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Associative induction of short-term potentiation in rat hippocampus 1986

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ASSOCIATIVE INDUCTION OF SHORT-TERM POTENTIATION IN RAT HIPPOCAMPUS By ANTHONY AUYEUNG B. Sc. (Pharm.), The U n i v e r s i t y of B r i t i s h Columbia, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology & Therapeutics, F a c u l t y of Medicine) 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 ® A n t h o n y Auyeung 1986 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 my1 department or by his 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 Pharmacology and Therapeutics The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date AUGUST 12 , 1986 - i i - ABSTRACT Tetanic s t i m u l a t i o n of e x c i t a t o r y a f f e r e n t pathways i n the hippocampus can lead to long-term p o t e n t i a t i o n (LTP) of the postsynaptic response. Simultaneous t e t a n i z a t i o n of two separate but convergent a f f e r e n t pathways can r e s u l t in a s s o c i a t i v e LTP, which e x h i b i t s a magnitude that i s greater than the sum of the i n d i v i d u a l p o t e n t i a t i o n s . Short-term p o t e n t i a t i o n (STP) of synaptic transmission also occurs i n the t e t a n i z e d pathway, and i s a well known presynaptic phenomenon at other neuronal j u n c t i o n s . Studies were con- ducted to examine i f STP i n the hippocampus could be induced a s s o c i a t i v e l y and to determine the nature of pre- and postsynaptic i n t e r a c t i o n s leading to t h i s a s s o c i a t i v e p o t e n t i a t i o n . Experiments were conducted on t r a n s v e r s e l y sectioned r a t hippocampal s l i c e s maintained i n - v i t r o . Population responses were recorded from the C A ^ pyramidal c e l l s and were evoked using s t i m u l a t i n g electrodes placed in stratum radiatum, stratum oriens and the alveus. Population e x c i t a t o r y postsynaptic p o t e n t i a l s (EPSP) were recorded with e x t r a c e l l u l a r recording e l e c t r o d e s placed at the a p i c a l dendrites of the CA-̂  c e l l s . I n d i v i d u a l c e l l u l a r EPSPs were recorded from the CA-̂  c e l l somata. To assess the in f l u e n c e of t e t a n i c c o n d i t i o n i n g on an untetanized a f f e r e n t pathway, s t i m u l a t i n g e l e c t r o d e s were placed i n d i f f e r e n t pathways to d e l i v e r t e t a n i c c o n d i t i o n i n g s t i m u l a t i o n to the CA^ c e l l s . Another s t i m u l a t i n g electrode was placed i n a separate t e s t pathway, which al s o converged on the same population of CA-̂  c e l l s . The t e s t pathway e i t h e r remained unstimulated or was stimulated once i n conjunction with each t e t a n i c c o n d i t i o n i n g t r a i n of s t i m u l a t i o n . C o n d i t i o n i n g t e t a n i d e l i v e r e d through s t r a t a oriens or radiatum i n - duced a s s o c i a t i v e STP of the t e s t response when paired with a s i n g l e t e s t s t i m u l a t i o n , but caused depression of the response when unpaired. A n t i - dromic t e t a n i z a t i o n of CA-̂  c e l l s at the alveus or a d e p o l a r i z a t i o n of these c e l l s by i n t r a c e l l u l a r current i n j e c t i o n s produced the same pattern of p o t e n t i a t i o n with the paired/unpaired paradigm. By d e l i v e r i n g a number of these paired t e s t - p l u s - c o n d i t i o n i n g t r a i n s i n r a p i d succession, the mag- nitude of the STP increased i n a graded manner, and the s i z e and time course resembled those of STP found at other j u n c t i o n s . At the maximum of ten p a i r i n g s used i n these s t u d i e s , the evoked a s s o c i a t i v e STP was succeeded by LTP. Presynaptic e x c i t a b i l i t y changes were assessed by monitoring the amount of current needed to f i r e an antidromic a c t i o n p o t e n t i a l from the Schaffer c o l l a t e r a l t e r m i n a l s . These are the a f f e r e n t terminals that form en passant synapses with the a p i c a l dendrites of the CA-̂  c e l l s . P a i r i n g a s i n g l e s t i m u l a t i o n of these terminals with a c o n d i t i o n i n g tetanus of other a f f e r e n t s r e s u l t e d i n STP of the t e s t EPSP, as w e l l as a p a r a l l e l decrease in the t e s t a f f e r e n t terminal e x c i t a b i l i t y . These changes are i n accord with a presynaptic mechanism of STP found i n the s p i n a l cord and neuro- muscular j u n c t i o n . The temporal overlap between the s i n g l e t e s t a f f e r e n t v o l l e y and the c o n d i t i o n i n g tetanus was found to be a determinant both of the magnitude and the p r o b a b i l i t y of STP i n d u c t i o n . The s i n g l e t e s t v o l l e y could precede the c o n d i t i o n i n g tetanus by up to 50 msec or f o l l o w the tetanus by up to 80 msec and s t i l l induce a degree of STP. However, the greatest amount of STP was produced by simultaneous t e s t and c o n d i t i o n i n g s t i m u l a t i o n s . These - i v - l e n i e n t temporal l i m i t s suggest an a l t e r e d e x c i t a b i l i t y s t a t e due to pre- and postsynaptic i n t e r a c t i o n s . Taken- together, the evidence i n d i c a t e s a postsynaptic i n i t i a t i n g s i t e f o r a s s o c i a t i v e STP and LTP i n the hippocampus. The i n i t i a l postsynaptic d e p o l a r i z a t i o n appeared to i n t e r a c t with an a f f e r e n t v o l l e y to a l t e r presyn- a p t i c terminal e x c i t a b i l i t y . I t i s proposed that a s u b l i m i n a l presynaptic r e l e a s e process f o l l o w s an a c t i o n p o t e n t i a l i n the t e r m i n a l . This sublim- i n a l process may be f a c i l i t a t e d by a s s o c i a t i v e i n t e r a c t i o n s between the postsynaptic d e p o l a r i z a t i o n and an a c t i o n p o t e n t i a l i n the presynaptic t e r - minal through an a l t e r e d presynaptic terminal e x c i t a b i l i t y . The a s s o c i a t i v e i n t e r a c t i o n s could lead to enhanced t r a n s m i t t e r r e l e a s e by subsequent a f f e r - ent v o l l e y s . The nature of t h i s s u b l i m i n a l process i s unknown, but several hypotheses were discussed. I t was concluded that a s s o c i a t i v e p o t e n t i a t i o n has a presynaptic locus of maintenance, and that STP and LTP i n the hippo- campus may be simply d i f f e r e n t m u l t i p l e s of the same u n i t p o t e n t i a t i o n event. However, the r e s u l t s do not r u l e out a p o s s i b l e a d d i t i o n a l post- synaptic locus f o r the maintenance of STP and LTP. Bhagavatula R. Sastry (Supervisor) - V - TABLE OF CONTENTS CHAPTER Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS ix 1 INTRODUCTION 1 2 REVIEW OF THE LITERATURE 4 2.1 Short-term p o t e n t i a t i o n 4 2.1.1 F a c i l i t a t i o n 6 2.1.2 Augmentation 7 2.1.3 P o s t - t e t a n i c p o t e n t i a t i o n 8 2.2 Locus of short-term p o t e n t i a t i o n 8 2.3 Mechanism of short-term p o t e n t i a t i o n 12 2.4 Ionic mechanism of short-term p o t e n t i a t i o n 14 2.5 Anatomy of the hippocampus 17 2.5.1 The dentate gyrus 20 2.5.2 The hippocampus proper 20 2.5.3 The f i e l d s of Amnion's horn 21 2.5.3.1 F i e l d CA 4 22 2.5.3.2 F i e l d CA3 22 2.5.3.3 F i e l d CA 2 22 2.5.3.4 F i e l d CA, 23 - v i - CHAPTER Page 2.5.4 Neuronal pathways of the hippocampus 24 2.5.5 A f f e r e n t s to the hippocampus 24 2.5.5.1 Perforant Path 24 2.5.5.2 A f f e r e n t s to the CA 3 region 26 2.5.5.3 A f f e r e n t s to the CA2 region 26 2.5.5.4 A f f e r e n t s to the CA-L region 1 27 2.5.6 E f f e r e n t s from the hippocampus 27 2.5.7 Interneurons 28 2.5.8 Lo n g i t u d i n a l a s s o c i a t i o n pathway 29 2.6 Short-term p o t e n t i a t i o n i n the hippocampus 29 2.7 A s s o c i a t i v e i n d u c t i o n of p o t e n t i a t i o n : a f f e r e n t c o o p e r a t i v i t y 30 3 METHODS 33 3.1 Preparation of s l i c e s 33 3.2 S l i c e bath 34 3.3 Per f u s i o n media 37 3.4 S t i m u l a t i o n systems 38 3.5 Recording systems 39 3.6 A s s o c i a t i v e induction of STP 40 3.6.1 C o n d i t i o n i n g by t e t a n i c s t i m u l a t i o n of f i b e r s 40 3.6.2 C o n d i t i o n i n g by i n t r a c e l l u l a r d e p o l a r i z i n g pulses 43 3.6.3 Temporal requirements governing the in d u c t i o n of STP 45 3.7 A s s o c i a t i v e i n d u c t i o n of Schaffer c o l l a t e r a l terminal e x c i t a b i l i t y changes 45 - v i i - CHAPTER Page 4 RESULTS 48 4.1 Stratum radiatum c o n d i t i o n i n g 48 4.2 Stratum o r i e n s c o n d i t i o n i n g 51 4.3 Alvear c o n d i t i o n i n g 51 4.4 I n t r a c e l l u l a r c urrent i n j e c t i o n s 53 4.5 Schaffer c o l l a t e r a l terminal e x c i t a b i l i t y changes 53 4.6 Temporal requirements f o r induction of STP 57 5 DISCUSSION 60 6 CONCLUSION 75 7 REFERENCES 76 - v i i i - LIST OF TABLES TABLE Page 1. P o s t - c o n d i t i o n i n g p o t e n t i a t i o n induced by p a i r i n g t e t a n i c t r a i n s of the alveus with a s i n g l e s t i m u l a t i o n of the t e s t input. 52 2. E f f e c t s of i n t r a c e l l u l a r l y i n j e c t e d d e p o l a r i z i n g current pulses on EPSPs evoked by s t i m u l a t i o n of stratum radiatum and stratum o r i e n s . 54 3. E f f e c t s of paired and unpaired c o n d i t i o n i n g t r a i n s on Schaffer c o l l a t e r a l terminal e x c i t a b i l i t y . 58 - ix - LIST OF FIGURES FIGURE Page 1. Anatomical l o c a t i o n of the hippocampus in the r a t b r a i n . 19 2. Anatomical diagram of a t r a n s v e r s e l y sectioned r a t hippocampal s l i c e showing the various a f f e r e n t , e f f e r e n t and i n t r i n s i c pathways. 25 3. Diagrammatic i l l u s t r a t i o n of the s l i c e bath used f o r e l e c t r o p h y s i o l o g i c a l recordings from in v i t r o hippocampal s l i c e s . 35 4. Experimental arrangement f o r a s s o c i a t i v e i n d u c t i o n of STP by c o n d i t i o n i n g of stratum radiatum and stratum o r i e n s . 41 5. Experimental arrangement f o r a s s o c i a t i v e induction of STP by alvear c o n d i t i o n i n g . 42 6. Experimental arrangement f o r a s s o c i a t i v e induction of STP by i n t r a c e l l u l a r i n j e c t i o n of d e p o l a r i z i n g c u r r e n t . 44 7. Experimental arrangement f o r e x c i t a b i l i t y t e s t i n g of S c h a f f e r c o l l a t e r a l terminal regions. 46 8. A s s o c i a t i v e induction of STP, LTR and the reduction i n the Schaffer c o l l a t e r a l terminal e x c i t a b i l i t y . 49 9. Induction of STP and LTP by paired c o n d i t i o n i n g d e p o l a r i -z a t i o n of a CAi neuron.. 55 10. The l i m i t s of the temporal r e l a t i o n s h i p between condi-t i o n i n g and t e s t s t i m u l i f o r the induction of asso-c i a t i v e p o t e n t i a t i o n . EPSP. 59 - X - ACKNOWLEDGEMENTS I would l i k e to thank Dr. B. R. Sastry f o r his ideas, i n s i g h t s and guidance during the preparation of t h i s t h e s i s . Thanks al s o go to Ms Joanne Goh and Mr. P a t r i c k May f o r t h e i r c o n t r i b u t i o n to some of the experiments included in t h i s t h e s i s , and f o r t h e i r support i n my moments of s t r u g g l e . I am g r a t e f u l f o r f i n a n c i a l a s s i s t a n c e from the Medical Research Council of Canada and the U n i v e r s i t y of B r i t i s h Columbia Graduate Summer Sch o l a r s h i p . - 1 - 1 INTRODUCTION The mammalian hippocampus i s under i n t e n s i v e study as the p o s s i b l e c o r t i c a l s t r u c t u r e subserving l e a r n i n g and memory (Goddard, 1980; McNaughton, 1983). The most promising phenomenon observed i s long-term p o t e n t i a t i o n (LTP) ( B l i s s and Gardner-Medwin, 1973; B l i s s and Ltfmo, 1973). Long-term p o t e n t i a t i o n i s induced by the t e t a n i c s t i m u l a t i o n of an a f f e r e n t pathway, leading to an enhanced p o s t - t e t a n i c response of the postsynaptic c e l l to a f f e r e n t s t i m u l a t i o n ( B l i s s and Gardner-Medwin, 1971). Since the i n i t i a t i n g event i s a short tetanus of several hundred m i l l i s e c o n d s , and the ensuing p o t e n t i a t i o n can l a s t f o r hours to days, ( B l i s s and Gardner-Medwin, 1973), LTP appears to share c e r t a i n key p r o p e r t i e s with l e a r n i n g and memory and could be the p h y s i o l o g i c a l substrate f o r both. A number of hypotheses have been advanced to e x p l a i n the mechanism behind LTP (Baudry and Lynch, 1980; C o l l i n g r i d g e , 1985; Malenka et- - a l : , 1986; Skrede and Malthe-Stfrenssen, 1981; Van Harreveld and F i f k o v a , 1975; Wigstrom and Gustafsson, 1985b), but i t s primary locus has yet to be deter- mined. The a s s o c i a t i v e nature of LTP was described by McNaughton et - -a-T; (1978), who showed that LTP can be produced in- - v i t r o by the simultaneous a c t i v a t i o n of separate converging a f f e r e n t pathways; the p o t e n t i a t i o n thus produced was greater than the sum of that produced s e p a r a t e l y by each a f - f e r e n t pathway. Tetanic s t i m u l a t i o n of an a f f e r e n t pathway to f i e l d CA^ produces LTP in t h i s area and increases the e x c i t a b i l i t y of other non-tetan- ized a f f e r e n t s here (Goh and Sastry, 1985). The mechanism of these i n t e r - actions i s unclear. I t appears that a t r a n s m i t t e r substance, potassium - 2 - r e l e a s e d during the tetanus, or d i r e c t p a r t i c i p a t i o n of the CA-̂  neuron, i s needed. This presynaptic i n t e r a c t i o n with the postsynaptic elements may play a r o l e in the a s s o c i a t i v e i n d u c t i o n of LTP. Short-term p o t e n t i a t i o n (STP) i s a p o s t - t e t a n i c p o t e n t i a t i o n of the p o s t s y n a p t i c response l a s t i n g up to a few minutes (Magleby and Zengel, 1975a, 1975b). I t has been observed at a l l e x c i t a b l e j u n c t i o n s examined, i n c l u d i n g those in the hippocampus (Feng, 1941b; Lloy d , 1949; Gloor et - a l ; , 1964). At p e r i p h e r a l nerve j u n c t i o n s , STP has been shown to be a presynap- t i c event mediated by a p o s t - t e t a n i c increase in evoked t r a n s m i t t e r r e l e a s e (del C a s t i l l o and Katz, 1954d). The same mechanism i s thought to mediate STP in the hippocampus (McNaughton, 1982). Because STP commonly precedes or accompanies LTP (McNaughton, 1982; Barrionuevo and Brown, 1983) and i t was shown that presynaptic terminals i n t e r a c t with each other (Goh and Sastry, 1985), i t i s p o s s i b l e that STP could a l s o be induced by a s s o c i a t i v e i n t e r - a c t i o n s . The p a r t i c i p a t i o n of the postsynaptic c e l l f o r the induction of a r e p o r t e d l y presynaptic phenomenon, namely STP, i s c e r t a i n l y worthy of inves- t i g a t i o n . Experiments were conducted in r a t hippocampal s l i c e s i n - v i t r o . Stu- dies were designed to examine whether STP of CA-̂  neuronal responses could be induced through a s s o c i a t i v e i n t e r a c t i o n s between a f f e r e n t inputs. If STP could be thus induced, then the temporal r e l a t i o n s h i p between the t e t a n i c c o n d i t i o n i n g and a f f e r e n t t e s t s t i m u l a t i o n s would be determined. Evidence in the l i t e r a t u r e i n d i c a t e s that t e t a n i c s t i m u l a t i o n of the c o n d i t i o n i n g input causes an e x t r a c e l l u l a r negative wave near the s y n a p t i c terminations of a separate a f f e r e n t t e s t pathway (Wigstrom and Gustafsson, 1985b). This - 3 - wave may be i n t e r p r e t e d as a d e n d r i t i c d e p o l a r i z a t i o n , which could be i n - volved i n the a s s o c i a t i v e i n d u c t i o n of LTP. Therefore, d i r e c t d e p o l a r i - z a t i o n of the postsynaptic c e l l could r e s u l t i n a c o n d i t i o n that favours i n d u c t i o n of a s s o c i a t i v e LTP. This p o s s i b i l i t y was examined with d e p o l a r i z - ing c urrents i n j e c t e d i n t o the postsynaptic c e l l to mimic the e f f e c t s of the c o n d i t i o n i n g tetanus. Short-term p o t e n t i a t i o n at the neuromuscular j u n c t i o n and s p i n a l cord i s known to be accompanied by h y p e r p o l a r i z a t i o n and decreased e x c i t a b i l i t y of the presynaptic terminal (Eccles and K r n j e v i c , 1959a, 1959b; Hubbard and W i l l i s , 1962). I n t r a c e l l u l a r recordings of hippocampal presynaptic termin- a l s would demonstrate d i r e c t l y that presynaptic changes occur during the a s s o c i a t i v e i n d u ction of STP. However, si n c e the impalement of the f i n e boutons i s not yet p o s s i b l e , the e x c i t a b i l i t y of presynaptic terminals was i n d i r e c t l y assessed using a method that Wall employed i n 1958 to measure primary a f f e r e n t terminal changes i n the s p i n a l cord. In Wall's (1958) terminology, an increase i n presynaptic e x c i t a b i l i t y i n d i c a t e s a d e p o l a r i - z a t i o n of the terminal membrane, and a decrease i n d i c a t e s a h y p e r p o l a r i - z a t i o n . - 4 - 2 REVIEW OF THE LITERATURE The nervous system's c a p a c i t y f o r f a c i l i t a t i o n of t r a n s m i t t e r r e l e a s e i s of great i n t e r e s t to n e u r o b i o l o g i s t s . In general, a c t i v a t i o n of a neu- ronal input w i l l a f f e c t i n some way the postsynaptic response to a subse- quent s t i m u l a t i o n of that input. Depending on the nature of the s t i m u l i and the e x c i t a b l e t i s s u e i n v o l v e d , the enhanced synaptic response may range from a f l e e t i n g to a profound p o t e n t i a t i o n . The synaptic enhancements due to high frequency r e p e t i t i v e s t i m u l a t i o n (tetanus or t e t a n i c s t i m u l a t i o n ) are e s p e c i a l l y i n t e r e s t i n g . The term " p o s t - t e t a n i c p o t e n t i a t i o n " (PTP) was f i r s t coined to describe a l l synaptic enhancements due to a tetanus (Feng, 1941b; Grumbach and Wilber, 1940). Eccles (1953) defined PTP as an i n - creased post-synaptic discharge e l i c i t e d homosynaptically and due to i n - creased presynaptic a c t i o n . With the discovery of long-term p o t e n t i a t i o n (LTP) ( B l i s s and Gardner-Medwin, 1971), which has a time course of tens of minutes to days ( B l i s s and Gardner-Medwin, 1973; B l i s s and L0mo, 1973), PTP has come to mean a sub-component of a p o s t - t e t a n i c short-term p o t e n t i a t i o n (STP) that l a s t s f o r no more than several minutes (Magleby and Zengel, 1982). 2.1 Short-term p o t e n t i a t i o n Short-term p o t e n t i a t i o n has been found i n a l l e x c i t a b l e systems s t u - died to date and i s considered a general phenomenon of synaptic p l a s t i c i t y (Eccles and K r n j e v i c , 1959a, 1959b; Feng, 1941a, 1941b; Hubbard and Schmidt, 1963; L l o y d , 1949; Martin and P i l a r , 1964; Walters and Byrne, 1984; Zengel et a l ; , 1980). There i s evidence i n the current l i t e r a t u r e to support three sub-components to p o s t - t e t a n i c STP: i ) f a c i l i t a t i o n i i ) augmentation i i i ) - 5 - p o s t - t e t a n i c p o t e n t i a t i o n . Their most s a l i e n t d i f f e r e n c e s l i e in t h e i r r e s - p e c t i v e time courses and time constants of decay (Magleby and Zengel, 1982). E a r l y work on f a c i l i t a t i o n and d e c u r a r i z a t i o n used nerve-muscle prepa- r a t i o n s from amphibians and mammals (Eccles et a l : , 1941; Feng, 1937, 1941a, 1941b). Spinal cord preparations were also favoured and many c r u c i a l f i n d - ings about STP were made here (Gasser and Grundfest, 1936; L l o y d , 1949; Wall and Johnson, 1958). In the i s o l a t e d h e a r t , a phenomenon that c l o s e l y resem- bles PTP was studied (Hadju and Szent-Gyorgyi, 1952). Evidence from the v i s u a l (Geldard, 1931; G r a n i t , 1955; Hughes et a l : , 1956), auditory (Hughes, 1954; Hughes and Ro s e n b l i t h , 1957) and o l f a c t o r y (MacLean e-t a l . , 1957) pathways showed that a form of PTP i s ope r a t i o n a l in these systems. P a i r e d - pulse f a c i l i t a t i o n was f i r s t described at the neuromuscular j u n c t i o n (NMJ) of the frog (Eccles e t a l . , 1941; Feng, 1941a, 1941b; Schaefer and Haass, 1939); a v a r i a n t of paired-pulse f a c i l i t a t i o n i s frequency f a c i l i t a t i o n , which some workers a s s e r t i s wholly d i f f e r e n t (Creager et a l - . , 1980). A r e l a t i v e l y newly recognized form of STP i s augmentation (Magleby and Zengel, 1976a), which has been nei t h e r f u l l y e l u c i d a t e d nor accepted by workers in t h i s f i e l d . Another new form of p o s t - t e t a n i c p o t e n t i a t i o n i s LTP, which was f i r s t observed i n the r a t hippocampus i n 1971 ( B l i s s and Gardner-Medwin, 1971). I t has als o been found at the crustacean NMJ (Sherman and Atwood, 1971), marine mollusc sensory neuron ( C a s t e l l u c c i and Kandel, 1976; C a s t e l l u c c i et a l : , 1970) and mammalian sympathetic gangl i a (Briggs et- - a l : , 1983, 1985), but has not yet been demonstrated at the mammalian NMJ. Regarding the d i s - t i n g u i s h i n g c h a r a c t e r i s t i c s , there i s no confusion of PTP and LTP because - 6 - the l a t t e r has a decidedly long time course of 30 minutes to many weeks ( B l i s s and Gardner-Medwin, 1973; B l i s s and Umo, 1973). 2.1.1 F a c i l i t a t i o n F a c i l i t a t i o n i s a two component enhancement of t r a n s m i t t e r r e l e a s e ( M a l l a r t and M a r t i n , 1967). I t i s present immediately upon the onset of a tetanus and i s evident f o r up to 600 msec i n t o a tetanus (Magleby, 1973a, 1973b; M a l l a r t and M a r t i n , 1967). Indeed, t e t a n i c s t i m u l a t i o n i s not essen- t i a l f o r f a c i l i t a t i o n ; a s i n g l e antecedent impulse can f a c i l i t a t e the r e s - ponse to a s t i m u l u s , provided the i n t e r v e n i n g i n t e r v a l i s not more than 50-100 msec. (Charlton and B i t t n e r , 1978b; Creager et- - a l ; , 1980; Larrabee and Bronk, 1947). This i s c a l l e d twin-pulse or paired-pulse f a c i l i t a t i o n and appears to be the elementary event behind frequency f a c i l i t a t i o n (Magleby and Zengel, 1975b; M a l l a r t and M a r t i n , 1967). I t was determined t h a t each impulse of a t e t a n i c t r a i n of s t i m u l a t i o n adds a l i n e a r component to the base r a t e of t r a n s m i t t e r r e l e a s e and to the f a c i l i t a t e d response; the magnitude of the aggregate p o t e n t i a t i o n ranged from 50% f o r frequency f a c i - l i t a t i o n to about 100% f o r paired-pulse f a c i l i t a t i o n (Magleby, 1973a, 1973b; Magleby and Zengel, 1975a; M a l l a r t and M a r t i n , 1967). Assuming that the magnitude and time course of each impulse were the same, M a l l a r t and Martin (1967) were able to describe f a c i l i t a t i o n i n the f r o g NMJ with two decay constants of 35 msec and 250 msec. This two component c h a r a c t e r i s t i c of f a c i l i t a t i o n has been found i n r a b b i t sympathetic gangl i a (Zengel et- - a l . - , 1980) and r a t hippocampus (Creager et a l ; , 1980). Katz and M i l e d i (1968) showed the calcium ( C a + + ) dependence of both paired-pulse f a c i l i t a t i o n and frequency f a c i l i t a t i o n and used these pheno- - 7 - mena to support t h e i r r e s i d u a l C a + + theory of f a c i l i t a t e d t r a n s m i t t e r r e l e a s e . Although both f a c i l i t a t i o n and PTP have been a t t r i b u t e d to r e s i - dual Ca , i t i s not f i r m l y e s t a b l i s h e d that both share a common a c t i v e pool of C a + + (Landau et - a l ; , 1973). For example, i n the squid gi a n t axon, f a c i l i t a t i o n may depend on the presence of a C a + + current r a t h e r than r e s i d u a l C a + + (Charlton and B i t t n e r , 1978a). Furthermore, f a c i l i t a t i o n — e s p e c i a l l y paired-pulse — can be evoked independently of PTP. The degree of f a c i l i t a t i o n and i t s time course remained the same even i f f a c i l i t a t i o n was induced during the maximal phase of PTP (Creager et - a l - ; , 1980; Magleby, 1973a). 2.1.2 Augmentation Augmentation i s d i f f e r e n t i a t e d from f a c i l i t a t i o n and PTP on the basis of i t s time course and decay constant (Magleby and Zengel, 1976b). Most of the work on augmentation has been done by Magleby and Zengel on the f r o g NMJ ++ blocked with high e x t r a c e l l u l a r magnesium (Mg ). In 1975(a), they pro- posed t h i s intermediate phase of synaptic enhancement on the strength of a decay constant of 7 seconds: longer than f a c i l i t a t i o n and shorter than PTP. Increasing the number of impulses i n the tetanus added l i n e a r l y to the s i z e of the augmentation without changing the time constant to any s i g n i f i - cant extent (Magleby and Zengel, 1976a). A m u l t i p l i c a t i v e e f f e c t of augmen- t a t i o n on f a c i l i t a t i o n and p o t e n t i a t i o n was proposed, as was a common i n - crease i n quantal content, m, to account f o r the enhancement (Magleby and Zengel, 1982; Zengel and Magleby, 1982). Magleby and Zengel have suggested in t h e i r various studies that augmentation was present i n the r e s u l t s of Larrabee and Bronk (1947), L i l e y (1956) and Landau et- al.-(1973). Notwith- - 8 - standing the common dependence on C a + + , d i f f e r e n t d i v a l e n t c a t i o n s have d i f f e r e n t i a l e f f e c t s on the stimulus-induced increases in t r a n s m i t t e r r e - lease (Zengel and Magleby, 1977, 1980). Corroborating evidence f o r the existence of augmentation i s s t i l l l i m i t e d and, t h e r e f o r e , the concept i s not yet g e n e r a l l y accepted. 2.1.3 P o s t - t e t a n i c p o t e n t i a t i o n P o s t - t e t a n i c p o t e n t i a t i o n can range from 3 minutes and up to 500% of control i n the frog NMJ (Magleby and Zengel, 1975a) to 10 minutes and over 700% of c o n t r o l i n the avian c i l i a r y ganglion ( M a l l a r t and Ma r t i n , 1967). There had been e a r l y hopes that PTP would prove to be the e l u s i v e l i n k bet- ween physiology and psychology as the substrate f o r such dynamic processes as l e a r n i n g and memory (Hughes, 1958). Since then, the focus f o r such a substrate has s h i f t e d to LTP (McNaughton et a l 1 9 7 8 ; Levy and Steward, 1979). Much of the work on STP has a c t u a l l y been d i r e c t e d at PTP; th e r e - f o r e , the f o l l o w i n g s e c t i o n s on the various aspects of STP by n e c e s s i t y deal l a r g e l y with PTP. 2.2 Locus of short-term p o t e n t i a t i o n In 1858, S c h i f f reported on the p o s t - t e t a n i c p o t e n t i a t i o n of tw i t c h tension i n the f r o g g a s t r o c n e m i u s - s c i a t i c p r e p a r a t i o n . Boehm (1894) ob- served a temporary d e c u r a r i z i n g e f f e c t of t e t a n i c s t i m u l a t i o n i n the h e a v i l y c u r a r i z e d s c i a t i c - g a s t r o c n e m i u s preparation. In 1912, Forbes reported the f a c i l i t a t i n g e f f e c t of a tetanus to one nerve on the p o s t - t e t a n i c response of another adjacent nerve. Feng e t • -a/h (1938) described the p o s t - t e t a n i c r e p e t i t i v e discharge of the f r o g neuromuscular j u n c t i o n ; however, the f a c i - l i t a t i n g e f f e c t co-occurs with a p r e v a i l i n g i n h i b i t i o n (depression). Other - 9 - workers found that a few t e t a n i of short duration induced a neuromuscular block, while a d d i t i o n a l t e t a n i could remove t h i s blockade (Brown and von E u l e r , 1938). There was much debate on the locus of t h i s PTP; some workers maintained that i t was a phenomenon of the muscle c o n t r a c t i l e elements (Walker, 1947), since d i r e c t s t i m u l a t i o n of c u r a r i z e d muscle appeared to produce PTP. However, other workers found that d i r e c t s t i m u l a t i o n did not produce PTP (Guttman et a l : , 1937), and concluded that PTP was an end-plate event at the NMJ. Meanwhile, i n the i s o l a t e d f e l i n e dorsal r o o t , Gasser and Grundfest (1936) made d e t a i l e d observations of p o s t - t e t a n i c changes which correspond to the same changes seen in f r o g nerve (Gasser and Graham, 1932). They des- c r i b e d two phases of p o s t - t e t a n i c h y p e r p o l a r i z a t i o n , a short f i r s t phase of several msec duration followed by a depression, and a prolonged second phase of h y p e r p o l a r i z a t i o n whose amplitude and duration were dependent upon the preceding tetanus: " A f t e r a maximal tetanus of 30 sec, the p o t e n t i a l i s + 0.6 to 0.7 mV, and the duration more than four minutes." (Gasser and Grundfest, 1936). Moreover, the a s s o c i a t e d p o s t - t e t a n i c spike p o t e n t i a l (presynaptic spike) was also g r e a t l y enhanced. Larrabee and Bronk (1938) reported what they c a l l e d "prolonged f a c i l i t a t i o n " i n the cat s t e l l a t e gang- l i o n . P o t e n t i a t i o n was observed with p r e g a n g l i o n i c tetanus, but not with antidromic p o s t g a n g l i o n i c tetanus, leading the authors to conclude that "prolonged f a c i l i t a t i o n " must occur p r e g a n g l i o n i c a l l y . Woolsey and Larrabee (1940) t e t a n i z e d the f e l i n e dorsal root and were able to show the same two phases of h y p e r p o l a r i z a t i o n , confirming that i n - creases in the frequency and duration of the tetanus lead to increases in - 10 - both amplitude and duration of the second h y p e r p o l a r i z a t i o n . A concomitant f a c i l i t a t i o n of the evoked i p s i l a t e r a l v e n t r a l root discharge l a s t e d f o r the duration of the second prolonged h y p e r p o l a r i z a t i o n . This f a c i l i t a t e d d i s - charge could not be induced f o l l o w i n g antidromic t e t a n i to the v e n t r a l r o o t . In 1947, Larrabee and Bronk again examined t h i s prolonged f a c i l i t a - t i o n and were convinced that a p r e g a n g l i o n i c s i t e was the locus of change. By recording from s i n g l e ganglion c e l l s as well as nerve trunks, i t was found that the l a r g e s t p o s t - t e t a n i c g a n g l i o n i c response was the r e s u l t of more ganglion c e l l s responding (Larrabee and Bronk, 1947). Experiments with converging nerve trunks showed that t e t a n i z a t i o n of one p r e g a n g l i o n i c input a c t u a l l y r e s u l t s in decreased e x c i t a b i l i t y of the p o s t g a n g l i o n i c c e l l . This same decrease in e x c i t a b i l i t y extended to responses to exogenous a c e t y l - c h o l i n e and l a s t e d f o r about 1 minute. Other workers have s i n c e disputed the decreased e x c i t a b i l i t y as a necessary r e s u l t of a tetanus (Charlton and B i t t n e r , 1978b; Martin and P i l a r , 1964). In a d d i t i o n , Larrabee and Bronk (1947) described paired-pulse f a c i l i t a t i o n without naming i t : a s i n g l e pre- g a n g l i o n i c v o l l e y was s u f f i c i e n t to p o t e n t i a t e the response to a succeeding v o l l e y , much as Feng (1940) had shown in the NMJ of the toad. Lloyd (1949) f u r t h e r advanced the causal r e l a t i o n s h i p between the p o s t - t e t a n i c p o s i t i v e a f t e r p o t e n t i a l ( h y p e r p o l a r i z a t i o n ) and PTP. He noted the p a r a l l e l s in the amplitude and time course between the p o s t - t e t a n i c a f f e r e n t impulse, the period of h y p e r p o l a r i z a t i o n and the p o t e n t i a t e d mono- synaptic r e f l e x . He proposed a presynaptic basis f o r PTP whereby a post- t e t a n i c h y p e r p o l a r i z a t i o n r e s u l t s in a l a r g e r presynaptic spike that in turn leads to greater t r a n s m i t t e r r e l e a s e . Eccles and R a i l (1951) argued that a - 11 - tetanus of 30-300 v o l l e y s i s followed by a b r i e f p o s t - t e t a n i c p o t e n t i a t i o n of the s y n a p t i c p o t e n t i a l , which occurs w i t h i n 200 msec post-tetanus at a time when the presynaptic spike i s a c t u a l l y smaller than the pre-tetanus c o n t r o l . Wall and Johnson (1958) examined Lloyd's r e s u l t s and hypothesis more d i r e c t l y by t e s t i n g the e x c i t a b i l i t y changes i n a f f e r e n t f i b e r s of a mono- synaptic r e f l e x loop ( W a l l , 1958). They found a marked decrease i n the e x c i t a b i l i t y of the a f f e r e n t f i b e r terminal a r b o r i z a t i o n s c o i n c i d e n t with the magnitude and duration of PTP. L i k e Eccles and R a i l (1951) and Lloyd (1949), Wall and Johnson found a discrepancy i n the immediate p o s t - t e t a n i c period: there was a delay before the p o t e n t i a t e d r e f l e x maximized, whereas the decrease i n a f f e r e n t terminal e x c i t a b i l i t y maximized almost immediate- l y . Given that the presynaptic terminals were indeed h y p e r p o l a r i z e d , they suggested that the i n t e n s i t y of t h i s h y p e r p o l a r i z a t i o n immediately post- tetanus i s ' s u f f i c i e n t to produce anodal block i n some branches of the a f f e - rent f i b e r s . A l t e r n a t e l y , the h y p e r p o l a r i z a t i o n can desynchronize the a f - f e r e n t impulses (del C a s t i l l o and Katz, 1954d) such that the height of the presynaptic spike thus generated w i l l be l e s s than that of the c o n t r o l . Subsequent studies in s p i n a l cord (Eccles and K r n j e v i c , 1959a, 1959b), squid giant axon (Takeuchi and Takeuchi, 1962) and mammalian NMJ (Hubbard and Schmidt, 1963) supported these f i n d i n g s . Furthermore, Hubbard and Schmidt (1963) showed that small increases i n presynaptic spike s i z e are capable of inducing PTP by v i r t u e of a l o g a r i t h m i c r e l a t i o n s h i p between the spike height and end-plate p o t e n t i a l (epp) amplitude. On the other hand, i n t r a - c e l l u l a r recordings at the nerve terminal of avian c i l i a r y g a n g l i a showed - 12 - n e i t h e r changes i n presynaptic membrane p o t e n t i a l nor increased amplitude of the presynaptic spike during paired-pulse f a c i l i t a t i o n and PTP (Martin and P i l a r , 1964). In s p i t e of these c o n f l i c t i n g r e s u l t s , the general consensus i s that f a c i l i t a t i o n and PTP are presynaptic events. 2.3 Mechanism o f - s h o r t - t e r m - p o t e n t i a t i o n The r e l a t i o n s h i p between p o s t - t e t a n i c h y p e r p o l a r i z a t i o n and increased postsynaptic response rested upon the e l u c i d a t i o n of synaptic t r a n s m i s s i o n . In 1934, Dale and Feldberg f i r s t gave evidence that a c e t y l c h o l i n e (ACh) was indeed the chemical r e s p o n s i b l e f o r synatpic transmission at mammalian NMJ. Continued e f f o r t s by various workers l e n t growing support to the idea of chemical transmission ( E c c l e s , 1948; Eccles et - - a l : , 1942; K u f f l e r , 1948), but the debate between e l e c t r i c a l and chemical transmission was not l a i d to r e s t u n t i l Eccles and MacFarlane's study (1949) of a n t i c h o l i n e s t e r a s e s and endplate p o t e n t i a l . The overwhelming evidence supported chemical transmission mediated by ACh. Del C a s t i l l o and Katz advanced t h e i r quantal theory of neuromuscular transmission i n 1954. Drawing from F a t t and Katz' (1952a, 1952b; 1953) observations of spontaneous miniature end-plate p o t e n t i a l s (mepp) at the f r o g NMJ, del C a s t i l l o and Katz observed i n the i s o l a t e d nerve-muscle prepa- r a t i o n bathed i n high magnesium (Mg + +) and/ or low calcium ( C a + + ) , that s t i m u l a t i n g the nerve r e s u l t e d i n epps whose minimal s i z e equals the mean amplitude of spontaneous mepps (del C a s t i l l o and Katz, 1954b). Most impor- t a n t l y , del C a s t i l l o and Katz noted that where the epps were not of the minimal s i z e , t h e i r amplitudes could be c l o s e l y p r e d i c t e d by whole number m u l t i p l e s of the mean mepp. Thus: m x mepp = epp - 13 - where mepp i s the elementary u n i t of t r a n s m i t t e r and quantal content (m) i s the number of such u n i t s per epp. Quantal content i s f u r t h e r defined as the product of the number of quanta a v a i l a b l e f o r r e l e a s e , n, and the probabi- l i t y of r e l e a s e , p; t h e r e f o r e , the mean quantal content i s : m = n x p When the presynaptic terminal i s hyperpolarized to or beyond a c r i t i c - al l e v e l , there i s an increased frequency of spontaneous mepps i n the form of b u r s t s , but the quantal u n i t of these mepps i s not increased (del C a s t i l l o and Katz, 1954d). During such a period of h y p e r p o l a r i z a t i o n , the amplitude of the epps increased, sometimes f o r a few seconds a f t e r the end of the h y p e r p o l a r i z a t i o n . In a d d i t i o n , evoked epps showed an increased amplitude that i s a t t r i b u t a b l e to increased quantal content. E a r l i e r , L i l e y and North (1953) had concluded that PTP i s a nerve terminal phenomenon that i s mediat- ed by an increased evoked r e l e a s e of ACh; increased end-plate s e n s i t i v i t y to the neurotransmitter appeared to have played very l i t t l e p a r t . Another observation explained by the quantal mechanism i s the post- t e t a n i c depression that occurs a f t e r an intense tetanus (Eccles and R a i l , 1951; L i l e y and North, 1953). Under c o n d i t i o n s of normal r e l e a s e , when t r a n s m i t t e r r e l e a s e i s not impaired by agents such as high e x t r a c e l l u l a r c oncentrations of Mg + +, a tetanus of 500 v o l l e y s i s followed by a depres- sion l a s t i n g several hundred msec (Eccles and R a i l , 1951). This i s succeed- ed by a period of PTP. However, i n high Mg , which diminishes t r a n s m i t - t e r r e l e a s e , no depression i s observed (del C a s t i l l o and Katz, 1954c; Feng, 1941b). L i l e y and North (1953) suggested a d e p l e t i o n of quanta during the tetanus to account f o r t h i s depression. I t appears that no s i g n i f i c a n t - 14 - changes i n p occurs during depression while there i s an associated decrease in n (del C a s t i l l o and Katz, 1954c). Del C a s t i l l o and Katz (1954c) a l s o showed that f a c i l i t a t i o n can be accounted f o r by an increased quantal content. To f u r t h e r e x p l a i n the quantal events during and post-tetanus, i t i s necessary to invoke the idea of t r a n s m i t t e r m o b i l i z a t i o n . Elmqvist and Quastel's (1965) f u n c t i o n a l model of t r a n s m i t t e r m o b i l i z a t i o n concurs with the evidence presented by others (Hubbard, 1963; Hubbard and W i l l i s , 1962). In t h i s model, the number of r e a d i l y a v a i l a b l e quanta i n the nerve terminal i s r e l a t i v e l y s m a l l . During a high i n t e n s i t y tetanus, t h i s pool of t r a n s - m i t t e r i s q u i c k l y depleted (Otsuka et a l ; , 1962). A second pool of t r a n s - m i t t e r , presumably not i n the form of packaged quanta, must then be mobil- i z e d i n t o the r e a d i l y a v a i l a b l e pool. Takeuchi (1958) estimates the time constant to r e s t o r e n at the p o s t - t e t a n i c NMJ to be 4-5 seconds. This d e p l e t i o n and subsequent r e s t o r a t i o n of quanta at the terminal thus e x p l a i n s the observation t h a t , although the amplitude and duration of PTP i s propor- t i o n a l to the i n t e n s i t y and number of impulses i n the tetanus, the l a t e n c y to peak p o t e n t i a t i o n a c t u a l l y increases with the i n t e n s i t y of the tetanus (del C a s t i l l o and Katz, 1954c; Eccles and R a i l , 1951; Feng, 1937; Feng et a l ; , 1939; Gasser and Graham, 1932; L l o y d , 1952, 1959). 2.4 I o n i c -mechanism-of-short-term-potentiation E a r l y work on neuromuscular preparations suggested that increased e x t e r n a l potassium (K +) had the same f a c i l i t a t o r y and d e c u r a r i z i n g e f f e c t s as t e t a n i c s t i m u l a t i o n (Feldberg and V a r t i a i n e n , 1934; Wilson and Wright, 1936; Feng and L i , 1941). Other i n v e s t i g a t o r s proposed that an increased - 15 - e x t r a c e l l u l a r K + was res p o n s i b l e f o r PTP (Rosenblueth and Morison, 1937; Grumbach and Wilber, 1940; Walker, 1948). Feng et - al-; (1939) sta t e d that r a i s e d e x t e r n a l K + cannot be involved since PTP i n the presence of potas- sium c h l o r i d e i s l e s s than that induced by t e t a n i c s t i m u l a t i o n alone. Fur- thermore, high K + leads to depression of the NMJ rather than p o t e n t i a t i o n (Feng and L i , 1941). L i l e y and North (1953) proposed another perspective: as i t was known that tetanus causes increased r e l e a s e of both ACh and K +, perhaps the c r i t e r i o n f o r p o t e n t i a t i o n i s an apparent decrease of i n t r a c e l - l u l a r K +, which can be mimicked by r a i s i n g external K +. The work on potassium proved i n c o n c l u s i v e , and with the e l u c i d a t i o n of the quantal mechanism of t r a n s m i t t e r r e l e a s e , the focus of a t t e n t i o n s h i f t e d to calcium. I t was known that C a + + and Mg + + have mutually 'antagon- i s t i c ' e f f e c t s at the NMJ: epps can be reduced and nerve transmission blocked by high e x t r a c e l l u l a r Mg + +; t h i s nerve block could be r e l i e v e d by r a i s i n g e x t r a c e l l u l a r C a + + (del C a s t i l l o and Engbaek, 1954). In the ab- sence of e x t r a c e l l u l a r C a + + , d e p o l a r i z a t i o n of the nerve terminal was i n i t s e l f i n s u f f i c i e n t to cause t r a n s m i t t e r r e l e a s e (Katz and M i l e d i , 1967). But i f the d e p o l a r i z a t i o n was coupled with an i o n t o p h o r e t i c pulse of C a + + l o c a l i z e d to the nerve t e r m i n a l s , then t r a n s m i t t e r r e l e a s e was evoked (Katz and M i l e d i , 1967). Hence, t r a n s m i t t e r r e l e a s e or m u l t i p l i c a t i o n of rel e a s e r e q u i r e s C a + + . Studies of C a + + uptake i n the squid giant axon using 45 ++ Ca showed stimulus-dependent uptake (Hodgkin and Keynes, 1957). Aequorin loaded squid axons emit l i g h t upon d e p o l a r i z a t i o n , showing a C a + + conductance whose time course i s unaffected by t e t r o d o t o x i n and t e t r a e t h y l - ammonium (Baker et a l ; , 1971; L I i n a s e t - a l ; , 1972). - 16 - E x a c t l y how calcium e f f e c t s t r a n s m i t t e r r e l e a s e i s unknown. I t has been proposed that a C a + + a c t i v a t e d complex, CaX, somehow increases the p r o b a b i l i t y of t r a n s m i t t e r r e l e a s e at the terminal membrane (del C a s t i l l o and Katz, 1954a). Assuming that r e l e a s e i s pr o p o r t i o n a l to the fo u r t h power of CaX (Dodge and Rahamimoff, 1967), Katz and M i l e d i (1968) advanced the r e s i d u a l calcium theory of f a c i l i t a t i o n at the f r o g NMJ. They proposed that the t r a n s i e n t increase i n C a + + conductance during the d e p o l a r i z a t i o n ,leads to an i n f l u x of C a + + that combines with X to form the a c t i v e complex CaX. It i s i n t e r e s t i n g to note that elevated l e v e l s of i n t r a c e l l u l a r C a + + per- s i s t beyond the duration of stimulus-evoked r e l e a s e ( M i l e d i and Parker, 1981). I f a subsequent stimulus invades the terminal w i t h i n a c e r t a i n i n - t e r v a l a f t e r the f i r s t stimulus, then the r e s i d u a l C a + + in the terminal w i l l be augmented by another i n f l u x of C a + + . The t r a n s m i t t e r thus r e - leased w i l l be pro p o r t i o n a l to the sum of the i n t r a c e l l u l a r C a + + (or CaX) r a i s e d to the f o u r t h power. A l t e r n a t e mechanisms f o r PTP i n v o l v i n g sodium (Na +) currents or i t s accumulation i n the presynaptic terminal are l a r g e l y unsupported. Post- t e t a n i c p o t e n t i a t i o n of the epp was i n d u c i b l e when a l l external Na + was replaced by i s o t o n i c calcium c h l o r i d e , and when voltage s e n s i t i v e Na + chan- nels were blocked by t e t r o d o t o x i n (Weinreich, 1971). I t i s p o s s i b l e that Na + plays a supportive r o l e , i n d i r e c t l y i n c r e a s i n g i n t r a c e l l u l a r calcium ions by competing at a common ion b u f f e r i n g system or f o r a l i m i t e d energy source f o r extr u s i o n (Birks and Cohen, 1968; Rahamimoff e t - - a l ; , 1980). I n t e r e s t i n g l y , M i s l e r and Hurlbut (1983) were able to induce PTP at the f r o g NMJ i n the absence of e x t r a c e l l u l a r C a + + . I n t r a c e l l u l a r recordings showed - 17 - that PTP o f mepp frequency and epp s i z e can be induced with r e p e t i t i v e stim- u l a t i o n i n 0 mM C a + + and 1-2 mM EGTA, provided that C a + + i s res t o r e d to the bathing medium immediately a f t e r the tetanus ( M i s l e r and Hurlbut, 1983). These authors also suggest a Na + dependent mechanism f o r PTP, but t h e i r s p e c u l a t i o n remains unsubstantiated. 2.5 Anatomy-of-the-hi ppocampus The hippocampus i s part of the o l d e s t c o r t i c a l s t r u c t u r e i n the mam- malian b r a i n . During embryonic development, c e l l s from the mantle l a y e r at the r o s t r a l end of the neural tube p r o l i f e r a t e . This c e l l mass migrates beyond the marginal l a y e r , e v e n t u a l l y surrounding the neural tube to become the c o r t i c a l grey matter ( C r e l i n , 1974). During p r o l i f e r a t i o n and migra- t i o n , the i s o c o r t e x separates from the mantle l a y e r to form the neocortex; the remaining a l l o c o r t e x , which i s the more p r i m i t i v e , maintains i t s a t t a c h - ment to the mantle layer ( F i l i m i n o f f , 1947). There i s also a t r a n s i t i o n a l cortex, c a l l e d the p e r i a l l o c o r t e x , that d i f f e r s i n c y t o a r c h i t e c t u r e from both neocortex and a l l o c o r t e x (Brodmann, 1909). In mammals, the a l l o c o r t e x i s found mostly around the brainstem in a c o r t i c a l convolution that Broca (1878) c a l l e d the l i m b i c lobe. This true a l l o c o r t e x i s subdivided i n t o paleocortex and a r e h i c o r t e x . The former com- p r i s e s the o l f a c t o r y bulb and associated s t r u c t u r e s ; the l a t t e r comprises the subiculum, the hippocampus proper (Amnion's horn), the dentate gyrus ( f a s c i a dentata), precommissural hippocampus and supracommissural hippo- campus (Schwerdtfeger, 1984). The obvious presence of o l f a c t o r y pathways led to the name "rhinencephalon", f o r i t was b e l i e v e d that o l f a c t i o n was the li m b i c lobe's only f u n c t i o n ( K o l l i k e r , 1896; Schaefer, 1898). Later work on - 18 - the l i m b i c system (MacLean, 1952; Papez, 1937) d i s p e l l e d the s i n g l e f u n c t i o n r o l e f o r t h i s o l d cortex. The hippocampal formation c o n s i s t s of the p e r i a l l o c o r t i c a l presubicu- lum, area r e t r o s p h e n i a l i s e, the parasubiculum and the ent o r h i n a l region (Chr o n i s t e r and White, 1975). Of the remaining a r c h i c o r t e x , only Amnion's horn and the f a s c i a dentata w i l l be considered as the hippocampus. This w i l l be f u r t h e r defined so that "hippocampus proper" w i l l denote only Amnion's horn, excluding the subiculum. The hippocampus i s b i l a t e r a l l y symmetrical, shaped l i k e commas or cashew nuts (Green, 1964; T e y l e r and DiScenna, 1984). They l i e d i r e c t l y under the neocortex with t h e i r dorsal ends connected by the commissural f i b e r t r a c t . The body of the hippocampus i s pressed against the medial wall of the i n f e r i o r horn of the l a t e r a l v e n t r i c l e . The ve n t r a l t a i l of the hippocampus f o l l o w s the l a t e r a l v e n t r i c l e toward the corpus callosum (Figure 1). Each hippocampus c o n s i s t s of two i n t e r d i g i t a t i n g a r c h i c o r t i c a l p a r t s , the cornu ammonis (CA) and the f a s c i a dentata (FD). The v e n t r i c u l a r s u r f a c e of the hippocampus i s covered by a white f i b e r l a y e r , the alveus. These f i b e r s are composed mainly of axons from c e l l s of the CA f i e l d s and converge to form the f i m b r i a on the medial surface of the hippocampus (Teyler and DiScenna, 1984). The c e l l s of the hippocampus are in three basic l a y e r s : molecular, p r i n c i p l e , and polimorph (Lorente de No, 1934). ' This i s in con- t r a s t to the s i x (Brodmann, 1909) or seven (Rose, 1926) lay e r s of the neo- cortex. I t i s i n t e r e s t i n g to note that Ramon y Cajal (1893) had a c t u a l l y described Amnion's horn as a seven layered c o r t i c a l s t r u c t u r e . - 19 - Figure 1 Anatomical l o c a t i o n o f the hippocampus i n the r a t b r a i n . The top diagram shows the p o s i t i o n o f the hippocampus with respect to the r e s t of the b r a i n and the bottom diagram i l l u s t r a t e s the various s u b f i e l d s of a t r a n s -verse s e c t i o n o f the hippocampus. Hip. f i s . - hippocampal f i s s u r e I n f r a - infrapyramidal blade o f the dentate granule c e l l l a y e r Supra - suprapyramidal blade o f the dentate granule c e l l l a y e r - 20 - 2.5.1 The-dentate-gyrus The dentate gyrus, as i t s name suggests, i s a V or U-shaped f o l d of cortex that caps the t h i n terminal edge of the hippocampus proper. The p r i n c i p l e c e l l s are the granule c e l l s (Ramon y C a j a l , 1893) found i n a l a y e r 5-10 c e l l s deep, the stratum granulosum. These granule c e l l s send dendrites that p o i n t towards the dentate h i l u s as well as dendrites that point to the v e n t r i c u l a r s u r f a c e . The granule c e l l axons course through the polymorph lay e r and converge about the h i l u s . In a d d i t i o n to polymorphic c e l l s i n t h i s l a y e r , there are i n h i b i t o r y basket c e l l s , which synapse with many granule c e l l s through a supragranular axon plexus (Ramon y C a j a l , 1893). Between the suprapyramidal and infrapyramidal blades of the FD i s the h i l u s , a t r a n s i t i o n region of polymorphic c e l l s and modified pyramidal c e l l s (Lorente de No, 1934). These pyramidal c e l l s are considered part of the t a i l of hippocampus proper and c o n s t i t u t e s the f i e l d CA^ (Lorente de No, 1934). Because the polymorphic l a y e r s of both CA and FD are c o n f l u e n t , the whole region demarcated by an imaginary l i n e drawn between the ends of the stratum granulosm has been c o l l e c t i v e l y l a b e l l e d area dentata (Blackstad, 1956; T e y l e r and DiScenna, 1984). However, c a r e f u l examination of the c y t o - morphology does not support t h i s c l a s s i f i c a t i o n (Ramon y C a j a l , 1893; Lorente de No, 1934). 2.5.2 The-hippocampus-proper The t h i n edge of the cornu ammonis terminates in the h i l u s of the f a s c i a dentata. The t r a n s i t i o n a l pyramidal c e l l s from the h i l u s g r a d u a l l y change to the pyramidal c e l l s of area CA^ (Lorente de No, 1934). Although the CA/, f i e l d i s d i f f i c u l t to detect (Blackstad, 1956; Chronister and - 21 - White, 1975), the cascade of pyramidal c e l l s from CA^ to form the stratum pyramidale of the hippocampus proper i s e a s i l y d i s c e r n i b l e . These pyramidal c e l l s are o r i e n t e d with t h e i r a p i c a l dendrites pointed towards the center of the hippocampus at the b l i n d end of the hippocampal f i s s u r e . The s t r a t i f i c a t i o n at the cornu ammonis i s more involved than that i n the dentate gyrus. A la y e r of axon f i b e r s , the alveus, l i e s next to the surface at the l a t e r a l v e n t r i c l e . Adjacent to the alveus i s a f i b e r plexus of polymorphic c e l l s , the stratum o r i e n s . The next two l a y e r s are the stratum pyramidale and the stratum lucidum, the l a t t e r l a y e r i s rather poor- l y defined i n the rodent hippocampus and i s considered as one with the stratum pyramidale (Lorente de No, 1934). The same a p p l i e s to the dense f i b e r plexus of the stratum radiatum and the stratum lacunosum, considered c o l l e c t i v e l y as stratum radiatum (Lorente de No, 1934). Next to the stratum radiatum i s the stratum moleculare (Lorente de No, 1934). The boundaries of s t r a t a radiatum and oriens mark the terminus of the hippocampus proper at the h i l u s . The boundary at the s u b i c u l a r end i s very sharply defined by the abrupt termination of the stratum pyramidale i n f i e l d CA, (Angevine, 1975; Blackstad, 1956; Ramon y C a j a l , 1893; Lorente de No, 1934). Because of anatomical and p h y s i o l o g i c a l d i f f e r e n c e , the hippocampus proper i s d i v i - ded i n t o several f i e l d s and s u b f i e l d s , each denoted by the l e t t e r s CA and a numerical or alpha-numerical term (Lorente de No, 1934). 2.5.3 The - f i elds of Ammon's- horn Figure 1 i l l u s t r a t e s the d i f f e r e n t s u b f i e l d s of Amnion's horn and the dentate gyrus in a transverse s e c t i o n of the r a t hippocampus. - 22 - 2.5.3.1 F i e l d - - G A / | A s mentioned above, t h i s poorly demarcated zone c o n s i s t s of t r a n s i t i o n a l pyramidal c e l l s in a l a y e r and a confluence of polymorphic c e l l s . The actual presence and appearance of t h i s f i e l d i s extremely v a r i a b l e across species (Geneser-Jensen, 1972). 2.5.3.2 F i e l d - GA^; This f i e l d i s subdivided i n t o CA 3 g, CA 3 b, and C A 3 c (Lorente de No, 1934). The pyramidal c e l l s of CA^ (along with those of CA 2) are the giant pyramids of the hippocampus (Lorente de No, 1934). The a p i c a l dendrites of the CA^ pyramid penetrate the stratum radiatum without l a t e r a l a r b o r i z a t i o n and terminate with 2-3 v e r t i c a l branches in stratum moleculare (Lorente de No, 1934). Thick spines at the proximal portion of these dendrites r e c e i v e e x c i t a t o r y inputs from the mossy f i b e r s of FD (Lorente de No, 1934). S u b f i e l d C A 3 c pyramids have such synapses at both the a p i c a l and basal d e n d r i t e s , whereas C A 3 a and CA-^ pyramids have only a p i c a l synapses with mossy f i b e r s (Lorente de No, 1934). The axon of the CA^ pyramid i s a t h i c k f i b e r that goes to the f i m - b r i a , where i t gives o f f a number of c o l l a t e r a l s (Ramon y C a j a l , 1893; Lorente de No, 1934). Most of these c o l l a t e r a l s are short, terminating in stratum o r i e n s or i n t e r p y r a m i d a l l y . The most notable i s a t h i c k c o l l a t e r a l discovered by Schaffer in 1892. This c h a r a c t e r i s t i c Schaffer c o l l a t e r a l penetrates the stratum pyramidale to run in the stratum radiatum and t e r m i - nates near the j u n c t i o n of CA^ a and CA^ b (Ramon y C a j a l , 1893; Schaffer, 1892). The Schaffer c o l l a t e r a l i s present in n e a r l y a l l C A 3 c axons, about 50% of CA.,, and n e a r l y absent i n CA Q axons (Lorente de No, 1934). 3D oa 2.5.3.3 F-i-el-d -CAg.- This i s a t r a n s i t i o n f i e l d of giant pyramidal c e l l s between CA^ and CA, (Lorente de No, 1934). These c e l l s are smal- - 23 - l e r than those of CA^ and lack both the d e n d r i t i c spines and the Schaffer c o l l a t e r a l s . These pyramids are arranged in i r r e g u l a r rows which form a very t h i n stratum pyramidale. Toward s u b f i e l d CA^ c, t h i s t h i n layer thickens with the i n t r o d u c t i o n of more pyramidal c e l l s . 2.5.3.4 Field--GA^. The CA^ f i e l d i s a l s o d i v i d e d into sub- f i e l d s a, b, and c (Lorente de N6\ 1934). The border between the s u b f i e l d s are not well defined, but the r e s p e c t i v e pyramidal c e l l s are morphologically d i f f e r e n t : amongst the CA^ a pyramids are s u b i c u l a r c e l l s ; C A ^ have the s m a l l e s t pyramidal c e l l s of a l l CA f i e l d s , and CA-^ pyramids are r e l a t i v e - l y large (Lorente de No, 1934). The a p i c a l dendrites of CA-̂  pyramids lack synaptic spines at the proximal p o r t i o n of the primary s h a f t (Ramon y C a j a l , 1893). In the stratum radiatum, these dendrites a r b o r i z e e x t e n s i v e l y i n t o very f i n e branches, where numerous f i n e spines form synapses with the Schaffer c o l l a t e r a l s (Lorente de No, 1934; Ramon y C a j a l , 1893). Basal den- d r i t e s form short t u f t s of i r r e g u l a r branches in the stratum oriens (Ramon y C a j a l , 1893). The CA-̂  primary axon i s a t h i n f i b e r that e s s e n t i a l l y courses through the stratum oriens to the f i m b r i a (Lorente de No, 1934). Axon c o l - l a t e r a l s may cross the stratum pyramidale to ramify i n the stratum r a d i a - tum. Long r e c u r r e n t c o l l a t e r a l s a r i s e from some CA^ axons, t r a v e r s i n g the stratum oriens to run in the alveus towards the f i m b r i a and the subiculum (Lorente de No, 1934). At the border of CA^ and the subiculum, the dense- l y packed stratum pyramidale abruptly stops; the pyramidal c e l l s d i s perse and the stratum oriens g r a d u a l l y t h i n s to a s i n g l e l a y e r in the subiculum (Angevine, 1975; C h r o n i s t e r and White, 1975). - 24 - 2.5.4 Neuronal-pathways-of the-hippocampus The o r d e r l y lamination of c e l l u l a r s t r u c t u r e i n the hippocampus make i t an i d e a l model f o r studying c o r t i c a l o r g a n i z a t i o n . The various s t r a t a of the hippocampus maintain t h e i r r e l a t i v e o r i e n t a t i o n a l l along the l o n g i t u d i - nal a x i s . In a d d i t i o n , the major intrahippocampal t r i - s y n a p t i c system i s organized i n p a r a l l e l planes that are roughly transverse to the l o n g i t u d i n a l a x i s (Andersen et - a-1 -., 1971). This l a m e l l a r o r g a n i z a t i o n has been c l e a r l y demonstrated with the i n v i t r o hippocampal s l i c e (Skrede and Westgaard, 1971) (Figure 2 ) . Although the t r i - s y n a p t i c system w i t h i n the hippocampus r a i s e s the p o s s i b i l i t y of f i b e r recruitment and a cascade e f f e c t of s i g n a l a m p l i f i c a t i o n (Teyler and DiScenna, 1984), there appears to be very l i t t l e divergence from the l a m e l l a r o r g a n i z a t i o n throughout the e n t i r e hippocampus (Andersen et a l : , 1971). 2.5.5 Afferents-to-the-hippocampus 2.5.5.1 P e r f or ant -Path.- The perforant path (PP) c a r r i e s the major e x c i t a t o r y input to the hippocampus (Lorente de No, 1934; Ramon y C a j a l , 1893). These f i b e r s o r i g i n a t e from the i p s i l a t e r a l medial and l a t e r a l ento- r h i n a l c o r t i c e s (EC), c r o s s i n g the hippocampal f i s s u r e to form synapses at the dendrites of the dentate granule c e l l s , as wel l as with the dendrites of some CA3 pyramids (Hjorth-Simonsen, 1973; Hjorth-Simonsen and Jeune, 1972). There i s a topographical s p e c i f i c i t y to the synapses i n that inputs from a given part of the EC p r o j e c t only to a l i m i t e d part of the stratum granulo- sum, but with equal d e n s i t y along the e n t i r e length of the f a s c i a dentata (L#mo, 1971). Another main e x c i t a t o r y input i s the commissural f i b e r s from the f i m b r i a ( B l a c k s t a d , 1956), which o r i g i n a t e from the c o n t r a l a t e r a l CA, Figure 2 Anatomical diagram of a t r a n s v e r s e l y sectioned r a t hippocampal s l i c e showing the various a f f e r e n t , e f f e r e n t and i n t r i n s i c pathways. Alv - alveus Comm - commissural input Ento - entorhinal cortex Fim - f i m b r i a HF - hippocampal f i s s u r e mf - mossy f i b e r s pp - perforant path Sch - S c h a f f e r c o l l a t e r a l s - 26 - and CA4 pyramidal c e l l s (Hjorth-Simonsen and Laurberg, 1977; Swanson et a l : , 1978). 2 .5 .5 .2 A f f e r e n t s - to -the- - G A 3-region; The axons of the dentate granule c e l l s form the mossy f i b e r a f f e r e n t s to f i e l d CA^. Th e i r t h i n (0.2 pm) f i b e r s make en - passant synapses at the proximal primary shaft of CA 3 dendrites (Blackstad e t - - a l : , 1970). Mossy f i b e r s from the supra- pyramidal granule c e l l s synapse with a p i c a l dendrites of the e n t i r e f i e l d CA 3, while the mossy f i b e r s from the infrapyramidal granules synapse only with the basal dendrites of s u b f i e l d C A 3 c (Lorente de No, 1934; Swanson et a l . , 1978). The synaptic elements at the a p i c a l dendrites are noteworthy: mossy f i b e r boutons, 3-6 urn in diameter and length, completely engulf the branched spines of the a p i c a l dendrite (Hamlyn, 1961). There i s als o evidence of mossy f i b e r synapses at the i n h i b i t o r y basket c e l l s of CA 3 ( F r o t s c h e r , 1985), which i s concrete evidence f o r feedforward i n h i b i t i o n (Douglas, 1978). The commissural input to CA^ o r i g i n a t e s in the homotopic region of the c o n t r a l a t e r a l hippocampus (Andersen and L0mo, 1966). These f i b e r s run through the f i m b r i a and terminate on the basal dendrites of the CA^ pyramids (Blackstad, 1956; Andersen and L$mo, 1966). There i s some evidence of p r o j e c t i o n s from the i p s i l a t e r a l and c o n t r a l a t e r a l f i e l d CA 4 (Schwerdtfeger and Sarvey, 1983). 2 .5 .5 .3 A f f e r e n t s - -to - the- -GA^ -region; The d i s t i n c t i o n of f i e l d CA 2 i s not embraced by a l l workers (Blackstad, 1956; Lorente de No, 1934; T e y l e r and DiScenna, 1984). However, from Lorente de No (1934), i t i s c l e a r that mossy f i b e r s do not make giant synapses with the a p i c a l d e n d r i t i c spines, since the l a t t e r are absent from CA ? pyramids. Local a f f e r e n t s - 27 - from CA-̂  axon c o l l a t e r a l s make synapses at the CA2 basal dendrites (Lorente de No, 1934); commissural p r o j e c t i o n s which have been shown to terminate at CA^ i n both s t r a t a radiatum and o r i e n s , can be i n t e r p r e t e d to have synapses at CA 2 by v i r t u e of the ambiguous d i s t i n c t i o n between these two CA f i e l d s ( B l a c k s t a d , 1956). 2.5.5.4 A f f e r e n t s - to the GA-̂  - region; The major input to CA^ i s from the i p s i l a t e r a l CA^ pyramids v i a the Schaffer c o l l a t e r a l s i n the stratum radiatum (Andersen, 1960; Andersen e-t- - a l ; , 1971; Lorente de No, 1934). These Schaffer c o l l a t e r a l s make numerous en•• passant synapses with the f i n e l y a r borized a p i c a l dendrites (Ramon y C a j a l , 1893). S t i m u l a t i o n of the i p s i l a t e r a l CAg pyramids or the Schaffer c o l l a t e r a l s e l i c i t s the gre a t e s t response from the CA-̂  pyramids (Andersen, 1960; Andersen et- - a l ; , 1971). Commissural p r o j e c t i o n s come from the c o n t r a l a t e r a l CA^ and CA^ pyramids, forming en• passant synapses at both basal and a p i c a l dendrites (Andersen et - a l ; , 1980; Bl a c k s t a d , 1956). The ma j o r i t y of these synapses are found at the a p i c a l dendrites i n the stratum radiatum. Lorente de No (1934) described c o l l a t e r a l s of CA^ that ascend from the alveus to terminate on the basal aspects of the CA-^-CA2 pyramids. Consequently, a c t i v a t i o n of CA-̂  pyramids generates an apparent synaptic p o t e n t i a l i n adjacent CA-̂  pyramids (Andersen, 1975). Autoradiographic evidence n e i t h e r supports nor r u l e s out the existence of t h i s short c o l l a t e r a l (Swanson e t - a l ; , 1978). 2.5.6 E f f e r e n t s from the-hippocampus Aside from commissural output to the c o n t r a l a t e r a l hippocampus, the only major e f f e r e n t from the hippocampus i s from CA-̂  pyramids to the s u b i - - 28 - culum (Lorente de No, 1934; Swanson e t - a l : , 1978). There i s anatomical e v i - dence of a r e c u r r e n t c o l l a t e r a l system from some parts of CA^ and a l l of CAj and CA 2 that terminates back in the entorhinal cortex (Hjorth- Simonsen, 1973; Lorente de No, 1934; Swanson et a l ; , 1978). However, s o l i d e l e c t r o p h y s i o l o g i c a l evidence f o r t h i s r e c u r r e n t network i s l a c k i n g . 2.5.7 Interneurons There are a number of interneuron types in the various s t r a t a of the hippocampus, the most prevalent being the basket c e l l s (Lorente de No, 1934; Ramon y C a j a l , 1893). These basket c e l l s are d i s t r i b u t e d w i t h i n the p r i n c i - ple c e l l l a y e r s of both CA and FD, in c l o s e proximity to the p r i n c i p l e c e l l s (Andersen et- a l : , 1964; Lorente de No, 1934). An axon of a basket c e l l a r b o r i z e s e x t e n s i v e l y , making 200-500 synapses with primary c e l l s w i t h i n a dense plexus of f i b e r s (Andersen et- - a l : , 1964; Strub-le -et a l : , 1978). In the FD, the axon terminals synapse at the soma and dendrites of granule c e l l s ( S t r u b l e et- - a l : , 1978), while those at the CA pyramids terminate at the soma, proximal dendrite and i n i t i a l segment of the axon (Blackstad and Flood, 1963; Kosaka, 1980; Seress and Ribak, 1983). E l e c t r o p h y s i o l o g i c a l evidence suggested that a r e c u r r e n t i n h i b i t o r y system was present in the CA f i e l d (Spencer and Kandel, 1961). Andersen et a l . (1963, 1964) located the s i t e of the i n h i b i t i o n at t h e c o m a of the pyramidal c e l l s and proposed a c i r c u i t of feedback i n h i b i t i o n through which adjacent p r i n c i p l e c e l l s i n h i b i t t h e i r neighbours v i a axon c o l l a t e r a l s to the basket c e l l s (Andersen et - a l ; , 1963, 1964; Kandel and Spencer, 1961). Immunoreactive s t a i n i n g of interneuron terminals f o r glutamic a c i d decar- boxylase (GAD) showed that these interneurons elaborate y-aminobutyric a c i d - 29 - (GABA) (Ribak et - -a-1.-, 1978; Seress and Ribak, 1983). The p o s s i b i l i t y of feedforward i n h i b i t i o n was suggested by the observation of very low t h r e s - hold interneurons in the FD (Buzsaki and E i d e l b e r g , 1981, 1982; Douglas et a l ; , 1983). L a b e l l i n g (Loy, 1978) and degeneration s t u d i e s (Frotscher and Zimmer, 1983) support d i r e c t commissural i n n e r v a t i o n of basket c e l l s from the c o n t r a l a t e r a l homotropic primary c e l l s . Electromicroscopy a l s o show i p s i l a t e r a l i n n e r v a t i o n of CAg basket c e l l s by mossy f i b e r s of the FD (Fr o t s c h e r , 1985). 2.5.8 Longitudinal-association-pathway Lorente de No (1934) f i r s t observed axon c o l l a t e r a l s from CA pyramidal c e l l s t h a t run p a r a l l e l to the long axis of the hippocampus. These c o l l a - t e r a l s a r i s e from CAg pyramids which lack a Schaffer c o l l a t e r a l (Lorente de No, 1934), forming a dense plexus on the a p i c a l side of the pyramidal c e l l s . The f i n e l y branched dendrites p r o j e c t along the axis to CAg g, CA 2 and CA^ C d e n d r i t e s , thereby l i n k i n g the r e l a t i v e l y i s o l a t e d lamin- ates of the t r i - s y n a p t i c system into a powerful i n t e r - l a m e l l a r pathway (Hjorth-Simonsen, 1973; Swanson et a-T:, 1978). The p h y s i o l o g i c a l s i g n i f i - cance of t h i s pathway has not been explored. 2.6 Short-term-potentiation in the hippocampus Gloor (1955) observed marked PTP in the f e l i n e hippocampus a f t e r s t i m u l a t i n g the amygdala. Increased spike amplitude of the hippocampal r e s - ponse p a r a l l e l e d the decrease in latency to spike generation. In a d d i t i o n , a decrease in e x c i t a t o r y t h r e s o l d and several phases of p o s t - t e t a n i c depres- sion were d e s c r i b e d . D i r e c t t e t a n i c s t i m u l a t i o n of a f f e r e n t s to the hippo- campal pyramidal c e l l , presumably through commissural f i b e r s v i a the f i m - - 30 - b r i a , led to PTP of the population EPSP and population spike, and decreased latency (Campbell and S u t i n , 1959). T e t a n i c s t i m u l a t i o n of the perforant path r e s u l t e d in PTP of pyramidal c e l l population EPSP and population spike (Gloor et- a l : , 1964); these authors a t t r i b u t e d the PTP to an increase in t r a n s m i t t e r r e l e a s e , a pre-synaptic event. More recent studies of STP in the hippocampus and f a s c i a dentata have revealed several components that are analogous to those found at the neuromuscular j u n c t i o n : f a c i l i t a t i o n (Creager et- - a l : , 1980), augmentation (McNaughton, 1982), and p o s t - t e t a n i c p o t e n t i a t i o n (Racine and Milgram, 1983). These authors agree that STP has a presynaptic locus and i s due to elevated t r a n s m i t t e r r e l e a s e (Abraham et al••, 1985; McNaughton, 1982). However, there i s no general agreement on the p h y s i o l o g i c a l s i g n i f i c a n c e of STP. Creager e t - - a l . (1980) suggested no s i g n i f i c a n c e in normal f u n c t i o n , perhaps some r o l e in e p i l e p t o g e n e s i s ; others suggest a p o s s i b l e r o l e f o r STP in short-term memory (Goddard, 1980; Racine and Milgram, 1983). The other point of concurrence i s that STP and long-term p o t e n t i a t i o n are decidedly d i f f e r e n t processes; that i s , STP does not l e a d , by graded increments or c r i t i c a l t h r e s o l d , to LTP (McNaughton, 1982; T e y l e r e t - a l . , 1982). 2.7 A s s o c i a t i v e - i n d u c t i o n - o f - p o t e n t i a t i o n : - - a f f e r e n t - c o o p e r a t i v i t y Long-term p o t e n t i a t i o n i s now the most promising candidate as a s u b s t r a t e f o r l e a r n i n g and memory. Several c h a r a c t e r i s t i c s of LTP are q u a l i t a t i v e l y s i m i l a r to some s a l i e n t features of l e a r n i n g and memory. For instance, LTP i s input s p e c i f i c , showing p o t e n t i a t i o n only at the pathways that have been t e t a n i z e d (homosynaptic p o t e n t i a t i o n ) (Andersen e t - a l : , 1977; Lynch et a l ; , 1977), and only a b r i e f event (tetanus) i s necessary f o r a - 31 - prolonged e f f e c t . Recently, LTP in the dentate gyrus and the hippocampus have been i n - duced v i a c o - a c t i v a t e d a f f e r e n t s (Barrionuevo and Brown, 1983; Lee, 1983; Levy and Steward, 1979; McNaughton et- a l ; , 1978). Long-term p o t e n t i a t i o n can be induced only i f a large number of a f f e r e n t f i b e r s to the t a r g e t c e l l s are t e t a n i z e d in synchrony, whereas STP can be produced by t e t a n i c s t i m u l a - t i o n of only one (McNaughton, 1983). Rather than t e t a n i c s t i m u l a t i o n of one a f f e r e n t pathway, a s s o c i a t i v e induction allows two or more separate but con- vergent pathways to cooperate. McNaughton et- a l ; , (1978) f i r s t showed c o o p e r a t i v i t y of c o - a c t i v e a f f e r e n t s i n the f a s c i a dentata. They t e t a n i z e d the l a t e r a l and medial e n t o r h i n a l pathways e i t h e r s e p a r a t e l y or simultaneously to produce LTP. I t should be noted that these separate but convergent pathways are independent- l y capable of p o t e n t i a t i o n . When the two pathways were t e t a n i z e d simul- taneously, the r e s u l t a n t LTP was equal to and often greater than the sum of the independently produced LTP. Furthermore, these authors showed that spike discharge of the postsynaptic granule c e l l was in i t s e l f i n s u f f i c i e n t f o r the induction of LTP. Their conclusion that LTP i s a cooperative pheno- menon a l s o r e s t s on the f i n d i n g that stimulus i n t e n s i t y , and not tetanus f r e - quency or duration, i s the determinant f o r LTP; high i n t e n s i t y tetanus can a c t i v a t e synchronously more f i b e r s of d i f f e r i n g thresholds to produce a l a r g e r LTP than a tetanus of lower i n t e n s i t y . This dependence of LTP magni- tude on stimulus i n t e n s i t y has since been found by other workers (Lee, 1983; Wigstrom and Gustafsson, 1983a) - 32 - Another v a r i a t i o n of a s s o c i a t i v e induction of LTP involves the p a i r i n g of a weak and a strong tetanus (Barrionuevo and Brown, 1983). In t h i s s t u - dy, both a weak input and a strong input were each located in the a f f e r e n t f i b e r s of the stratum radiatum that converge on a population of CA^ pyramidal c e l l s . Stimulation of these non-overlapping pathways a f f e c t e d d i f f e r e n t i a l l y the c o n t r o l response to a c o n t r o l stimulus at the weak i n - put: t e t a n i c s t i m u l a t i o n of the strong input r e s u l t e d in heterosynaptic depression (Lynch e t a l : , 1977); t e t a n i c s t i m u l a t i o n of the weak input r e - s u l t e d in PTP. However, when both pathways were stimulated simultaneously, the weak co n t r o l stimulus e x h i b i t e d PTP and LTP. Kelso and Brown (1986) have shown that the two t e t a n i w i l l not induce a s s o c i a t i v e LTP i f separated by 200 msec. Levy and Steward (1983) have described the temporal r e q u i r e - ments f o r a s s o c i a t i v e LTP and long-term depression. These authors showed that LTP can be induced without temporal overlap, but the separation between weak and strong inputs cannot exceed 5-20 msec, otherwise, long-term depres- sion masks any manifestations of LTP. Douglas (1978) had reported a 2-3 msec i n t e r v a l f o r a s i m i l a r induction of LTP. Thus, i t appears that a s s o c i a t i v e i n d u c t i o n of p o t e n t i a t i o n has seve- r a l c r i t e r i a : 1. A c r i t i c a l t e t a n i c stimulus i n t e n s i t y must be exceeded, the stimulus i n t e n s i t y being greater than the minimal needed to produce an EPSP (McNaughton et - a 1 ~., 1978); 2. The a s s o c i a t i v e l y potentiated pathway does not have to produce LTP by i t s e l f (Barrionuevo and Brown, 1983); 3. The p a i r i n g or a s s o c i a t i o n of the separate a f f e r e n t s must take place within a narrow temporal window (Douglas, 1978; Kelso and Brown, 1986; Levy and Steward, 1983); 4. Spike discharge of the postsynaptic c e l l i s not neces- - 33 - sary f o r a s s o c i a t i v e induction of LTP (Douglas, 1978; Lee, 1983; McNaughton e t - a l ; , 1978; Wigstrom e t - a l ; , 1983) 3 METHODS 3.1 Preparation of s i i c e s Male Wistar r a t s (75-125 g) were placed on top of an i c e pack i n s i d e a des s i c a t o r j a r . The d e s s i c a t o r j a r was closed and a mixture of anesthetic gas c o n t a i n i n g 2% halothane in 95% 0 2 and 5% C0 2 (carbogen) was fed i n t o the c o n t a i n e r . The i c e pack lowered the body temperature to 31-32°C (mea- sured r e c t a l l y ) , presumably decreasing the body's metabolic demands. A f t e r 20-30 minutes, the r a t was taken from the d e s s i c a t o r j a r and the top of the s k u l l exposed v i a an a n t e r o p o s t e r i o r i n c i s i o n of the s k i n . The s k u l l p l a t e s were c a r e f u l l y removed and the dura mater s l i t open to expose the b r a i n . A copious amount (about 10 mL) of c o l d (4°C) normal perfusate was poured onto the brain to cool i t as well as to c l e a r the f i e l d of blood. The brain was separated from the s p i n a l cord with a th i n blade at the pontine l e v e l . The ol f a c t o r y t r a c t s were then severed, followed by severance of the o p t i c nerves, and the whole brain was then removed from the c r a n i a l v a u l t . Again about 5 mL of c o l d medium was poured over the brain before one or both hippocampi were d i s s e c t e d f r e e . The f r e e d hippocampus was placed on the c u t t i n g platform with i t s long axis perpendicular to the blade of the Mcllwain t i s s u e chopper. The hippo- campus was chopped i n t o transverse s l i c e s of 500 urn t h i c k and t r a n s f e r r e d as - 34 - a whole to a nylon net submerged in a p e t r i dish of c o l d medium saturated with carbogen. The s l i c e s were then c a r e f u l l y separated with a s p a t u l a and about s i x s l i c e s were arranged on the nylon net; the remaining s l i c e s were disc a r d e d . A second nylon mesh was placed over the s l i c e s , e f f e c t i v e l y anchoring them in a sandwich arrangement. This prevented movement of the s l i c e s during the experiment. These sandwiched s l i c e s were taken from the p e t r i d i s h and placed in the s l i c e chamber, where they were perfused with normal medium at a r a t e of 3 ml/min. Elapsed time between the s t a r t of sur- gery and placement in the s l i c e chamber did not exceed 3 minutes. While in the s l i c e chamber, the s l i c e s were submerged in carbogen saturated normal medium; about 0.5 mm of perfusate covered the top surface of the s l i c e s . In a d d i t i o n , a constant stream of humidified carbogen flowed over the top of the medium. A piece of p a r a f i l m was placed over the chamber opening f o r the duration of the one hour e q u i l i b r a t i o n period to maintain an oxygen-saturated atmosphere. During e q u i l i b r a t i o n , the bath was kept at room temperature (24°C). A l l experiments were done at a bath temperature of 32 ± 0.2°C. Flow r a t e of the medium remained unchanged. To minimize v a r i a - b i l i t y due to d i f f e r e n t durations of ex - vivo p e r f u s i o n , only one s l i c e from each animal was used per experiment. Only those s l i c e s which e x h i b i t e d a s t a b l e response to a t e s t stimulus (60-100 uA, 0.2 msec duration, negative pulses) over a 30 minute period were used. 3.2 S l i c e - b a t h The s l i c e bath has been described in d e t a i l in a p u b l i c a t i o n from t h i s l a b o r a t o r y (Murali Mohan and Sastry, 1984). Figure 3 gives a diagrammatic i l l u s t r a t i o n of the in - v i t r o s l i c e bath. B a s i c a l l y , the bath has an alumi- - 35 - Fi gure 3 Diagrammatic i l l u s t r a t i o n o f the s l i c e bath used f o r e l e c t r o p h y s i o l o g i c a l recordings from i n v i t r o hippocampal s l i c e s . GP - grounding pin GW - ground wire HA - humidified a i r HB - heater block IN - inner net LM - medium l i n e s LS - s u c t i o n l i n e MF - manifold ON - outer net SC - s l i c e chamber SS - s e c u r i n g screw TP - temperature probe TR - temperature r e g u l a t o r  - 37 - num heating block placed beneath the c i r c u l a r chamber where the hippocampal s l i c e s were perfused. A temperature sensing device attached to the block fed back to a r e g u l a t o r where the bath temperature was set and d i s p l a y e d . A length of polyethylene tubing was threaded through holes bored in the alumi- num block. One end of t h i s tubing was i n s e r t e d through a hole d r i l l e d into the side of the chamber; t h i s i s the i n l e t . P erfusing medium fed i n t o the other end of the tubing from a r e s e r v o i r was heated v i a the aluminum block to approximately bath temperature before entering the chamber. Waste medium flowed beyond the c i r c u l a r chamber into a long trench where i t was a s p i r a t e d by gentle s u c t i o n . The p o s i t i o n of the suction tube determined the depth of the perfusate at the chamber. Humidified carbogen was blown over the s l i c e s through a tube f i x e d to the top of the bath and the whole bath was f i x e d to a s t a i n l e s s s t e e l p l a t e v i a two screws. The switching of perfusates was f a c i l i t a t e d by a manifold between the r e s e r v o i r ( s ) and the bath i n l e t tubing. Changing the p e r f u s i n g medium was accomplished simply by clamping shut a l l but the d e s i r e d tubing to the mani- f o l d . The normal and p i c r o t o x i n media were contained in d i f f e r e n t r e s e r - v o i r s ; each medium was independently saturated with carbogen at the r e s e r - v o i r s . 453 Perfusion-media The normal medium was of the f o l l o w i n g composition: NaCl, 120 mM; KC1, 3.1 mM; NaH 2P0 4, 1.3 mM; NaHC0 3 > 26 mM; C a C l 2 , 2 mM; MgCl 2, 2 mM; dextrose 10 mM. The pH of t h i s medium was s t a b l e at 7.4 while aerated with carbogen. The p i c r o t o x i n medium contained the same components with the f o l l o w i n g changes: NaH?P0,, 0 mM; C a C l ? , 4 mM; MgCl ? 4 mM; p i c r o - 38 - toxin 0.01 mM. It has been reported that the GABA^ antagonist, p i c r o t o x i n , can f r e e the i n - v i t r o hippocampal s l i c e of GABAergic i n h i b i t i o n , thereby f a c i l i t a t i n g the i n d u c t i o n of LTP (Wigstrom and Gustafsson, 1983b, 1985a). However, p i c r o t o x i n also induces e p i l e p t i f o r m a c t i v i t y i n the hippocampus ( H a b l i t z , 1984) which n e c e s s i t a t e d the increase of both d i v a l e n t c a t i o n s in the per- f u s i n g medium: calcium and magnesium were elevated to s t a b i l i z e the c e l l u - l a r membrane. The higher CaCl,, concentration l e d to s o l u b i l i t y d i f f i c u l - t i e s that were remedied by the omission of NaH2P0^. Notwithstanding the omission, the p i c r o t o x i n medium maintained i t s b u f f e r i n g c a p a c i t y , showing a steady pH of 7.4 while aerated with carbogen. 3.4 Stimulation-systems In most experiments, the e x t r a c e l l u l a r s t i m u l a t i o n e l e c t r o d e s used were m e t a l l i c (SNEX 100, Rhodes E l e c t r o n i c s , r e s i s t a n c e 1-2 Mfi). The con- c e n t r i c c o n f i g u r a t i o n of these b i p o l a r e l e c t r o d e s minimized current spread beyond the stimulus s i t e . Current pulses were generated by a 2 channel Grass Instruments S88 s t i m u l a t o r . Pulses from each channel were passed through a Grass Instruments PSIU6 constant current stimulus i s o l a t i o n u n i t before reaching t h e i r r e s p e c t i v e s t i m u l a t i n g e l e c t r o d e s . A l l s t i m u l a t i o n pulses were negative square waves. For i n t r a c e l l u l a r d e p o l a r i z a t i o n , a monopolar glass e l e c t r o d e f i l l e d with 1 M KC1 and 1.6 M K c i t r a t e served the dual purpose of s t i m u l a t i n g and recording (see f o l l o w i n g s e c t i o n ) . A 4 M N a C l - f i l l e d glass microelectrode or a f i n e - t i p monopolar tungsten e l e c t r o d e was used to stimulate the Schaffer c o l l a t e r a l terminal regions i n e x c i t a b i l i t y t e s t i n g . - 39 - 3.5 Recording-systems Recording microelectrodes were p u l l e d from f i b r e - f i l l e d c a p i l l a r y tubing ( b o r o s i l i c a t e g l a s s , O.D. 1.5 mm, I.D. 1.0 mm, Frederick Haer and Co.) using a Narishige PE-2 microelectrode p u l l e r . E x t r a c e l l u l a r micro- el e c t r o d e s were f i l l e d with 4 M NaCl and had t i p s of approximately 1 urn. T y p i c a l r e s i s t a n c e was 1-2 Mfi. E x t r a c e l l u l a r s i g n a l s were a m p l i f i e d by e i t h e r a World P r e c i s i o n Instruments DAM-5A d i f f e r e n t i a l p r e a m p l i f i e r or a Medical Systems Neurolog AC-preamplifier and AC-DC a m p l i f i e r . The a m p l i f i e d s i g n a l s were then d i s p l a y e d on a Data P r e c i s i o n DATA 6000 waveform ana- l y z e r . Evoked responses were stored and averaged by the analyzer and hard copies of the averaged records (4-8 sweeps) were p l o t t e d on paper by a Hewlett-Packard 7470A graphics p l o t t e r . I n t r a c e l l u l a r r e c o r d i n g / c u r r e n t i n j e c t i o n e l e c t r o d e s were als o p u l l e d from the same c a p i l l a r y tubing with the Narishige p u l l e r . E l e c t r o d e t i p s were sub-micron in s i z e and were f i l l e d with 1 M KC1 and 1.6 M K c i t r a t e . T y p i c a l r e s i s t a n c e s were 40-50 Mfi. Signals were a m p l i f i e d with a World P r e c i s i o n Instruments (WPI) M-707 i n t r a c e l l u l a r a m p l i f i e r . Current pulses generated by the Grass Instruments S88 sti m u l a t o r were d i r e c t e d to the cur- rent i n j e c t i o n c i r c u i t b u i l t i n t o the WPI M-707 a m p l i f i e r . Current pulses and i n t r a c e l l u l a r responses were monitored on a Tektronix type 5113 dual beam storage o s c i l l o s c o p e . Records were e i t h e r captured on p o l a r o i d f i l m or p l o t t e d using the Hewlett-Packard 7470A graphics p l o t t e r ( i n some e x p e r i - ments, the a m p l i f i e d responses were fed to the DATA 6000 u n i t where 4-8 sweeps were averaged and p l o t t e d ) . - 40 - 3.6 A s s o c i a t i v e - induction of STP 3.6.1 C o n d i t i o n i n g - b y - t e t a n i c s t i m u l a t i o n - o f f i b e r s These experiments were done in p i c r o t o x i n - c o n t a i n i n g medium. The e f - f e c t s of t e t a n i c c o n d i t i o n i n g t r a i n s on the induction of STP were examined. A b i p o l a r t e s t s t i m u l a t i n g e l e c t r o d e (S2) was placed in the stratum r a d i a - tum of the CA3 region to stimulate the Schaffer c o l l a t e r a l s (Figures 4, 5) . The s t i m u l a t i n g current of the t e s t input ( S 2 ) was adjusted to pro- duce a "weak" (200-600 yV) population EPSP as recorded i n the a p i c a l d e n d r i - t i c area of CA^. The c o n d i t i o n i n g e l e c t r o d e was placed i n one of three p o s s i b l e p o s i t i o n s : in the stratum radiatum on the s u b i c u l a r side of the reco r d i n g e l e c t r o d e (Figure 4); in the stratum oriens (Figure 4); and in the alveus (Figure 5) to stimulate the CA-̂  pyramidal c e l l s a n t i d r o m i c a l l y . This c o n d i t i o n i n g input was the "strong" input (S^) which produced r e l a - t i v e l y l arge responses at the rec o r d i n g s i t e : stratum radiatum EPSP was 1-3 mV; stratum oriens EPSP was 2-5 mV, with a superimposed population spike of 0.5-1 mV; and the alv e a r antidromic compound a c t i o n p o t e n t i a l was 3-7 mV. The recording e l e c t r o d e was placed i n the a p i c a l d e n d r i t i c region of CA^ where a t e s t impulse from S 2 produced the maximal EPSP (Figures 4, 5). The 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 n g the S-̂  and S 2 inputs were placed so that non-overlapping a f f e r e n t s would be stimulated. This c r i t e r i o n was t e s t e d by paired pulse experiments. The second t e s t response to paired pulse s t i m u l a t i o n ( i n t e r s t i m u l u s i n t e r v a l of 50 msec) of S 2 was poten- t i a t e d over c o n t r o l . I f the "strong" input (S^) was a c t i v a t e d 50 msec before a s i n g l e pulse of S 2, then the t e s t response showed e i t h e r no change or a s l i g h t decrease i n amplitude (heterosynaptic depression [Lynch e t - a l ; , Figure 4 Experimental arrangement f o r a s s o c i a t i v e induction of STP by c o n d i t i o n i n g of stratum radiatum and stratum o r i e n s . Conditioning t r a i n s c o n s i s t e d o f 10 pulses at 100 Hz (1-10 t r a i n s at 5 second i n t e r v a l s ) and were e i t h e r d e l i -vered alone or in conjunction with a s i n g l e stimulus of the t e s t (S2) input at 1 msec f o l l o w i n g the i n i t i a t i o n of each t r a i n . In some e x p e r i -ments, the temporal r e l a t i o n s h i p between the c o n d i t i o n i n g t r a i n and the t e s t EPSP f o r STP i n d u c t i o n was determined. The t e s t s t i m u l a t i o n (S2) was given at various i n t e r v a l s between -100 and +100 msec with r e s p e c t to the onset of the c o n d i t i o n i n g t r a i n ( S i 0 , stratum o r i e n s ) . R - e x t r a c e l l u l a r r e c o r d i n g e l e c t r o d e Sio - s t i m u l a t i n g e l e c t r o d e f o r stratum oriens c o n d i t i o n i n g S\r - s t i m u l a t i n g e l e c t r o d e f o r stratum radiatum c o n d i t i o n i n g S2 - s t i m u l a t i n g e l e c t r o d e f o r evoking stratum radiatum t e s t population EPSP Figure 5 Experimental arrangement f o r a s s o c i a t i v e induction of STP by alvear c o n d i -t i o n i n g . As in the case of stratum radiatum and stratum o r i e n s c o n d i t i o n -ing, alvear c o n d i t i o n i n g a l s o c o n s i s t e d o f t r a i n s o f 10 pulses at 100 Hz (1-10 t r a i n s given every 5 seconds). These c o n d i t i o n i n g t r a i n s were e i t h e r d e l i v e r e d alone or p a i r e d with a s i n g l e stimulus o f the stratum radiatum t e s t input (S2) at 1 msec f o l l o w i n g the onset o f the t r a i n . R - e x t r a c e l l u l a r r e c o r d i n g e l e c t r o d e 51 - s t i m u l a t i n g e l e c t r o d e f o r alv e a r c o n d i t i o n i n g 52 - s t i m u l a t i n g e l e c t r o d e f o r evoking t e s t stratum radiatum population EPSP - 43 - 1977]). This i n d i c a t e s no overlap between S 2 and S-^; overlapping a f - f e r e n t s r e s u l t e d i n a potentiated S 2 response with the S 2 paired pulse experiments (see Figure 8A_). Only non-overlapping inputs were used in the experiments. Since a l l experiments were done in 10 pM p i c r o t o x i n , the pos- s i b i l i t y of an i n h i b i t i o n masking a potentiated S 2 response was mini- mized. The frequency of S^ and S 2 s t i m u l a t i o n s was 0.1 Hz so that each stimulus a l t e r n a t e d at 5 second i n t e r v a l s . The c o n d i t i o n i n g tetanus was d e l i v e r e d through S^ as t r a i n s of impulses. Each t r a i n c o n s i s t e d of 10 impulses at 100 Hz. One, f i v e or ten t r a i n s were given; the frequency of m u l t i t r a i n c o n d i t i o n i n g was 0.2 Hz (one t r a i n every 5 seconds). Each t r a i n was e i t h e r d e l i v e r e d alone (without the concomitant a c t i v a t i o n of the t e s t input, S 2) or paired with a s i n g l e s t i m u l a t i o n of S 2 at 1 msec a f t e r the onset of the c o n d i t i o n i n g t r a i n . A f t e r t e t a n i c c o n d i t i o n i n g , S^ and S 2 were returned to the pre-tetanus frequency of 0.1 Hz. 3.6.2 Gonditioning b y - i n t r a c e l l u l a r d e p o l a r i z i n g - pulses The r o l e of postsynaptic c e l l d e p o l a r i z a t i o n i n the a s s o c i a t i v e induc- t i o n of STP and LTP was examined using i n t r a c e l l u l a r recording (see Figure 6 f o r experimental arrangement). Two separate t e s t inputs were used here: one in stratum oriens and one in stratum radiatum. An i n t r a c e l l u l a r r e c o r d - ing e l e c t r o d e at the stratum pyramidale of the CA-̂  area recorded s i n g l e c e l l EPSPs. Stimulus i n t e n s i t y f o r each t e s t input was adjusted to produce a "weak" EPSP of about 30% of maximum. Control s t i m u l a t i o n frequency f o r each input was once every 15 seconds; they were a l t e r n a t e d so that there was an i n t e r v a l of 7.5 seconds between each successive s t i m u l a t i o n . - 44 - R / D Experimental arrangement f o r a s s o c i a t i v e i n d u c t i o n of STP by i n t r a c e l l u l a r i n j e c t i o n of d e p o l a r i z i n g c u r r e n t . I n t r a c e l l u l a r d e p o l a r i z i n g pulses (3-10 nA, 75-200 msec duration) were e i t h e r given alone or paired with a s i n g l e s t i m u l a t i o n of the stratum radiatum t e s t EPSP (S2) at 1 msec f o l l o w i n g onset of the pulse. The stratum oriens t e s t EPSP ( S i ) served as a c o n t r o l and was not paired with any d e p o l a r i z i n g i n j e c t i o n s . R/D - i n t r a c e l l u l a r e l e c t r o d e f o r r e c o r d i n g and i n j e c t i o n of d e p o l a r i z i n g current pulses 51 - s t i m u l a t i n g e l e c t r o d e f o r evoking t e s t stratum o r i e n s EPSP 52 - s t i m u l a t i n g e l e c t r o d e f o r evoking t e s t s t r a t u m r a d i a t u m EPSP - 45 - Instead of a separate input f o r the c o n d i t i o n i n g tetanus t r a i n s , suprathreshold d e p o l a r i z i n g pulses were i n j e c t e d i n t o the impaled CA-̂  c e l l through the i n t r a c e l l u l a r e l e c t r o d e . These d e p o l a r i z a t i o n s (75-200 msec duration; 3-10 nA; one, f i v e or ten d e p o l a r i z i n g commands at 0.2 Hz) were used to mimic the e f f e c t s of s y n a p t i c a l l y driven d e p o l a r i z a t i o n s induced by t e t a n i of inputs. The d e p o l a r i z a t i o n s were given e i t h e r alone or p a i r e d with one s t i m u l a t i o n of the stratum radiatum t e s t input at 1 msec a f t e r the onset of the d e p o l a r i z a t i o n . The stratum oriens input was never paired with any d e p o l a r i z i n g current i n j e c t i o n s , thus serving as a secondary c o n t r o l in the experiment. 3.6.3 Temporal-requirements-governing-the-induction of STP The temporal r e l a t i o n s h i p between the c o n d i t i o n i n g tetanus and the t e s t stimulus was examined by varying the i n t e r v a l between the onset of t e t a n i c s t i m u l a t i o n and the s t i m u l a t i o n of the t e s t input. The t e s t input (S 2) was evoked by s t i m u l a t i o n of stratum radiatum while the c o n d i t i o n i n g input (S^) was evoked by s t i m u l a t i o n of stratum oriens (Figure 4 ) . Each input was stimulated at 0.1 Hz ( a l t e r n a t i n g every 5 seconds) except during the c o n d i t i o n i n g tetanus, which was f i x e d at f i v e t r a i n s at 0.2 Hz, each t r a i n c o n s i s t i n g of 10 pulses at 100 Hz. The t e s t stimulus ( S 2 ) e i t h e r preceded or succeeded the onset of each t e t a n i c c o n d i t i o n i n g t r a i n by an i n t e r v a l of 0-100 msec. 3.7 A s s o c i a t i v e - •induction of- - -Schaffer- - c o l l a t e r a l - terminal - - e x c i t a b i l i t y changes Changes in presynaptic terminal e x c i t a b i l i t y were assessed using the method of Wall (1958). Figure 7 shows the experimental arrangement. A - 46 - Figure 7 Experimental arrangement f o r e x c i t a b i l i t y t e s t i n g of Schaffer c o l l a t e r a l terminal r e g i o n s . The e x t r a c e l l u l a r r e c o r d i n g e l e c t r o d e (R) was placed i n the CA3 c e l l body lay e r to monitor a l l - o r - n o n e a c t i o n p o t e n t i a l s i n s i n g l e CA3 neurons. The CA3 neuron recorded from was a c t i v a t e d by s t i m u l a t i o n at the Schaffer c o l l a t e r a l terminal regions (S2) l o c a t e d at the a p i c a l d e n d r i t i c region of CAi. The e x c i t a b i l i t y o f the terminal regions ( d e t e r -mined by the amount of current r e q u i r e d to discharge the c e l l in 1-2 of 3 consecutive attempts) was monitored before and a f t e r c o n d i t i o n i n g . Condi-t i o n i n g t r a i n s . were d e l i v e r e d e i t h e r to stratum o r i e n s ( S i 0 ) or stratum radiatum ( S i r ) arid c o n s i s t e d of 1, 5 or 10 t r a i n s o f 10 pulses a t 100 Hz given every f i v e seconds. The c o n d i t i o n i n g was e i t h e r given alone or p a i r e d with a s i n g l e suprathreshold stimulus of the t e s t f i b r e at 1 msec f o l l o w i n g the onset o f each t r a i n . It was confirmed that the c o n d i t i o n i n g t r a i n s d i d not a c t i v a t e the experimental c e l l . - 47 - monopolar glass microelectrode ( S 2 ) or a f i n e - t i p monopolar tungsten e l e c - trode was p o s i t i o n e d i n the stratum radiatum of f i e l d CA-̂ , presumably i n the area of Schaffer c o l l a t e r a l synapses at the a p i c a l d e n d r i t e s . Stimula- t i o n of the Schaffer c o l l a t e r a l terminals through the S 2 e l e c t r o d e r e s u l t s in antidromic all-or-none a c t i o n p o t e n t i a l s (AP) in the CA3 pyramidal soma. An e x t r a c e l l u l a r recording e l e c t r o d e placed i n the CA3 stratum pyramidale recorded these a c t i o n p o t e n t i a l s . An e l e c t r o d e to a c t i v a t e a "strong" input (S^) was p o s i t i o n e d i n stratum oriens or stratum radiatum i n order to provide the c o n d i t i o n i n g tetanus. S t i m u l a t i o n of the "strong" input alone d id not induce an antidromic a c t i o n p o t e n t i a l in the c e l l recorded from in f i e l d CA 3. Te t a n i c s t i m u l a t i o n s of S-̂  (10 pulses at 100 Hz; one, f i v e or ten t r a i n s ; one t r a i n every f i v e seconds) were d e l i - vered alone or paired with a s i n g l e s t i m u l a t i o n of S 2 (a suprathreshold stimulus i n t e n s i t y was used) at 1 msec a f t e r the onset of the tetanus. Schaffer c o l l a t e r a l terminal e x c i t a b i l i t y was measured as the amount of current r e q u i r e d to generate an a c t i o n p o t e n t i a l i n the CA^ neuron in 1 or 2 of 3 consecutive attempts by s t i m u l a t i o n through the t e s t ( S 2 ) e l e c - trode. An increase in e x c i t a b i l i t y would be r e f l e c t e d as a decrease i n the amount of current required to discharge the c e l l and v i c e versa. A bas e l i n e l e v e l of current to produce the antidromic a c t i o n p o t e n t i a l by s t i m u l a t i o n through the t e s t ( S 2 ) el e c t r o d e was determined once every 2 to 3 minutes over 15 minutes before any c o n d i t i o n i n g s t i m u l a t i o n s were given. Three a t - tempts were made f o r every stimulus i n t e n s i t y t e s t e d . - 48 - 4 RESULTS 4.1 Stratum-radiatum-conditioning The e f f e c t s of strong c o n d i t i o n i n g t e t a n i on a weak population r e s - ponse were examined i n these experiments. T e t a n i c s t i m u l a t i o n of the "strong" c o n d i t i o n i n g input (S^) without any concomitant a c t i v a t i o n of the "weak" t e s t input ( S 2 ) was i n s u f f i c i e n t i n i t s e l f to induce any p o t e n t i a - t i o n of the t e s t input ( S 2 ) . A l l unpaired t e t a n i c c o n d i t i o n i n g t r a i n s produced a degree of depression i n the t e s t response (85 ± 4% SEM of co n t r o l at 60 seconds post-10 unpaired t e t a n i c t r a i n s of stratum radiatum, 7 of 8 expts., no change i n 1 of 8) (Figure 8A). P o t e n t i a t i o n of t h i s population EPSP was induced i f the c o n d i t i o n i n g tetanus was paired with one s t i m u l a t i o n of the "weak" t e s t input at 1 msec a f t e r the onset of each t r a i n . A f t e r one paired t r a i n , there was a b r i e f , small p o t e n t i a t i o n of the t e s t response l a s t i n g about two minutes. Increasing the number of paired t r a i n s to f i v e markedly increased the s i z e of the STP induced (Figure 8A_). Repeated p a i r - ings of 1-5 t r a i n s led to repeated STP with no apparent changes in the dura- t i o n of the p o t e n t i a t i o n ; depression d id not set in a f t e r repeated p a i r - ings. The STP, measured at 60 seconds post-10 paired t r a i n s , was 184 ± 8% SEM of c o n t r o l and l a s t e d 2-3 minutes (6 of 8 expts., no change in 2 of 8). This STP was followed by LTP (162 ± 5% SEM of c o n t r o l at 15 minutes post-10 paired t r a i n s , 6 of 8 expts., no change in 2 of 8 ) . Note that depression due to unpaired t r a i n s did not increase p r o p o r t i o n a t e l y with the STP. - 49 - Figure 8 A s s o c i a t i v e induction of STP, LTP and the reduction in the Schaffer c o l l a -t e r a l terminal e x c i t a b i l i t y . (A) The schematic diagram on the l e f t i l l u s -t r a t e s the experimental arrangement. A b i p o l a r t e s t s t i m u l a t i n g e l e c t r o d e (S2) was p o s i t i o n e d i n the stratum radiatum and a b i p o l a r c o n d i t i o n i n g s t i m u l a t i n g e l e c t r o d e ( S i ) was p o s i t i o n e d i n another area of the stratum radiatum. A r e c o r d i n g microelectrode ( c o n t a i n i n g 4 M NaCl) was p o s i t i o n e d in the a p i c a l d e n d r i t i c area of CAi neurones to monitor the t e s t EPSP evoked at 0.2 Hz ( s t i m u l a t i o n strength was adjusted to obtain a response between 300-600 yV). The c o n d i t i o n i n g s t i m u l a t i o n strength was adjusted to evoke a population EPSP of 1-3 mV i n s i z e . I f a twin s t i m u l a t i o n of S? (50 ms i n t e r v a l ) r e s u l t e d in a f a c i l i t a t i o n of the second population EPSP (see i n s e t , l e f t ) and i f a s t i m u l a t i o n of S i preceding S2 s t i m u l a t i o n by 50 ms r e s u l t e d in no f a c i l i t a t i o n o f the second population EPSP (see i n s e t , r i g h t ) , then the S i and S2 s t i m u l a t i o n s were presumed to a c t i v a t e sepa-ra t e input f i b r e s . In a l l experiments, the e f f e c t of unpaired c o n d i t i o n i n g t r a i n s (UC; i . e . , the t e s t s t i m u l a t i o n was o f f during the c o n d i t i o n i n g ; each c o n d i t i o n i n g t r a i n contained 10 pulses at 100 Hz) and of pa i r e d c o n d i t i o n i n g t r a i n s (PC; i . e . , the t e s t s t i m u l a t i o n was on 1 ms a f t e r the onset o f each t r a i n ) were examined on the t e s t population EPSP. During the f i r s t 3 min-utes a f t e r UC or PC, the response was monitored every 15 seconds and at a l l other times at 30 second i n t e r v a l s . The graph on the r i g h t shows r e s u l t s from one experiment. Note STP a f t e r 1 and 5 PCs, and LTP a f t e r 10 PCs. (B) E f f e c t s of the c o n d i t i o n i n g on the e x c i t a b i l i t y of the terminal region o f a Sc h a f f e r c o l l a t e r a l . A monopolar t e s t s t i m u l a t i n g e l e c t r o d e (S2) was p o s i t i o n e d i n the a p i c a l d e n d r i t i c area of the CAi neurones to a c t i v a t e (0.2 ms negative pulses, 3-10 yA, 0.2 Hz) the terminal regions of Schaffer c o l l a t e r a l s so that antidromic a l l - o r - n o n e a c t i o n p o t e n t i a l s (see in s e t ) could be recorded from the CA3 c e l l bodies. A c o n d i t i o n i n g stimu-l a t i o n e l e c t r o d e ( S j ) was p o s i t i o n e d i n the stratum radiatum and the unpaired (UC) and p a i r e d (PC) c o n d i t i o n i n g t r a i n s were a p p l i e d as des c r i b e d i n Au I t was confirmed that the c o n d i t i o n i n g s t i m u l a t i o n d i d not a c t i v a t e the t e s t Schaffer c o l l a t e r a l . During the PC, the s t i m u l a t i o n s t r e n g t h to a n t i d r o m i c a l l y a c t i v a t e the t e s t S c h a f f e r c o l l a t e r a l was increased to 2 times co n t r o l to make sure that the f i b r e was a c t i v a t e d during PC. A s i m i -l a r a c t i v a t i o n of the t e s t f i b r e without the presence of the c o n d i t i o n i n g produced no changes i n the e x c i t a b i l i t y of the t e s t f i b r e ( r e s u l t s not shown). The amount o f c u r r e n t r e q u i r e d to produce an a l l - o r - n o n e a c t i o n p o t e n t i a l was taken as that which induced a spike i n 1-2 of 3 con s e c u t i v e attempts. In the graph to the r i g h t o f the schematic diagram, recordings taken at 30 second i n t e r v a l s were p l o t t e d . Note that 1 and 5 PCs induced a 3 minute decrease while 10 PCs induced a prolonged decrease i n the e x c i t a b i -l i t y of the t e s t f i b r e t e r m i n a l . Results i n (A) and (B_) were from d i f f e r e n t experiments.  - 51 - 4.2 S t r a t u m o r i e n s - c o n d i t i o n i n g The e f f e c t s of stratum oriens c o n d i t i o n i n g on the t e s t stratum r a d i a - tum EPSP were q u i t e s i m i l a r to those seen above. Unpaired c o n d i t i o n i n g t r a i n s to the stratum oriens input (5 t r a i n s of 10 pulses at 100 Hz, one t r a i n every 5 seconds) led to depression of the t e s t EPSP (94 ± 2% SEM of c o n t r o l at 60 seconds post-5 unpaired t r a i n s , n = 23; see Figure 10). P a i r - ing the c o n d i t i o n i n g tetanus with one stratum radiatum s t i m u l a t i o n at 1 ms f o l l o w i n g the onset of each t r a i n led to STP (population EPSP as a % of con- t r o l at 60 seconds post-5 paired t r a i n s : 121 ± 4 SEM, 5 of 5 e x p t s . ) . Since the p h y s i c a l separation between the c o n d i t i o n i n g and t e s t inputs did not appear to a f f e c t the a s s o c i a t i v e induction of p o t e n t i a t i o n , i t i s p o s s i - ble to exclude presynaptic terminal i n t e r a c t i o n s as necessary c r i t e r i a f o r a s s o c i a t i v e i n d u c t i o n . 4.3 A1vear - c o n d i t i o n i n g The r o l e of the postsynaptic c e l l in a s s o c i a t i v e induction of STP and LTP i s unclear. Using alvear s t i m u l a t i o n to a n t i d r o m i c a l l y a c t i v a t e CA^ c e l l s , i t was p o s s i b l e to d e p o l a r i z e the CA-̂  c e l l through a c t i o n p o t e n t i a l discharge that did not involve t r a n s s y n a p t i c responses. The CA-̂  c e l l was stimulated with antidromic t e t a n i c t r a i n s in the same paired and unpaired manner as the previous experiments. As with the above experiments, only the paired c o n d i t i o n i n g t e t a n i induced p o t e n t i a t i o n ; 10 paired c o n d i t i o n i n g t r a i n s produced both STP and LTP (Table 1). Unlike the s y n a p t i c a l l y driven t e t a n i , however, unpaired antidromic t e t a n i did not produce any s i g n i f i c a n t depression of the t e s t response (98 ± 3 % SEM of c o n t r o l at 60 seconds post-10 paired t r a i n s , 6 of 6 e x p t s . ) . - 52 - Table l . Post-Conditioning P o t e n t i a t i o n Induced by P a i r i n g T e t a n i c Trains of the Alveus with a S i n g l e S t i m u l a t i o n of the Test Input. . A lveus Time post-10 paired c o n d i t i o n i n g t r a i n s 60 s 15 min Test population EPSP 146 ± 7 SEM 133 ± 5 SEM as a % of c o n t r o l n = 6 n = 4 - 53 - 4.4 I i n t r a c e l l u l a r current - i n j e c t i o n s Postsynaptic c e l l d e p o l a r i z a t i o n was f u r t h e r examined with i n t r a c e l l u - l a r c u r rent i n j e c t i o n s . A l l stratum oriens e x t r a c e l l u l a r responses from the unpaired s t i m u l a t i o n s e x h i b i t e d only depression a f t e r current i n j e c t i o n (Table 2). In the case of the stratum radiatum responses, the EPSPs f o l l o w - ing p a i r i n g showed p o t e n t i a t i o n whereas the unpaired EPSPs were depressed (Table 2 ) . This pattern of p o t e n t i a t i o n was s i m i l a r to that of other e x p e r i - ments i n v o l v i n g t e t a n i c c o n d i t i o n i n g t r a i n s : i n c r e a s i n g the number of paired d e p o l a r i z i n g commands r e s u l t e d in p r o g r e s s i v e l y l a r g e r STP. With ten paired c o n d i t i o n i n g commands, STP was superimposed on LTP (Figure 9, Table 2). This evidence c l e a r l y suggests the involvement of the postsynaptic c e l l in a s s o c i a t i v e i n d u c t i o n . 4.5 S c h a f f e r c o l l a t e r a l - t e r m i n a l - e x c i t a b i l i t y - c h a n g e s Because STP has been shown to be a presynaptic phenomenon in other e x c i t a b l e j u n c t i o n s (Eccles and K r n j e v i c 1959a, 1959b; Magleby and Zengel, 1975a, 1975b), i t i s p e r t i n e n t to the argument to examine concomitant pre- s y n a p t i c changes during a s s o c i a t i v e l y - i n d u c e d p o t e n t i a t i o n . Unpaired t e t a n - i c c o n d i t i o n i n g t r a i n s to stratum radiatum did not change the e x c i t a b i l i t y of the Schaffer c o l l a t e r a l terminals (amount of current to f i r e c e l l as a % of c o n t r o l : 98 ± 3 SEM at 60 seconds post-5 unpaired t r a i n s , 7 of 7 expts.; 99 ± 2 SEM at 60 seconds post-10 unpaired t r a i n s , 7 of 7 e x p t s . ) . S i m i l a r - l y , unpaired t e t a n i to stratum oriens a l s o r e s u l t e d in no a l t e r a t i o n s in terminal e x c i t a b i l i t y (amount of current to discharge c e l l as a % of con- t r o l : 99 ± 3 SEM at 60 seconds post-5 unpaired t r a i n s , 6 of 6 e x p t s . ) . However, as can be seen in Figure 8, paired t e t a n i c c o n d i t i o n i n g led to a - 54 - Table 2. E f f e c t s of i n t r a c e l l u l a r l y i n j e c t e d d e p o l a r i z i n g current pulses on EPSPs evoked by s t i m u l a t i o n o f stratum radiatum and stratum o r i e n s . A. Stratum radiatum t e s t EPSP Number of c o n d i t i o n i n g pulses 1 5 10 UP P UP P UP P 1 min post-conditioning (EPSP as % of c o n t r o l ) Range 89 Mean±SEM -n 1 129-140 135 * 6 2 72-89 82 ± 3 5 96-186 71-78 134 ± 10 74 ± 1 9 4 95-218 145 ± 14 9 15 min post-conditioning (EPSP as % of control) Range 102 Mean±SEM -n 1 90-112 98 ± 5 2 91-113 100 * 6 5 89-109 87-112 103 ± 4 97 ± 5 9 4 99-162 122 ± 8 9 B . Stratum o r i e n s t e s t EPSP Number of c o n d i t i o n i n g p u l s e s 1 5 10 UP UP UP 1 min post-conditioning (EPSP as % of control) Range Mean±SEM n 86 - 87 87 ± 1 2 71 - 88 82 ± 2 9 71 - 86 76 ± 2 9 15 min post-conditioning (EPSP as % of control) Range Mean±SEM n - 91 - 110 101 * 4 9 88 - 111 98 ± 5 9 P = each c o n d i t i o n i n g d e p o l a r i z i n g command was p a i r e d with one s t i m u l a t i o n o f the t e s t stratum radiatum EPSP at 1 msec f o l l o w i n g the onset o f the cond i t i o n i n g p u l s e . UP a c o n d i t i o n i n g d e p o l a r i z i n g commands were given alone and not p a i r e d with t e s t input s t i m u l a t i o n . - 55 - Figure 9 Induction of STP and LTP by paired c o n d i t i o n i n g d e p o l a r i z a t i o n of a CA^ neuron. To evoke EPSPs in the CAi neuron, b i p o l a r t e s t s t i m u l a t i n g e l e c -trodes were po s i t i o n e d i n stratum radiatum and stratum o r i e n s . A re c o r d i n g i n t r a c e l l u l a r microelectrode in the CAi neuron was used to record the t e s t EPSPs (a.: the c a l i b r a t i o n s represent 10 msec and 5 mV) and to apply the c o n d i t i o n i n g d e p o l a r i z i n g commands (3-10 nA, 75-200 msec, 1-10 commands at 0.2 Hz) (fj: the square wave i s 0 r.lnA and 75 msec). The s t i m u l a t i o n strengths were adjusted to evoke EPSPs at 30% of maximum s i z e . The s t i m u l a -t i o n of stratum radiatum (every 15 seconds) and stratum oriens (every 15 seconds) was arranged i n such a way that there was a 7.5 second delay bet-ween the two s t i m u l a t i o n s . During the unpaired c o n d i t i o n i n g d e p o l a r i z a t i o n (UC), the t e s t EPSPs by stratum o r i e n s and stratum radiatum were not evoked and during the paired c o n d i t i o n i n g d e p o l a r i z a t i o n (PC) the stratum radiatum-induced EPSP was evoked 1 msec a f t e r the onset of the d e p o l a r i z i n g command while the stratum o r i e n s was not sti m u l a t e d . When more than one UC or PC was a p p l i e d , they were given at 0.2 Hz. Stratum radiatum s t i m u l a t i o n at 0.2 Hz without the presence of the c o n d i t i o n i n g d e p o l a r i z a t i o n of the CAi neuron did not r e s u l t i n a change in the s i z e o f the EPSP ( r e s u l t s not shown). Note STP, and LTP of the stratum radiatum-induced, but not of the stratum oriens-induced, EPSP f o l l o w i n g the PC. In the graphs, EPSPs were recorded at 30 second i n t e r v a l s . A f t e r UC and PC, however, recordings were taken at 15 second i n t e r v a l s f o r 3 minutes. The ' r e s t i n g ' membrane poten-t i a l of the neuron at the beginning of the experiment was -65 mV and at the end of the experiment was -61 mV. This i s a t y p i c a l experiment; s i m i l a r r e s u l t s were found i n s i x c e l l s .  - 57 - graded decrease in e x c i t a b i l i t y that p a r a l l e l e d the p o s t - t e t a n i c p o t e n t i a - t i o n s in both time course and magnitude (amount of current to discharge c e l l as a % of c o n t r o l : 185 ± 6 SEM at 60 seconds, post-5 t r a i n paired condi- t i o n i n g by radiatum, 6 of 7 expts., no change in 1 of 7; 138 ± 6 SEM at 60 seconds post-5 t r a i n paired c o n d i t i o n i n g by o r i e n s , 5 of 6 expts., no change in 1 of 6). Tetanic t r a i n s to c o n d i t i o n i n g inputs in e i t h e r stratum oriens or stratum radiatum were able to induce the decrease in terminal e x c i t a b i - l i t y . Increasing the number of paired c o n d i t i o n i n g t r a i n s to ten led to a prolonged decreased in e x c i t a b i l i t y that was a s s o c i a t e d with LTP of the t e s t EPSP (amount of current to discharge c e l l as a % of c o n t r o l : 154 ± 5 SEM at 15 minutes post-10 t r a i n paired c o n d i t i o n i n g by radiatum, 5 of 6 expts., no change in 1 of 6) (Figure 8 ) . A summary of a l l the r e s u l t s i s shown in Table 3. 4.6 Temporal -requirements-for - induction-of-STP For these experiments, the number of pair e d c o n d i t i o n i n g t r a i n s was f i x e d at f i v e because t h i s paradigm was one that most r e l i a b l y induced STP with no LTP. Two parameters were v a r i e d here: the order in which each t e s t and c o n d i t i o n i n g inputs were paired and the i n t e r v a l between the two. In Figure 10, the x-axis i s the i n t e r s t i m u l u s i n t e r v a l between the onset of the c o n d i t i o n i n g t r a i n and the t e s t stimulus. Negative i n t e r s t i m u l u s i n t e r v a l s i n d i c a t e that the t e s t stimulus preceded the onset of the c o n d i t i o n i n g t r a i n s while p o s i t i v e i n t e r v a l s i n d i c a t e the duration between the onset of the c o n d i t i o n i n g t r a i n and the succeeding t e s t stimulus. In the experiments where the t e s t stimulus succeeded the onset of the c o n d i t i o n i n g tetanus, there was s i g n i f i c a n t STP up to an i n t e r s t i m u l u s - 58 - Table 3 . E f f e c t s of p a i r e d and unpaired c o n d i t i o n i n g t r a i n s on S c h a f f e r c o l l a t e r a l terminal e x c i t a b i l i t y . 60 s post-5 t r a i n s 15 min post-10 t r a i n s unpaired paired unpaired p a i r e d stratum radiatum c o n d i t i o n i n g ( t h r e s h o l d 98 ± 3 185 ± 6 99 ± 2 154 ± 5 as a % of c o n t r o l ) n = 7 n = 6 n = 7 n = 5 stratum o r i e n s c o n d i t i o n i n g ( t h r e s h o l d 99 ± 3 138 ± 6* as a % of c o n t r o l ) n = 6 n = 5 * Results are expressed as mean ± SEM. - 5 9 - 90 L- i t t i i i i i i i i -100 -80 -60 -40 -20 0 20 40 60 80 100 INTERSTIMULUS INTERVAL (ms) Figure 10 The l i m i t s of the temporal r e l a t i o n s h i p between c o n d i t i o n i n g and t e s t stimu-li f o r the i n d u c t i o n of a s s o c i a t i v e p o t e n t i a t i o n . The t e s t EPSP was evoked by s t i m u l a t i o n of stratum radiatum and the c o n d i t i o n i n g was achieved through s t i m u l a t i o n of stratum o r i e n s (5 t r a i n s , 10 pulses i n each t r a i n at 100 Hz, one t r a i n every 5 seconds). One t e s t stimulus was p a i r e d with each o f the f i v e c o n d i t i o n i n g t r a i n s at every i n t e r s t i m u l u s i n t e r v a l examined. The c o n d i t i o n i n g - t e s t i n t e r v a l was v a r i e d berween -100 to +100 ms. A negative delay i n d i c a t e s that the t e s t stimulus preceded the onset of the c o n d i t i o n -ing t r a i n and a p o s i t i v e delay i n d i c a t e s the time at which the t e s t popula-t i o n EPSP was evoked f o l l o w i n g the onset of the c o n d i t i o n i n g tetanus. Each point on the graph ( f i l l e d c i r c l e s ) represents mean ± SEM of the t e s t popu-l a t i o n EPSP magnitude measured at 1 minute post-5 paired t r a i n s . The one point represented by the f i l l e d diamond shows a s i g n i f i c a n t depression of the t e s t EPSP at 1 minute post-5 unpaired t r a i n s of stratum o r i e n s ( i . e . , t e s t EPSP was not evoked during c o n d i t i o n i n g ) , n i s shown i n parentheses above each poin t on the graph. - 60 - i n t e r v a l of 80 msec. The curve i s bimodal with a dip in the p o t e n t i a t i o n at the 40-50 msec i n t e r v a l . Maximal p o t e n t i a t i o n was induced when both t e s t and c o n d i t i o n i n g s t i m u l i are a c t i v a t e d simultaneously. Unpaired t r a i n s were given to the c o n d i t i o n i n g stratum oriens input to confirm non-overlap with the t e s t input at stratum radiatum, as well as to show that t e t a n i c condi- t i o n i n g t r a i n s alone did not induce STP. The amount of STP induced by r e v e r s i n g the order of the s t i m u l i ap- pears to drop o f f markedly with respect to i n c r e a s i n g the i n t e r s t i m u l u s i n t e r v a l . S i g n i f i c a n t p o t e n t i a t i o n was l i m i t e d to an i n t e r s t i m u l u s i n t e r v a l of -50 msec. The s i g n i f i c a n c e of these r e s u l t s are examined in the f o l l o w - ing s e c t i o n . 5 DISCUSSION The r e s u l t s show that STP and LTP in the hippocampal s l i c e can be i n - duced a s s o c i a t i v e l y without t e t a n i c s t i m u l a t i o n of the p o t e n t i a t e d pathway. Studies in the current l i t e r a t u r e found a s s o c i a t i v e i n d u c t i o n of p o t e n t i a - t i o n only in t e t a n i z e d pathways (Barrionuevo and Brown, 1983; Lee, 1983; Levy and Steward, 1979; McNaughton et- a l ; , 1978). In the present e x p e r i - ments, the synaptic response to a s i n g l e a f f e r e n t v o l l e y i s p o t e n t i a t e d f o r a v a r i a b l e duration when i t c o i n c i d e s with a c o n d i t i o n i n g t e t a n i z a t i o n given at a separate converging a f f e r e n t pathway. This c o i n c i d e n t a l s t i m u l a t i o n of the s i n g l e a f f e r e n t v o l l e y must occur w i t h i n an asymmetrical temporal window around the c o n d i t i o n i n g tetanus. - 61 - No attempt was made to subdivide the STP induced by the present method in t o subcomponents such as augmentation and PTP, as was done with STP in other systems (Magleby and Zengel, 1975b, 1976a). Like the homosynaptic nature of tetanus-induced PTP ( E c c l e s , 1953), only that a f f e r e n t pathway paired with the c o n d i t i o n i n g stimulus i s p o t e n t i a t e d . This a s s o c i a t i v e l y induced STP increases in amplitude with the number of paired c o n d i t i o n i n g t r a i n s in a manner s i m i l a r to PTP. However, the duration of p o t e n t i a t i o n does not seem to increase with the number of c o n d i t i o n i n g t r a i n s , a property that suggests augmentation. Since only 1, 5 and 10 paired c o n d i t i o n i n g t r a i n s were examined, i t i s p o s s i b l e that the graded nature of the STP was not f u l l y evident, e s p e c i a l l y i f the induction of LTP masked a prolonged duration of STP. Previous studies of STP in s p i n a l cord, neuromuscular j u n c t i o n and squid giant axon a l l concluded that STP was mediated p o s t - t e t a n i c a l l y v i a an increase in t r a n s m i t t e r r e l e a s e (del C a s t i l l o and Katz, 1954c; Eccles and K r n j e v i c , 1959a, 1959b; Lloy d , 1949; Magleby and Zengel, 1975b; Takeuchi and Takeuchi, 1962). In the hippocampus, where quantal a n a l y s i s of t r a n s m i t t e r r e l e a s e i s s t i l l an equivocal procedure (Johnston and Brown, 1984), the locus of STP i s a l s o b e l i e v e d to be presynaptic (McNaughton, 1982; Racine and Mil gram, 1983). In the present experiments where the c o n d i t i o n i n g e l e c t r o d e was placed in the same stratum as the t e s t e l e c t r o d e , there was the p o s s i b i l i t y of some undetected i n t e r a c t i o n of presynaptic terminals which might account f o r the p o s t - c o n d i t i o n i n g p o t e n t i a t i o n . This communication may be through ephaptic i n t e r a c t i o n s , as was suggested f o r i n t e r a c t i o n s between somata and dendrites - 62 - (Richardson et.- - a l : , 1984; Turner et- a l ; , 1984), e l e c t r o t o n i c coupling (MacVicar and Dudek, 1981, 1982), the r e l e a s e of ions (Goh and Sastry, 1985; Weight and E r u l k a r , 1976) or neurotransmitter (Alger and T e y l e r , 1978; Goh and Sastry, 1985). However, the placement of the c o n d i t i o n i n g e l e c t r o d e i n an anatomically separate stratum precludes the p o s s i b i l i t y t h at the post- c o n d i t i o n i n g e f f e c t s are due e n t i r e l y to presynaptic terminal or f i b e r i n t e r a c t i o n s , e s p e c i a l l y since no axoaxonic synapses have been found in t h i s area. Thus, i t i s i n t e r e s t i n g that the r e s u l t a n t a s s o c i a t i v e STP and LTP from experiments using stratum oriens and stratum radiatum c o n d i t i o n i n g e l e c t r o d e s are remarkably s i m i l a r . Without invoking h y p o t h e t i c a l e x c i t a t o r y interneurons or i n t e r - s t r a t a a s s o c i a t i o n a l pathways, the most l o g i c a l me- d i a t o r of the c o n d i t i o n i n g e f f e c t s i s the postsynaptic CA-̂  c e l l (McNaughton, 1982; McNaughton et a-1:, 1978; Robinson and Racine, 1982). I t i s evident that the postsynaptic c e l l i s capable of mediating heterosynaptic e f f e c t s , s i n c e unpaired stratum oriens c o n d i t i o n i n g t r a i n s cause a hetero- synaptic depression of the EPSP to stratum radiatum t e s t s t i m u l a t i o n (see Figure 10). Notwithstanding the postsynaptic nature of heterosynaptic de- p r e s s i o n (Dunwiddie and Lynch, 1978; Lynch et-.- a l : , 1977; Sastry et a l ; , 1984), the point i s that a c t i v a t i o n of the CA^ c e l l v i a the b a s i l a r den- d r i t e s w i l l have an e f f e c t that can be observed at the a p i c a l d e n d r i t e s . The p i v o t a l r o l e of the postsynaptic c e l l in the present experiments i s in p a r t i a l agreement with the r e s u l t s of others who nonetheless found that postsynaptic c e l l spike discharge was not necessary f o r induction of p o t e n t i a t i o n (Douglas, 1978; Wigstrom e t - a l - . , 1982). In the alveus c o n d i t - i o n i n g experiments (see Table 1), i t was a necessary c o n d i t i o n of antidromic - 63 - dromic s t i m u l a t i o n that the CA-̂  c e l l does spike. S i m i l a r l y , with the i n t r a c e l l u l a r d e p o l a r i z a t i o n experiments, i t was necessary to use supra- t h r e s o l d currents in order to induce a s s o c i a t i v e p o t e n t i a t i o n . Lee (1983) examined the a b i l i t y of antidromic alvear tetanus to induce a s s o c i a t i v e p o t e n t i a t i o n in the CA^ c e l l and found that LTP i s not enhanced when an orthodromic tetanus i s paired with an antidromic t r a i n . This led him to conclude that postsynaptic c e l l discharge i s not e s s e n t i a l . A major d i f - ference between that study and the present one i s that the former was done in normal pe r f u s i n g medium whereas the l a t t e r included 10 nm p i c r o t o x i n . In the absence of p i c r o t o x i n , the r e c u r r e n t or feed-forward i n h i b i t o r y neurons to the CA^ c e l l s would s u r e l y be a c t i v a t e d , thereby shunting the d e p o l a r i - z a t i o n of the soma and probably the d e n d r i t e s . The p r o b a b i l i t y of a s u f f i - c i e n t l y large d e p o l a r i z a t i o n reaching the subsynaptic d e n d r i t i c s i t e would be correspondingly low. Hence, Lee (1983) observed no enhancement with alvear c o n d i t i o n i n g . The need f o r c e l l spike discharge in the present experimental schemes i s obvious i f the i n i t i a t i n g event f o r a s s o c i a t i v e induction i s at the subsynaptic zone in the d e n d r i t e s . Orthodromic s t i m u l a - t i o n of a f f e r e n t s r e s u l t s in synaptic transmission that f i r s t l y d e p o l a r i z e s the subsynaptic d e n d r i t e . The e l e c t r o t o n i c spread of t h i s d e p o l a r i z a t i o n to adjacent d e n d r i t i c s i t e s could be independent of c e l l spike discharge. In the present s t u d i e s , where s i n g l e a f f e r e n t v o l l e y s are paired with condi- t i o n i n g t r a i n s , the subsynaptic s i t e must be invaded by a s u f f i c i e n t l y large d e p o l a r i z a t i o n f o r the i n i t i a t i n g event to occur. Unlike orthodromic c o n d i - t i o n i n g t e t a n i , the antidromic t e t a n i do not d e p o l a r i z e the dendrites f i r s t ; t h i s i s a l s o true of the i n t r a c e l l u l a r c u rrent i n j e c t i o n s . In a d d i t i o n , the - 64 - v o l l e y s do not de p o l a r i z e the dendrites s u f f i c i e n t l y f o r a s s o c i a t i v e induc- t i o n . Therefore, i t i s e s s e n t i a l to a c t i v a t e the CA-̂  c e l l above the th r e s h o l d f o r an act i o n p o t e n t i a l , and to do so repeatedly with a prolonged tetanus to ensure that the decrementally propagated d e p o l a r i z a t i o n a c t u a l l y invades the de n d r i t e s . In agreement with the above l i n e of reasoning are the r e s u l t s from i n t r a c e l l u l a r c u rrent i n j e c t i o n experiments. Indeed, there seems to be an exaggerated need f o r the postsynaptic c e l l to f i r e a c t i o n p o t e n t i a l s ; the l e f t i n s e t i n Figure 9 shows that the CA^ c e l l a c t u a l l y f i r e s repeatedly with a d e p o l a r i z i n g current i n j e c t i o n ofO'.lnA f o r 75 msec. Yet the current i n j e c t i o n s needed to induce a s s o c i a t i v e STP and LTP were several orders of magnitude greater than the i n t e n s i t y needed to f i r e the c e l l . This apparent discrepancy can be e a s i l y explained i f one examines the premise f o r a s s o c i a - t i v e p o t e n t i a t i o n , which re q u i r e s that a number of a f f e r e n t f i b e r s i n t e r a c t more or le s s simultaneously. This means that a great number of a f f e r e n t terminals — and presumably subsynaptic d e n d r i t i c s i t e s — must be a c t i - vated. Since the number of a c t i v a t e d synapses to any one CA-̂  c e l l i s f i n i t e , i t may be necessary to d e p o l a r i z e the c e l l with several times the t h r e s o l d current to ensure the d e p o l a r i z a t i o n of the maximum number of den- d r i t i c i n i t i a t i n g s i t e s . A l t e r n a t e l y , the maximum number of i n i t i a t i n g s i t e s on the dendrites of one c e l l may be too small f o r a s s o c i a t i v e induc- t i o n , r e q u i r i n g the f i r i n g of the CA-̂  c e l l so that a d d i t i o n a l c e l l s could be r e c r u i t e d through ephaptic i n t e r a c t i o n s (Richardson e t - a l ; , 1984; Turner et a l ; , 1984) or e l e c t r o t o n i c coupling (MacVicar and Dudek, 1981, 1982). Whatever the mechanism, i t i s c l e a r that the postsynaptic c e l l ( s ) i n i t i a t e s the induction - 65 - of a s s o c i a t i v e p o t e n t i a t i o n but cannot be the only causal event. To induce a s s o c i a t i v e STP and LTP, the a f f e r e n t pathway must be stimu- l a t e d at l e a s t once in conjunction with the c o n d i t i o n i n g stimulus. Presuma- bly , the postsynaptic d e p o l a r i z a t i o n sets up a synaptic environment that i s conducive to p o t e n t i a t i o n . It can be argued that the c r u c i a l event f o r a s s o c i a t i v e p o t e n t i a t i o n occurs at the presynaptic terminals; the input s p e c i f i c i t y of the a s s o c i a t i v e l y - i n d u c e d p o t e n t i a t i o n supports t h i s view. Since the s i t e of the c o n d i t i o n i n g input i s inconsequential to which a f - f e r e n t pathway i s p o t e n t i a t e d , the d e p o l a r i z a t i o n of the subsynaptic s i t e can only be i n t e r p r e t e d as a g e n e r a l i z e d change in the postsynaptic c e l l , namely the d e n d r i t e s . Otherwise, subsequent s t i m u l a t i o n of any a f f e r e n t pathway would have produced a po t e n t i a t e d postsynaptic response. This i s c l e a r l y not the case. The e x c i t a b i l i t y changes of the Schaffer c o l l a t e r a l terminals i n d i c a t e a c r i t i c a l r o l e f o r the presynaptic f i b e r . P o s t - t e t a n i c p o t e n t i a t i o n at the neuromuscular j u n c t i o n and s p i n a l cord i s accompanied by a p a r a l l e l period of h y p e r p o l a r i z a t i o n at the presynaptic terminal (Gasser and Graham, 1932; Gasser and Grundfest, 1936; Larrabee and Bronk, 1938). Upon reaching the hyperpolarized t e r m i n a l , a presynaptic a c t i o n p o t e n t i a l would be r e l a t i v e l y l a r g e r in amplitude, thereby r e l e a s i n g more t r a n s m i t t e r per a c t i o n p o t e n t i a l (Eccles and K r n j e v i c , 1959a, 1959b; Hubbard and Schmidt, 1963; Lloyd, 1949). Another consequence of t h i s h y p e r p o l a r i z a t i o n i s a decrease in the e x c i t a b i l i t y of the presynaptic terminal as defined by Wall (1958) (Wall and Johnson, 1958). This decreased e x c i t a b i l i t y i s a l s o observed i n the present study (see Figure 4B, Table 3). - 66 - Note that only the paired c o n d i t i o n i n g t r a i n s produced any changes in e x c i t a b i l i t y of the Schaffer c o l l a t e r a l t e r m i n a l s . E q u a l l y important i s that unpaired c o n d i t i o n i n g t r a i n s did not a l t e r e x c i t a b i l i t y , thereby r e a f - f i r m i n g the lack of l a s t i n g consequences due to any d i r e c t i n t e r a c t i o n s bet- ween the t e s t and c o n d i t i o n i n g inputs. The graded nature of the e x c i t a b i - l i t y decrease i s r e a d i l y seen. The p a r a l l e l between Schaffer terminal e x c i t a b i l i t y changes and the a s s o c i a t i v e l y - i n d u c e d STP and LTP suggests a causal r e l a t i o n s h i p . Assuming that the decreased e x c i t a b i l i t y r e f l e c t s a proportionate h y p e r p o l a r i z a t i o n of the t e r m i n a l s , one can i n f e r that t h i s h y p e r p o l a r i z a t i o n w i l l lead to increased t r a n s m i t t e r r e l e a s e per a f f e r e n t v o l l e y . The r e s u l t s presented are f a r from c o n c l u s i v e , but they do suggest a p l a u s i b l e presynaptic mechanism f o r a s s o c i a t i v e l y - i n d u c e d STP; s i m i l a r decreases in a f f e r e n t terminal e x c i t a b i l i t y have been found to accompany LTP in the hippocampus (Sastry, 1982). A d e f i n i t i v e quantal a n a l y s i s of potent- iat e d t r a n s m i t t e r r e l e a s e in the hippocampus has yet to be done, but i n - creased quantal content was found i n the c r a y f i s h neuromuscular j u n c t i o n during LTP (Baxter et • al-;, 1985). However, the r e l a t i o n s h i p between pre- synaptic changes and hippocampal LTP i s more tenuous. What f a c t o r or process does the postsynaptic c e l l e l a b orate, and how does t h i s t r a n s l a t e i n t o a presynaptic change This e l u s i v e l i n k between the pre- and postsynaptic elements may be one or more of several p o s s i b l e candidates. In the stratum pyramidale of f i e l d C A ^ e x t r a c e l l u l a r potas- sium [ K + ] Q may reach a maximum of 12 mM during a tetanus (Benninger et a l ; , 1980). Alger and T e y l e r (1978) found a good c o r r e l a t i o n between post- t e t a n i c e x t r a c e l l u l a r potassium [ K + ] and the amplitude of the poten- - 67 - t i a t e d CA-̂  population s p i k e ; no such c o r r e l a t i o n was found between [ K + ] q and the population EPSP. However, the p o t e n t i a t i o n that they c a l l STP i s at l e a s t one order of magnitude greater i n duration than the STP s t u - died here and would be more properly c a l l e d LTP. Their conclusion that e l e - vated [ K + ] Q increases the e x c i t a b i l i t y of the postsynaptic c e l l i s i n agreement with other mechanisms suggesting enhanced responsiveness of the postsynaptic c e l l a f t e r t e t a n i c s t i m u l a t i o n (Abraham et a l : , 1985; F r i t z and Gardner-Medwin, 1976). The same study showed th a t [ K + ] Q at the den- d r i t e s i s also elevated a f t e r a tetanus; such an i o n i c environment at the terminal region could increase terminal e x c i t a b i l i t y by d e p o l a r i z a t i o n r a t h e r than decrease i t . Indeed, Goh and Sastry (1985) found t h a t untetan- ized a f f e r e n t terminals adjacent to t e t a n i z e d terminals a c t u a l l y e x h i b i t increased e x c i t a b i l i t y . This t h e o r e t i c a l impasse can be surmounted by examination of the i o n i c mechanism underlying p o s t - t e t a n i c h y p e r p o l a r i z a t i o n at p e r i p h e r a l neuronal j u n c t i o n s . Of the two phases of p o s t - t e t a n i c h y p e r p o l a r i z a t i o n (Gasser and Graham, 1932; Gasser and Grundfest, 1936), the second prolonged phase e x h i - b i t s a duration of several minutes, which c o r r e l a t e s w e l l with that of a s s o c i a t i v e STP. This second phase has been presumed to be the r e s u l t of an a c t i v a t e d e l e c t r o g e n i c sodium pump (Na +,K +-ATPase) (Nakajima and Takahashi, 1966; Rang and R i t c h i e , 1968a, 1968b). The a c t i v i t y of t h i s o u a b a i n - s e n s i t i v e Na +,K +-ATPase i s d i r e c t l y dependent upon the i n t r a c e l - l u l a r sodium concentration [Na ]^ ( R i t c h i e and Straub, 1957; Thomas, 1972), and to a l e s s e r extent, on an elevated [ K + ] Q (Rang and R i t c h i e , 1968a, 1968b). I t was found t h a t both Na +,K +-ATPase and p o s t - t e t a n i c - 68 - h y p e r p o l a r i z a t i o n e x h i b i t the same sigmoidal dependence on [ K + ] Q (McDougal and Osborn, 1976). Tetanic s t i m u l a t i o n of g a r f i s h o l f a c t o r y nerve and r a b b i t vagus nerve increases Na +,K +-ATPase a c t i v i t y as measured by inorganic phosphate e f f l u x , which then returns to r e s t i n g l e v e l s p o s t - t e t a n - i c a l l y with an exponential r a t e constant of about 4 minutes ( R i t c h i e and Straub, 1978). The r a t e of i n o r g a n i c phosphate e f f l u x a l s o increases with i n c r e a s i n g stimulus frequency ( R i t c h i e and Straub, 1978). In l i g h t of the above mechanism, a s s o c i a t i v e i n d u c t i o n of STP may involve the f o l l o w i n g s e r i e s of events: a c o n d i t i o n i n g tetanus or i n t r a c e l - l u l a r current i n j e c t i o n d e p o l a r i z e s the postsynaptic d e n d r i t e s , which r e - lease a large amount of potassium. The t r a n s i e n t increase i n [ K + ] Q does not exert enough d r i v e to a c t i v a t e the Na +,K +-ATPase at the t e s t pre- synaptic t e r m i n a l s ; i n f a c t , the e x c i t a b i l i t y of the terminals may be i n - creased. During t h i s phase of increased e x c i t a b i l i t y , the invasion of these terminals by a s i n g l e a f f e r e n t v o l l e y may induce an exaggerated Na +,K +- ATPase response to the consequent Na + i n f l u x , thereby h y p e r p o l a r i z i n g the terminals and preterminal axons. This h y p e r p o l a r i z a t i o n would be analogous to that induced by t e t a n i c s t i m u l a t i o n of the a f f e r e n t f i b e r s , leading to a f a c i l i t a t i o n of t r a n s m i t t e r r e l e a s e to subsequent t e s t s t i m u l a t i o n s of the a f f e r e n t s . The h y p e r p o l a r i z a t i o n i s merely the priming event f o r some other process, such as an increase i n the pool of a v a i l a b l e t r a n s m i t t e r (Hubbard, 1963) or b e t t e r propagation of a preterminal a c t i o n p o t e n t i a l to the t e r m i - n a l , t hat mediates the p o t e n t i a t i o n ; h y p e r p o l a r i z a t i o n presumably elevates t h i s process to a s t a t e of s u b l i m i n a l a c t i v a t i o n to await s u p r a - a c t i v a t i o n by the a f f e r e n t impulse. Increasing the number of paired c o n d i t i o n i n g - 69 - t r a i n s or current i n j e c t i o n s i n t o CA-̂  neurons could increase the [ K + ] Q so as to r e c r u i t more presynaptic terminals to the s u b l i m i n a l l e v e l of a c t i - v a t i o n . The expression of the p o t e n t i a t e d r e l e a s e would then f o l l o w mechanisms along the l i n e s of the r e s i d u a l calcium theory (Katz and M i l e d i , 1968). I t must be borne in mind that the h y p e r p o l a r i z a t i o n i s i n f e r r e d from the decreased terminal e x c i t a b i l i t y and, i t i s p o s s i b l e to have an apparent decrease in e x c i t a b i l i t y without h y p e r p o l a r i z a t i o n . For example, a small d e p o l a r i z a t i o n , such as that induced by a s l i g h t l y elevated [ K + ] Q , can lead to i n a c t i v a t i o n of voltage dependent sodium channels at the t e r m i n a l . To generate an antidromic a c t i o n p o t e n t i a l , a higher d e p o l a r i z i n g current must be passed to a c t i v a t e those a v a i l a b l e sodium channels that are not r i g h t at the t e r m i n a l . Furthermore, the e x c i t a b i l i t y i t s e l f i s i n f e r r e d from higher current i n t e n s i t i e s needed to f i r e an antidromic a c t i o n poten- t i a l . The higher current may be needed to overcome an increase in r e s t i n g conductance of any one of several ions. Other p o s s i b l e mechanisms f o r the observed induction of STP may i n - volve c a l c i u m - a c t i v a t e d potassium channels at the presynaptic terminal ( M a l l a r t , 1984; Sastry, 1979), calcium-dependent c h l o r i d e channels (Owen et a l ; , 1984) or voltage-dependent calcium channels (MacVicar, 1984). In the periphery, C a + + f l u x e s at the presynaptic terminal during normal and f a c i - l i t a t e d t r a n s m i t t e r r e l e a s e has been well e s t a b l i s h e d (Hodgkin and Keynes, 1957; Katz and M i l e d i , 1967, 1968). Therefore, i t i s hardly s u r p r i s i n g that C a + + or Ca + +-mediated currents can be involved in a s s o c i a t i v e STP. Some workers have suggested the a c t i v a t i o n of a Ca -mediated p r o t e i n kinase C - 70 - in the presynaptic terminal to account f o r LTP (Malenka et - - a l . , 1986b). This kinase can be s e l e c t i v e l y a c t i v a t e d by c e r t a i n phorbol e s t e r s to pro- duce LTP in the hippocampus; the LTP thus produced cannnot be d i s t i n g u i s h e d from tetanus induced LTP, and nei t h e r LTP can be induced when maximal poten- t i a t i o n has been achieved by e i t h e r method (Malenka et-al.- , 1986b). P r o t e i n kinase C i s found i n presynaptic terminals ( G i r a r d et- a l ; , 1985) and may c a t a l y z e the phosphorylation of several p r o t e i n s during LTP (Nelson and Routtenberg, 1985, Browning e t a l ; , 1979). In the hippocampal pyramidal c e l l , c e r t a i n phorbol e s t e r s can block a Ca + +-mediated potassium current (Malenka e t - a l ; , 1986a); s i m i l a r channels have been found in pre- synaptic nerve terminals (Bartschat and B l a u s t e i n , 1985). The blockade of t h i s outward K + conductance could delay membrane r e p o l a r i z a t i o n and lead to a prolonged C a + + c u r r e n t , which would increase t r a n s m i t t e r r e l e a s e . S i m i l a r l y , the delayed r e p o l a r i z a t i o n may prolong sodium channel i n a c t i v a - t i o n , r e s u l t i n g i n an apparent decrease in terminal e x c i t a b i l i t y . In addi- t i o n , there i s evidence that phorbol e s t e r s increase calcium currents by some a c t i o n on p r o t e i n kinase C (DeRiemer et a l ; , 1985). However, phorbol e s t e r s do not induce p o s t - t e t a n i c p o t e n t i a t i o n independently of LTP (Malenka e t - a l ; , 1986b). C o l l i n g r i d g e (1985) has proposed a postsynaptic inward current to ac- count f o r LTP. This hypothesis involves a glutatmate receptor subtype, the s o - c a l l e d N-methyl-D-aspartate (NMDA) receptor located on the postsynaptic dendrites (Baudry and Lynch, 1981). I t i s suggested that a voltage depend- ent magnesium (Mg + +) blockade of a NMDA-receptor coupled conductance i s l i f t e d upon d e p o l a r i z a t i o n of the postsynaptic c e l l (Mayer et - a-1., 1984; - 71 - Nowak et - al.-, 1984). Subsequent r e l e a s e of t r a n s m i t t e r , presumably g l u t a - mate (Storm-Mathisen, 1977), from the presynaptic terminal then causes a greater l a t e current through the NMDA receptor-coupled ion channel to f u r - ther enhance the postsynaptic response ( H a r r i s et- a l ; , 1984; Wigstrom and Gustafsson, 1984, 1985b). This inward current i s blocked by the NMDA anata- g o n i s t 2-amino-5-phosphonovalerate (APV), which al s o blocks the induction of LTP (Col 1 ingridge e t - a l ; , 1983; H a r r i s et a l , 1984; Wigstrom and Gustafsson, 1984). This mechanism thus accounts f o r the need to de p o l a r i z e the post- synaptic c e l l f o r a s s o c i a t i v e induction of LTP. The presynaptic element could be a c t i v a t e d by p o s s i b l e autoreceptors f o r glutamate (Col 1 ingridge et al-.-, 1983; McBean and Roberts, 1981). I f these autoreceptors are als o NMDA rec e p t o r s , then presynaptic d e p o l a r i z a t i o n ++ by the a f f e r e n t v o l l e y may be req u i r e d to remove the Mg i n h i b i t i o n before current flow can occur. Assuming that C a + + flows through these channels (Dingledine, 1983a, 1983b), the ext r a C a + + in the terminal would act as the r e s i d u a l C a + + in Katz and M i l e d i ' s (1968) theory f o r transmit- t e r r e l e a s e . Again, the increased conductance may shunt the e x t r a c e l l u l a r current i n j e c t i o n s , thereby n e c e s s i t a t i n g higher current i n t e n s i t i e s to f i r e an antidromic a c t i o n p o t e n t i a l . A f t e r the conclusion of the present s t u d i e s , Wigstrom e t a l ; (1986) published a paper examining the a s s o c i a t i v e induction of LTP using i n t r a c e l - l u l a r d e p o l a r i z a t i o n in conjunction with s i n g l e a f f e r e n t v o l l e y s . These authors agree that the l e v e l of postsynaptic c e l l d e p o l a r i z a t i o n , rather than spike a c t i v i t y , i s the determinant f a c t o r at the postsynaptic c e l l to induce LTP. However, they propose a postsynaptic locus f o r the induction of - 72 - LTP based on the v o l t a g e - s e n s i t i v e NMDA r e c e p t o r - a c t i v a t e d current (Wigstrom et- - a l ; , 1985). This current i s apparent a f t e r a s i n g l e high i n t e n s i t y s t i m u l a t i o n or a b r i e f tetanus (Wigstrom e t - a l . , 1985; Wigstrom and Gustafsson, 1984), and i s blocked by the NMDA antagonist 2-amino-5-phosphono- v a l e r a t e (APV). In t h e i r scheme of events, d e p o l a r i z a t i o n of the subsynap- t i c membrane containing NMDA receptors, i n conjunction with t r a n s m i t t e r released by a f f e r e n t s t i m u l a t i o n , i s the a s s o c i a t i v e event that r e s u l t s in potenti a t e d postsynaptic responses. This i s a very a t t r a c t i v e hypothesis, f o r these authors also showed that the c o n d i t i o n i n g tetanus and the s i n g l e a f f e r e n t s t i m u l a t i o n can be separated by a period of 40 msec (Wigstrom and Gustafsson, 1985b). This temporal separation can be a t t r i b u t e d to a r e s i d u a l c u r r r e n t passing through the NMDA receptor channel and having a time course of about 50 msec f o r a s i n g l e v o l l e y . (Wigstrom et al.-, 1985). These authors suggest t h a t a temp- oral overlap of the NMDA cur r e n t with the c o n d i t i o n i n g tetanus i s e s s e n t i a l f o r a s s o c i a t i v e i n d u c t i o n . Such a mechanism would explain, the present ob- ser v a t i o n that a t e s t stimulus can precede the c o n d i t i o n i n g tetanus by 50 msec and s t i l l induce STP. S i m i l a r l y , a c o n d i t i o n i n g tetanus would be expected to generate a current of longer duration; hence, the t e s t stimulus can f o l l o w a c o n d i t i o n i n g tetanus by up to 80 msec and s t i l l produce STP. The great e s t amount of p o t e n t i a t i o n i s induced by simultaneous t e s t and co n d i t i o n i n g s t i m u l a t i o n s . Other workers have al s o found that the a s s o c i a - t i v e i n d u c t i o n of LTP re q u i r e s temporal overlap of t e s t and c o n d i t i o n i n g s t i m u l a t i o n s (Kelso and Brown, 1986; Levy and Steward, 1983). - 73 - Wigstrom and Gustafsson's theory f o r the a s s o c i a t i v e induction of LTP (1985b) does not account f o r the decreased terminal e x c i t a b i l i t y i n the pre- sent study. This decrease i n terminal e x c i t a b i l i t y i s an important l i n k between STP in the hippocampus and that i n other systems. Although hyper- p o l a r i z a t i o n of the Schaffer c o l l a t e r a l terminals and ac c e l e r a t e d Na ,K - ATPase a c t i v i t y was not demonstrated d i r e c t l y in the present study, t h e i r reported r o l e s i n PTP at other e x c i t a b l e j u n c t i o n s suggest equivalent r o l e s in the hippocampus. With respect to the temporal l i m i t s of a s s o c i a t i v e STP — namely the 40 msec separation between a t e s t stimulus and the succeeding c o n d i t i o n i n g tetanus, i t i s conceivable that each a f f e r e n t v o l l e y induces an increment of subliminal a c t i v a t i o n at the presynaptic t e r m i n a l , much as each a f f e r e n t v o l l e y induces an inward current mediated by the NMDA channel. This increment of subliminal a c t i v a t i o n could be t r i g g e r e d by the t r a n s i e n t + + ++ increase i n Na ,K -ATPase a c t i v i t y or i n t r a c e l l u l a r Ca through v o l - tage dependent channels, but only i f a t e t a n i c d e p o l a r i z a t i o n of the post- synaptic c e l l occurs within a short time. Since the exact nature of t h i s subliminal a c t i v a t i o n i s unknown, any number of p o s s i b i l i t i e s could be advanced. Notwithstanding t h i s u n c e r t a i n t y , the r e s u l t s in t h i s t h e s i s s t r o n g l y support a c r u c i a l presynaptic event f o r the a s s o c i a t i v e induction of STP as well as LTP. Up u n t i l now, STP and LTP in the hippocampus have been con- sidered t o t a l l y separate phenomena. The proposed d e n d r i t i c i n i t i a t i o n s i t e f o r the a s s o c i a t i v e induction of STP i s unusual because STP has already been shown to be a presynaptic event at other neuronal j u n c t i o n s . Furthermore, a s i n g l e s t i m u l a t i o n of a f f e r e n t s has not p r e v i o u s l y been shown to induce any - 74 - short-term synaptic e f f i c a c y changes. On the other hand, a s s o c i a t i v e l y induced STP in the hippocampus may be due to a postsynaptic mechanism i n - v o l v i n g NMDA receptor a c t i v a t i o n as suggested f o r LTP (Col 1ingridge, 1985; Wigstrb'm and Gustafsson, 1985b). With respect to LTP, a great number of hypotheses regarding i t s mechanism focus on the postsynaptic c e l l f o r both i n d u c t i o n and maintenance. Although there i s evidence f o r increased t r a n s - m i t t e r r e l e a s e during LTP (Dolphin et a l ; , 1982; Lynch e t - a l . - , 1985; Skrede and Malthe-St&renssen, 1981), a p l a u s i b l e i n t e r a c t i o n between pre- and post- synaptic c e l l s has only j u s t been advanced (Wigstrom and Gustafsson, 1985b). Unfortunately, even t h i s l a s t hypothesis ignores the p o s s i b l e r o l e of presynaptic causal events such as the l a s t i n g decrease in Schaffer c o l - l a t e r a l terminal e x c i t a b i l i t y , which i s a s u b s t a n t i a l l i n k between LTP and presynaptic changes. The present r e s u l t s also suggest a p o s s i b l e postsynaptic i n i t i a t i n g s i t e f o r LTP. This evidence i n d i c a t e s s i m i l a r c o n d i t i o n s underlying the two forms of p o t e n t i a t i o n and suggests a common mechanism of induction and main- tenance. The f a c t that a s s o c i a t i v e LTP can be induced in the same fa s h i o n as STP r a i s e s i n t e r e s t i n g p o s s i b i l i t i e s regarding the locus of each poten- t i a t i o n ; perhaps a s s o c i a t i v e STP in the hippocampus (or c e n t r a l nervous sys- tem) i s uniquely d i f f e r e n t from that in the periphery and tetanus-induced STP; there i s also the p o s s i b i l i t y that some fundamental u n i t of p o t e n t i a - t i o n i s r e s p o n s i b l e f o r both a s s o c i a t i v e STP and LTP, the d i f f e r e n c e between the two being only a matter of d u r a t i o n . - 75 - 6 CONCLUSIONS Ever since B l i s s and Gardner-Medwin (1971) f i r s t reported LTP in the hippocampus, the working hypothesis f o r LTP has always been that LTP does not share a basic mechanism with STP ( B l i s s and L0mo, 1973; McNaughton, 1982; Abraham e t - a l ; , 1985); the former i s b e l i e v e d by some i n v e s t i g a t o r s to have a postsynaptic locus, the l a t t e r a presynaptic l o c u s . In s p i t e of the recent f i n d i n g s that a s s o c i a t i v e induction of LTP depends on c o i n c i d e n t pre- and postsynaptic a c t i v i t y , the study of synaptic p o t e n t i a t i o n in the hippocampus remains l a r g e l y focussed on the postsynaptic c e l l . The present study shows that STP can a l s o be induced by a s s o c i a t i v e i n t e r a c t i o n s between the pre- and postsynaptic c e l l s . This STP demonstrates a magnitude and duration that p a r a l l e l a presynaptic decrease in e x c i t a b i l i t y , which has been shown to accompany STP in other s y n a p t i c j u n c t i o n s . A novel f i n d i n g i s that STP and LTP can be induced without t e t a n i c s t i m u l a t i o n of the a f f e r e n t f i b e r s . By i n c r e a s i n g the number of i n t e r a c t i o n s between the pre- and post- synaptic elements, greater STP can be induced. With ten p a i r s of such i n t e r a c t i o n s , STP i s followed by LTP. The s i t e of a c t i o n of the c o n d i t i o n - ing tetanus i s narrowed to the postsynaptic c e l l ; the c o n d i t i o n i n g e f f e c t i t s e l f i s shown to be analogous to a g e n e r a l i z e d d e p o l a r i z a t i o n of the post- s y n a p t i c c e l l and can be mimicked by i n t r a c e l l u l a r i n j e c t i o n s of d e p o l a r i z - ing c u r r e n t . Temporal separation between the a c t i v a t i o n of the pre- and postsynaptic c e l l s was examined, and p o s s i b l e mechanisms f o r the presynaptic changes were suggested. These f i n d i n g s suggest that STP induction i s not confined to the presynaptic terminals and may share a common a s s o c i a t i v e - 76 - induction with LTP. Conversely, LTP may have a very strong presynaptic component f o r induction and maintenance. 7 REFERENCES ABRAHAM, W. C , BLISS, T. V. P. and GODDARD, G. V. (1985). 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