e t h e p o s s i b i l i t y o f co m p r e s s i v e damage t o t h e d e n d r i t i c r e g i o n . The b l a d e was th e n " r o l l e d " a l o n g the c u r v e d edge a c r o s s and t h r o u g h t h e s l i c e i n one smooth m o t i o n . A few drops o f f r e s h l y oxygenated c o l d medium was i m m e d i a t e l y p l a c e d on t h e c u t s l i c e and t h e p r o c e d u r e r e p e a t e d f o r t h e ne x t s l i c e . 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 s s u e , o n l y t h r e e t o f o u r s l i c e s were c u t per d i s s e c t i o n , 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 once c u t t i n g was completed. The time r e q u i r e d f o r c u t t i n g s l i c e s i n t h e above manner ( t o t a l o f 3-5 min) was not found t o compromise s l i c e 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 i n t h e p e t r i d i s h under t h e same c o n d i t i o n s were found t o e x h i b i t e l e c t r o p h y s i o l o g i c a l a c t i v i t y comparable t o s l i c e s removed d i r e c t l y f r o n t h e t i s s u e chopper t o t h e r e c o r d i n g chamber. S l i c e s were a l l o w e d a p e r i o d o f a t l e a s t 45 min f o r r e c o v e r y and e q u i l i b r a t i o n t o b a t h c o n d i t i o n s . I n some c a s e s , t h e c o n c e n t r a t i o n o f e x t r a c e l l u l a r c a l c i u m i n t h e p e r f u s i n g medium was e l e v a t e d from 1.6mM t o 2.5-3mM t o enhance t h e r e c o v e r y o f c u t t i s s u e e l e m e n t s , as o u t l i n e d i n t h e p r o c e d u r e o f Benardo e t a l . (1982). The evoked a c t i v i t y i n t h e C A l r e g i o n o f d i s s e c t e d s l i c e s was examined p r i m a r i l y by e x t r a c e l l u l a r r e c o r d i n g t e c h n i q u e s i n response 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 maximal - 137 - so m a t i c and/or d e n d r i t i c response t o SR s t i m u l a t i o n (up t o 60V s t i m u l u s i n t e n s i t y ) was r e c o r d e d a t a s e q u e n t i a l s e r i e s o f s i t e s e x t e n d i n g t o e i t h e r s i d e o f the c u t from t h e l a t e r a l b o r d e r o f CA2 t o t h e s u b i c u l a r r e g i o n . When t h e c u t r e g i o n o f s t r a t u m p y r a m i d a l e was s t i l l a t t a c h e d t o t h e s l i c e , 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 a t a l l p o i n t s a l o n g the C A 2 - s u b i c u l a r a x i s ( d e n d r i t i c e l e c t r o d e d i r e c t l y below t h e so m a t i c r e c o r d i n g s i t e ) . I f t h e e x c i s e d p o r t i o n was removed from the s l i c e , 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 on e i t h e r s i d e o f t h e c u t r e g i o n , w h i l e 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 t h r o u g h t h e e n t i r e e x t e n t o f t h e c u t . In o r d e r t o a c h i e v e maximal 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 a f f e r e n t i n p u t s , t h e s t i m u l a t i n g e l e c t r o d e was u s u a l l y moved w i t h t h e r e c o r d i n g e l e c t r o d e t o m a i n t a i n a c o n s t a n t s t i m u l a t i n g - r e c o r d i n g e l e c t r o d e d i s t a n c e o f a p p r o x i m a t e l y 400pm. In o t h e r c a s e s , t h e s t i m u l a t i n g 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 kept c o n s t a n t w h i l e t h e r e c o r d i n g e l e c t r o d e was moved. The p o s i t i o n o f the k n i f e c u t , l o c a t i o n o f each r e c o r d i n g s i t e , and t h e maximal evoked s o m a t i c or d e n d r i t i c response a t each r e c o r d i n g l o c a t i o n were noted i n a d e t a i l e d diagram o f t h e s l i c e , 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 s e s s i o n f o r h i s t o l o g i c a l a n a l y s i s . S l i c e s were f i r s t p l a c e d i n a 10% f o r m a l i n / 1% Ca+2-acetate s o l u t i o n f o r a t l e a s t 24 hours and t r a n s f e r r e d t o a 20% suc r o s e - p h o s p h a t e b u f f e r e d s a l i n e s o l u t i o n (pH 7.4) 5 hours b e f o r e s e c t i o n i n g . S l i c e s were i n d i v i d u a l l y p l a c e d on t h e f l a t s u r f a c e o f a p r e - c u t f r o z e n drop o f O.C.T. Compound (Tissue-Tek) on t h e c u t t i n g s t a g e of a c r y o s t a t ( T i s s u e - t e k ; 12-14 degrees c e n t i g r a d e ) and 22pm s e r i a l s e c t i o n s c u t and mounted on g l a s s s l i d e s . S l i c e t i s s u e was the n s t a i n e d - 138 - with c r e s y l v i o l e t and m i c r o s c o p i c a l l y examined to assess the extent of the k n i f e cut at a l l l e v e l s of the s l i c e and the r e l a t i o n of neuronal elements to p r e v i o u s l y recorded a c t i v i t y . 5-3. R e s u l t s - 139 - S p i k e A m p l i t u d e and H a l f w i d t h R e p r e s e n t a t i v e examples of a n t i d r o m i c and o r t h o d r o m i c p o t e n t i a l s r e c o r d e d a t the soma and v a r i o u s l o c a t i o n s a l o n g t h e a p i c a l d e n d r i t i c t r e e are i l l u s t r a t e d i n F i g 5.1. A l l s t i m u l u s evoked s p i k e s were found t o e x h i b i t the maximal 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 a t the l e v e l o f t h e p y r a m i d a l c e l l body. In t h e d e n d r i t i c r e g i o n , s p i k e s d i s p l a y e d a g r a d u a l d e c l i n e i n a m p l i t u d e and i n c r e a s e i n h a l f w i d t h w i t h d i s t a n c e from th e b o r d e r of s t r a t u m p y r a m i d a l e ( F i g 5.1). The SO evoked EPSP was a l s o l a r g e s t i n t h e s o m a t i c r e g i o n , d e c l i n i n g i n a m p l i t u d e and r a t e o f r i s e a l o n g the a p i c a l d e n d r i t e ( F i g 5 . I B ) . C o n v e r s e l y , t h e a m p l i t u d e and r a t e o f r i s e o f t h e EPSP evoked by SR s t i m u l a t i o n was g r e a t e s t i n t h e r e g i o n o f t h e a p i c a l d e n d r i t e , d e c r e a s i n g w i t h p r o x i m i t y t o t h e c e l l body l a y e r ( F i g 5.1C). Data such as t h a t shown i n F i g 5.1 were c o l l e c t e d a t a l l l e v e l s o f t h e a p i c a l d e n d r i t i c t r e e and used t o c a l c u l a t e t h e average v a l u e o f evoked p o t e n t i a l c h a r a c t e r i s t i c s a l o n g the d e n d r o-somatic a x i s ( F i g s 5.2-5.4). In each c a s e , th e average v a l u e s are p l o t t e d a g a i n s t t h e a n a t o m i c a l d i s t a n c e of t h e r e c o r d i n g s i t e from th e b o r d e r of s t r a t u m p y r a m i d a l e ( s o m a t i c r e c o r d i n g s t a k e n as "0"um). In F i g s 5.2 and 5.4 a s c a l e d s c h e m a t i c diagram of a r a t p y r a m i d a l neuron and t h e average v a l u e s o f r e s t i n g membrane p o t e n t i a l or i n p u t r e s i s t a n c e a t each r e c o r d i n g s i t e are a l s o p r o v i d e d . These c a l c u l a t i o n s r e v e a l t h a t b o t h a n t i d r o m i c and o r t h o d r o m i c i n t r a c e l l u l a r s p i k e s d i s p l a y a p r o g r e s s i v e d e c l i n e i n a m p l i t u d e w i t h d i s t a n c e from th e c e l l body l a y e r ( F i g 5.2A-C). However, the 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 o f t y p i c a l 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 r e c o r d e d i n p y r a m i d a l c e l l somata and a p i c a l d e n d r i t e s 65, 165 and 265pm from the b o r d e r of s t r a t u m p y r a m i d a l e . S p i k e d i s c h a r g e was evoked by 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) or s t r a t u m r a d i a t u m ( C ) . Note t h e g r a d u a l change i n s p i k e a m p l i t u d e and h a l f w i d t h w i t h r e c o r d i n g d i s t a n c e from the c e l l body l a y e r . In each c a s e , s e v e r a l sweeps are shown superimposed f o r s t i m u l u s i n t e n s i t i e s near t h r e s h o l d f o r s p i k e a c t i v a t i o n . Somatic r e c o r d i n g s were t a k e n from t h r e e s e p a r a t e c e l l s , w h i l e d e n d r i t i c r e c o r d i n g s a t each l o c a t i o n a l o n g the c e l l a x i s a r e 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 f o r a l l r e c o r d i n g s . Stim. Site A. Recording Site (p) - 142 - FIG 5.2 P l o t s o f the average a m p l i t u d e o f s t i m u l u s evoked s p i k e s r e c o r d e d i n p y r a m i d a l c e l l somata 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 from the b o r d e r o f s t r a t u m p y r a m i d a l e (mean +/- sem; measured from 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 a r e t a k e n as Ojnm d i s t a n c e . S p i k e s were evoked by 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) or s t r a t u m r a d i a t u m ( C ) . D. Schematic diagram o f t h e r a t p y r a m i d a l c e l l drawn t o s c a l e w i t h t h e b o r d e r o f t h e a l v e u s and hippocampal f i s s u r e denoted by dark l i n e s . Average r e s t i n g membrane p o t e n t i a l s (RMP) f o r t h e p y r a m i d a l c e l l impalements i n p l o t s A-C are shown i n lower p a r t o f D. The number o f impalements (n) f o r each p o i n t a r e shown at t h e base o f t h e g r a p h s . Average v a l u e s were c a l c u l a t e d from t h e l a r g e s t evoked s p i k e a t each l o c a t i o n , and v a l u e s w i t h o u t s t a n d a r d e r r o r b a r s are t h o s e i n which t h e s t a n d a r d e r r o r l i e s w i t h i n t h e b o r d e r o f t h e i l l u s t r a t e d p o i n t . A. B. C. Stim. Site Alveus Stratum Oriens Stratum Radiatum 8̂0H f 70H < 6 0 CD £ 50 H C L if) 4 0 H * I i i n 9 2 4 3 10 10 0 100 200 300 0 i 4 3 7 10 12 2 I - 1 1 100 200 3 0 0 9 2 4 5 I ' 0 100 23 6 4 200 300 D. cr 8 2 4 E ffi o ° gj o 4 6 9 12 3 I r 0 100 200 300 Apical Dendritic Recording Site (/ J ) - 144 - am p l i t u d e a l o n g t h e d e n d r i t i c t r e e o c c u r r e d i n a n o n - l i n e a r manner. A sh a r p i n i t i a l drop i n s p i k e a m p l i t u d e was obser v e d w i t h i n lOOum from s t r a t u m p y r a m i d a l e , f o l l o w e d by a " p l a t e a u " and a second r a p i d d e c l i n e i n d i s t a l d e n d r i t i c impalements 150-200um from t h e c e l l l a y e r . These r e s u l t s a re not due t o v a r i a b i l i t y i n t h e q u a l i t y o f d e n d r i t i c impalements, as t h e average v a l u e o f r e s t i n g membrane p o t e n t i a l f o r each r e c o r d i n g l o c a t i o n f e l l w i t h i n t h e range of 62-68mV ( F i g 5.2D). R a t h e r , a comparison o f s p i k e a m p l i t u d e t o a s c a l e d diagram o f t h e p y r a m i d a l c e l l s u g g e s t s t h a t p o i n t s o f t r a n s i t i o n i n s p i k e c o n f i g u r a t i o n c o r r e l a t e t o t h e a n a t o m i c a l l o c a t i o n o f major b r a n c h p o i n t s of t h e a p i c a l d e n d r i t i c t r e e . A p r o g r e s s i v e i n c r e a s e i n s p i k e w i d t h w i t h d i s t a n c e a l o n g t h e a p i c a l d e n d r i t e i s a l s o e v i d e n t i n a p l o t o f t h e average h a l f w i d t h o f s p i k e s evoked from each o f t h e t h r e e s t i m u l a t i o n s i t e s ( F i g 5.3). Once a g a i n , t h e change i n s p i k e h a l f w i d t h i s s i m i l a r f o r b o t h a n t i d r o m i c ( F i g 5.3A) and o r t h o d r o m i c s p i k e s ( F i g 5.3B,C), w i t h a g r a d u a l i n c r e a s e i n s p i k e w i d t h t h r o u g h t h e p r o x i m a l - m i d a p i c a l d e n d r i t i c r e g i o n and a more pronounced i n c r e a s e i n d i s t a l d e n d r i t i c l o c a t i o n s . S i n c e t h e m a j o r i t y o f th e s e v a l u e s were o b t a i n e d from t h e a c t i o n p o t e n t i a l s i n F i g 5.2, t h e ob s e r v e d change i n s p i k e h a l f w i d t h cannot be e x p l a i n e d on t h e b a s i s o f a v a r i a b i l i t y i n r e s t i n g membrane p o t e n t i a l o f c e l l u l a r impalements. The c h a r a c t e r i s t i c s o f a c t i o n p o t e n t i a l s evoked by i n j e c t i o n o f d e p o l a r i z i n g c u r r e n t a l s o v a r i e d a c c o r d i n g t o t h e r e c o r d i n g s i t e a l o n g t h e p y r a m i d a l c e l l a x i s ( F i g 5.4). R e p r e s e n t a t i v e photographs o f the c u r r e n t evoked response ( F i g 5.4A) demonstrate t h a t t h e f a s t Na+ s p i k e was evoked w i t h t h e - 145 - FIG. 5.3 P l o t s o f the average h a l f w i d t h ( w i d t h a t h a l f a m p l i t u d e ) o f s t i m u l u s evoked s p i k e s r e c o r d e d i n p y r a m i d a l c e l l somata 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 from t h e b o r d e r o f s t r a t u m p y r a m i d a l e (mean +/- sem; h a l f a m p l i t u d e measured from 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 a re t a k e n as Oum d i s t a n c e . S p i k e s were evoked by s t i m u l a t i o n o f the a l v e u s ( A ) , s t r a t u m o r i e n s (B) or s t r a t u m r a d i a t u m ( C ) . The number o f impalements (n) f o r each p o i n t a r e shown a t the base o f graphs. Stim. Site Alveus Stratum Oriens Stratum Radiatum CD TD Z5 E < o u _C CD CO A. 1.5 H ° E 1.0 5 J ' "O CD CO 5 J  J n 7 I 5 3 10 8 3 I B. 0 100 2 0 0 3 0 0 0 C. 4 1 4 4 7 10 12 3 I • C I 4 4 100 2 0 0 3 0 0 0 100 12 20 5 3 1 , 1 2 0 0 3 0 0 Apical Dendritic Recording Site ( p ) - 147 - FIG. 5.4 A. O s c i l l o s c o p e t r a c e s o f t y p i c a l c u r r e n t 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 r e c o r d e d i n p y r a m i d a l c e l l somata and a p i c a l d e n d r i t e s 100 and 165um from the b o r d e r of s t r a t u m p y r a m i d a l e . B. P l o t s o f the average a m p l i t u d e (B) and h a l f w i d t h ( w i d t h a t h a l f a m p l i t u d e ) of the c u r r e n t evoked s p i k e r e c o r d e d i n p y r a m i d a l c e l l somata 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 from the b o r d e r o f s t r a t u m p y r a m i d a l e (mean +/- sem). Somatic r e c o r d i n g s are t a k e n as Oum d i s t a n c e . A m p l i t u d e s were measured from r e s t i n g membrane p o t e n t i a l f o r t h e f i r s t c u r r e n t evoked s p i k e i n e i t h e r Type 1 or Type 2 d i s c h a r g e . The number o f impalements (n) f o r each p o i n t are shown at the base of each graph. Spike Amplitude (mV) ^ > (ji CD ->J CO (X) o o o o o =3 I i _ J I I o- , = ro ro cn CD o o ro o o > 9. o - 1 o o CD C L O JO CD O O 13 iQ CO CD Spike Width at Half Amplitude (msec) b CJI ^ • • • i i i i i i i i i _ o ro b O n tf> ro ro o o OJ o ro -•—i CO o 3 Q o' O CD C L O S O o CD C L o OS - 8 f r T - - 149 - g r e a t e s t 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 r e g i o n o f t h e p y r a m i d a l c e l l . S p i k e s evoked i n 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 s then d i s p l a y e d 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 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 l a y e r ( F i g 5.4A). S i n c e a s i m i l a r response was found f o r s p i k e s evoked i n Type 1 or 2 d i s c h a r g e , t h e f i r s t evoked s p i k e i n e i t h e r case was used t o c a l c u l a t e t h e average a m p l i t u d e and h a l f w i d t h o f t h e c u r r e n t evoked s p i k e a l o n g the dendro-somatic a x i s ( F i g 5.4B). A l t h o u g h t h e average v a l u e s i n t h e p l o t s o f F i g 5.4B,C were d e r i v e d from r e l a t i v e l y few c e l l impalements, the r e s u l t s do i n d i c a t e a d e c l i n e i n s p i k e a m p l i t u d e and i n c r e a s e i n h a l f w i d t h a l o n g t h e a p i c a l d e n d r i t e , w i t h t h e g r e a t e s t change i n s p i k e c o n f i g u r a t i o n i n d i s t a l d e n d r i t i c l o c a t i o n s . V o l t a g e T h r e s h o l d o f Orthodromic S p i k e D i s c h a r g e V o l t a g e t h r e s h o l d f o r o r t h o d r o m i c s p i k e a c t i v a t i o n a l s o v a r i e d a c c o r d i n g t o t h e r e c o r d i n g p o s i t i o n a l o n g t h e dendro-somatic a x i s o f t h e p y r a m i d a l c e l l ( F i g 5.5A). In t h e case o f SR s t i m u l a t i o n , s p i k e t h r e s h o l d was found t o be l o w e s t i n t h e s o m a t i c r e g i o n , g r a d u a l l y i n c r e a s i n g w i t h d i s t a n c e a l o n g t h e a p i c a l d e n d r i t e . I n c o n t r a s t , s p i k e t h r e s h o l d i n response t o SO s t i m u l a t i o n was g r e a t e s t a t t h e soma, e x h i b i t i n g a p r o g r e s s i v e d e c r e a s e t h e g r e a t e r the d i s t a n c e o f t h e r e c o r d i n g s i t e from s t r a t u m p y r a m i d a l e . These r e s u l t s a r e not due t o a v a r i a b l e q u a l i t y o f c e l l u l a r impalement, as average v a l u e s o f r e s t i n g membrane p o t e n t i a l and i n p u t r e s i s t a n c e f e l l w i t h i n an a c c e p t a b l e range f o r a l l r e c o r d i n g l o c a t i o n s ( F i g 5.5C). A v a r i a b i l i t y o f d e n d r i t i c s p i k e t h r e s h o l d f o r two forms o f s y n a p t i c i n p u t would a g a i n suggest t h a t t h e a c t i o n p o t e n t i a l - 150 - FIG 5.5 P l o t s o f t h e average v o l t a g e t h r e s h o l d f o r s p i k e d i s c h a r g e evoked by s t i m u l a t i o n o f s t r a t u m o r i e n s o r s t r a t u m r a d i a t u m a t v a r y i n g d i s t a n c e s a l o n g the dendro-somatic a x i s , o f th e p y r a m i d a l c e l l . V o l t a g e t h r e s h o l d a t each l o c a t i o n was t a k e n as t h e average peak a m p l i t u d e o f t h e evoked 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 (measured from 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 are t a k e n as Oum d i s t a n c e . B. Schematic diagram o f t h e r a t p y r a m i d a l neuron drawn t o s c a l e below t h e p l o t s i n A. C. Average r e s t i n g membrane p o t e n t i a l (RMP) and i n p u t r e s i s t a n c e f o r t h e impalements shown i n A. The number of impalements (n) f o r s t r a t u m r a d i a t u m evoked EPSPs are shown a t the t o p o f A w h i l e o t h e r s are g i v e n a t t h e base o f each graph. Average v a l u e s w i t h o u t s t a n d a r d e r r o r b a r s are t h o s e i n which t h e s t a n d a r d e r r o r l i e s w i t h i n t h e boundary o f the i l l u s t r a t e d p o i n t . - 151 - A. Stim. Site •Stratum Radiatum oStr. Oriens n 34 2 5 4 10 12 22 6 4 I B. C. ° E 2 0 CD ^ TD TD a o CL to E E 10 CL CO CD 0 J § * 5 i 5 1 4 3 7 5 o O 2 I I St. Oriens ^ 68 n CL cr 60 J Pyr Radiatum I * * 5 19 2 4 4 3 8 13 2 4 0 CD CJ . CI Q- ~ £ ^ 20 or 23 2 2 5 2 v 3 4 10 0 100 200 300 Apical Dendritic Recording Site ( / J ) - 152 - r e c o r d e d w i t h i n an a p i c a l d e n d r i t e i s g e n e r a t e d at a s i t e d i s t a l t o t h e r e c o r d i n g e l e c t r o d e . In t h i s r e g a r d , i t i s worth n o t i n g t h a t t h e o n l y l o c a t i o n i n which s p i k e t h r e s h o l d f o r e i t h e r s y n a p t i c i n p u t was e q u i v a l e n t was i n t h e r e g i o n o f t h e p y r a m i d a l c e l l body ( F i g 5.5A). S t i m u l u s Evoked S p i k e L a t e n c y S i n c e d u a l impalements of p y r a m i d a l c e l l s were not o b t a i n e d i n t h e p r e s e n t s t u d y , s p i k e o n set l a t e n c y c o u l d not be measured a t d i f f e r e n t r e g i o n s o f a g i v e n p y r a m i d a l neuron. In a d d i t i o n , t h e l a t e n c y f o r s p i k e d i s c h a r g e c o u l d v a r y d r a m a t i c a l l y a c c o r d i n g t o t h e i n f l u e n c e o f such f a c t o r s as l a t e n c y j i t t e r o r s t i m u l u s i n t e n s i t y , p r e v e n t i n g a d i r e c t comparison between s e p a r a t e p y r a m i d a l c e l l impalements. T h e r e f o r e , i t was n e c e s s a r y t o e s t i m a t e t h e l a t e n c y f o r s p i k e d i s c h a r g e by comparing the i n t r a c e l l u l a r response t o t h e l a t e n c y o f evoked 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 at b o t h th e s o m a t i c and d e n d r i t i c l e v e l . P r e v i o u s s t u d i e s have shown t h a t i n t r a s o m a t i c s p i k e d i s c h a r g e o f C A l p y r a m i d a l c e l l s c o r r e l a t e s i n t ime t o t h e n e g a t i v e - g o i n g phase or peak of t h e 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 response i n s t r a t u m p y r a m i d a l e ( R i c h a r d s o n e t a l . 1984; T a y l o r and Dudek 1984; Turner e t a l . 1984). The l a t e n c y of t h e p o p u l a t i o n s p i k e can t h u s be used as an i n d i c a t i o n o f the l a t e n c y o f i n t r a s o m a t i c s p i k e g e n e r a t i o n , and as such, compared t o t h a t o f s p i k e d i s c h a r g e i n t h e a p i c a l d e n d r i t e . A second e s t i m a t e of t h e l a t e n c y 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 was o b t a i n e d by d e t e r m i n i n g the r e l a t i o n s h i p between the i n t r a d e n d r i t i c s p i k e and e x t r a c e l l u l a r waveform i n t h e immediate v i c i n i t y o f t h e d e n d r i t i c impalement (see 5.2 Methods). I n t h i s way, - 153 - i n t r a d e n d r i t i c AP d i s c h a r g e c o u l d be compared t o t h e l a t e n c y o f e x t r a d e n d r i t i c s p i k e waveforms r e c o r d e d on l a m i n a r p r o f i l e s o f 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 (Chapter 3 ) . The 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 and 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 evoked by s t i m u l a t i o n o f the a l v e u s , s t r a t u m o r i e n s or r a d i a t u m i s shown i n F i g 5.6 ( a l l r e c o r d s o b t a i n e d from a s i n g l e d e n d r i t i c impalement and hippocampal s l i c e ) . A comparison of i n t r a d e n d r i t i c s p i k e a c t i v a t i o n t o t h e f i e l d p o t e n t i a l response r e c o r d e d a t t h e c e l l l a y e r r e v e a l e d t h a t d e n d r i t i c s p i k e s were evoked at a l o n g e r peak l a t e n c y than t h e f a l l i n g edge or peak of t h e p o p u l a t i o n s p i k e i n s t r a t u m p y r a m i d a l e ( t o p two t r a c e s i n F i g 5.6). The d i f f e r e n c e i n l a t e n c y between t h e s e two responses was found t o i n c r e a s e d i r e c t l y w i t h t h e d i s t a n c e of t h e d e n d r i t i c r e c o r d i n g s i t e from th e b o r d e r o f s t r a t u m p y r a m i d a l e . A comparison o f t h e d e n d r i t i c s p i k e t o t h e e x t r a d e n d r i t i c f i e l d p o t e n t i a l s r e c o r d e d i m m e d i a t e l y f o l l o w i n g w i t h d r a w a l from th e impalement i s shown i n t h e l o w e s t s e t o f r e c o r d s o f F i g 5.6. In each c a s e , i n t r a d e n d r i t i c s p i k e s were found t o a l i g n w i t h t h e b i p h a s i c p o s i t i v e / n e g a t i v e e x t r a c e l l u l a r s p i k e p o t e n t i a l r e c o r d e d i n t h e d e n d r i t i c r e g i o n d u r i n g s u p r a t h r e s h o l d s t i m u l a t i o n o f t h e p y r a m i d a l c e l l p o p u l a t i o n (Chapter 3 ) . S p e c i f i c a l l y , t h e onset of t h e i n t r a d e n d r i t i c s p i k e c o r r e s p o n d e d t o t h a t o f t h e 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 , w h i l e t h e b r e a k p o i n t o f decay o f the s p i k e a l i g n e d w i t h t h e t e r m i n a t i o n o f t h e n e g a t i v e component of t h e d e n d r i t i c f i e l d p o t e n t i a l (denoted by d o t t e d l i n e s i n F i g 5.6). W h i l e th e p o s i t i v e / n e g a t i v e component o f the SR evoked e x t r a c e l l u l a r EPSP i s not as e v i d e n t i n t h i s p a r t i c u l a r example, t h e onset of 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 o f t h e l a t e n c y o f 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 t o 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 r e c o r d e d i n s t r a t u m p y r a m i d a l e ( p o p u l a t i o n s p i k e ) and s t r a t u m r a d i a t u m ( e x t r a d e n d r i t i c ) i m m e d i a t e l y f o l l o w i n g w i t h d r a w a l from t h e d e n d r i t i c impalement. P o t e n t i a l s were evoked by 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) or s t r a t u m r a d i a t u m ( C ) . S t i m u l u s i n t e n s i t i e s were s e t t o t h r e s h o l d f o r i n t r a d e n d r i t i c s p i k e a c t i v a t i o n ( s e v e r a l sweeps shown) and f i e l d p o t e n t i a l s averaged over 10 s u c c e s s i v e sweeps. D o t t e d l i n e s i n d i c a t e 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 s and e x t r a d e n d r i t i c f i e l d p o t e n t i a l s . I n t r a d e n d r i t i c r e c o r d i n g s were o b t a i n e d from a s i n g l e impalement 200um from the b o r d e r o f s t r a t u m p y r a m i d a l e . S t r a t u m R a d i a t u m 5 msec - 156 - EPSP can be seen t o c o r r e s p o n d t o t h e r i s i n g edge of t h e i n t r a d e n d r i t i c s p i k e a t 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 S p i k e F r a c t i o n a t i o n P r e v i o u s i n v e s t i g a t o r s have r e p o r t e d t h a t t h e 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 neurons c o u l d be s e p a r a t e d i n t o m u l t i p l e components, a response r e f e r r e d t o as a " f r a c t i o n a t i o n " o f t h e s p i k e waveform (Wong e t a l . 1979). T h i s r e s u l t was t a k e n as e v i d e n c e f o r s p i k e d i s c h a r g e a t r e g i o n a l "hot s p o t s " of d e n d r i t i c membrane e x h i b i t i n g a low t h r e s h o l d f o r s p i k e a c t i v a t i o n . I n t h e p r e s e n t s t u d y , a f r a c t i o n a t i o n o f the d e n d r i t i c s p i k e was o c c a s i o n a l l y o b s e r v e d i n 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 s 200-300um from th e b o r d e r of s t r a t u m p y r a m i d a l e . T h i s p a t t e r n o f s p i k e g e n e r a t i o n c o u l d be evoked by s t i m u l a t i o n o f t h e a l v e u s , s t r a t u m o r i e n s , or s t r a t u m r a d i a t u m , w i t h t h e f r a c t i o n a t e d s p i k e evoked at a l a t e n c y beyond th e peak of t h e 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 a t the c e l l body l a y e r ( F i g 5.7A-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 r e l a t e d t o t h e l a t e n c y t o s p i k e d i s c h a r g e , w i t h a g r e a t e r degree o f f r a c t i o n a t i o n o bserved f o r t h o s e s p i k e s evoked w e l l beyond th e peak of t h e p o p u l a t i o n s p i k e or on t h e f a l l i n g edge of t h e i n t r a d e n d r i t i c EPSP. The f r a c t i o n a t e d s p i k e appeared t o be comprised of two p r i n c i p l e a l l - o r - n o n e components. The f i r s t was a s m a l l p o t e n t i a l o f 9-25mV a m p l i t u d e t h a t gave r i s e t o a second, l a r g e r s p i k e w i t h an a m p l i t u d e t h a t c o u l d v a r y w i t h t h e i n t e n s i t y o f s t i m u l a t i o n ( F i g 5.7A-C). I f evoked at j u s t - t h r e s h o l d s t i m u l u s i n t e n s i t y , t h e second component was o f s m a l l a m p l i t u d e and c l e a r l y d i s t i n c t i n a r i s i n g from th e f a l l i n g edge of t h e f i r s t - 157 - FIG. 5.7 F r a c t i o n a t i o n o f i n t r a d e n d r i t i c s p i k e s r e c o r d e d 230pm from t h e bord e r o f s t r a t u m p y r a m i d a l e . A-C. S p i k e d i s c h a r g e evoked t h r o u g h 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) or s t r a t u m r a d i a t u m (C) i s compared t o t h e 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 response r e c o r d e d i n s t r a t u m p y r a m i d a l e . I n each c a s e , s e v e r a l sweeps are superimposed f o r i n c r e a s i n g i n t e n s i t i e s o f s t i m u l a t i o n . Note f r a c t i o n a t i o n o f s p i k e s i n t o two p r i n c i p l e a l l - o r - n o n e components, and t h e i n c r e a s e i n a m p l i t u d e o f the second component a t h i g h e r s t i m u l u s i n t e n s i t i e s . D,E. F r a c t i o n a t i o n of t h e d e n d r i t i c s p i k e i n response t o 10Hz r e p e t i t i v e s t i m u l a t i o n o f the a l v e u s (D) or s t r a t u m r a d i a t u m ( E ) . D u r i n g s t i m u l a t i o n , t h e f u l l a m p l i t u d e d e n d r i t i c s p i k e i n c r e a s e d i n peak l a t e n c y and d e c r e a s e d i n a m p l i t u d e t o e v e n t u a l l y f r a c t i o n a t e i n t o a two component waveform. 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 t h e s t i m u l u s t r a i n i s e v i d e n t . The a n t i d r o m i c response i n A was 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 impalement from t h a t o f B and C. Stim. Site Alveus Single Sweeps Stratum Oriens Stratum Radiatum B. C. 2 msec - 159 - p o t e n t i a l . As s t i m u l u s i n t e n s i t y was i n c r e a s e d , t h e second p o t e n t i a l c o u l d e x h i b i t t h e p e c u l i a r c h a r a c t e r i s t i c o f i n c r e a s i n g i n a m p l i t u d e , p r o g r e s s i v e l y a r i s i n g w i t h a s h o r t e r onset l a t e n c y u n t i l t he break between t h e two components c o u l d no l o n g e r be d i s c e r n e d ( b e s t i l l u s t r a t e d f o r the case o f a l v e a r and SO s t i m u l a t i o n i n F i g 5.7A,B). F r a c t i o n a t i o n o f t h e s p i k e c o u l d o f t e n o c c u r i n d i s t a l d e n d r i t i c impalements d u r i n g r e p e t i t i v e s t i m u l a t i o n o f e f f e r e n t or a f f e r e n t pathways ( F i g 5.7D,E). In e i t h e r c a s e , a d e n d r i t i c s p i k e evoked i n f u l l form on t h e f i r s t p u l s e would i n c r e a s e s l i g h t l y i n peak l a t e n c y d u r i n g t h e s t i m u l u s t r a i r i , w i t h the second component g r a d u a l l y d e c l i n i n g i n a m p l i t u d e t o r e v e a l an i n i t i a l s m a l l a l l - o r - n o n e p o t e n t i a l . As shown i n F i g 5.7D, t h e second l a r g e r p o t e n t i a l c o u l d be l o s t d u r i n g r e p e t i t i v e s t i m u l a t i o n , w h i l e t h e f i r s t component d e c l i n e d o n l y s l i g h t l y t o be evoked a t a c o m p a r a t i v e l y c o n s t a n t a m p l i t u d e t h r o u g h t h e m a j o r i t y o f t h e s t i m u l u s t r a i n . I n t h e s t u d y o f Wong e t a l . (1979), 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 a c h i e v e d t h r o u g h 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 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 . S i m i l a r r e s u l t s c o u l d be o b t a i n e d i n t h e p r e s e n t s t u d y , and a g a i n t h e f r a c t i o n a t i o n c o u l d be r e l a t e d t o t h e l a t e n c y o f t h e evoked d e n d r i t i c s p i k e . I n t h i s c a s e , the onset l a t e n c y f o r s p i k e d i s c h a r g e i n response t o SR s t i m u l a t i o n would i n c r e a s e w i t h membrane h y p e r p o l a r i z a t i o n a t b o t h t h e s o m a t i c and d e n d r i t i c l e v e l , 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 a f r a c t i o n a t i o n i n t o t h e s e p a r a t e components d e s c r i b e d above. - 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 Elements by K n i f e Cuts i n t h e C A l Region In o r d e r t o determine whether d e n d r i t i c s p i k e a c t i v a t i o n c o u l d o c c u r i n d e p e n d e n t l y o f t h a t a t t h e soma, attemp t s were 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 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 c e l l s by a k n i f e c u t a c c o r d i n g t o t h e t e c h n i q u e o f Benardo e t a l . (1982). B r i e f l y , t h i s was performed t h r o u g h use 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 r a z o r b l a d e c h i p g l u e d t o a wooden dowel mounted on a m i c r o - m a n i p u l a t o r . S l i c e t i s s u e was c u t w h i l e r e s t i n g upon t h e s u r f a c e o f t h e n y l o n net w i t h i n t h e r e c o r d i n g chamber by l o w e r i n g the b l a d e a c r o s s t h e p r o x i m a l - m i d s t r a t u m r a d i a t u m under d i r e c t m i c r o s c o p i c o b s e r v a t i o n (see Methods f o r d e t a i l s ) . I n i t i a l a t t e m p t s t o c u t s l i c e t i s s u e f o l l o w e d t h i s p r o c e d u r e , and SR evoked 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 were r e c o r d e d from w i t h i n and on e i t h e r s i d e o f t h e c u t t o a s s e s s t h e c h a r a c t e r i s t i c s o f evoked d e n d r i t i c a c t i v i t y . However, s e v e r a l d i f f i c u l t i e s were enc o u n t e r e d i n . t r y i n g t o a c h i e v e s e p a r a t i o n of t h e c e l l body r e g i o n w i t h o u t damaging r e m a i n i n g s l i c e t i s s u e . The m a j o r i t y of problems were r e l a t e d t o the f a c t t h a t the n y l o n net w i t h i n t h e r e c o r d i n g chamber d i d not p r o v i d e adequate s u p p o r t f o r o b t a i n i n g a complete c u t o f t h e s l i c e . For i n s t a n c e , t h e net c o n f i g u r a t i o n of t h e n y l o n f i b e r s s u p p o r t i n g the s l i c e would r e s u l t i n o n l y one or a few n y l o n s t r a n d s c r o s s i n g beneath t h e i n t e n d e d l i n e o f c u t i n t h e C A l r e g i o n , o f f e r i n g l i t t l e or no background s u p p o r t f o r which t o push th e b l a d e a g a i n s t . The p l i a b i l i t y o f t h e n y l o n s t r a n d s a l s o a l l o w e d t h e net s u r f a c e t o bend beneath t h e p r e s s u r e o f t h e b l a d e , r e q u i r i n g f u r t h e r f o r w a r d advance of t h e k n i f e w i t h t h e p o s s i b i l i t y o f c o m p r e s s i v e damage t o s u r r o u n d i n g - 161 - t i s s u e . I n f a c t , 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 d i s p l a y e d e v i d e n c e of damage t o the " i n t a c t " r e g i o n s o f a c u t s l i c e , i n c l u d i n g evoked p o t e n t i a l s o f low a m p l i t u d e or t h e r e p e t i t i v e d i s c h a r g e o f p o p u l a t i o n s p i k e responses at t h e c e l l body l a y e r . H i s t o l o g i c a l a n a l y s i s r e v e a l e d t h a t t h e o n l y means of o b t a i n i n g complete s e p a r a t i o n o f t h e c e l l body l a y e r u s i n g t h i s t e c h n i q u e was t o make s m a l l r e p e a t e d c u t s a l o n g the a x i s o f t h e b l a d e a t t h e bottom of t h e s l i c e . However, t h i s p r o c e d u r e o f t e n s e v e r e d t h e n y l o n net s u p p o r t i n g the s l i c e , r e s u l t i n g i n a sudden l o s s o f net t e n s i o n and e x t e n s i v e j a r r i n g o f s l i c e t i s s u e . The i n a b i l i t y t o s u c c e s s f u l l y s e c t i o n s l i c e s i n t h i s manner r e q u i r e d t h e development of another t e c h n i q u e f o r c u t t i n g the s l i c e p r i o r t o placement w i t h i n t h e r e c o r d i n g chamber. I n t h i s c a s e , t h e s l i c e was p l a c e d i n a s m a l l drop of oxygenated medium w i t h i n a p l a s t i c p e t r i d i s h and c u t by a s c a l p e l b l a d e under m i c r o s c o p i c o b s e r v a t i o n . T h i s t e c h n i q u e was found t o be f a r s u p e r i o r t o t h a t d e s c r i b e d above, a l l o w i n g complete and r e l i a b l e s e p a r a t i o n o f t h e c e l l 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 n e u r o n a l t i s s u e . A t h i n s e c t i o n (22um) from a hippocampal s l i c e c u t i n t h i s manner and s t a i n e d w i t h c r e s y l v i o l e t i s shown i n F i g 5.8B, d e m o n s t r a t i n g t h e e x c i s i o n of p y r a m i d a l c e l l b o d i e s from th e a p i c a l d e n d r i t i c r e g i o n . SR evoked p o t e n t i a l s r e c o r d e d a t t h e i n d i c a t e d l o c a t i o n s are shown i n F i g 5.8C. A r e c o r d i n g e l e c t r o d e p l a c e d i n s t r a t u m p y r a m i d a l e or r a d i a t u m i n t h e i n t a c t r e g i o n o f a c u t s l i c e r e v e a l e d t h a t SR evoked p o t e n t i a l s were comparable t o t h a t o f c o n t r o l hippocampal t i s s u e , w i t h maximal evoked EPSPs of 12mV and p o p u l a t i o n s p i k e s of up t o 27mV w i t h no e v i d e n c e of m u l t i p l e p o p u l a t i o n s p i k e d i s c h a r g e . However, t h e peak a m p l i t u d e o f t h e evoked EPSP and p o p u l a t i o n s p i k e p r o g r e s s i v e l y d e c l i n e d - 162 - FIG. 5.8 T h i n s e c t i o n s (22um) of c r e s y l v i o l e t s t a i n e d hippocampal t i s s u e from an i n t a c t s l i c e and a s l i c e w i t h a k n i f e c u t a c r o s s r e g i o s u p e r i o r f o r i s o l a t i o n o f t h e a p i c a l d e n d r i t e s from somata o f C A l p y r a m i d a l c e l l s . The major s u b f i e l d s o f t h e hippocampal f o r m a t i o n a re shown i n A, i n c l u d i n g t h e hippocampal r e g i o n s o f C A l and CA3, and the d e n t a t e gyrus (DG). The s t a i n e d somata o f C A l p y r a m i d a l c e l l s a r e r e p r e s e n t e d by t h e dark band o f t i s s u e i n s t r a t u m p y r a m i d a l e . The d i f f u s e and s c a t t e r e d s t a i n i n g o f c e l l s i n s t r a t u m r a d i a t u m can be a t t r i b u t e d i n l a r g e p a r t t o g l i a l e l e m e n t s . I n B, t h e removal o f p y r a m i d a l c e l l somata by t h e k n i f e c u t i s i n d i c a t e d by t h e l o s s o f s t r a t u m p y r a m i d a l e i n C A l . R e c o r d i n g e l e c t r o d e p o s i t i o n s a re marked i n s t r a t u m p y r a m i d a l e (dark t r i a n g l e s ) and s t r a t u m r a d i a t u m (open c i r c l e s ) . C. St r a t u m r a d i a t u m evoked p o t e n t i a l s r e c o r d e d a t the l o c a t i o n s i n d i c a t e d i n B from s t r a t u m p y r a m i d a l e and s t r a t u m r a d i a t u m , moving from l e f t t o r i g h t . The s t i m u l a t i n g e l e c t r o d e was p l a c e d i n s t r a t u m r a d i a t u m on t h e f i m b r i a l s i d e o f t h e r e c o r d i n g e l e c t r o d e and moved w i t h t h e r e c o r d i n g e l e c t r o d e t o m a i n t a i n a c o n s t a n t d i s t a n c e of a p p r o x i m a t e l y 400um. D. An enlargement o f the s t r a t u m r a d i a t u m evoked f i b e r p o t e n t i a l r e c o r d e d i n s t r a t u m r a d i a t u m a t t h e p o s i t i o n i n d i c a t e d by t h e a s t e r i s k i n B and C. A. -yv- -y - | f 5 mV • 5 msec i 5 msec I mV - 1 6 4 - as t h e r e c o r d i n g e l e c t r o d e was moved towards t h e edge of t h e c u t ( F i g 5.8C). A r a p i d d e c l i n e i n t h e a m p l i t u d e of t h e evoked EPSP i n s t r a t u m r a d i a t u m was t h e n o b s e r v e d w i t h i n t h e c u t r e g i o n over a p p r o x i m a t e l y 100-150pm from the b o r d e r of t h e i n c i s i o n . In almost a l l s l i c e s examined, th e maximal a m p l i t u d e o f "EPSPs" evoked beyond t h i s p o i n t w i t h s t i m u l u s i n t e n s i t i e s o f up t o 60V was i n t h e range o f 0.2-0.4mV. However, l a r g e f i b e r p o t e n t i a l s (up t o 4mV) c o u l d be evoked and r e c o r d e d i n s t r a t u m r a d i a t u m a l o n g the e n t i r e e x t e n t o f t h e c u t , d e m o n s t r a t i n g t h a t a f f e r e n t s y n a p t i c p r o j e c t i o n s were s t i l l i n t a c t ( F i g 5.8D). T h e r e f o r e , t h e l a c k o f 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 r a d i a t u m c o u l d not be 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 r e g i o n t h a t might have been s u s t a i n e d d u r i n g the c u t t i n g p r o c e d u r e . In some s l i c e s , a p o s t s y n a p t i c p o t e n t i a l c o u l d be evoked over a l i m i t e d d i s t a n c e w i t h i n t h e c u t r e g i o n of t h e s l i c e . However, subsequent h i s t o l o g i c a l a n a l y s i s r e v e a l e d t h e p r e s e n c e of s c a t t e r e d o r d i s p l a c e d p y r a m i d a l c e l l somata a t t h e edge of the i n c i s i o n d i r e c t l y above the p o s i t i o n o f the evoked a c t i v i t y . The evoked p o t e n t i a l s i n t h i s case c o u l d t h u s c o r r e s p o n d t o a c t i v i t y o f p y r a m i d a l c e l l d e n d r i t e s s t i l l a t t a c h e d t o t h e c e l l body r e g i o n . I t appeared t h e r e f o r e , t h a t the v a s t m a j o r i t y 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 had not s u r v i v e d s e p a r a t i o n from the c e l l body r e g i o n , p r e v e n t i n g an a n a l y s i s o f s p i k e a c t i v a t i o n i n an i s o l a t e d d e n d r i t e . - 165 - 5-4. D i s c u s s i o n P r e v i o u s s t u d i e s on t h e evoked a c t i v i t y o f the p y r a m i d a l c e l l have r e p o r t e d e v i d e n c e f o r the i n i t i a l a c t i v a t i o n of a s p i k e i n a p i c a l d e n d r i t i c membrane, w i t h a subsequent e l e c t r o t o n i c c o n d u c t i o n o f t h e d e n d r i t i c s p i k e t o t h e s o m a t i c r e g i o n (Andersen e t a l . 1960; Andersen and Lomo 1966; Cragg and Hamlyn 1955; S c h w a r t z k r o i n 1977; Spencer and Kandel 1961b). One would thus e x p e c t t o f i n d e v i d e n c e i n the c o n f i g u r a t i o n o f 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 and decay towards the c e l l body r e g i o n . I n f a c t , a c o m p a r a t i v e i n t r a c e l l u l a r a n a l y s i s o f s p i k e c h a r a c t e r i s t i c s a l o n g t h e de n d r o - s o m a t i c a x i s r e v e a l e d a p r o g r e s s i v e change i n s e v e r a l parameters o f s p i k e d i s c h a r g e , i n c l u d i n g s p i k e a m p l i t u d e , h a l f w i d t h , v o l t a g e 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 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 A l o n g the Dendro-Somatic A x i s One consequence of an e l e c t r o t o n i c c o n d u c t i o n of a s p i k e from t h e d e n d r i t i c r e g i o n would be 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 h a l f w i d t h w i t h p r o x i m i t y t o the c e l l body l a y e r . However, s p i k e s evoked from each o f t h e s t i m u l u s pathways or t h r o u g h c u r r e n t i n j e c t i o n d i s p l a y e d 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 h a l f w i d t h w i t h d i s t a n c e from s t r a t u m p y r a m i d a l e , o p p o s i t e t o t h a t e x p e c t e d f o r a s o m a t o p e t a l c o n d u c t i o n o f the d e n d r i t i c s p i k e . The i n i t i a l a c t i v a t i o n o f a s p i k e from a g e n e r a t o r zone (hot spot) i n d e n d r i t i c membrane would a l s o p r e d i c t a low v o l t a g e t h r e s h o l d f o r s p i k e a c t i v a t i o n a t one or more p o i n t s a l o n g the dendro-somatic a x i s . An a n a l y s i s of s y n a p t i c a l l y - 166 - evoked s p i k e t h r e s h o l d d i d r e v e a l a g r a d u a l change i n t h e v o l t a g e t h r e s h o l d f o r s p i k e d i s c h a r g e a l o n g the 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 . However, the p r o g r e s s i o n o f change i n s p i k e t h r e s h o l d was d i s t i n c t l y d i f f e r e n t f o r two forms o f evoked s y n a p t i c d e p o l a r i z a t i o n o f the p y r a m i d a l c e l l . V o l t a g e t h r e s h o l d f o r s p i k e d i s c h a r g e i n response t o SR s t i m u l a t i o n was g r e a t e s t i n t h e m i d - d i s t a l a p i c a l d e n d r i t e , d e c l i n i n g towards t h e p y r a m i d a l c e l l l a y e r . In 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 by 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 t h r e s h o l d i n t h e c e l l body, w i t h v o l t a g e t h r e s h o l d a p p a r e n t l y d e c r e a s i n g w i t h d i s t a n c e a l o n g t h e a p i c a l d e n d r i t e . A s t r i c t i n t e r p r e t a t i o n of t h e s e r e s u l t s would i m p l y t h a t (1) an a p i c a l d e n d r i t i c s p i k e i s most e f f e c t i v e l y evoked by an EPSP of b a s a l d e n d r i t i c o r i g i n , and (2) t h a t a s p i k e a t any g i v e n l o c a t i o n a l o n g t h e a p i c a l d e n d r i t e can be evoked a t two e n t i r e l y d i f f e r e n t v o l t a g e t h r e s h o l d s ( r e c a l l t h a t complementary t h r e s h o l d measurements a r e t a k e n from t h e same d e n d r i t i c impalement; c f 5.1). N e i t h e r c o n c l u s i o n would appear t o conform t o the u s u a l u n d e r s t a n d i n g of v o l t a g e - d e p e n d e n t a c t i v a t i o n o f s p i k e d i s c h a r g e i n n e u r o n a l membrane. An a l t e r n a t i v e e x p l a n a t i o n f o r t h e v a r i a b i l i t y i n s p i k e t h r e s h o l d i s t h a t a d e n d r i t i c s p i k e i s g e n e r a t e d a t a s i t e 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 . I n f a c t , t h e o n l y l o c a t i o n i n t h e p y r a m i d a l c e l l i n which s p i k e d i s c h a r g e was evoked from 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 was i n t h e r e g i o n o f the c e l l body l a y e r . The above r e s u l t s might t h e n be e x p l a i n e d by the i n i t i a l a c t i v a t i o n o f a s p i k e i n t h e s o m a t i c r e g i o n , and a subsequent r e t r o g r a d e s p i k e i n v a s i o n of d e n d r i t i c membrane. An a c t i o n p o t e n t i a l c o n d u c t i n g back t h r o u g h t h e d e n d r i t i c t r e e would t h e n superimpose upon t h e c o i n c i d e n t l e v e l o f - 167 - d e p o l a r i z a t i o n w i t h i n t h e a p i c a l d e n d r i t e , g i v i n g r i s e t o an apparent l a c k o f v o l t a g e t h r e s h o l d i n d e n d r i t i c l o c a t i o n s . The r e s u l t s o b t a i n e d i n an a n a l y s i s o f t h e l a t e n c y f o r s p i k e d i s c h a r g e a l s o c o n t r a d i c t t h e concept of i n i t i a l s p i k e a c t i v a t i o n i n d e n d r i t i c l o c a t i o n s o f the p y r a m i d a l c e l l . A l t h o u g h s t r i c t measures of s p i k e l a t e n c y were not o b t a i n e d , a comparison of the i n t r a d e n d r i t i c s p i k e t o 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 p r o v i d e s an i n d i r e c t e s t i m a t e of t h e l a t e n c y f o r s p i k e d i s c h a r g e a t d i f f e r e n t l e v e l s o f the d e n d r o - s o m a t i c a x i s . Should d e n d r i t i c s p i k e a c t i v a t i o n precede t h a t a t t h e s o m a t i c l e v e l , one would e x p e c t th e d e n d r i t i c s p i k e t o be evoked at a s h o r t e r l a t e n c y t h a n th e p o p u l a t i o n s p i k e i n s t r a t u m p y r a m i d a l e , a p o t e n t i a l r e f l e c t i n g the average l a t e n c y f o r s o m a t i c s p i k e d i s c h a r g e . However, i n t r a d e n d r i t i c s p i k e s were evoked beyond the peak o f t h e p o p u l a t i o n s p i k e a t t h e c e l l l a y e r , s u g g e s t i n g t h a t s p i k e s r e c o r d e d i n d e n d r i t i c l o c a t i o n s were evoked subsequent t o t h a t i n t h e s o m a t i c r e g i o n . F u r t h e r m o r e , i n t r a d e n d r i t i c s p i k e s a l i g n e d w i t h t h e e x t r a d e n d r i t i c 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 r e c o r d e d i n s t r a t u m r a d i a t u m 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 o f t h e p y r a m i d a l c e l l p o p u l a t i o n . As d e s c r i b e d i n Chapter 3, t h i s p o t e n t i a l was found t h r o u g h c u r r e n t - s o u r c e d e n s i t y a n a l y s i s t o conduct w i t h i n c r e a s i n g l a t e n c y t h r o u g h th e d e n d r i t i c r e g i o n w i t h d i s t a n c e from th e c e l l body l a y e r . T h e r e f o r e , t h e c h a r a c t e r i s t i c s o f evoked d e n d r i t i c s p i k e a m p l i t u d e , h a l f w i d t h , and onset l a t e n c y a l o n g t h e d e n d ro-somatic a x i s o f t h e p y r a m i d a l c e l l a re d i r e c t l y opposed t o the e x p e c t e d r e s u l t f o r t h e i n i t i a l a c t i v a t i o n o f a s p i k e from w i t h i n t h e d e n d r i t i c a r b o r i z a t i o n . In a d d i t i o n , an a n a l y s i s o f the v o l t a g e t h r e s h o l d f o r s p i k e a c t i v a t i o n i n t h e d e n d r i t e d i d not r e v e a l - 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 a low v o l t a g e t h r e s h o l d f o r s p i k e d i s c h a r g e (hot s p o t s ) . I n f a c t , t h e o n l y 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 f o r s p i k e d i s c h a r g e i n response t o e i t h e r s y n a p t i c or c u r r e n t evoked d e p o l a r i z a t i o n s was found i n t h e r e g i o n o f t h e c e l l body l a y e r . A l t h o u g h d e n d r i t i c "hot s p o t s " may have been i s o l a t e d or remote t o t h e d e n d r i t i c impalements o b t a i n e d , a l l d a t a a re c o n s i s t e n t w i t h t h e i n i t i a l a c t i v a t i o n o f a s p i k e i n t h e s o m a t i c r e g i o n of the p y r a m i d a l c e l l , and a subsequent r e t r o g r a d e c o n d u c t i o n of t h e s p i k e t h r o u g h t h e d e n d r i t i c a r b o r i z a t i o n . Some of t h e s t r o n g e s t e v i d e n c e f o r t h i s i n t e r p r e t a t i o n comes from the c l o s e c o r r e l a t i o n o f i n t r a c e l l u l a r s p i k e d i s c h a r g e t o t h e s p i k e components o f 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 t a l l l e v e l s of t h e dendro-somatic a x i s . 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 t h r e e pathways c o r r e s p o n d e d t o the f i e l d p o t e n t i a l waveform shown t h r o u g h c u r r e n t - s o u r c e d e n s i t y a n a l y s i s t o conduct from the c e l l body l a y e r . The c o r r e l a t i o n between i n t r a - and e x t r a c e l l u l a r s p i k e p o t e n t i a l s i s f u r t h e r noted i n t h e p a t t e r n o f t r a n s i t i o n i n s p i k e c o n f i g u r a t i o n a l o n g t h e den d r o - s o m a t i c a x i s . At a p p r o x i m a t e l y 100pm from s t r a t u m p y r a m i d a l e , t h e e x t r a c e l l u l a r s p i k e component changed from a n e g a t i v e - g o i n g p o p u l a t i o n s p i k e t o a p o s i t i v e / n e g a t i v e d e n d r i t i c p o t e n t i a l (Chapter 3 ) . T h i s l o c a t i o n c o r r e s p o n d s t o t h e approximate p o i n t a t which i n t r a d e n d r i t i c s p i k e s e x h i b i t 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 h a l f w i d t h w i t h r e s p e c t t o s p i k e s r e c o r d e d i n t h e so m a t i c r e g i o n o f t h e p y r a m i d a l c e l l . S i m i l a r l y , t h e l o s s o f t h e n e g a t i v e component o f t h e e x t r a c e l l u l a r d e n d r i t i c s p i k e i n d i s t a l s t r a t u m r a d i a t u m c o r r e l a t e s t o t h e second r a p i d d e c l i n e - 169 - i n a m p l i t u d e and i n c r e a s e i n h a l f w i d t h o f t h e i n t r a d e n d r i t i c s p i k e . A n a t o m i c a l measurements o f the r a t p y r a m i d a l c e l l i n d i c a t e t h a t t h e s e p o i n t s o f t r a n s i t i o n c o r r e s p o n d t o t h e approximate l o c a t i o n o f major b r a n c h p o i n t s o f t h e a p i c a l d e n d r i t i c s t r u c t u r e (Chapter 3 ) . The change i n s p i k e c o n f i g u r a t i o n i n p r o x i m a l s t r a t u m r a d i a t u m may th e n c o r r e s p o n d t o a c o n d u c t i o n o f t h e s p i k e p a s t t h e p o i n t o f b i f u r c a t i o n o f the p r o x i m a l d e n d r i t i c s h a f t . S i m i l a r l y , t h e change i n s p i k e c h a r a c t e r i s t i c s i n d i s t a l s t r a t u m r a d i a t u m may i n d i c a t e s p i k e c o n d u c t i o n p a s t secondary d e n d r i t i c b r a n c h p o i n t s i n d i s t a l d e n d r i t i c 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 waveform i s i n f a c t t h e e x p e c t e d r e s u l t f o r the case o f a s p i k e c o n d u c t i n g p a s t t h e b r a n c h p o i n t o f a n e u r o n a l s t r u c t u r e ( G o l d s t e i n and R a i l 1974). However, r e g a r d l e s s o f t h e mechanism u n d e r l y i n g the change i n s p i k e c h a r a c t e r i s t i c s a l o n g the d e n d r o - s o m a t i c a x i s , t h e c l o s e c o r r e l a t i o n between i n t r a - and e x t r a c e l l u l a r s p i k e p r o p e r t i e s i n d i c a t e t h a t r e s u l t s o b t a i n e d from f i e l d p o t e n t i a l p r o f i l e s a r e r e p r e s e n t a t i v e o f the a c t i v i t y i n i n d i v i d u a l p y r a m i d a l c e l l e l e m e n t s . I n t r a c e l l u l a r d a t a would t h u s s u p p o r t t h e c o n c l u s i o n s d e r i v e d form c u r r e n t - s o u r c e d e n s i t y a n a l y s i s , i n d i c a t i n g a r e t r o g r a d e s p i k e c o n d u c t i o n t h r o u g h t h e a p i c a l d e n d r i t i c r e g i o n (Chapter 3 ) . D e n d r i t i c S p i k e F r a c t i o n a t i o n Beyond a p p r o x i m a t e l y 200jum from t h e b o r d e r o f s t r a t u m p y r a m i d a l e ( a p p r o x i m a t e l y h a l f the d i s t a n c e t o t h e hippocampal f i s s u r e ) , d e n d r i t i c s p i k e s c o u l d e x h i b i t a " f r a c t i o n a t i o n " i n waveform. S p i k e f r a c t i o n a t i o n has p r e v i o u s l y been t a k e n as e v i d e n c e f o r m u l t i p l e s i t e s o f s p i k e g e n e r a t i o n i n d e n d r i t i c - 170 - membrane (Wong et a l . 1979). In t h e p r e s e n t s t u d y , s p i k e f r a c t i o n a t i o n was o b s e r v e d f o r d e n d r i t i c s p i k e s evoked t h r o u g h b o t h a n t i d r o m i c or o r t h o d r o m i c s t i m u l a t i o n , w i t h a l l components of t h e f r a c t i o n a t e d s p i k e evoked beyond the peak of t h e p o p u l a t i o n s p i k e r e c o r d e d i n s t r a t u m p y r a m i d a l e . Thus, i t would appear t h a t f r a c t i o n a t e d d e n d r i t i c s p i k e s had been evoked subsequent t o AP d i s c h a r g e a t t h e c e l l body l a y e r . These r e s u l t s a l s o i n d i c a t e t h a t an a n t i d r o m i c s p i k e c o n d u c t i n g from the s o m a t i c r e g i o n can e x h i b i t a f r a c t i o n a t i o n o f waveform s i m i l a r t o t h a t o b s e r v e d f o r o r t h o d r o m i c s p i k e d i s c h a r g e . Thus, t h e f r a c t i o n a t i o n o f an o r t h o d r o m i c s p i k e does not n e c e s s a r i l y i n d i c a t e t h e p r e s e n c e of m u l t i p l e s i t e s f o r s p i k e a c t i v a t i o n i n t h e 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 cannot r u l e out t h e p o s s i b l e p r e s ence o f d e n d r i t i c hot s p o t s , as r e t r o g r a d e s p i k e a c t i v a t i o n o f a low t h r e s h o l d d e n d r i t i c r e g i o n might be " r e f l e c t e d " i n n e i g h b o r i n g d e n d r i t i c branches as a f r a c t i o n a t e d s p i k e (Parnas 1979). C l o s e e x a m i n a t i o n of t h i s phenomenon r e v e a l e d t h a t a f r a c t i o n a t e d s p i k e c o u l d be s e p a r a t e d i n t o e s s e n t i a l l y two components - a s m a l l a l l - o r - n o n e p o t e n t i a l of r e l a t i v e l y c o n s t a n t a m p l i t u d e , and a second s p i k e o f v a r i a b l e a m p l i t u d e . The b a s i c form of t h e " f r a c t i o n a t e d " s p i k e t h u s resembles t h e case of a f a s t p r e - p o t e n t i a l u n d e r l y i n g the a c t i v a t i o n o f a second l a r g e r s p i k e ( c f 4.9 and 4.10). In f a c t , f r a c t i o n a t e d s p i k e s d i s p l a y e d s i m i l a r evoked c h a r a c t e r i s t i c s t o t h a t o f FPP d i s c h a r g e , b e i n g uncovered 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 or 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 the s p i k e was a l s o s t r o n g l y c o r r e l a t e d t o t h e l a t e n c y of s p i k e a c t i v a t i o n , a parameter of AP d i s c h a r g e d i r e c t l y r e l a t e d t o t h e r e c o r d i n g - 171 - d i s t a n c e from s t r a t u m p y r a m i d a l e . A g r e a t e r f r a c t i o n a t i o n o f t h e s p i k e was found at c o m p a r a t i v e l y l o n g l a t e n c i e s t o o n s e t , w i t h more s e p a r a t i o n o f s p i k e components when evoked upon the f a l l i n g edge of t h e EPSP. I t i s i m p o r t a n t t o note t h a t s p i k e s evoked at t h i s l a t e n c y f a l l c l o s e t o t h e approximate time f o r a c t i v a t i o n o f t h e i n h i b i t o r y p o s t s y n a p t i c conductance ob s e r v e d at t h e c e l l body r e g i o n ( D i n g l e d i n e and Langmoen 1980) or perhaps i n d e n d r i t i c membrane ( A l g e r and N i c o l l 1984). F r a c t i o n a t i o n o f t h e evoked s p i k e may t h u s c o r r e s p o n d t o an i n h i b i t i o n of s p i k e a m p l i t u d e as a r e t r o g r a d e l y conducted d e n d r i t i c s p i k e s h i f t s i n peak l a t e n c y t o w i t h i n t h e time of i n h i b i t o r y c u r r e n t movements a l o n g t h e d e n d ro-somatic a x i s . Such a mechanism c o u l d e x p l a i n t h e c o r r e l a t i o n between s p i k e f r a c t i o n a t i o n and r e c o r d i n g d i s t a n c e from th e c e l l l a y e r , as w e l l as t h e i n t e n s i t y dependence o f the a m p l i t u d e o f a 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 s p i k e f r a c t i o n a t i o n , and t o a s s e s s t h e s i m i l a r i t y o f t h i s form of s p i k e a c t i v a t i o n t o FPP d i s c h a r g e . 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 In o r d e r t o d e t e r m i n e whether an a c t i o n p o t e n t i a l c o u l d be evoked i n d e n d r i t i c membrane i n d e p e n d e n t l y o f t h a t a t the c e l l body, a t t e m p t s were made t o i s o l a t e a p i c a l d e n d r i t e s from the c e l l body l a y e r by p l a c i n g a k n i f e c u t i n t h e C A l r e g i o n . To a c c o m p l i s h t h i s , a t e c h n i q u e was developed by which t h e c u t would ensure a complete and r e l i a b l e s e p a r a t i o n o f p y r a m i d a l c e l l somata and a p i c a l d e n d r i t e s , as c o n f i r m e d t h r o u g h subsequent t h i n s e c t i o n i n g and h i s t o l o g i c a l a n a l y s i s o f s l i c e t i s s u e . An a n a l y s i s o f evoked a c t i v i t y i n c u t s l i c e s r e v e a l e d - 172 - m i n i m a l damage t o p y r a m i d a l c e l l s t r u c t u r e s o u t s i d e t h e r e g i o n of t h e c u t or t o a f f e r e n t f i b e r s p r o j e c t i n g t h r o u g h t h e d e n d r i t i c f i e l d under t h e c u t . In c o n t r a s t , l i t t l e or no p o s t s y n a p t i c a c t i v i t y c o u l d be r e c o r d e d from i s o l a t e d d e n d r i t i c elements beyond a p p r o x i m a t e l y 150um from t h e b o r d e r o f t h e k n i f e c u t . A n a t o m i c a l measurements would i n d i c a t e t h a t t h i s d i s t a n c e i s w i t h i n t h e range of l a t e r a l b r a n c h i n g o f t h e 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 (Chapter 3 ) . Evoked a c t i v i t y a t t h e edge of a c u t c o u l d t h e n c o r r e s p o n d t o d e n d r i t i c s t r u c t u r e s a t t a c h e d t o t h e c e l l b o d i e s o f p y r a m i d a l c e l l s i n t h e i n t a c t p o r t i o n of s t r a t u m p y r a m i d a l e . In f a c t , i n t h e few s l i c e s d e m o n s t r a t i n g evoked a c t i v i t y w i t h i n t h e r e g i o n of t h e c u t , h i s t o l o g i c a l a n a l y s i s r e v e a l e d the presence o f p y r a m i d a l c e l l somata a t t h e edge of t h e c u t above t h e l o c a t i o n o f t h e evoked p o t e n t i a l . The r e s u l t s would thus i n d i c a t e t h a t most i f not a l l i s o l a t e d a p i c a l d e n d r i t e s had not s u r v i v e d s e p a r a t i o n from t h e c e l l 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 have r e p o r t e d o b t a i n i n g an i s o l a t i o n of a p i c a l d e n d r i t e s and somata of p y r a m i d a l c e l l s , and have r e c o r d 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 from w i t h i n t h e " i s o l a t e d " d e n d r i t e (Benardo e t a l . 1982; Masukawa and P r i n c e 1984). However, t h e t e c h n i q u e used i n t h e s e s t u d i e s f o r c u t t i n g the s l i c e c o u l d not be s u c c e s s f u l l y employed i n t h e p r e s e n t work, and pr o v e d t o be inadequate or a t l e a s t u n r e l i a b l e i n o b t a i n i n g complete s e p a r a t i o n of d e n d r i t i c s t r u c t u r e s from t h e c e l l body r e g i o n . I t may t h u s be p o s s i b l e t h a t d e n d r i t i c r e c o r d i n g s i n t h e s e s t u d i e s were o b t a i n e d from " i n t a c t " d e n d r i t e s s t i l l a t t a c h e d t o somata not 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 d i f f i c u l t i e s a s s o c i a t e d w i t h t h i s t e c h n i q u e , t h e d e m o n s t r a t i o n of evoked a c t i v i t y from w i t h i n an i s o l a t e d d e n d r i t e would - 173 - r e q u i r e a d d i t i o n a l a n a t o m i c a l c o n f i r m a t i o n o f c e l l body l o s s t h r o u g h i n j e c t i o n o f a s u i t a b l e c e l l s t a i n . T h e r e f o r e , u s i n g the t e c h n i q u e s p r e s e n t l y a v a i l a b l e , i t would appear t h a t p y r a m i d a l c e l l d e n d r i t e s cannot s u r v i v e s e p a r a t i o n from th e c e l l body r e g i o n , and t h e p o s s i b i l i t y o f d e n d r i t i c s p i k e a c t i v a t i o n independent o f t h a t i n t h e c e l l body has not y e t been a d e q u a t e l y t e s t e d . The p r e s e n t s t u d y has f u r t h e r c h a r a c t e r i z e d t h e p r o p e r t i e s o f 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 s o m a t i c and d e n d r i t i c membranes of the C A l p y r a m i d a l c e l l . A d e t a i l e d a n a l y s i s o f s p i k e a c t i v i t y has demonstrated a g r a d u a l change i n the p r o p e r t i e s of s p i k e a m p l i t u d e , h a l f w i d t h , v o l t a g e t h r e s h o l d and onset l a t e n c y a l o n g the p y r a m i d a l c e l l d e n d r o -somatic a x i s . However, the d i r e c t i o n o f change i n t h e s e c h a r a c t e r i s t i c s i s d i r e c t l y opposed t o t h e r e s u l t s p r e d i c t e d by a model o f s p i k e g e n e r a t i o n w i t h i n the d e n d r i t i c a r b o r i z a t i o n . The i n t r a c e l l u l a r c h a r a c t e r i s t i c s o f s p i k e d i s c h a r g e t h u s c o i n c i d e w i t h t h e r e s u l t s o b t a i n e d t h r o u g h c u r r e n t - s o u r c e d e n s i t y a n a l y s i s (Chapter 3 ) , i n d i c a t i n g t h a t t h e i n i t i a l s i t e f o r Na+ s p i k e g e n e r a t i o n i n t h e p y r a m i d a l neuron i s i n t h e r e g i o n o f t h e soma-axon h i l l o c k . 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 d e n d r i t i c membrane the n f o l l o w s t h a t i n t h e soma as t h e r e s u l t o f a r e t r o g r a d e s p i k e i n v a s i o n o f t h e d e n d r i t i c a r b o r i z a t i o n . - 174 - 6-0. GENERAL SUMMARY AND DISCUSSION The S i t e o f O r i g i n o f P y r a m i d a l C e l l D e n d r i t i c S p i k e s The p r e s e n t s t u d y has s e r v e d t o 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 d e n d r i t i c s p i k e s i n C A l p y r a m i d a l neurons o f mammalian hippocampus. A comprehensive a n a l y s i s o f f i e l d p o t e n t i a l s , c u r r e n t - s o u r c e d e n s i t y , and i n t r a c e l l u l a r a c t i v i t y i n d i c a t e t h a t t h e i n i t i a l s i t e o f s p i k e d i s c h a r g e i n t h e p y r a m i d a l c e l l f o r bo t h a n t i d r o m i c or o r t h o d r o m i c s t i m u l a t i o n i s i n t h e r e g i o n o f the soma-axon h i l l o c k . Evoked s p i k e s i n d e n d r i t i c l o c a t i o n s then a r i s e t h r o u g h a r e t r o g r a d e c o n d u c t i o n o f the s p i k e from t h e c e l l l a y e r t h r o u g h t h e d e n d r i t i c a r b o r i z a t i o n . F u r t h e r m o r e , t h e s i m i l a r i t y i n t h e c h a r a c t e r i s t i c s o f Na+ s p i k e s evoked by d e p o l a r i z i n g c u r r e n t t o t h o s e evoked by a n t i - o r o r t h o d r o m i c 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 s i t e o f o r i g i n f o r a l l evoked Na+ s p i k e s r e c o r d e d i n the p y r a m i d a l c e l l d e n d r i t e . These r e s u l t s a re d i r e c t l y opposed t o the e x i s t i n g h y p o t h e s i s r e g a r d i n g d e n d r i t i c s p i k e a c t i v a t i o n i n t h e p y r a m i d a l c e l l . A c c o r d i n g t o t h i s model, d e n d r i t i c s p i k e s a re evoked from w i t h i n t h e d e n d r i t i c t r e e a t r e g i o n a l "hot s p o t s " of membrane e x h i b i t i n g a low t h r e s h o l d f o r s p i k e d i s c h a r g e (Andersen and Lomo 1966; Spencer and Kandel 1961b; Traub and L l i n a s 1979; Wong et a l . 1979). 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 can g i v e r i s e t o t h e i n i t i a l a c t i v a t i o n o f a s p i k e a t the d e n d r i t i c l e v e l t h a t e l e c t r o t o n i c a l l y c o n ducts t o t h e c e l l body t o appear i n t h e soma as a f a s t p r e - p o t e n t i a l (Andersen and Lomo 1966; S c h w a r t z k r o i n 1977; Wong e t a l . 1979). A s p i k e evoked w i t h i n t h e d e n d r i t e can thus 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 the p r o b a b i l i t y f o r AP d i s c h a r g e a t t h e axon h i l l o c k . S e v e r a l r e s u l t s o f t h e - 1 7 5 - p r e s e n t s t u d y argue a g a i n s t the v a l i d i t y o f .the above h y p o t h e s i s . For i n s t a n c e , c u r r e n t - s o u r c e d e n s i t y a n a l y s i s r e v e a l e d 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 evoked i n t h e soma-axon h i l l o c k r e g i o n o f p y r a m i d a l c e l l s , and not i n d e n d r i t i c l o c a t i o n s . I n a d d i t i o n , t h e observed change i n t h e c o n f i g u r a t i o n of the i n t r a c e l l u l a r s p i k e a l o n g the d e n d r i t i c a x i s was o p p o s i t e t o t h a t p r e d i c t e d f o r the e l e c t r o t o n i c c o n d u c t i o n o f a s p i k e from the d e n d r i t e t o t h e c e l l body. F i n a l l y , i n t r a d e n d r i t i c s p i k e s were evoked f o l l o w i n g the peak of the p o p u l a t i o n s p i k e r e c o r d e d i n s t r a t u m p y r a m i d a l e , and a l i g n e d w i t h t h e e x t r a d e n d r i t i c f i e l d p o t e n t i a l shown t h r o u g h CSD a n a l y s i s t o conduct from th e c e l l body l a y e r . T h e r e f o r e , t h e e v i d e n c e does not s u p p o r t th e c o n t e n t i o n o f a d e n d r i t i c s i t e of o r i g i n f o r s p i k e s r e c o r d e d a t t h e d e n d r i t i c l e v e l o f the p y r a m i d a l c e l l . R a t h e r , the r e s u l t s i n d i c a t e t h a t evoked d e n d r i t i c s p i k e s 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 o f t h e d e n d r i t e by a s p i k e i n i t i a t e d i n the r e g i o n o f t h e soma-axon h i l l o c k . I m p l i c a t i o n s o f t h e P r e s e n t Study The f i n d i n g s p r e s e n t e d i n t h i s work q u e s t i o n our c u r r e n t u n d e r s t a n d i n g o f p y r a m i d a l c e l l p h y s i o l o g y . For i n s t a n c e , t h e f a c t t h a t t h e d e n d r i t i c s p i k e i s evoked f o l l o w i n g t h a t a t t h e c e l l body i m p l i e s i m m e d i a t e l y t h a t d e n d r i t i c s p i k e a c t i v a t i o n does not c o n t r i b u t e d i r e c t l y t o the g e n e r a t i o n o f a s p i k e i n t h e axon h i l l o c k . The r e t r o g r a d e c o n d u c t i o n o f a s p i k e from the c e l l 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 r e f l e c t t h e e l e c t r o t o n i c decay o f a s p i k e from th e d e n d r i t e towards t h e p y r a m i d a l c e l l body. I n f a c t , FPPs of s i m i l a r a m p l i t u d e and - 176 - waveform were r e c o r d e d at b o t h th e s o m a t i c and d e n d r i t i c l e v e l , and t h e change i n c o n f i g u r a t i o n of t h e s p i k e a l o n g th e d e n d r i t e was o p p o s i t e t o t h a t p r e d i c t e d f o r a s o m a t o p e t a l decay of an a c t i o n p o t e n t i a l a r i s i n g from w i t h i n t h e d e n d r i t i c t r e e . A l t h o u g h the mechanism u n d e r l y i n g FPP d i s c h a r g e was not d e t e r m i n e d , a l t e r n a t i v e e x p l a n a t i o n s have been proposed i n p r e v i o u s s t u d i e s , i n c l u d i n g the e l e c t r o t o n i c c o n d u c t i o n of a s p i k e a c r o s s a gap j u n c t i o n l o c a t e d a t e i t h e r t h e s o m a t i c or d e n d r i t i c l e v e l (Andrew e t a l . 1982; M a c V i c a r and Dudek 1981). The r e s u l t s a l s o p r e s s f o r a c r i t i c a l e v a l u a t i o n of the e v i d e n c e f o r low t h r e s h o l d hot s p o t s f o r Na+ s p i k e a c t i v a t i o n i n d e n d r i t i c membrane of t h e p y r a m i d a l c e l l . D e n d r i t i c hot s p o t s were o r i g i n a l l y proposed t o account f o r t h e apparent d e n d r i t i c s i t e o f o r i g i n o f s p i k e s i n t h e p y r a m i d a l c e l l , and the i n t e r p r e t a t i o n t h a t s o m a t i c f a s t p r e - p o t e n t i a l s 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 decayed d e n d r i t i c s p i k e (Andersen and Lomo 1966; Spencer and K andel 1961b). A 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 t a k e n as f u r t h e r e v i d e n c e f o r m u l t i p l e s i t e s o f s p i k e g e n e r a t i o n i n d e n d r i t i c r e g i o n s (Wong e t a l . 1979). However, as d i s c u s s e d above, s p i k e s were found t o o r i g i n a t e a t t h e s o m a t i c l e v e l o f the p y r a m i d a l c e l l , and the r e t r o g r a d e c o n d u c t i o n and c o n f i g u r a t i o n o f t h e s p i k e a l o n g the d e n d r o -somatic a x i s suggest t h a t t h e s o m a t i c FPP does not r e p r e s e n t a d e n d r i t i c s p i k e . In a d d i t i o n , f r a c t i o n a t e d s p i k e s were evoked f o l l o w i n g the . peak of the p o p u l a t i o n s p i k e i n s t r a t u m p y r a m i d a l e and by a n t i d r o m i c s t i m u l a t i o n , s u g g e s t i n g t h a t t h e f r a c t i o n a t e d s p i k e had a r i s e n subsequent t o s p i k e d i s c h a r g e at t h e c e l l body l a y e r . F u r t h e r m o r e , s p i k e d i s c h a r g e a t d e n d r i t i c hot s p o t s was not d e t e c t e d on c u r r e n t - s o u r c e - 177 - d e n s i t y p r o f i l e s , and measurement o f the v o l t a g e t h r e s h o l d f o r s p i k e d i s c h a r g e a l o n g t h e dendro-somatic a x i s d i d not r e v e a l r e g i o n s o f p a r t i c u l a r l y low t h r e s h o l d f o r s p i k e a c t i v a t i o n i n t h e a p i c a l d e n d r i t e . I t might be argued t h a t g e n e r a t i o n o f a s p i k e a t a d e n d r i t i c hot s p o t o n l y o c c u r s d u r i n g p e r i o d s o f i n t e n s e a c t i v a t i o n , such as t h a t found d u r i n g e p i l e p t i f o r m d i s c h a r g e o f t h e p y r a m i d a l c e l l p o p u l a t i o n . However, p r e l i m i n a r y a n a l y s i s o f m u l t i p l e p o p u l a t i o n s p i k e d i s c h a r g e has r e v e a l e d a s i m i l a r p a t t e r n o f s p i k e a c t i v a t i o n as t h a t found under normal c o n d i t i o n s , w i t h each s p i k e i n t h e b u r s t o r i g i n a t i n g a t t h e c e l l body l a y e r . I t i s p o s s i b l e t h a t s p i k e d i s c h a r g e a t a d e n d r i t i c hot spot was s i m p l y not d e t e c t e d i n t h e p r e s e n t s t u d y . N e v e r t h e l e s s , a l t e r n a t i v e e x p l a n a t i o n s can be o f f e r e d f o r d a t a s u p p o r t i n g t h e e x i s t e n c e o f hot s p o t s i n d e n d r i t i c membrane. T h e r e f o r e , i t may not be n e c e s s a r y t o i n v o k e t h e s p e c i a l c h a r a c t e r i s t i c o f d e n d r i t i c hot s p o t s t o account f o r c e r t a i n a s p e c t s o f p y r a m i d a l c e l l p h y s i o l o g y . The p r o p e r t i e s o f t h e evoked d e n d r i t i c s p i k e would f u r t h e r q u e s t i o n t h e p o s s i b l e means o f c o n d u c t i o n o f a s p i k e a l o n g the dendro-somatic a x i s . P r e v i o u s i n v e s t i g a t i o n s have shown a b l o c k a d e o f t h e 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 i c s p i k e t h r o u g h a p p l i c a t i o n o f t e t r o d o t o x i n (TTX) or QX-314 i n j e c t i o n (Benardo e t a l . 1982; Wong et a l . 1979), s u g g e s t i n g t h a t the d e n d r i t i c s p i k e i s a c t i v e l y g e n e r a t e d t h r o u g h a v o l t a g e - d e p e n d e n t Na+ conductance. However, the r e s u l t s o f the p r e s e n t s t u d y can a l s o be e x p l a i n e d on t h e b a s i s o f a p a s s i v e e l e c t r o t o n i c c o n d u c t i o n o f the s p i k e a l o n g t h e d e n d r i t i c a x i s . As d i s c u s s e d i n Chapter 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 by a c u r r e n t s o u r c e / s i n k c o u l d be i n t e r p r e t e d as a p a s s i v e d e p o l a r i z a t i o n and subsequent - 178 - a c t i v e s p i k e g e n e r a t i o n as a s p i k e conducted towards t h e r e c o r d i n g l o c a t i o n . However, a b i p h a s i c 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 and c u r r e n t s o u r c e / s i n k would a l s o be ex p e c t e d f o r c a p a c i t a n c e c u r r e n t f l o w a s s o c i a t e d w i t h t h e e l e c t r o t o n i c c o n d u c t i o n o f a h i g h f r e q u e n c y component ( i e a s p i k e ) a l o n g membrane w i t h a c o m p a r a t i v e l y l o n g time c o n s t a n t (Nobles 1966). The g r a d u a l change i n waveform o f t h e s p i k e t h r o u g h the d e n d r i t i c f i e l d would 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 decay o f t h e i n t r a c e l l u l a r s p i k e a t each l o c a t i o n . P a s s i v e e l e c t r o t o n u s c o u l d t h u s account f o r the b i p h a s i c p o s t i v e / n e g a t i v e s p i k e p o t e n t i a l c o n d u c t i n g t h r o u g h t h e d e n d r i t i c r e g i o n on l a m i n a r p r o f i l e s o f 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 . The presence o f a c u r r e n t s i n k i n t h e p r o x i m a l s t r a t u m r a d i a t u m may however 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 o f the s p i k e t h r o u g h the p r o x i m a l d e n d r i t i c r e g i o n . I n t r a c e l l u l a r d a t a would a l s o s u p p o r t t h e c o n t e n t i o n o f a 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 t h r o u g h t h e m a j o r i t y o f th e d e n d r i t i c a r b o r i z a t i o n . I n t r a c e l l u l a r s p i k e s d e c l i n e d i n a m p l i t u d e and i n c r e a s e d 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 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 o n l y a t t h e l e v e l o f t h e p y r a m i d a l c e l l body. The l a c k o f a v o l t a g e t h r e s h o l d f o r s p i k e a c t i v a t i o n i n t h e d e n d r i t e c o u l d be e x p l a i n e d by an e l e c t r o t o n i c l l y conducted s p i k e s u p e r i m p o s i n g upon t h e e x i s t i n g 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 the c e l l a x i s . I n f a c t , t h e a m p l i t u d e o f t h e d e n d r i t i c s p i k e c o u l d be a l t e r e d w i t h t h e l e v e l o f membrane p o l a r i z a t i o n , s u g g e s t i n g no c l e a r r e v e r s a l p o t e n t i a l f o r t h e d e n d r i t i c s p i k e (see F i g 4.8). The e v i d e n c e f o r a Na+-dependence o f d e n d r i t i c s p i k e d i s c h a r g e may r e l a t e t o the methods used i n a p p l y i n g Na+ ch a n n e l - 1 7 9 - b l o c k e r s . I n t h e s t u d y i n which TTX was a p p l i e d t o d e n d r i t i c membrane, t h e drug was p e r f u s e d a c r o s s t h e e n t i r e s l i c e (Wong e t a l . 1979), 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 have o c c u r r e d s e c o n d a r i l y t o i n h i b i t i o n o f s o m a t i c s p i k e d i s c h a r g e . S i m i l a r l y , i n j e c t i o n o f t h e a n e s t h e t i c QX-314 i n t o a d e n d r i t i c impalement (Benardo e t a l . 1982) cannot n e c e s s a r i l y be t a k e n as e v i d e n c e f o r a b l o c k a d e o f t h e s p i k e a t t h e d e n d r i t i c l e v e l , as t h e drug may have d i f f u s e d t o the r e g i o n of the soma-axon h i l l o c k (or p r o x i m a l d e n d r i t e ) . A t e s t f o r t h e i o n i c b a s i s o f t h e d e n d r i t i c s p i k e may t h e r e f o r e 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 Na+ c h a n n e l b l o c k e r s 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 c e l l . The P o s s i b l e S i g n i f i c a n c e o f D e n d r i t i c S p i k e s t o P y r a m i d a l C e l l F u n c t i o n The r e t r o g r a d e c o n d u c t i o n o f a s p i k e t h r o u g h t h e d e n d r i t i c 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 consequence t o p y r a m i d a l c e l l f u n c t i o n . For i n s t a n c e , t h e l a r g e d e p o l a r i z a t i o n i n d uced by a s p i k e i n v a d i n g the d e n d r i t e would r a p i d l y d e p o l a r i z e t h e m a j o r i t y o f t h e d e n d r i t i c s t r u c t u r e over a s h o r t p e r i o d o f t i m e . Through a c t i v a t i o n 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 c h a n n e l s u n r e l a t e d t o s p i k e d i s c h a r g e , such a d e p o l a r i z a t i o n c o u l d s e r v e t o i n c r e a s e t h e e f f e c t i v e l e n g t h c o n s t a n t o f t h e c e l l , e nhancing 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 l a y e r ( L l i n a s and S u g i m o r i 1984). A l t e r n a t i v e l y , a s p i k e c o n d u c t i n g t h r o u g h t h e r e g i o n o f a f f e r e n t t e r m i n a t i o n might a l s o l i m i t t he time c o u r s e of s y n a p t i c d e p o l a r i z a t i o n by r e d u c i n g the d r i v i n g f o r c e f o r inward s y n a p t i c c u r r e n t s . The r e t r o g r a d e i n v a s i o n o f a s p i k e w i l l c e r t a i n l y i n f l u e n c e t h e form o f e x t r a c e l l u l a r p o t e n t i a l s a l o n g t h e c e l l a x i s , a l t e r i n g the shape o f t h e e x t r a c e l l u l a r - 180 - v o l t a g e g r a d i e n t i n t h e C A l r e g i o n ( R i c h a r d s o n e t a l . 1984b). The d e n d r i t i c s p i k e w i l l t h e r e f o r e modify the 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 c u r r e n t f l o w , and w i l l be an i m p o r t a n t c o n s i d e r a t i o n i n d e t e r m i n i n g t h e e p h a p t i c i n f l u e n c e of f i e l d p o t e n t i a l s upon t h e p y r a m i d a l c e l l a t b o t h t h e s o m a t i c and d e n d r i t i c l e v e l ( R i c h a r d s o n e t a l . 1984a,b; Turner e t a l . 1984). The r e t r o g r a d e c o n d u c t i o n o f t h e s p i k e t h r o u g h t h e d e n d r i t e might a l s o c o n t r i b u t e t o t h e m u l t i p l e s p i k e d i s c h a r g e of p y r a m i d a l 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 . Under normal c o n d i t i o n s , p y r a m i d a l c e l l d i s c h a r g e evoked by s t i m u l a t i o n o f a f f e r e n t or e f f e r e n t pathways i s r e s t r i c t e d t o s i n g l e s p i k e a c t i v a t i o n by a r e c u r r e n t i n h i b i t o r y feedback system (Andersen e t a l . 1969; D i n g l e d i n e and Langmoen 1980; S c h w a r t z k r o i n and P r i n c e 1980). However, i n t h e pr e s e n c e of p h a r m a c o l o g i c a l agents known t o b l o c k t h e a c t i o n o f i n h i b i t o r y s y n a p t i c i n p u t s , s y n a p t i c d e p o l a r i z a t i o n can e l i c i t a l a r g e p aroxysmal d e p o l a r i z i n g s h i f t (PDS) c a p a b l e of e v o k i n g r e p e t i t i v e s o m a t i c s p i k e a c t i v a t i o n ( S c h w a r t z k r o i n and P r i n c e 1980; S c h w a r t z k r o i n and Wyler 1979). The PDS i s Ca+2-dependent, and thought t o a r i s e i n p a r t from t h e a c t i v a t i o n o f vo l t a g e - d e p e n d e n t Ca+2 c h a n n e l s i n d e n d r i t i c membrane ( S c h w a r t z k r o i n and P r i n c e 1980; S c h w a r t z k r o i n and Wyler 1979; Wong and P r i n c e 1979). In f a c t , p r e v i o u s work has shown t h a t a c t i v a t i o n o f a d e n d r i t i c Na+ s p i k e by a n t i d r o m i c s t i m u l a t i o n under t h e s e c o n d i t i o n s g i v e s r i s e t o Ca+2 s p i k e d i s c h a r g e a t t h 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 P r i n c e 1980). T h e r e f o r e , one f u n c t i o n o f the i n h i b i t o r y feedback network t o t h e p y r a m i d a l c e l l may be t o p r e v e n t the a c t i v a t i o n o f d e n d r i t i c Ca+2 s p i k e s by t h e Na+ s p i k e c o n d u c t i n g t h r o u g h t h e d e n d r i t e from t h e r e g i o n - 181 - of t h e c e l l l a y e r . The p r e s e n t s t u d y has demonstrated t h a t d e n d r i t i c s p i k e s o f t h e p y r a m i d a l c e l l 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 o f t h e d e n d r i t e by a s p i k e i n i t i a t e d i n t h e r e g i o n of t h e soma-axon h i l l o c k , a f i n d i n g t h a t w i l l r e q u i r e major r e v i s i o n s i n our p r e s e n t concept of p y r a m i d a l c e l l p h y s i o l o g y . A s i m i l a r p a t t e r n of a c t i v i t y has been shown t o e x i s t i n o t h e r CNS neurons, i n c l u d i n g the d e n t a t e g r a n u l e c e l l ( J e f f e r y s 1979), c e r e b e l l a r P u r k i n j e c e l l ( L l i n a s 1975; L l i n a s and S u g i m o r i 1980a,b), and t h e Mauthner c e l l o f t h e g o l d f i s h (Furshpan and Furakawa 1962). The p r o p e r t i e s o f Na+ s p i k e d i s c h a r g e i n t h e p y r a m i d a l c e l l a re t h u s comparable t o a d i v e r s e group of c e l l t y p e s , and e l u c i d a t i o n o f the r o l e of d e n d r i t i c s p i k e s i n d e t e r m i n i n g p y r a m i d a l c e l l a c t i v i t y may have wi d e s p r e a d i m p l i c a t i o n s t o our b a s i c u n d e r s t a n d i n g of n e u r o n a l f u n c t i o n . - 182 - REFERENCES ALGER, B.E. AND NICOLL, R.A. (1982) F e e d - f o r w a r d d e n d r i t i c i n h i b i t i o n i n r a t hippocampal p y r a m i d a l c e l l s s t u d i e d i n v i t r o . J . P h y s i o l . London 328: 105-123. ALLEN, G.I., ECCLES, J . , NICOLL, R.A., OSHIMA, T. AND RUBIA, F . J . (1977) The i o n i c mechanisms concerned i n g e n e r a t i n g the IPSPs o f hippocampal p y r a m i d a l c e l l s . P r o c . R. Soc. Lond. B 198: 363-384. ANDERSEN, P. (1959) I n t e r h i p p o c a m p a l i m p u l s e s . I . 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