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Long-lasting potentiation and depression of synaptic responses in the hippocampus Goh, Joanne Wan Yoong 1984

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LONG-LASTING POTENTIATION AND DEPRESSION OF SYNAPTIC RESPONSES IN THE HIPPOCAMPUS By JOANNE WAN YOONG GOH B.Sc. (Pharm.), The University of Brit ish Columbia, 1981 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) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1984 © J o a n n e Wan Yoong Goh, 1984 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . .Department o f Pharmacology & Therapeutics The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date April 2, 1984 - i i -ABSTRACT Tetanic stimulations when applied to the Schaffer col laterals or the commissural input to CA^ neurones in the mammalian hippocampus results in a long-lasting potentiation (LLP) of synaptic transmission across the tetanized synapse (Andersen et a l . , 1977; Schwartzkroin and Wester 1975). The induction of LLP is dependent on the extracellular C a + + concentration (Dunwiddie and Lynch, 1979; Wigstrom et a l . , 1979) but whether i t is generated pre- and/or postsynaptically is at present unresolved. Transient homo- and heterosynaptic depressions of the CA-^  population spike have been reported following low frequency (1-20 Hz) tetani to hippocampal inputs (Lynch et a l . , 1977; Alger et a l . , 1978) which precede the observation of homosynaptic LLP (Dunwiddie and Lynch, 1978). The present studies were conducted to 1) test the hypothesis of Baudry and Lynch (1980a) who think that LLP can be explained postsynaptically by a C a + + dependent increase in the number of ++ transmitter receptors; 2) examine the effects of verapamil (a Ca antagonist that blocks CA^ neuronal C a + + currents [Gr i f f i th and Brown, 1982] without interfering with transmitter release [Nachshen and Blaustein, 1979]) on the induction of LLP and homo- and heterosynaptic depressions; 3) investigate the C a + + dependence of a decrease in presynaptic terminal exci tabi l i ty that is associated with LLP (Sastry, 1982) and 4) examine the suggestion of Collingridge et a l . , (1983b) that N-methyl-DL-aspartate preferring amino acid receptors are involved in the induction of LLP. - i i i -The studies conducted led to the conclusions that 1) LLP is probably generated presynaptically and depression of synaptic responses is loca l -ized to the postsynaptic neurone; 2) verapamil appears to antagonize Ca+ +-dependent depression postsynaptically but i t does not block the induction of C a + + dependent LLP; 3) the decrease in exc i tabi l i ty of Schaffer col lateral terminal regions which correlates with LLP is C a + + dependent but insensitive to verapamil and 4) N-methyl-DL-aspartate preferring receptors are not involved in the induction of LLP. Bhagavatula R. Sastry Supervisor - iv -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES v i i LIST OF FIGURES v i i i ABBREVIATIONS x ACKNOWLEDGEMENTS xi INTRODUCTION 1 LITERATURE SURVEY 4 Anatomy of the hippocampus 4 Major afferents to the hippocampus and dentate gyrus 5 a. Perforant and alvear paths 5 b. Commissural input 5 c. Septal input 6 Intrinsic connections of the hippocampus and dentate gyrus 6 Inhibitory cel ls of the hippocampal formation 7 Electrophysiology of CA^  and CA^ pyramidal cel ls 8 Synaptic transmitter(s) between Schaffer col laterals and CA^ neurones 10 "Antagonists" of excitatory amino acids in hippocampus 13 Long-lasting potentiation of synaptic responses 14 Evidence for a presynaptic role in LLP 21 Evidence for a postsynaptic role in LLP 23 - V -Homo- and heterosynaptic depressions 26 Effects of verapamil in the hippocampus , 28 Role of inhibition in LLP 29 Paired pulse and frequency potentiation in hippocampus 30 METHODS 31 SI ice preparation 31 Recording and stimulating systems 32 Induction of LLP and homo- and heterosynaptic depressions 33 Effects of drug application to the whole bath on the CA^ population spike 35 Localized applications of L-glutamate to the CA^ and CA3 areas 36 Iontophoretic application of drugs 38 Antidromic stimulation of single CA^ cel ls 41 RESULTS 42 Tetanus-induced LLP and homo- and heterosynaptic depressions 42 Iontophoretic C a + + applications 54 L-Glutamate 59 a. Application by push-pull cannula 59 b. Iontophoretic application 61 N-Methyl-DL-aspartate 67 Amino acid "antagonists" and LLP 69 a. a-Aminoadipate 69 b. 2-Amino-5-phosphonovalerate 71 - vi -DISCUSSION 76 Tetanus-induced LLP and homo- and heterosynaptic depressions 76 L-Glutamate 81 N-Methyl-DL-aspartate 85 Amino acid "antagonists" and LLP 87 CONCLUSIONS 89 REFERENCES 91 — v i i — LIST OF TABLES TABLE Page I. Effects of verapamil (0.33 jiM) on the induction of long-lasting potentiation. 51 II. Effects of verapamil (0.33 yM) on homo-and heterosynaptic depressions. 52 III. Effects of C a + + on the CAi population spike. 58 IV. Effect of glutamate application at the apical dendritic zone of CA^. 62 - vi i i -LIST OF FIGURES FIGURE Page 1. Anatomical i l lustrat ion of a transverse section of the hippocampal formation. 2 2. Population responses in the CA^ area evoked by Schaffer col lateral stimulation. 15 3. Long-lasting potentiation of the CAj neuronal population spike induced by a tetanic stimulation (100 Hz, 1 s) of the Schaffer col lateral input. 18 4. Placement of stimulating and recording electrodes for induction of long-lasting potentiation and/or homo- and heterosynaptic depressions of the CA^ population spike. 34 5. Placement of stimulating and recording electrodes, and push-pull cannula for local glutamate applications in the CA^ and CA3 regions. 37 6. Experimental arrangements for examining actions of iontophoretically applied drugs at the CAi ce l l body and apical dendritic/ synaptic regions. 39 7. The masking of long-lasting potentiation by a low frequency (20 Hz, 200 pulses), but not by a high frequency (400 Hz, 200 pulses), tetanus. 43 8. The blockade of homosynaptic and heterosyn-aptic depressions of the CA^ population spike by verapamil (0.33 yM, 3 min, applied to the whole bath). 45 9. Failure of verapamil (0.33 yM, 3 min, applied to the bath) to block the induction of tetanus-induced (400 Hz, 200 pulses) long-lasting potentiation. 47 10. The blockade of homo- and heterosynaptic depressions by verapamil (0.33 uM, 3 min, applied to the bath). 49 - ix -11. Depression of the CA^ population spike and the population "EPSP" by Ca applied near the cel l body area. 56 12. Actions of local applications of glutamate in the C A 3 and CA} areas on Schaffer col lateral stimulation-induced population spike in CA^. 60 13. Effects of iontophoretically applied glutamate at the terminal regions of Schaffer col laterals on the antidromic threshold of a single C A 3 neurone. 64 14. Glutamate-induced depression of the population spike when applied at the U\\ cel l body layer. 65 15. Potentiation of glutamate-induced depression of the CAi population spike by a tetanic stimulation to the Schaffer co l latera ls . 66 16. Effects of N-methyl-DL-aspartate (NMA) (200 yM, 1 min, applied to the bath) on the CAj population spike and the threshold for ant i -dromic activation of single C A 3 ce l l s from the terminal regions of Schaffer co l latera ls . 68 17. The induction of long-lasting potentiation by bath application of DL-a-aminoadipate (5 mM, 3 min). 70 18. Effects of 2-amino-5-phosphonovalerate (APV) (A) and verapamil on the CA^ population spike produced by the stimulation of Schaffer col laterals at 0.1 Hz. 72 19. Failure of verapamil (0.33 yM, 3 min, applied to the bath) to block the induction of tetanus-induced (100 Hz, 1 s) long-lasting potentiation. 73 20. The masking of long-lasting potentiation of the Schaffer col lateral stimulation-induced CAi population spike by APV. 74 21. Schematic diagram of a CA^ neurone and Schaffer col lateral input to i l lust rate proposed sites of Ca+ +-dependent depression and Ca+ +-dependent LLP. 90 - X -ABBREVIATIONS otAA a-aminoadipate APB 2-amino-4-phosphonobutyrate APV 2-amino-5-phosphonovalerate Comm commissural input DGG Y-D-glutamylglycine DLH DL-homocysteate EPSP excitatory postsynaptic potential GABA y-aminobutyric acid GDEE glutamic acid diethyl ester IPSP inhibitory postsynaptic potential LLP long-lasting potentiation NMA N-methyl-DL-aspartate PTP post-tetanic potentiation Sch Schaffer col laterals - xi -ACKNOWLEDGEMENTS I am grateful to my supervisor, Dr. B. R. Sastry, for his guidance and encouragement throughout this study. I thank Mr. S. S. Chirwa, Dr. H. Maretic and Dr. P. Mural i Mohan for their help during the course of this investigation and Mr. C. Caritey for development of the in vitro s l i ce chamber. Financial support from the Medical Research Council of Canada is gratefully acknowledged. To my - 1 -INTRODUCTION It has been reported in l i terature that brief trains of high frequency tetani to the Schaffer col lateral (Sch) input in the mammalian hippocampus results in a post-tetanic long-lasting potentiation (LLP) of synaptic transmission across the Sch-CA^ neuronal synapse (Schwartzkroin and Wester, 1975). The induction of LLP is Ca+ +-dependent (Dunwiddie and Lynch, 1979; Wigstrom et a l . , 1979), but whether LLP is generated pre- and/or postsynaptically is at present unclear. The present studies were conducted on the Sch and/or commissural (Comm) input-CA-^ neuronal system in transversely sectioned rat hippocampal s l ices in vitro (see F ig . 1 for anatomical arrangements). The transmitter between Sch terminals and CA^ neurones has been thought to be glutamate and/or aspartate (Storm-Mathisen, 1977a). Baudry and Lynch (1980) hypothesized that C a + + influx into CA^ neurones results in an uncovering of new glutamate receptors and this increase in the number of subsynaptic glutamate receptors can account for LLP. If the hypothesis is correct, and i f transmitter released during tetanic stimulation activates subsynaptic receptors to trigger the events mentioned above, then an exogenous application of glutamate in the CA^ area should e l i c i t LLP and a blockade of the receptors during tetanic stimulation should prevent the induction of LLP. Therefore, experiments were conducted to examine the effects of exogenous glutamate application to the CA^ and CAg areas, the latter application to mimic a tetanic stimulation to the Sch input (since glutamate is an excitatory amino acid). There is a decrease in exc i tabi l i ty of axon terminal regions that is associated with and may be responsible for LLP (Sastry, 1982), so the - 2 -FIG. 1 Anatomical i l lust rat ion of a transverse section of the hippocampal formation. The hippocampal formation consists of the hippocampus (also called Amnion's horn or Cornu Ammonis) which is a C-shaped structure that folds t ight ly into another C-shaped structure, the dentate gyrus (also called Fascia dentata). The hippocampus is divided into four subfields: CAi, CA 2, CA3 and CA 4 . Enlargement on right shows layered structure of the hippocampal formation. PROS - prosubiculum HF - hippocampal f issure Sch - Schaffer col lateral Comm - commissural input MF - mossy f ibre PP - perforant path input B - basket ce l l • represent pyramidal cel ls of the hippocampus • represent granule cel ls of the dentate gyrus - 3 -threshold for antidromic activation of single Sch terminal regions was also monitored during and following glutamate application to the synaptic zone to determine i f glutamate has a presynaptic effect leading to LLP. The amino acid "antagonist", a-aminoadipate (aAA) which has been reported to block the actions of the endogenous transmitter (Fagni et a l . , 1983) was examined on LLP induction to further test the hypothesis of Baudry and Lynch (1980). Homo- and heterosynaptic depressions of the CA^ population spike have been reported following low frequency (1-20 Hz) tetani to inputs in the hippocampus (Lynch et a l . , 1977; Alger et a l . , 1978). It has recently been demonstrated that these depressions are l ike ly due to a C a + + accumulation in C/\^ neurones (Chirwa et a l . , 1983). Verapamil is a C a + + antagonist that blocks C a + + spikes (Dingledine, 1983) and currents (Gr i f f i th and Brown, 1982) in CA^ neurones but i t does not alter transmitter release (Nachshen and Blaustein, 1979; Norris et a l . , 1983). Since both LLP and depression co-occur and are Ca + +-dependent, i t would be d i f f i c u l t to analyze one without the other interfering. It was thought that verapamil may preferential ly block the actions of C a + + postsynaptically and therefore separate LLP (presuming that LLP is presynaptic) from the depression so i t was for this reason that the drug was u t i l i zed . It has been suggested (Collingridge et a l . , 1983b) that N-methyl-DL-aspartate (NMA) receptors are involved in the induction of LLP. It was, therefore, decided to examine the effects of 2-amino-5-phosphonovalerate (APV, reported to be a selective NMA antagonist; Collingridge et a l . , 1983a) on tetanus-induced LLP and - 4 -exogenously applied NMA (to the bath or iontophoretically onto Sch-CA^ synaptic zone) on the CA-^  population spike and Sch terminal exc i tab i l i t y . LITERATURE SURVEY Anatomy of the hippocampus The hippocampal formation (consisting of the hippocampus, dentate gyrus and subiculum) is contained in the medial wall of the inferior horn of the lateral ventr ic le. This structure extends from the region of the splenium of the corpus callosum to the t ip of the ventr icle. The hippo-campal formation, which represents phylogenetically the oldest type of cortex, is folded into the ventricle along the hippocampal sulcus. The hippocampus, a C-chaped structure that can be subdivided into four regions (CA^|-a k c j , CA^, CA^r-a k c - j , and CA^ subfields based on anatomi-cal and hisotological properties of cel ls) (Lorente de Ntf, 1934), folds t ightly into another C- or V-shaped structure, the dentate gyrus, with the CA^ region of the former extending into the hi lar region of the latter (see Fig . 1). The hippocampus-dentate gyrus system can be divided into 7 layers (Cajal , 1911): Stratum oriens, Stratum pyramidale, Stratum radiatum, Stratum lacunosum, Stratum moleculare, Stratum granulosum and Stratum polymorphe in a dorsal to ventral order (see Fig . 1). The main cel l type in the hippocampus is the pyramidal cel l (Blackstad, 1956; Golgi, 1886; Lorente de No", 1934) while that in the dentate gyrus is the granule cel l (Golgi, 1886; Lorente de No", 1934), but Golgi type II (basket) ce l ls which have synaptic connections with both pyramidal and granule cel ls - 5 -(C aj a l , 1968) and polymorphic cel ls in the dentate gyrus (Lorente de No, 1934) are also present. Major afferents to the hippocampus and dentate gyrus a. Perforant and alvear paths The main afferent input to the hippocampal formation is the perforant path originating from the lateral entorhinal cortex which innervates the prosubicul urn, CAp C ^ , CA^, and primarily the dentate gyrus (Lorente de No', 1934). Perforant path fibres terminate in the outer and middle portions of the Stratum moleculare (Hjorth-Simonsen, 1972). In addition to the perforant path, another input from the medial entorhinal cortex, the alvear path enters the hippocampus along the dorsal surface to innervate the prosubiculum and CA-^ regions (Lorente de No, 1934). b. Commissural input Using lesion-induced degeneration (Blackstad, 1956), autoradiographic (Gottlieb and Cowan, 1973) and electrophysiological (Andersen, 1960; Deadwyler et a l . , 1975) studies, the connections of the commissural system have been determined. The commissural input arises from the CA^ and CA^ regions of the contralateral hippocampus and innervates a l l regions in the hippocampus and dentate gyrus (Gottlieb and Cowan, 1973; Hjorth-Simonsen and Laurberg, 1978). The commissural f ibres that originate in the contralateral hippocampus enter the ipsi lateral hippocampus via the fimbria near CA^. The commissural fibres to CA^ terminate on both apical (Stratum radiatum) and basal dendrites (Stratum oriens) of the pyramidal cel ls (Andersen, 1960; Gottlieb and Cowan, 1973), although there is evidence from degeneration studies for a greater density of terminations on the apical dendrites (Blackstad, 1958). - 6 -Consistent with this observation, a larger negative extracellular f ie ld is recorded in the apical dendrites than in the basal dendrites by stimula-tion of the commissural input (Cragg and Hamlyn, 1957). c. Septal input Studies on the septo-hippocampal projection to date have not as yet resulted in a clear cut picture regarding the precise origin(s) and termination(s) in the hippocampal formation of the pathway. However, neuroanatomical (Raisman, 1966; Siegel and Tassoni, 1971), histochemical (Fonnum, 1970; Mellgren and Srebro, 1973; Shute and Lewis, 1967; Storm-Mathisen, 1970) and pharmacological (Dudar, 1975, 1977; Kuhar, 1975; Kuhar et a l . , 1972; Rommelspacher et a l . , 1974; Smith, 1972, 1974) studies support the existence of a septo-hippocampal projection which may use acetylcholine as a transmitter. There is evidence that the hippocampus receives a d is t inct , well organized substance P innervation and i t has been suggested that many of these fibres either arise in or pass through the septal area (Vincent et a l . , 1981). Intrinsic connections of the hippocampus and dentate gyrus Axons of granule cel ls in the dentate form the mossy f ibre system which projects to dendrites of cel ls in areas CA^ and CA^ of the hippocampus (Cajal, 1968). The axons of CAg pyramidal cel ls go to the fimbria, a major efferent pathway of the hippocampus as well as giving off Schaffer col laterals (Cajal, 1911; Schaffer, 1892) which cross the Strata pyramidale and radiatum to enter Stratum lacunosum where they constitute horizontal f ibres . The bundles of horizontal fibres of the Stratum lacu-nosum are made up of these col laterals and their region of termination is onto pyramidal cel ls in CA l f l and C A l b (Lorente de No", 1934). The - 7 -axons given off by pyramids of CA^  are thin and course through the alveus before entering the fimbria leading out of the hippocampus (Lorente de No", 1934). Some of the axons of the CA^ pyramids have a long recur-rent col lateral which courses in the alveus or Stratum oriens towards the prosubiculum (Lorente de No", 1934). The trisynaptic loop that has been described (dentate-CA^-CA^) (see Fig . 1) suggests that an activation of the perforant path input onto dentate granule cel ls wil l affect sequen-t i a l l y CA3 and CA1 ce l l s . Inhibitory cel ls of the hippocampal formation With the exception of Golgi type II (basket) c e l l s , a l l inputs and synaptic connections described so far in the hippocampus and dentate area are excitatory in nature. The histological ly identif ied basket cel l (Cajal , 1911) has been shown to be inhibitory in nature (Andersen et a l . , 1964b; Spencer and Kandel, 1961c). The basket cel l is postulated to receive excitatory inputs from a large number of principal axon c o l l a -terals and this recurrent c i rcui t ry provides inhibitory feedback to the principal cel ls (Andersen et a l . , 1964b; Spencer and Kandel, 1961c). The basket cel ls have GABAergic processes that come into contact with the somata of many principal cel ls (Andersen, 1975; Andersen et a l . , 1964a; Curtis et a l . , 1970). Until recently, the only mode of inhibition that has been demonstrated in the hippocampal system is recurrent feedback inhibit ion. Buzsaki and Eidelberg (1981, 1982) have provided evidence for feed-forward inhibit ion in the dentate gyrus. Whether this process is mediated by the same basket cel ls that are responsible for recurrent inhibition is not clear at present. There appears to be a subset of inhibitory neurones distinct from the classical basket cel l that mediates - 8 -recurrent as well as feed-forward inhibition in the hippocampus (Knowles and Schwartzkroin, 1981). There is no l i terature up to the present time demonstrating the existence of presynaptic inhibition in the hippocampal formation. Electrophysiology of CA-^  and CA3 pyramidal cel ls The pyramidal cel ls in hippocampus consist of a soma, an axon and two dist inct dendritic systems (basilar and apical) on opposite sides of the soma (see F ig . 1). The electrophysiological properties of these cel ls have been thoroughly investigated in vivo by Kandel and associates (Kandel and Spencer, 1961; Kandel et a l . , 1961; Spencer and Kandel, 1961a; Spencer and Kandel, 1961b). It is reported that the pattern of spontan-eous act iv i ty of these cel ls consists of both single spikes as well as bursts (Kandel and Spencer, 1961). Solitary spikes are followed by a depolarizing after -potent ia l , and bursting act iv i ty once triggered may be sustained by a summation of the depolarizing after-potentials of subse-quent spikes (Kandel and Spencer, 1961). In the majority of neurones studied, spike in i t iat ion occurred upon depolarization to a constant level even with different modes of activation (Spencer and Kandel, 1961a). There exists in some neurones the presence of a fast prepotential which has been thought to play a role in impulse generation (Spencer and Kandel, 1961b). With the development of the hippocampal s l ice preparation, many of the characteristics of CA^  pyramidal cel ls in vivo have been shown to be present in vitro as well (Schwartzkroin, 1975). Studies in vivo (Kandel and Spencer, 1961) and in vitro (Wong and Prince, 1978) have shown that hippocampal CAg pyramidal neurones are capable of discharging spontaneous bursts of action potentials. The - 9 -bursting activity may be triggered by a post-spike depolarization (Kandel and Spencer, 1961) mediated by a slow voltage-sensitive C a + + current (Johnston et a l . , 1980). Following repetit ive f i r ing in CA^ and CA-^  neurones, a prolonged (1-3 min) hyperpolarization is observed (Alger and N ico l l , 1980; Gustafsson and Wigstrb'm, 1981; Hablitz, 1981; Hotson and Prince, 1980; Nicoll and Alger, 1981; Schwartzkroin and Stafstrom, 1980; Segal, 1981). This hyperpolarization has been reported to be due to a C a + + -activiated K+ conductance (Alger and N ico l l , 1980; Hotson and Prince, 1980; Nicoll and Alger, 1981; Schwartzkroin and Stafstrom, 1980). It has also been claimed to be due to the activation of a Na+-pump (Segal, 1981). It is possible that both these processes are involved, as evidenced by Gustafsson and wigstrom (1981) and Schwartzkroin and Stafstrom (1980) who report two types of afterhypolarization, a C a + + -dependent K + conductance and another undetermined component which is not „ ++ sensitive to Ca antagonists. An extracellular f ie ld potential can be evoked in the CA-^  area by synaptic activation of the apical dendrites in these neurones. An analy-sis of this f ie ld potential has been carried out in vivo by Andersen (1960a). He reports that upon recording on the hippocampal surfaces direct ly above the CA^ region the response constitutes a positive wave with one or more spikes (usually negative) superimposed on the positive wave. Depth recordings showed that the i n i t i a l part of the positive wave (presumed to be the presynaptic volley) retained i ts polarity while the later , major part of this wave reversed i ts polar i ty at the ce l l body layer and was recorded as negative in the apical dendrites. The deep - 10 -negative wave was considered to be a summated EPSP e l ic i ted by a direct afferent volley. The spike latency was shortest in the region of the apical dendrites, suggesting that i t was init iated close to this s i te . The CA-^  dendritic membrane was thought to be composed of a part respon-sible for generation of the deep negative wave and another part for in i t iat ion of the spike and its propagation along the dendrite. Synaptic transmitter(s) between Schaffer col laterals and CA-^  neurones Glutamic acid, which occurs in re lat ively high concentrations in the brain (Johnson, 1972) is believed to be the excitatory transmitter between Sch f ibres and CA^ neurones (see Storm-Mathisen, 1977a for review). Iontophoretically applied glutamate in the hippocampal s l ice e l i c i t s a powerful excitation at both the cel l bodies (Schwartzkroin and Andersen, 1975) as well as the dendrites of pyramidal cel ls (Dudar, 1974; Schwartzkroin and Andersen, 1975), the most sensitive areas corresponding to the regions of distribution of excitatory synapses. Intracellular studies in hippocampal neurones with glutamate reveal that the amino acid is an effective excitatory agent which mediates a depolarization of the cel lular membrane (Dingledine, 1983; Schwartzkroin and Andersen, 1975; Segal, 1981) that is associated with a decrease in input resistance (Dingledine, 1983; Segal, 1981). The conductance changes during glutamate excitation appear to be mediated mainly by sodium and to a lesser extent calcium (Dingledine, 1983). In support of th is , Zanotto and Heinemann (1983) observed that glutamate application at the somatic regions of CA^ cel ls induce reductions in both extracellular _ ++ ++ Ca and Na concentrations here, although there is no reason to feel that al l depletion is due to CA-^  neurones only. - l i -l t has been reported that following repetit ive f i r ing in hippocampal ce l ls induced by depolarizing current pulses (Gustafsson and Wigstrom, 1981; Hotson and Prince, 1980; Schwartzkroin and Stafstrom, 1980), e p i -leptogenic agents (Alger and N ico l l , 1980; Habl i tz , 1981; Schwartzkroin and Stafstrom, 1980), synaptic excitation (Nicoll and Alger, 1981) or glutamate (Segal, 1981), there is an afterhyperpolarization that results . The hyperpolarization appears to have two components (Gustafsson and Wigstrom, 1981; Schwartzkroin and Stafstrom, 1980) which may be mediated by a Ca + + -act ivated potassium conductance (Alger and Nico l l , 1980; Gustafsson and Wigstrom, 1981; Hotson and Prince, 1980; Johnston et a l . , 1980; Nicoll and Alger, 1981; Schwartzkroin and Stafstrom, 1980) and/or ++ activation of a Na -pump (Segal, 1981). There are reports suggesting that glutamate-induced excitations are antagonized by glutamic acid diethyl ester (GDEE) in a relat ively specif ic way, and that this drug also inhibits synaptic excitation (Haldeman et  a l . , 1972; McLennan, 1974). However, i ts usefulness as an antagonist has been questioned by others (Curtis et a l . , 1972; Krnjevic, 1974; Zieglgansberger and P u i l , 1973) and i t is necessary to search for other antagonists with spec i f ic i ty and af f in i ty for glutamate receptors. In addition to a low af f in i ty uptake system for glutamate, there exists a high af f in i ty uptake mechanism (Bennett et a l . , 1973; Logan and Snyder, 1972). The high af f in i ty system, in contrast to the low af f in i ty uptake, shows a marked dependence on Na ions (Bennett et a l . , 1973). Glutamate accumulated in the presence of Na+ is released in a C a + + -dependent manner in response to high extracellular K + , whereas glutamate accumulated in the absence of Na does not show such a release (Mulder and Snyder, 1974). 3 In hippocampal s l ices incubated with H-glutamate, autoradiographic studies indicate that the label becomes concentrated in Stratum radiatum and oriens of CA-j-CA^, these sites being target areas of i p s i - and contralateral projections of CA3 and CA^ neurones (Iversen and Storm-Mathisen, 1976; Storm-Mathisen and Iversen, 1979). Surgical interruption of Comm and Sch fibres to Stratum oriens and radiatum of CA^  led to a marked decrease (80-90%) in glutamate uptake from the CA^ area (Storm-Mathisen, 1977b). Although the above observations suggest that uptake of glutamate is speci f ica l ly associated with nerve endings in CA-^ , and may act as an excitatory transmitter here, i t should be kept in mind that glutamate and aspartate seem to travel by the same ion transport system (Balcar and Johnston, 1972; Roberts and Watkins, 1975) and uptake studies may not distinguish between these two amino acids. More evidence in support of glutamate and/or aspartate as a transmitter between Schaffer coll ateral-CA-^ neuronal synapses is provided by Wieraszko and Lynch (1979) who found that exogenous H-glutamate is released upon stimula-++ tion of Sch in a Ca -dependent manner. In the same connection, 3 Malthe-S0renssen et a l . (1979) loaded s l ices with D- H-aspartate (a false transmitter considered to be a marker for glutamate releasing neurones that is not metabolized) and demonstrated a Ca+ +-dependent stimulus evoked release of transmitter by stimulation of Sch. Kainic acid-induced destruction of CA^ cel ls (which give r ise to the Schaffer col laterals) results in a decrease of both aspartate and glutamate in the dorsal hippocampus (Fonnum and Walaas, 1978). The incorporation of from C-glucose into aspartate and glutamate as well as a Ca -dependent release of these amino acids was increased by stimulation of the Stratum radiatum (where Sch and Comm fibres can be found) (Corradetti et a l . , 1983). The evidence available so far suggests that either aspartate or glutamate (or possibly both) is the excitatory neuro-transmitter between the Sch-CA-^ neuronal synapse but further work is required to determine if one or the other (or both) mediate synaptic transmission at this s i te . "Antagonists" of excitatory amino acids in hippocampus As discussed previously, the excitatory transmitter(s) between Sch-CA-L neuronal synapses is probably L-glutamate and/or L-aspartate. Various "antagonists" of these amino acids have been studied in different areas of the central nervous system, including the hippocampus. On the basis of studies in the spinal cord, amino acid receptors were c lass i f ied and antagonists to these receptors were f i r s t described (McLennan, 1981; watkins, 1981a, 1981b). Recently, Fagni et a l . (1983) have c lass i f ied excitatory amino acid receptors in the hippocampus. Four dist inct types of amino acid receptors have been proposed to exist in the CA-^  region of the hippocampus: 1) an N-methyl-DL-aspartate (NMA) preferring receptor activated by NMA and D-glutamate; 2) a kainate preferring receptor; 3) an L-glutamate/aspartate preferring receptor; 4) a DL-homocysteate (DLH) activated receptor, proposed to be the synaptic receptor (Fagni et a l . , 1983). The following antagonists were examined in the CA-^  region of the hippocampus: 1) D-(-)-a-aminoadipate (<xAA) has been reported to block excitations induced by NMA (Col 1ingridge et a l . , 1983a; Fagni et a l . , - 14 -1983), DLH and endogenous transmitter of Sch/Comm afferents (Fagni et a l . , 1983); 2) ( ± ) - 2 - a m i n o - 5 - p h o s p h o n o v a l e r a t e (APV) blocks NMA selectively and is more potent than oAA in this respect (Collingridge et a l . , 1983a); 3) Y-D-glutamylglycine (DGG) inhibits the responses to NMA, kainate and aspartate (Collingridge et a l . , 1983a); 4) D-(-)-2-amino-4-phosphono-butyric acid (D-APB) blocks kainate and NMA responses but L-APB had no antagonistic effects and seemed to be an agonist instead (Collingridge et a l . , 1983a). Fagni et a l . (1983) however, reported that NMA and kainate are not blocked by APB whereas the DLH activated and synaptically excited receptors are. One should be cautious in interpreting the results of Fagni et a l . (1983) because these authors used DL-APB and not D-APB in their studies. 4) L-Glutamic acid diethyl ester (GDEE), according to Col 1ingridge et a l . (1983a) does not show specif ic antagonism of any amino acid excitations. In contrast, Spencer et a l . (1976) provided evidence for GDEE antagonism of glutamate and aspartate induced responses. Long-lasting potentiation of synaptic responses Long-lasting potentiation (LLP) of synaptic responses has been observed in vivo following high frequency e lectr ical stimulation in the hippocampal dentate area in both anesthetized (Bliss and L#mo, 1973) as well as unanesthetized (Bliss and Gardner-Medwin, 1973) rabbits. In these experiments, LLP was manifested either as a decrease in latency of the population spike, an increase in size of the population spike, or an enhancement in the amplitude of the population EPSP (or any combination of the three). F ig . 2 shows population responses recorded at the CA^ cel l body layer and apical dendrites in response to Sch stimulation. Extra-ce l lu lar ly recorded potentials which appear positive can be a result of - 15 -FIG. 2 Population responses in the CA^ area evoked by Schaffer col lateral stimulation. On the left is a diagram of a CA^ neurone and on the right are responses monitored at the cel l body layer and dendritic regions. Record A shows an extracellular population "EPSP" recorded at the cel l body layer. Record B_ shows a population spike interrupting the population "EPSP" when stimulus intensity is increased and the bottom trace {C) is a population "EPSP ,P recorded at the apical dendritic/synaptic area. Al l three records were obtained from the same experiment. The responses were evoked at 0.2 Hz and each record is an average of four consecutive sweeps. Posit iv i ty is upwards. - 16 -10ms local ( i . e . , at the recording site) hyperpolarization or distant ( i . e . , away from the recording site) depolarization of the neuronal membrane. Conversely, a negative potential can either be due to a local depolari -zation or a distant hyperpolarization. The positive wave (Fig. 2A) is due to the population excitatory postsynaptic potential ("EPSP") recorded at the cel l body layer which is l ike ly a mixture of f ie lds generated by the pure EPSP from the dendrites (recognized as an extracellular posi t iv i ty at the somata) and the local inhibitory postsynaptic potential (IPSP). In this record, the stimulus intensity was insuff icient to evoke a syn-chronous population spike. However, in F i g i 2B, i t is seen that an increased stimulation strength produces an interruption of the population "EPSP" by a sharp negatively going population spike. The magnitude of the population spike is an index of the number of postsynaptic neurones a c t i -vated synchronously by the stimulus. The trace in Fig. 2C i l lustrates a population "EPSP" recorded at the apical dendritic/synaptic region (100-200 urn from CA^ cel l body layer). In this case, the wave is nega-tive as i t is at the source for the dendritic EPSP and in addition recog-nizes the distant IPSP (from the somata) as being negative here. LLP has also been demonstrated in the intact rat dentate area (Douglas and Goddard, 1975). This phenomenon can be e l ic i ted in the in vitro s l i ce preparation in the dentate, CA^, as well as CA-^  areas of guinea pig and rat hippocampus (Alger and Teyler, 1976; Andersen et a l . , 1977; Lynch et a l . , 1976; Schwartzkroin and Wester, 1975; Yamamoto and Chujo, 1978) (see Fig . 3). The hippocampus has been thought to be involved in processes such as learning and memory (Best and Best, 1976; Coleman and Lindsley, 1977; - 18 -FIG. 3 Long-lasting potentiation of the CA^ neurona.1 population spike induced by a tetanic stimulation (100 Hz, 1 s) of the Schaffer col lateral input. The f i r s t record shows the control population spike and the following three are the post-tetanic responses at the times indicated above each trace. Responses were evoked at 0.1 Hz by stimulation of Sch. Negativity is down. Each trace is an average of four consecutive sweeps. - 19 -Green, 1964; Isaacson, 1974; O'Keefe and Nadel, 1978; Olds et a l . , 1972; Penfield and Milner, 1958; Scovi l le and Milner, 1957; Segal and Olds, 1973). However, the precise role of the hippocampus and the mechanism(s) responsible for learning are not c lear. It has been reported that hippocampal neurones show a marked increase in act iv i ty during classical conditioning which continues to grow over the course of training (Berger and Thompson, 1978; Berger et a l . , 1976). On the basis of these findings, hippocampal LLP has been considered as a possible mechanism for learning and memory (Berger and Thompson, 1978; Chung, 1977; Teyler, 1976). ++ There is evidence in the l i terature which suggests that LLP is Ca -dependent (Dunwiddie and Lynch, 1979; Wigstrom et a l . , 1979) and is not related to short-term forms of p last ic i ty l ike frequency f a c i l i t a t i o n , paired pulse fac i l i ta t ion and post-tetanic potentiation that can be e l i -cited in low C a + + concentrations (Dunwiddie and Lynch, 1979). Further-more, Wigstrom and Swann (1980) have demonstrated that the C a + + agonist _ ++ Sr supports synaptic transmission and LLP in the hippocampus. A ++ transient exposure of hippocampal s l ices to elevated Ca concentrations results in a condition that resembles LLP (Turner et a l . , 1982) and 45 + + Ca uptake and retention as well as total intracel lular content of C a + + was signif icantly increased for prolonged periods of time following return to the control medium (Baimbridge and Mi l le r , 1981). The transmitter(s) between Schaffer col laterals and CA-^  neurones is thought to be glutamate and/or aspartate (see Storm-Mathisen, 1977a for review). If transmitter released during tetanic stimulations acts on pre- and/or postsynaptic receptors to trigger events leading to LLP, then - 20 -a blockade of these receptors during the tetanus should result in fa i lure to induce LLP. Some amino acid "antagonists" have been tested on the development of LLP. Collingridge et a l . (1983b) report that y-D-glutamyl-glycine (DGG) and 2-amino-5-phosphonovalerate (APV), presumed by them to be selective antagonists of N-methyl-DL-aspartate (NMA) (Collingridge et  a l 1 9 8 3 a ) , block LLP. It has subsequently been demonstrated that DGG (Dolphin, 1983) masks, rather than antagonizes LLP development. Krug et_ a l . (1982) fe l t that glutamic acid diethylester (GDEE), which has been reported to antagonize glutamate and aspartate induced excitations in dentate granule cel ls (Spencer et a l . , 1976), blocked the induction of LLP. They (Krug et a l . , 1982) observed a marked depression of the control response for prolonged periods of time by GDEE and proceeded to examine the development of LLP in the presence of this depression. Unfortunately, they did not follow the response beyond the recovery time of the drug-induced depression, and i t could well be that LLP was concealed, but not blocked by the "antagonist". DL-2-Ami no-4-phosphonobutyrate (DL-APB), presumed by the authors to be a glutamate antagonist, was also reported to block LLP while not antagonizing the effects of applied glutamate (Dunwiddie et a l . , 1978), and perhaps blocked LLP through other actions rather than a blockade of the amino acid receptors. The exact mechanism involved and the locus for LLP are at present unclear and there is evidence for both the pre- and postsynaptic sites in the development and maintenance of LLP. Several theories have been proposed as mechanisms for LLP: 1) an increase in the number of sub-synaptic transmitter receptors brought about by activation of a C a + + -dependent membrane bound protease (Baudry and Lynch, 1980a); 2) increased - 21 -transmitter release from presynaptic terminals (Andersen et a l . , 1977; Dolphin et a l . , 1983; Sastry, 1982; Skrede and Malthe-Sj&renssen, 1981); 3) morphological changes in postsynaptic dentrites leading to increased subsynaptic excitabi l i ty (Desmond and Levy, 1981; Fifkova and Van Harreveld, 1977; Horwitz, 1981; Lee et a l . , 1980; Van Harreveld and Fifkova, 1975); 4) calmodulin (an intracel lu lar Ca + + -buffer ing protein), plays an important role in the induction of LLP (Baimbridge and Mi l le r , 1979; Finn et a l . , 1980); 5) N-methyl-DL-aspartate (NMA) pre-ferr ing receptors (Col 1ingridge et a l . , 1983b) in response to transmitter released during tetanic stimulations, triggers events leading to LLP. Evidence for a presynaptic role in LLP LLP e l ic i ted in the CA^ area of the hippocampus has been demon-strated to be input specif ic (Andersen et a l . , 1977; Lynch et a l . , 1976). It has been demonstrated that the induction of LLP is dependent on the extracellular C a + + concentration (Dunwiddie and Lynch, 1979; Wigstrom et a l . , 1979). Trains of high frequency tetani (100 Hz) that do not evoke synchronous population spikes in dentate granule cel ls can e l i c i t LLP (Bliss and Ltfmo, 1973). Intracellular recordings in the somata of CA^  neurones reveal that the membrane potential does not change during LLP (Andersen et a l . , 1977). A hyperpolarization of CA-^  ce l l s that markedly reduced or abolished postsynaptic discharges during afferent input tetanization could s t i l l e l i c i t LLP (Wigstrbm et a l . , 1982). All the observations above, although suggestive of a presynaptic mechanism for LLP, cannot rule out a possible postsynaptic change localized to the CA^ dendrites in the synaptic zone. An alteration limited to the postsynaptic dendritic membrane can account for the input spec i f ic i ty of the pheno-- 22 -menon. Similarly , a membrane potential change localized to this area may not be recognized at the somatic recording s i te . Tetanizing at high frequencies or hyperpolarization of CA^  ce l l s during the tetanus can result in minimal activation of the subsynaptic membrane. It is possible that the hyperpolarizing pulse which was applied at the soma (Wigstrom et al•, 1982) did not penetrate into the distal dendrites where the synapses are located even though the authors claim that i t did. The high frequency tetanic stimulations which f a i l to produce synchronous discharges in CA^ cel ls wi l l invariably result in release of some transmitter into the synaptic c left and it may be this transmitter that acts on the subsynaptic membrane to bring about changes leading to LLP. Additional evidence in support of a presynaptic mechanism for LLP include a decrease in axon terminal exc i tabi l i ty associated with LLP (an effect that is not seen at the non-terminal regions) (Sastry, 1982), an increased secretion of proteins into extracellular medium during LLP which is limited to the potentiated region of the hippocampal s l i ce (Duffy et a l . , 1981), an increased resting and evoked release of exogenously loaded D-( H)aspar-tate following tetanization in the hippocampus (Skrede and Malthe-Stfrenssen, 1981). Recently, Dolphin et a l . (1983) demonstrated that LLP 3 of the perforant path is associated with increased H-glutamate release 3 newly synthesized from infused H-glutamine. Quantal analysis of exc i -tatory postsynaptic potentials (EPSPs) in hippocampus has shown that frequency potentiation in the dentate granule cel ls may be explained by an increase in the number of quanta released by the presynaptic f ibres (Yamamoto, 1982). Furthermore, a similar analysis of LLP in the crayfish neuromuscular junction has shown that the phenomenon can be explained by - 23 -an entirely presynaptic mechanism (Baxter and Brown, 1983). In addition, Voronin (1980) has demonstrated that there is an increase in the number of released quanta but no change in quantal size during LLP, indicating a presynaptic locus of control. Jack et a l . (1981a, 1981b) have postulated that at synapses of primary afferents and motoneurones in the spinal cord, the amount of transmitter released is suff ic ient to saturate al l sub-synaptic receptors so that increased transmitter release wil l not result in a larger response. They also suggested that PTP is due not to an increase in transmitter release but to an activation of more presynaptic terminals. If these situations exist in the hippocampus, then quantal analysis would not y ie ld meaningful results . On the other hand, the ^opposite situation exists at the neuromuscular junction, where the number of subsynaptic receptors present greatly exceeds the number required for synaptic transmission, i . e . , spare receptors are present. In this case, an increase in transmitter release wi l l result in fac i l i ta t ion of the response. Evidence for a postsynaptic role in LLP It has been suggested that LLP is due to a postsynaptic change (Baudry and Lynch, 1980a; Desmond and Levy, 1981; Douglas et a l . , 1982; Fifkova and Van Harreveld, 1977; Lee et a l . , 1980; McNaughton et a l . , 1978; Van Harreveld and Fifkova, 1975). The induction of LLP is dependent on the presence of extracellular C a + + (Dunwiddie and Lynch, 1979; Wigstrom et a l . , 1979). It is interesting, however, that high frequency tetanic stimulations (100 Hz) to an input (which is favourable for the observation of LLP) results in l i t t l e or no C a + + influx into the CA^ ce l ls (Chirwa et a l . , 1983) whereas low frequency (20 Hz) tetani (which favour the - 24 -observation of homo- and heterosynaptic depressions) cause a much greater CA^ neuronal influx of the divalent ion (Chirwa et a l . , 1983; Morris et  a l . , 1983). Dunwiddie et a l . (1978) feel that synaptic transmission is required for in i t iat ion of LLP. The drug used to block the postsynaptic response (DL-2-amino-4-phosphonobutyric acid, which they presumed to be a glutamate antagonist), however did not block the effects of iontophore-t i c a l l y applied glutamate (the suspected transmitter) to the dendrites of pyramidal c e l l s . It is possible that the drug had inhibitory actions on the presynaptic terminals and the val id i ty of these results as evidence for a postsynaptic mechanism is questionable. A Ca + + -act ivated protease seems to be involved in the regulation of hippocampal glutamate receptors (Baudry and Lynch, 1980b; Baudry et a l . , 1981a, 1981b; Vargas and Costa, 1981) . Baudry and Lynch (1980a) put forth the hypothesis that CAX neuronal accumulation of Ca that takes place during the tetanic stimulation of the inputs induces an activation of a membrane-bound protease leading to the uncovering of new glutamate receptors which are responsible for LLP. According to these authors, LLP can be due to an increase in the binding of glutamate to new receptors. They, therefore, presume that an excess of transmitter release is present at these synapses than can be captured by the control number of receptors. There is evidence for an increase in glutamate binding sites following tetanic stimulations in hippocampal sl ices (Baudry et a l . , 1980c; Lynch et a l . , 1982) that is irreversible (Baudry et a l . , 1983). Following exposure of isolated hippocampal membranes to elevated C a + + concentrations, a similar increase in glutamate binding is observed (Baudry and Lynch, 1979). Lynch et a l . (1983) reported that intracel lular injections of EGTA - 25 -into CAj ce l ls block the induction of LLP. If the results of Jack et  a l . (1981a, 1981b) can be extrapolated to the hippocampus, then an increase in glutamate receptors could account for LLP. These authors postulated that at the synapses between primary afferents and motoneurones in the spinal cord, the amount of transmitter released from the presynaptic terminals exceeds the quantity required for occupation of a l l the sub-synaptic receptors. In this case, i t is logical to assume that an in -crease in synaptic transmission can be achieved by increasing the number of subsynaptic transmitter receptors. Other l ines of evidence have been interpreted to be postsynaptic explanations for LLP. There seems to be a co-operativity of afferents in the induction of LLP (Lee, 1983; McNaughton et a l . , 1978; Robinson and Racine, 1982). This co-operativity has been thought to mean a postsynaptic locus for LLP (Douglas et a l . , 1982; McNaughton, 1982; McNaughton et a l . , 1978; Robinson and Racine, 1982). There is no reason, however, to assume that interactions among inputs is entirely postsynaptic as they could just as l ikely be pre-synaptic. In fac t , i t has been recently demonstrated that co-operativity is not due to an increased postsynaptic discharge during conditioning, but rather to interactions among afferents (Lee, 1983). Although there seems to be input spec i f ic i ty of LLP in the CA-^  region (Andersen et a l . , 1977; Lynch et a l . , 1977), this spec i f ic i ty is absent in the CA^ area (Misgeld et a l . , 1979; Yamamoto and Chujo, 1978). LLP in the dentate area e l i c i ted by perforant path tetanization was prevented or reduced by stimulation of other afferents that resulted in an inhibit ion of the granule c e l l s . In the same vein, Wigstrom and Gustafsson (1983a) demon-strate a fac i l i ta ted LLP during blockade of inhibit ion in the CA^ area - 26 -(presumed by the authors to be postsynaptic inhibi t ion) . It has recently been reported that homosynaptic LLP is enhanced by simultaneously tetaniz -ing the homo- and a heterosynaptic input (Wigstrom and Gustafsson, 1983b). As mentioned previously, pre- and/or postsynaptic interactions of inputs may account for the absence of input spec i f ic i ty in CAg as well as for the inhibitory or heterosynaptic modulation of LLP in the dentate and CA^ areas. Anatomical changes in dendritic morphology following tetanic stimu-lations have been interpreted to represent a postsynaptic change involved in LLP (Lee et a l . , 1980; Desmond and Levy, 1981; Fifkova and Van Harreveld, 1977; Horwitz, 1981; Van Harreveld and Fifkova, 1975). It has been reported that there is a swelling of dendritic spines in dentate granule cel ls after tetanic stimulations to the perforant path (which results in an increase in surface area) (Fifkova and Van Harreveld, 1977; Van Harreveld and Fifkova, 1975). One would expect an increase in surface area of the dendritic membrane to increase capacitance and hence decrease the exc i tabi l i ty of the processes so i t is puzzling why these authors suggest that this mechanism may be responsible for LLP. In fact, spread-ing depression has been shown to be characterized by swelling of spines (Van Harreveld and Khattab, 1967). Lee et a l . (1980) demonstrated that brief bursts of high frequency stimuli produce an increase in the density of dendritic shaft synapses and a decrease in dendritic spine var iab i l i t y , suggesting an increase in efficacy for synaptic transmission. Homo- and heterosynaptic depressions Following tetanic stimulations of an input to the CA-^  area of hippo-campus, one observes a transient but re lat ively prolonged homosynaptic - 27 -( i . e . , to the same input) (Andersen et a l . , 1980; Barrionuevo et a l . , 1980; Dunwiddie and Lynch, 1978) as well as heterosynaptic ( i . e . , to an untetanized input) depression of evoked responses (Abraham and Goddard, 1983; Alger et a l . , 1978; Dunwiddie and Lynch, 1978; Lynch et a l . , 1977). The extent and/or time course of the depression seems to be f r e -quency dependent, low stimulation frequencies (1-20 Hz) favouring the observation of homo- and heterosynaptic depressions while high frequencies (100-500 Hz) favouring the observation of LLP (Barrionuevo et a l . , 1980; Dunwiddie and Lynch, 1978). LLP, therefore, seems to be favoured when there is minimal postsynaptic activation while depression is preferred in the presence of enhanced postsynaptic discharge. Indeed, i t has been demonstrated that there is massive frequency fac i l i ta t ion during low frequency tetani (Andersen and Ltfmo, 1967; Creager et a l . , 1980) that is reduced or abolished during high frequency stimulations (Andersen and Ltfmo, 1967; Bl iss and Ltfmo, 1973), the latter observation presumably being due to a depletion of releasable transmitter or to a blockade of evokable release. This frequency fac i l i ta t ion can be sustained for the duration of the tetanus. The early stage of the fac i l i ta t ion appears to be generated presynaptically (Creager et a l . , 1980) by an increase in transmitter release (Yamamoto, 1982). However, the later phase of poten-t iat ion during a prolonged low frequency tetanus is thought to originate postsynaptically (Creager et a l . , 1980). During high stimulus frequencies the late fac i l i ta t ion is absent, whereas during low frequency tetani , one sees this fac i l i ta t ion quite clearly (Creager et a l . , 1980; Dunwiddie and Lynch, 1978). Since low frequency stimulations are optimal for e l i c i t ing - 28 -homo- and heterosynaptic depressions, i t is logical to assume that changes which take place to result in these depressions are postsynaptic in nature. During low frequency tetanic stimulations, there is a marked depletion of extracellular C a + + in the region of the postsynaptic cel l body (Benninger et a l . , 1980; KrnjeviC et a l . , 1980). This depletion, a l -though present at the dendritic/synaptic areas is not as prominent as in the cel l body area (Krnjevic et a l . , 1980), suggesting that C a + + influx during tetanus is primarily into the soma of postsynaptic c e l l s . In this connection, intracel lular measurements of Ca concentration during low and high frequency tetani reveal that there is a marked increase in int ra -cel lu lar C a + + during the low frequency (20 Hz) tetanus (Chirwa et a l . , 1983; Morris et a l . , 1983) and there is much less or no Ca accumu-lation during high frequency (100 Hz) tetanus (Chirwa et a l . , 1983). Whether or not C a + + is involved in the in i t iat ion of homo- and hetero-synaptic depressions is unknown and should be examined. It is possible that both LLP and the depressions are postsynaptic but due to different ++ levels of Ca accumulation into CA-^  neurones. Low concentrations of ++ intracel lu lar Ca may trigger events leading to LLP while high concen-trations can result in homo- and heterosynaptic depressions. Effects of verapamil in the hippocampus Verapamil, an agent that is known to block C a + + currents in a variety of tissues (Fleckenstein, 1977) also exerts C a + + blocking effects in the hippocampus. It has been shown that the agent blocks C a + + currents and spikes in CA^  neurones (Dingledine, 1983; Gr i f f i th ++ and Brown, 1982) and veratridine-induced Ca entry in synaptosomes while i t has no effect on transmitter release (Nachshen and Blaustein, - 29 -1979; Norris et a l . , 1983). Verapamil, at low doses (approximately 1 u M ) , blocks C a + + channels selectively but at higher concentrations (30-200 uM) may exert local anaesthetic effects by blocking active Na+ channels (Norris et a l . , 1983). There is recent evidence to indicate that the agent exerts its effects through an intracel lular site (Reynolds et  a l . , 1983). Role of inhibition in LLP There is evidence for (Douglas et a l . , 1982; Misgeld et a l . , 1979; Wigstrom and Gustafsson, 1983a; Yamamoto and Chujo, 1978) as well as against (Haas and Rose, 1982) the involvement of inhibitory processes in the induction of LLP. Since presynaptic inhibit ion has not as yet been demonstrated in the hippocampus, the authors who feel that a postsynaptic inhibition plays a role in LLP (Douglas et a l . , 1982; Misgeld et a l . , 1979; Wigstrom and Gustafsson, 1983a; Yamamoto and Chujo, 1978) presumed that the locus for the inhibit ion and, hence, LLP are postsynaptic. Recently, i t has been demonstrated that in addition to the classic recur-rent inhibit ion mediated by basket cel ls (Andersen et a l . , 1964; Spencer and Kandel, 1961c), feed forward inhibition is present in the dentate gyrus (Buzsaki and Eidelberg, 1981, 1982) as well as the hippocampal CA^ region (Buzsaki and Eidelberg, 1982; Knowles and Schwartzkroin, 1981). Furthermore, LLP can be generated in these interneurones (Buzsaki and Eidelberg, 1982). Whether the LLP of inhibitory mechanism(s) affects the in i t iat ion and/or maintenance of LLP of excitatory inputs remains to be explored. - 30 -Paired pulse and frequency potentiation in hippocampus When afferents in the hippocampus are stimulated twice in rapid succession, there is a fac i l i ta t ion of the EPSP, population spike and unitary f i r i ng to the second of the responses (Andersen, 1960a, 1960b; Bliss and Gardner-Medwin, 1973; Creager et a l . , 1980; Dunwiddie and Lynch, 1979; Fujita and Sakata, 1962; Ltfmo, 1971; Steward et a l . , 1977). This form of potentiation is thought to be generated presynapti-ca l l y because i t is present homo- but not heterosynaptically (Creager et a l . , 1980) and quantal analysis reveals that i t can be explained entirely by an increase in the number of released quanta (Yamamoto, 1982). The presynaptic volley is unchanged during the second response, suggesting that the fac i l i ta t ion is not due to an increase in the number of activated fibres (Creager et a l . , 1980). Lowering the extracellular C a + + or increasing Mg + + concentration increased the potentiation, an effect that ++ may be related to a Ca -dependent depletion of transmitter by the f i r s t impulse thereby reducing the amount available for the second response (Creager et a l . , 1980). The exact mechanism(s) responsible for paired pulse potentiation are presently unclear and need to be investigated further. Frequency fac i l i ta t ion which develops slowly during the course of repetit ive low frequency (1-30 Hz) stimulation has been seen in the hippo-campus (Andersen, 1960a, 1960b; Andersen and Ltfmo, 1967; Bl iss and Garder-Medwin, 1973; Creager et a l . , 1980; Dudek et a l . , 1976; Dunwiddie and Lynch, 1979; Gloor et a l . , 1964; Lrfmo, 1966). This late potentiation, which is dist inct from early fac i l i ta t ion is thought to be postsynaptic because i t can be evoked by a heterosynaptic test pulse given - 31 -during the tetanus (Creager et a l . , 1980). It is interesting that follow-ing a low rather than high frequency tetanus, homosynaptic (Andersen et  a l . , 1980; Barrionuevo et a l . , 1980; Dunwiddie and Lynch, 1978) as well as heterosynaptic depressions (Alger et a l . , 1978; Dunwiddie and Lynch, 1978; Lynch et a l . , 1977) can be seen, suggesting that frequency f a c i l i -tation may help in mediating events leading to the depressions. The late fac i l i ta t ion has been postulated to be due to increased extracellular K + accumulation resulting in an enhancement of the exci tabi l i ty of post-synaptic elements (Creager et a l . , 1980). Indeed, i t has been demon-strated that low frequency tetani to inputs in the hippocampus result in markedly elevated concentrations of extracellular K+ (Benninger et a l . , 1980; Fr i tz and Gardner-Medwin, 1976; Krnjevic et a l . , 1980). METHODS Slice preparation All experiments were conducted on transversely sectioned rat (wistar or Sprague-Dawley male, 100-150 g) hippocampal s l i ces . The animal was anaesthetized with a mixture of halothane and 0£ or halothane, ^ 0 and 0£ while being cooled on an ice pack for 30-35 min. Rectal temperature just prior to surgery was 31-32°C. An incision of the skin covering the head was made sagi t ta l ly to expose the skul l . The skull was then care-fu l ly removed, the brainstem sectioned just caudal to the pons and the brain scooped out. The hippocampus from one hemisphere was located and excised, and transverse sl ices of 400-500 yM were made with a Mcllwain tissue chopper. Prior to being transferred to the s l ice chamber, the sl ices were placed in oxygenated (95% 0 ? , 5% C0 ?) standard medium - 32 -(NaCl: 120 mM, KC1: 3.1 mM, NaHC03: 26 mM, NaH2P04 1.3 mM, CaC l 2 : 2 mM, MgCl 2 : 2mM, dextrose: 10.0 mM, pH 7.4 while bubbled with 95% 0 2 , 5% C02) maintained at 4°C to decrease metabolic rate and oxygen requirements. The whole procedure from the time of incision of the skin to the time when the s l ices were submerged in cold medium took 2 min or less. The sl ices were then transferred quickly to the s l i ce chamber and sandwiched between two nylon meshes to minimize movement. They were submerged in the standard medium and temperature was maintained at 32 ± 0.5°C throughout al l experiments. A mixture of 95% 0 2 , 5% C02 was constantly bubbling into the standard medium for perfusion and was also delivered to the sl ices via an additional line above them. Flow rate of medium was maintained at 3 ml/min. It has been reported that prolonged incubation of brain s l ices leads to a gradual alteration in the act iv i ty of Na + , K+-ATPase (Kovachich and Mishra, 1981). Since the properties of sl ices incubated for different durations might be dif ferent , only one s l i ce per animal was used, even though they survived for more than 12 hr in the bath. All s l ices were incubated for 1 hr to allow for equi l ibra-tion before any recordings were in i t iated. Recording and stimulating systems Signals from recording electrodes (fibre f i l l e d glass borosil icate microelectrodes, Frederick Haer and Co. , t ip 1-2 um, f i l l e d with 4 M NaCl, resistance 1-2 Mfi ) were fed to a DAM-5A amplifier (WPI Instruments) or a Neurolog System amplifier before being displayed on a Tektronix storage oscilloscope or a Data 6000 (Data Precision) signal processor. Quanti-tative records of population responses were obtained by averaging 4-8 consecutive sweeps and were either captured on polaroid f i lm, plotted out - 33 -on a Hewlett Packard 7404A str ip chart recorder, or plotted on a Hewlett Packard 7470A graphics plotter. To monitor f i r i n g rates of CA^ or CAg c e l l s , the amplified signal was led to a ratemeter and the output connec-ted to a Grass polygraph or to a Hewlett Packard 7404A recorder to obtain records. Stimulation of the Sch and/or Comm inputs to evoke population res -ponses at 0.1-0.2 Hz in the CA^ area was achieved using bipolar concen-t r i c metal stimulating electrodes (SNEX 100, Rhodes Electronics, res i s -tance 1-2 ) and antidromic activation of single CAg ce l ls was done using either a monopolar glass electrode similar to the recording elec-trode or a 4 M NaCl f i l l e d barrel of a 7 barrel iontophoresis electrode. Negative pulses were used to evoke a l l responses. A Grass S88 stimulator was ut i l ized for delivering stimulation pulses through either a Grass SIU5 stimulus isolation unit or a Grass SIU6 constant current unit for popula-tion responses as well as antidromic single cel l activation. A Digitimer model DS2 stimulator controlled by a Digitimer D4030 programmer was also used to evoke population responses. Induction of LLP and homo- and heterosynaptic depressions Control population spikes were recorded in the CA^ cel l body layer with a glass microelectrode and evoked by Schaffer col lateral and/or commissural stimulation (see Fig . 4) at 0.1-0.2 Hz with negative pulses. The size of the control population spike was chosen to be relat ively constant (1-2 mV) in each series of experiments so that changes due to tetanic stimulation or drug applications in different sl ices could be compared. In some experiments, the population "EPSP" in the apical den-d r i t i c region (100-200 urn from CA, ce l l body layer) was recorded in - 34 -R R- recording electrode S-stimulating electrode FIG. 4 Placement of stimulating and recording electrodes for induction of long-lasting potentiation and/or homo- and heterosynaptic depressions of the CAi population spike. Separate stimulating electrodes were placed on Schaffer col laterals and commissural input to synaptically activate apical and basilar dendrites, respectively, of presumably the same population of CA^  neurones that were recorded from. To induce LLP, a high frequency tetanus (100 Hz, 1 s or 400 Hz, 200 pulses) was delivered to an input and to induce homo- and heterosynaptic depressions, a low frequency tetanus (20 Hz, 200 pulses) was given. - 35 -addition to the population spike in the cel l body layer in response to Sch stimulation. To induce LLP, a high frequency tetanic stimulation (100 Hz, 1 s or 400 Hz, 200 pulses) was applied to Sch or Comm input at control stimula-tion strengths. To examine homo- and heterosynaptic depressions of the population spike, a 20 Hz, 200 pulses tetanus was given. Post-tetanic recordings were made at regular intervals (2-5 min) for 60 min and 4 - 8 consecutive responses were averaged routinely for quantitative studies. The actions of verapamil ( 0 . 3 3 uM, 3 min) to the whole bath were exa-mined on the depression-inducing (20 Hz) as well as the LLP inducing (400 Hz) tetani . The application of the agent was commenced at 2 min 30 s before the tetanus was delivered and terminated 30 s later . In cases where verapamil produced a suppression of the response, stimulation strengths were adjusted to obtain pre-drug control population spike sizes just prior to tetanus and control stimulation strengths were resumed immediately post-tetanus. As in the previous experiments, the post-tetanic responses were followed for 60 min. Effects of drug application to the whole bath on the CA^ population spike Schaffer col laterals were stimulated at 0 . 1 - 0 . 2 Hz ( 0 . 1 - 0 . 3 ms nega-tive pulses, 2-6 V) with a bipolar concentric electrode (SNEX 100, Rhodes Electronics, resistance 1-2 Mo. ). The population spike in response to the stimulation was recorded in the cel l body layer of CA-^  with a glass electrode f i l l e d with 4 M NaCl. The same electrode was used to monitor the f i r i ng rate of CA-^  c e l l s . Stimulation strength to evoke the spike was adjusted to e l i c i t responses of constant size (1-2 mV) in each experi-ment to allow room for potentiation as well as comparison of results. - 36 -Following the 1 hr incubation of the s l i ces , an evoked population spike was obtained and control records were taken for at least 30 min to ensure their s tab i l i t y . Either DL- (3 mM, 5 min) or D-(l mM, 5 min) a-Amino-adipate (aAA; Sigma Chemicals), N-methyl-DL-aspartate (NMA, 200 yM, 1 min; Sigma Chemicals) or verapamil (0.33 yM, 3 min; Knoll) was then infused into the bath before reinst i tut ing the standard medium. In some experiments, NMA (200 yM, 1 min) and verapamil (4 yM, 4 min) were applied concurrently. The verapamil application was in i t iated 2 min before and continued for 1 min after NMA was terminated. In addition, the effects of Ca + + - f ree medium (1 mM Mn + + , 3 mM Mg + + ; applied for 3 min prior to, continued during and 1 min after NMA) was also examined. Post-drug popu-lation spikes were monitored for 60 min. Al l records were taken at 2-5 min intervals and 4-8 consecutive sweeps were averaged for each record. Fir ing rate of CA^  ce l l s was plotted on a Grass polygraph. Localized applications of L-glutamate to the CA-^  and CAg areas See F ig . 5 for experimental set-up. The Sch-induced population spike and f i r ing rate of CA-^  ce l l s at the CA^ cel l body layer were evoked and recorded as described in the previous section. In some experiments, a recording electrode ( f i l l e d with 4 M NaCl, t ip 1-2 ym, resistance 1-2 Mn ) was placed in the CAg cel l body layer to record f i r ing rate of CAg c e l l s . Following 30 min of controls, localized application of oxygenated L-glutamate (Sigma Chemicals, 5 mM, 0.51 ml/min by an infusion pump) for 5 s was achieved using a push-pull cannula placed as close as possible to the tissue without touching it (with the aid of a binocular microscope) with the edge of the outside barrel (withdrawing barrel , internal diameter 1.5 mm) extending 1.5 mm beyond the inside barrel (injecting barrel , - 37 -FIG. 5 Placement of stimulating and recording electrodes, and push-pull cannula for local glutamate applications in the CA^ and C A 3 regions. The electrode in CAi was used to record both population spike and the f i r i n g rate of CA^  c e l l s . The f i r i n g rate of C A 3 neurones was monitored by the recording electrode positioned in that area. The push-pull cannula was positioned either over the CA^ or CA3 ce l l body layer for localized perfusions of glutamate. The bipolar stimulating electrode was inserted into the axonal region of the Schaffer col laterals to evoke a population spike at 0 . 2 Hz in CA^ with negative pulses. - 38 -internal diameter 1.0 mm). The glutamate solution was made up with stan-dard medium and the dye phenol Red (5 mg% ) was added to visualize the application (through a microscope) to ensure that the drug was contained within the borders of the withdrawing barrel and was not diffusing out lateral ly beyond this point to other parts of the s l i c e . Buckle and Haas (1982) have fa i led to show any effects of Phenol Red (1%) on neuronal properties or responses to stimulation in the hippocampal s l i c e . Selec-tive glutamate applications were made f i r s t in the CAg region and then in the CA^ region of each s l ice (15-30 min later ) . In some experiments, glutamate was applied only to the CA-^  region. Records of population spikes taken were an average of several sweeps and f i r i n g rates of CA^  and CAg ce l ls were plotted on a Grass polygraph. Iontophoretic application of drugs See F ig . 6A, 6B for experimental set-up. To apply C a + + , L -g luta-mate, NMA or APV near the CA^ cel l bodies or the apical dendrites where Schaffer col laterals synapse with CA^ neurones (100-200 ym from cell body layer), seven barrel microelectrodes (3 side barrels f i l l e d with one of the above drugs, 3 side barrels f i l l e d with 4 M NaCl and central barrel f i l l e d with 4 M NaCl; t ip diameter 2-3 ym) were positioned in the appro-priate areas. Iontophoretic applications of drugs were done using a 6 channel Neurophore BH2 (Medical Systems Corp.) iontophoresis unit. Concentrations of drugs are as follows: CaC^ (Sigma Chemicals), 0.2 M; L-glutamate (Sigma Chemicals), 100 mM; APV (Cambridge Research Biochemicals L td . ) , 20 mM in 160 mM NaCl, pH 8.0; NMA (Sigma Chemicals), 100 mM. Population spikes evoked by Schaffer col lateral stimulation (0.1 Hz) were recorded in the cell body layer using the central barrel of the 7 - 39 -FIG. 6 Experimental arrangements for examining actions of iontophoretically applied drugs at the CA^ cel l body and apical dendritic/synaptic regions. A. Glutamate, APV and C a + + were applied at the CAj cel l body layer and their effects examined on the Schaffer col lateral (Sch) stimulation-induced population spike in CA^. Glutamate and APV were ejected by passing negative charge and Ca was applied using positive charge from appropriate barrels of a seven barrel micropipette. Stimulating electrode was placed on Sch to deliver negative pulses. One 4 M NaCl barrel of a seven barrel microelectrode was used to monitor CA^ population spike as well as f i r i n g rate of CAi neurones. B. Glutamate, APV, NMA and C a + + were applied at the apical dendritic/synaptic area of CA^  to examine their effects on the Sch stimulation-induced population spike and "EPSP". Iontophoresis of the drugs was carried out as described above. To test drug effects on synaptic transmission, both the dendritic population "EPSP" and the ce l l body population spike were recorded using one 4 M NaCl f i l l e d barrel of the seven barrel iontophoresis electrode and a single barrel glass electrode respectively. To antidromically activate a single CA3 cel l from the terminal regions of Sch, either a single barrel glass electrode or one 4 M NaCl f i l l e d barrel of the seven barrel iontophoresis electrode was used for stimulation and a single barrel glass electrode was placed in the C A 3 ce l l body layer to record the evoked all -or-none action potential . - 40 -R-recording electrode S-stimulating electrode R or S - 41 -barrelled electrode in experiments where drugs were iontophoresed onto the cel l bodies. In experiments where the drugs were applied to the dendritic regions, both the population EPSP at the zone of application (using the central barrel of the 7 barrel electrode) as well as the population spike in the ce l l body layer using a single barrel electrode f i l l e d with 4 M NaCl, t ip 1-2 ym) were recorded. APV, NMA and L-glutamate were held in the barrels with "backing" currents of positive charge while C a + + was retained using "backing" currents (negative charge) of 10-15 nA. To apply APV, NMA and glutamate, currents (negative charge) of 100, 10-100 and 200 nA, respectively, were passed through the appropriate drug solutions. To iontophorese C a + + , a positive charge of 2-100 nA was applied. The "backing" currents as well as the iontophoresis of the drugs were automa-t i c a l l y "current balanced". The effects of current were examined by pass-ing a negative charge (in the case of APV, NMA and L-glutamate) and a positive charge (in the case of Ca + + ) through the NaCl barrels. The actions of verapamil (0.33 yM, 3-4 min) to the bath were examined on APV and glutamate-induced alterations in CAj responses. The verapa-mil infusion was ini t iated 1 min before iontophoresis of APV or glutamate. Antidromic stimulation of single CAg ce l ls See Fig . 6B for experimental set-up. The technique used for excita-b i l i t y testing of terminal regions of axons is a modification of the method f i r s t described by Wall (1958). To antidromically activate single CAg neurones a glass microelectrode or a 4 M NaCl f i l l e d barrel of a 7 barrel microelectrode (3 side barrels f i l l e d with 100 mM L-glutamate, 3 side barrels and central barrel f i l l e d with 4 M NaCl for stimulation and current balancing) was placed at the terminal regions (100-200 ym from - 42 -CA^  cel l body layer) of Schaffer co l latera ls . The stimulating electrode was moved vert ica l ly to a point where the threshold for activation ( i . e . , the f ibre is activated in an all -or-none manner 50% of the time) was mini-mal. A glass recording electrode was placed in the cell body layer of C A 3 to record the al l -or -nothing spike. The stimulation strengths required to f i re the cell ranged from 6-12 uA (0.1 ms negative pulses, used S88 Grass stimulator with SIU6 constant current unit) . Control thresholds were monitored for at least 10-15 min to ensure s tab i l i t y . The effects of iontophoretic glutamate (200 nA, 3 min) at the stimula-tion site and NMA (200 uM, 1 min to the whole bath) were examined on the antidromic threshold. In addition, the actions of verapamil (4 uM, 4 min) and C a + + - f r e e (Mn + + 1 mM, Mg + + 3 mM; 5 min) medium were examined on the NMA-induced exci tabi l i ty change at the Sch terminals. In these experiments, verapamil application was in i t iated 2 min and Ca + + - f ree medium 3 min prior to NMA (200 uM, 1 min) application. All post-drug responses were monitored for 30-60 min. RESULTS Tetanus-induced LLP and homo- and heterosynaptic depressions A high frequency tetanic stimulation of Sch or Comm (400 Hz, 200 pulses) results in LLP of the Sch or Comm induced population spike in CAp respectively (Figs. 7, 8, 9 and Table I). The LLP of Sch-induced population spike could be masked by a low frequency (20 Hz, 200 pulses) tetanus given to either Comm or Sch input (Figs. 7, 8 and Table II). The masking of the LLP was only temporary and i ts time course was roughly parallel to the co-occurring depression of the population spike not FIG. 7 The masking of long-lasting potentiation by a low frequency (20 Hz, 200 pulses), but not by a high frequency (400 Hz, 200 pulses), tetanus. In A and B, LLP of the CAi population spike was induced by a 400 Hz tetanus of the Schaffer col lateral input. A second tetanus (20 Hz in A and 400 Hz in B_) was given to the commissural input. Controls were taken for at least 30 min and a l l points in the graph were expressed as a percent of the mean of al l control points. In A and B_,4—•shows the population spike evoked by the stimulation of Sch and • • shows the response produced by Comm stimulation. In £ , c u r v e s •—•and • — • show the sizes of population spikes expressed as a percent of control recorded during a 400 Hz and 20 Hz tetanus, respectively, given to Sch input. - 44 -a. to 3 a. O a. 300 O ^20 0 z o o 0 100 SCH COM 400 H i 20 HI 200 PULSES 200 PULSES T T / ... B 300 a. -J co O or z H O Z z o < " 3 O a. 200 100 SCH 400 Hi 200 PULSES COM 400 Hi 200 PULSES T 20 40 60 MIN 80 100 20 40 60 MIN 80 100 0. CO Z o 700 500 < u . O 300 U O se 1 0 0 40 80 120 160 NO. OF PULSE 200 FIG 8 The blockade of homosynaptic and heterosynaptic depressions of the CA^ population spike by verapamil (0.33 yM, 3 min, applied to the whole bath). In A and Q, the population spike was induced by the stimulation of Sch (•—•) and Comm (•—•) inputs. LLP was induced by a 400 Hz tetanus given to the Sch input. A 20 Hz tetanus given to the Sch input during LLP produced heterosynaptic depression and masked the LLP (A). Verapamil counteracted the heterosynaptic depression and unmasked LLP (B). The horizontal bar above verapamil shows the duration of i ts application in the bathing medium. POPULATION SPIKE A OF CONTROL CD POPULATION SPIKE 7o OF CONTROL - 47 -FIG. 9 Failure of verapamil (0.33 uM, 3 min, applied to the bath) to block the induction of tetanus-induced (400 Hz, 200 pulses) long-lasting potentiation. The effect of verapamil on the development of LLP is shown in A and on the control population spike "in B. In /V, the curves<k>—-4and«—mare for Sch-induced and Comm-induced CAi population spikes, respectively. In B^ , each point on the curve shows the mean from 12 experiments. The SEM is too small to be v is ib le on the graph. POPULATION SPIKE CO /o OF CONTROL S t POPULATION SPIKE > /o OF CONTROL = 1 1 1 s s I i • < m 73 i P> *> • 1 ' \ — CO — -Ji • a . oo 1 r- \ f W w T: • • 2 It ;. |1 ; - 49 -FIG 10 The blockade of homo- and heterosynaptic depressions by verapamil ( 0 . 3 3 uM, 3 min, applied to the bath). Homo- and heterosynaptic depressions induced by a 20 Hz (200 pulses) tetanus to Sch are shown in A and the effect of verapamil on these depressions ' in B. In the graph .•—•indicates the Sch-induced population spike and •—•indicates the Comm-induced population spike. POPULATION SPIKE CD POPULATION SPIKE % OF CONTROL % OF CONTROL r o 3 Z CO o o r o rn CO to CO © ° K (/> m c n o TABLE I Effects of verapamil (0.33 yM) on the induction of long-lasting potentiation Sch-induced population spike Comm-induced population spike  (% of control)* (% of control)* 30 min post-tetanus 30 min post-tetanus 400 Hz tet Sch 243 ± 56 (n = 7) 103 ± 4 (n = 7) (as in F ig . IA and B) 400 Hz tet Sch * 494 ± 95 (n = 5) 108 ± 4 (n = 5) with verapamil (as in F ig . 3A) *A11 values are mean ± SEM. **Significantly different from control (p < 0.01, paired t - t e s t ) . TABLE II Effects of verapamil (0.33 pM) on homo- and heterosynaptic depressions Sen-induced population spike (% of control) Comm-induced population spike (% of control)* n 5 min 10 min 20 min 5 min 10 min 20 min (post-tetanus) (post-tetanus) 20 Hz (Sch) 8 56 * 6 77 * 3 79 ± 3 47 * 6 64 * 5 70 * 3 (as in F ig . 4A) 20 Hz tet (Sch) 8 93 * 7 118 ± 16 139 * 21 81 ±" 10 89 * 7 101 * 5 with verapamil (p < 0.01) (p < 0.05) (p < 0.02) (p < 0.02) (p < 0.02) (p < 0.01) (as in F ig . 48) 20 Hz tet (Sch) 10 53 ± 10 80 * 7 94 ± 7 59 ± 18 68 * 12 88 ± 13 during 400 Hz induced LLP (as in F ig . 2A) 20 Hz tet (Sch) 8 98 * 4 101 * 4 102 * 3 103 * 5 1 0 1 * 4 114* 7 with verapamil dur- (p < 0.01) (p < 0.05) (p < 0.05) (p < 0.05) ing LLP (as in F ig . 2B) *A11 values are mean * SEM. p values are obtained using unpaired t - test between verapamil-treated and untreated responses. - 53 -previously subjected to a high frequency tetanus (Figs. 7, 8 and Table III). Tetanic stimulation of one input, therefore, does not permanently reverse LLP of the population spike produced by another input on presuma-bly the same population of CA-^  neurones. As can be seen in F ig . 10, homo- and heterosynaptic depressions occur together suggesting a common link in their i n i t ia t ion . During LLP of Sch-induced population spike, i f Comm input was tetanized at 400 Hz, there was no recognizable homo- or heterosynaptic depression (Fig. 7). Although the response to Comm stimu-lation is in LLP, the LLP of Sch-induced population spike was pract ical ly unaffected. In this connection i t is interesting that a 20 Hz tetanus produced homosynaptic depression while masking the LLP by heterosynaptic depression (Fig. 7). Such a depression was not seen when a high frequency tetanus was given (Fig. 7). As can be seen in F ig . 7C, the population spike does not follow the high frequency tetanus but is under frequency fac i l i ta t ion during a 20 Hz tetanus, indicating that CA^  neurones res -ponding to the tetanus may bring about homo- and heterosynaptic depres-sions. It is also clear that LLP and these depressions appear to be due to different mechanisms. Verapamil (0.33 yM, applied for 3 min) produced dramatic effects on homo- and heterosynaptic depressions (Figs. 8 and 10). The dose and duration of verapamil were chosen after studying dose-response re lat ion -ships with 0.083-100 yM (Maretic", Muralimohan and Sastry, unpublished ++ results) . This "Ca antagonist" clearly interfered with the masking of LLP, as well as homo- and heterosynaptic depressions. In fact , homo-synaptic depression was, in most cases, followed by LLP if verapamil was applied during the 20 Hz tetanus (Fig. 10 and Table II). It i s , there-- 54 -fore, possible that the LLP was co-occurring during homosynaptic depres-sion and i ts presence could be revealed by minimizing the depression. It is interesting that verapamil, in doses that counteract the depression, fa i led to interfere with the development of LLP although i t has been postulated in l i terature that LLP in i t iat ion can be dependent on a CA-^  cel l accumulation of C a + + (Baudry and Lynch, 1980a). In fact , verapamil in doses up to 100 yM does not block LLP development (Maretic', Muralimohan and Sastry, unpublished resul ts ) . One striking observation of verapamil's effect is i l lustrated in F ig . 9A where post-tetanic potentiation (PTP) produced by a 400 Hz tetanus was many folds greater than when the tetanus was given without the presence of verapamil (Figs. 7A, 7B, 8A, and 8B). Perhaps, the depression of CA-^  neuronal response which may be present during the time course of PTP (see weak heterosynaptic depression in Figs. 7A, 7B, 8A and 8B) is capable of masking the real level of PTP. On the other hand, verapamil may fac i l i ta te PTP by as yet unknown presynaptic mechani sms . F ig . 9B shows the effect of verapamil on the control response. The agent produced a weak increase in the population spike in some of the experiments. Iontophoretic C a + + applications Since homo- and heterosynaptic depressions occurred together and since verapamil, a C a + + blocker, interferes with these depressions, i t was of interest to examine if C a + + i t se l f produced a depression of the CA-^  ++ population response. Ca consistently produced a depression of the Sch-induced CA^ ^ population spike when applied in the CA^ cel l body area, an effect that was observed after termination of i ts application - 55 -(Fig. 11, Table III). The depression (10-40%of control response lasted for 2-30 min in the majority of cases but in 3 experiments was present unti l recordings were terminated (60 min). It is possible that Ca suppressed the population spike by enhancing the inhibitory postsynaptic potential (IPSP) of CA^ ce l ls or by producing a hyperpolarization so that the spike in individual cel ls might not be triggered. It i s , how-ever, interesting that the population "EPSP" recorded in the ce l l body layer was also suppressed (Fig. 11, Table III). The "EPSP" recorded in the CA^  cel l body area is l ike ly a mixture of f ie lds generated by the local IPSP and by the EPSP from the dendrites. If C a + + were to increase the IPSP or to hyperpolarize the c e l l s , the total population "EPSP" could have been larger but i t was not. It is further interesting that Ca applied at the cel l body region suppressed the "EPSP" recorded at the dendritic/synaptic area (Fig. 11, Table III). This observation also precludes the poss ib i l i t y of a charge screening effect by C a + + to be responsible for the depression. Perhaps, the C a + + application on the cell bodies of CA-^  neurones results in an intracel lular accumulation of the ion which induces changes that lead to a decreased responsiveness of the subsynaptic membrane to released transmitter. ++ When Ca (2-100 nA) was applied on the CA-^  apical dendrites, there was no lasting change in the dendritic "EPSP" in most cases (Table III). It has been reported that an application of elevated extracellular C a + + to the whole s l i ce induces LLP (Turner et a l . , 1982). Since our studies indicate that a selective application of the divalent ion onto somata produce a depression instead of LLP, i t was thought that the site of LLP generation is at the synaptic zone, so i t is puzzling why C a + + - 56 -FIG. 11 Depression of the CA^ population spike and the population "EPSP" by C a + + applied near the cel l body area. Rows A, jB and show population spike recorded at the somatic area, population "EPSP" recorded at the cel l body and apical dendritic/synaptic area, respectively. The left column shows controls and the right column shows responses taken +5 min after the termination of a 5 min iontophoretic application of Ca (100 nA), at the cel l body area. A, J3 and C_ were from 3 different experiments. Polarity : posi t iv i ty up. - 57 -Ca 100nA APPLIED FOR 5 min AT CELL BODY LAYER CONTROLS T 5min POST-CALCIUM 10 ms TABLE III Effects of C a + + on the CAj population responses Cell body Cell body Dendritic population spike* population "EPSP"* population "EPSP"* C a + + (2-100 nA) Depressed 5/6 Depressed 7/9 Depressed 4/4 applied at the cel l No effect .1/6 No effect 2/9. No effect 0/4 body area Increased 0/6 Increased 0/9 Increased 0/4 C a + + (2-100 nA) Depressed 4/18 applied at the — — No effect 12/18 dendritic area — — Increased 2/18 *The values show the number of responses affected (numerator) vs. total responses tested (denominator); "no change" in the response was determined as < 10% deviation from control s ize. - 59 -application to the dendritic area fa i led to produce either LLP or depres-sion. Perhaps, the agent produced both effects that nu l l i f ied each other so that neither was observed. L-Glutamate a. Application by push-pull cannula Glutamate application in the CA^ area with a push-pull cannula resulted in LLP of the population spike in CA-^  evoked by Schaffer col lateral stimulation (115-183% of control , 4 of 9 experiments). The same application of the amino acid in the CA-^  area resulted in a depres-sion of the pre-drug response (38-79% of control , lasted 5-30 min, 7 of 9 experiments). F ig . 12 i l lustrates one experiment where both LLP and the depression were seen in the same s l i c e . The depression of the response after application in the CA-^  region was seen more often than LLP after administration in the CA^ region. Perhaps, the Schaffer col lateral axons connecting the CA^ cel l bodies to the dendritic region of the cel ls recorded from were cut during the s l ic ing procedure and hence action potentials from activated CA^ cel l bodies could not be communicated to the terminals of these f ibres . It is also possible that glutamate applied to the CAg area did not reach and activate those ce l ls whose axons were stimulated to evoke the population spike in the CA-^  area. Also i l l u s -trated in F ig . 12 is the f i r i n g rate of a CA^ neurone during the amino acid application in the CA^ as well as the CA^ areas. During gluta-mate application in the CA^ area, the f i r i ng rate of the CA^ cel l was not altered, indicating an absence of postsynaptic activation to d is -charge. In contrast to the lack of effect on CA^ neurones observed with the above application, a localized application to the CA, region pro-- 60 -CONTROL J GIu (CA3) FIRING RATE OF A CAj NEURONE 15min POST-DRUG CA3Glu p50 I spikes/s GIu (CA.j) 15 min POST- 1 DRUG [J CA1 GIu 2mV r100 spikes/s «-0 10 ms 1 min FIG. 12 • . Actions of local applications of glutamate in the C A 3 and CA^ areas on Schaffer col lateral stimulation-induced population spike in CA^. Top record shows the pre-drug control population spike. The middle record is the post-drug response at f i f teen minutes following C A 3 glutamate application and the bottom trace i l lust rates the population spike at f i f teen minutes post-glutamate applied in the CA^ region. Each record is an average of four consecutive sweeps. Negativity is down. On the right are traces of the f i r ing rate of a CA^ neurone during each of the above applications of the amino acid. Horizontal bars immediately below each trace indicate the duration of drug appl icat ion. - 61 -duced a marked increase in cel l f i r ing (9 of 9 experiments). To confirm that CAg cel ls were activated by glutamate application in the CAg area, a recording electrode was placed in the ce l l body layer and f i r i n g rate was recorded during the application. In al l cases (3 of 3 experi -ments), there was a drastic increase in the f i r i n g rate of CAg neu-rones. The depression of responses to Sch stimulation seen after exposure of the CAj area to glutamate was not associated with a reduction of the cell response to the amino acid because repeated administration of the agent on the same ce l ls resulted in similar increases in f i r i n g rate (this procedure was carried out on each s l ice at the termination of each experi-ment, n = 11). In 2 experiments, glutamate was applied only to the CA^ region and in these cases, a post-application depression of the response rather than LLP was seen, b. Iontophoretic application Glutamate iontophoresis in the synaptic regions of CA-^  (100-200 yM from ce l l body layer; 200 nA, 3 min) resulted in an increase in the majority of cases (122-211 % of control at 30 min post-drug; 5 of 7 experiments), a decrease (76% of control at 30 min post drug; 1 of 7 experiments) or no change (1 of 7 experiments) of the Sch-induced popula-tion spike (Table IV). Concurrent verapamil application (0.33 yM, 3 min) during the iontophoresis of glutamate resulted in similar observations as obtained previously (increased in 6 of 10 experiments, 121-267 of con-t r o l ; decreased in 4 of 10 experiments, 71-94 of control at 30 min post-drug) (Table IV). This observation is puzzling as one would expect depression to be counteracted and LLP unmasked by treatment with the C a + + antagonist. Glutamate was presumably affecting both presynaptic TABLE IV Effect of glutamate application at the apical dendritic zone of CAi Glutamate 200 nA on dendrites  (population spike% of control) 2 min 30 min (post-glutamate) Glutamate 200 nA in presence of verapamil 0.33 pM  (population spike % of control) 2 min 30 min (post-glutamate) 97 193 100 267 33 211 62 121 78 122 92 167 127 109 46 138 36 136 42 125 60 — 50 150 59 .76 36 91 50 100 63 88 78 94 59 71 n 8 7 10 10 7 68 135 63 131 SEM 11 19 7 18 - 63 -terminals of Sch as well as the dendrites of CA^  neurones when applied in the synaptic zone and different effects ( i . e . , LLP vs depression) may be present but only the predominant effect is manifested. Since both LLP and the depression may be co-occurring, i t would be d i f f i c u l t to quantify either one without the other interfering. Also, there is no way of knowing the magnitudes of LLP vs depression in each s l i ce . Perhaps, i f one could separate these two out, clearer results would be obtained. To determine i f glutamate had effects on the terminal regions of Schaffer col laterals when applied in the synaptic zone of CA^ the change in threshold for antidromic activation of CAg cel ls was deter-mined during the application of the amino acid as well as after termina-tion of the drug. During glutamate application, the stimulation strength required to evoke all -or-none action potentials in Sch terminals was reduced to 31-92 % of control (n = 1 9 ) , presumably due to depolarization (see F ig . 1 3 ) . Following termination of the iontophoresis, however, there was a slight increase in threshold in the majority of cases (104-137% of control , 6 of 9 experiments at 15 min post-drug) (see Fig . 13) and no change in 3 of 9 experiments. A depression of the population spike was induced by application of glutamate (200 nA, 3 min) at the CA^ cel l body layer (65 ± 15% of con-t r o l , n = 8 at 5 min post-drug) (Fig. 1 4 ) , an effect that was more pro-nounced if a 100 Hz, 1 s tetanus was given during glutamate application (4 ± 3% of control , n = 4 at 5 min post-drug) (Fig. 1 5 ) . Verapamil ( 0 . 3 3 uM, 3 min) clearly counteracted the depression of the population spike pro-duced by the glutamate application (92 ± 17% of control , n = 5 at 5 min post-drug) (Fig. 14) and unmasked LLP that was induced by the 100 Hz, 1 s - 6 4 -20 L • • • • • 0 5 10 15 20 25 30 MIN FIG. 13 Effects of iontophoretically applied glutamate at the terminal regions of Schaffer col laterals on the antidromic threshold of a single CAo neurone. Abscissa represents the time scale and the ordinate shows percent of control antidromic threshold of the single f ibre determined by stimu-lation at the drug ejection s i t e . Horizontal bar above the graph shows duration of drug eject ion. - 65 -O cn K z o o u. o LU X cZ CO 5 3 D_ o Q. VERAPAMIL 0-33uM (—) Glu 200nA 120 100 80 60 40 10 MIN POST T GLU 20 J 30 FIG. 14 GTutamate-induced depression of the population spike when applied at the •CAj ce l l body layer. The curve- • • — • i l l u s t r a t e s the actions of iontophoretically applied glutamate (200 nA, 3 min at the CA^ cel l body layer) and the curve •—•shows the results following the same glutamate application in the presence of verapamil (0.33 yM, 4 min). Horizontal bars above the graph- show the durations of application of the indicated drugs. Vertical bars and each point on the curves are SEM (n = 8). - 66 -VERAPAMIL 0-33uM (•—) GIu 200nA • I • B 0 10 20 30 MIN POST-GLU FIG. 15 Potentiation of glutamate-induced depression of the CA^ population spike by a tetanic stimulation to the Schaffer co l la tera ls . The curve • — 4 i 11 ustrates the effects of a 100 Hz, 1 s tetanus to Sch delivered 30 s before termination of glutamate (200 nA, 3 min at the CAi ce l l body layer) iontophoresis. The curve •—•represents identical treatment as given previously but in the presence of verapamil (0.33 yM, 4 min). Note that LLP induced by the tetanic stimulation was unmasked by treatment with verapamil. Horizontal bars above the graph show the durations of application of the drugs indicated and the arrow shows the time at which the tetanus was given. Vertical bars on each point on curves are SEM; in some cases they were too small to be v is ib le (n - 4). - 67 -tetanus delivered during glutamate application (179 ± 44%.of control, n = 5 at 5 min post-tetanus) (Fig. 15). N-Methyl-DL-aspartate NMA (200 yM, 1 min) application resulted in a total abolition of the CA-j^  population spike during the drug perfusion which was followed by a potentiation of the response after termination of NMA (210 ± 25% SEM of control , n = 6 at 15 min post NMA) which recovers to control size in 30-40 min and is succeeded by a depression (50 ± 7% of pre-drug control, n = 6 at 60 min post-NMA) (Fig. 16A). The NMA dose of 200 yM was chosen after doing dose-response relationships (25-400 yM) and i t seemed to be the concentration that produced the most consistent and rel iable results , although similar observations were made with the other doses. The C A 3 antidromic spike resulting from Sch terminal region stimulation could not be evoked using 5-10 times control threshold during and for 5-10 min following the same NMA application. Subsequent to this period of refrac-toriness, the threshold is sustained at a level above control for the duration of the experiment (110-171% of pre-drug control , n = 4 at 60 min post-NMA) (Fig. 16B). Iontophoretic application (10-100 nA, 1 min) of NMA at the synaptic zone of CA^ also resulted in an i n i t i a l potentiation of the population spike (145-160% of control , 2 of 5 experiments at 10 min post-NMA) followed by a depression (10-70% of control , 5 of 5 experiments at 30 min post-NMA). Although low doses (10-40 nA) of NMA were suff icient to produce the depression (n = 3), higher doses (100 nA) were required to e l i c i t the early potentiation (n = 2) of the population spike. When NMA (200 yM, 1 min) is applied in the presence of verapamil (4 yM, 1 min), the results indicate that the late depression is blocked - 68 -CONT. 0 20 40 60 min post - NMA VERAPAMIL 4liM i . CONT. 0 20 40 60 min post - NMA MnlmM, Mg 3mM Y A Mn1mM,Mg3r Y A CONT. 0 20 40 60 min post - NMA FIG. 16 Effects of N-nethy1-DL-aspartate (NMA) (200 uM, 1 min; applied to the bath) on the CA^ population spike and the threshold for antidromic activation of single C A 3 c e l l s from the terminal regions of Schaffer co l la tera ls . The actions of NMA on the population spike are shown in A (n = 6 ) . An increase in antidromic threshold of single Sch terminal regions that lasts for a prolonged period of time is seen following termination of the above application (B_) (n = 4) . When NMA is applied during verapamil (4 uM, 4. min) the late depression of the population spike is blocked (C) (n = 6) but the associated increase in Sch antidromic threshold is s t i l l present (f)) in = 5). NMA application in the presence of Ca + + - f ree medium (Mn + + 1 mM, Mg + + 3 mM) blocks both the early potentiation and late depression of the population spike (E) (n = 5) as well as the increase in Sch antidromic threshold (f) (n = 9)~ The upward arrows in each graph indicate the time when NMA (200 uM, 1 min) application commences and the downward arrows show time of termination of the drug. Horizontal bars above graphs C, fJ, E and F_ show duration of application of the specif ied drugs. Vert ical bars at each point on curves repres'ent SEM; in some instances they were too small to be v i s ib le . - 69 -(186 ± 20% of pre-drug control , n = 6 at 60 min post-drug) (Fig. 16C) whereas the increase in antidromic threshold post-drug is comparable to the previous experiment in the absence of verapamil (119-150% , n = 5 at 60 min post-NMA) (Fig. 16D). The concentration of 4 yM was chosen for verapamil after doing dose-response relationships on the drug (0.083-100 yM; Maretic, Muralimohan and Sastry, unpublished resul ts ) . It was found to be an optimal concentration for blockade of homo- and hetero-synaptic depressions induced by low frequency (20 Hz) tetanic stimulations without blocking the induction of LLP. The exclusion of C a + + (substituted by Mn + + ImM, Mg + + 3 mM) from the bathing medium results in a blockade of the potentiation (97 ± 7% of pre-drug control , n = 5 at 15 min post-NMA) as well as the depression (87 ± 7% of pre-drug control, n = 5 at 60 min post-NMA) of the population spike induced by the amino acid (Fig. 16E). In addition, this treatment also abolishes the increase in Sch terminal threshold (100 ± 4% of pre-drug control , n = 9 at 60 min post-NMA) (Fig. 16F). Amino acid "antagonists" and LLP a. a-Aminoadipate DL-aAA when applied to the whole s l i ce (3 mM, 5 min) depressed the CA^ population spike during its application and for 5-15 min after termination (4 s l i ces ) . The agent increased the f i r ing rate of CAg neurones during i ts application (4 of 4 experiments). Following the drug-induced depression, LLP of the population spike could be observed (144-178% of control response at 20 min post-drug; n = 4) (see F ig . 17). The D- isomer of the drug was also tested and similar results were found - 70 -during D L - c x - A A 5 mM 16 ms ^ ^ • ^ ^ V . .^V I 1 - 6 m V C O N T R O L 1 2 3min 2 5 15 30 min post D L - O K - A A F I R I N G R A T E O F A C A 3 N E U R O N E f % AM / A K W U T ^ D L-0 . - A A 3 min V * W r100 "5. «A F I G . 17 • The induction of long-Tasting potentiation by bath application of DL-a-aminoadipate ( 5 mM, 3 min). The trace on the top le f t shows a control population spike recorded at the CA^ ce l l body layer evoked by stimulation of Schaffer co l la tera ls at 0 . 2 Hz. The next three records show the response in the presence of the drug application at the times indicated. The subsequent responses were recorded after termination of drug application at the specif ied times. Each record is an average of f ive consecutive sweeps. Pos i t i v i t y is upwards. The bottom trace shows the f i r i ng rate of a C A 3 neurone during the above application of drug. - 71 -in 2 s l i ces , although D-aAA seemed to be more potent than the racemate (used 1 mM, 5 min). b. 2-Amino-5-phosphonovalerate The population spike and the population "EPSP" in response to ionto-phoretic APV at the dendritic area of CA-^  were affected in similar fashion by drug treatments, as well as by the tetanic st imuli . Quanti-tat ively , however, the population spike was affected more profoundly rather than the population "EPSP" as would have been expected. The effects on the population spike were, therefore, quantitated and drawn as graphs in Figs. 18-20. It is l ike ly that the population of neurones from which recordings were made was common to the population spike recordings as well as the dendritic "EPSP" recordings, because the drugs applied at the dendritic area produced effects on both the "EPSP" and the population spike. It is unlikely that the APV application in the dendrites spreads to the C/\^ ce l l body area because Collingridge et a l . (1983b), using the same application parameters, reported that the drugs did not reach the cel l body regions. Verapamil (0.33 pM, 3 min) applied to the whole bath produced a weak but not a significant increase in the control population spike (Fig. 18B). A 100 Hz, 1 s tetanus of the Schaffer col laterals produced LLP of the population spike in a l l 5 s l ices tested (Fig. 19A). This LLP was similar i f the tetanus was given in the presence of verapamil (Fig. 19B), i n d i -cating that verapamil did not counteract LLP development. The population spike, 5 min following the 100 Hz tetanus, was larger i f the tetanus was given in the presence of verapamil (see Fig . 19A and 19B), presumably - 72 -FIG. 18 Effects of 2-amino-5-phosphonovalerate (APV) (A) and verapamil (B) on the CAj population spike produced by the stimulation of Schaffer col laterals at 0.1 Hz. APV (100 nA, 5 min) was applied iontophoretically in the apical dendritic area 100-200 vm away from the CAj ce l l body layer (n = 8). Verapamil (0.33 uM) was applied to the whole bath for 3 min (n = 12). Vertical bars at each point on curves represent SEM. - 73 -o o o LL. o a. to 2 O 3 a. o a. 2 0 0 r 180 160 140 120 100 80 B 100HZ 1s 200r 180 160 140 120 100 80 100H2-1s y VERAPAMIL 0-33 i f 10 30 50 _l 70 10 30 50 70 MIN MIN FIG. 19 Failure of verapamil ( 0 . 3 3 uM, 3 induction of tetanus-induced (100 of the CA\ population spike was Schaffer col laterals when given presence (B, n = 4) of verapamil. min, applied to the bath) to block the Hz, 1 s) long- last ing potentiation. LLP produced by a 100 Hz (1 s) tetanus of either in the absence (A, n = 5) or - 74 -MIN MIN FIG 20 The masking of long- lasting potentiation of the Schaffer col lateral stimulation-induced CAi population spike by APV. In A, a 100 Hz (1 s) tetanus of Schaffer col laterals was given during the last 10 s of a 5 min application of APV (100 nA) in the apical dendritic area (n = 13). In E5, same procedure as in A was carried out, except that verapamil (0.33 uM, 3 min) was applied 1 min prior to the in i t ia t ion of APV application (n = 8). - 75 -because verapamil counteracted the depression of the population spike that occurs together with the LLP. When the LLP-inducing tetanus was given during the application of APV (100 nA, 5 min), as reported by Collingridge et a l . (1983b), LLP could not be visualized (Fig. 20A). In fact , the population spike was depressed to 50-90 % of control 20 min following APV application in 9 of 13 s l i ces . The response was increased by 12.5% in one s l i c e . The depressant action of APV was seen in the tetanized s l i c e s , but not in control s l ices (compare F ig . 20A with Fig . 18A). Verapamil (0.33 yM, 3 min), which counteracts homo- and heterosynaptic depressions, nu l l i f ied the depression of the population spike produced by APV and brought out LLP (Fig. 20B). In these experiments, the population spike was 120-300% of control at 30 min post-APV application when examined in 8 experiments. It, therefore, appears that verapamil counteracted the depression of the population spike produced by APV and the 100 Hz tetanus and hence unmasked the LLP. During the application of APV (100 nA, 5 min) in non-tetanized s l i ces , the CA^ population spike was depressed to 65-80% of i ts control size in 5 out of 8 experiments and in 2 cases the response was increased by 6% . In 6 of 8 experiments, the response was back to control levels in 3-5 min after APV application was terminated. Thirty minutes following the drug application, however, the response was increased to 107-148% of control in 5 experiments, unchanged in 1 and depressed to 82-88% in the remaining 2 experiments. F ig . 18A shows the effects of APV on the control population spike. - 76 -DISCUSSION In the present investigations, there are reasons to use verapamil as a C a + + blocker. In hippocampal CA-^  neurones, the depolarization and conductance changes produced by NMA were counteracted by Co , Mn , C d + + , D600 and verapamil (Dingledine, 1983). Gr i f f i th and Brown (1982) also demonstrated that in voltage clamped conditions, Ca currents in the same cel ls were blocked by removing extracellular C a + + , adding ++ ++ Cd , Mn or verapamil. In cultured spinal neurones as well , ++ verapamil appears to block Ca action potentials (MacDonald and Schneiderman, 1983). It, therefore, appears that verapamil is capable of blocking amino acid-induced C a + + currents as well as C a + + currents ++ involved in Ca spikes. Hence, there is strong evidence that this drug ++ is a Ca antagonist. Al l the above authors used a verapamil concentration of 100 yM. This dose of the drug does not prevent the induction of LLP (Muralimohan and Sastry, unpublished observations). It is not understood why Dingledine (1983), Gr i f f i th and Brown (1982) and MacDonald and Schneiderman (1983) used 100 yM verapamil even though it is known in l i terature that in synaptosomes (Norris et a l . , 1983) and in other tissues (Fleckenstein, 1977), concentrations around 1 yM were suggested to be selective in blocking Ca channels while concentrations above 30 yM reportedly have "nonspecific" effects including on Na+ channels (Norris et a l . , 1983). Moreover, Reynolds et a l . , (1983) found ++ that the binding of verapamil to Ca binding sites on cerebral cortical membranes was selective in concentrations less than 1 yM. The Ca -dependent release of transmitter appears to be unaltered by vera-pamil in concentrations around 1 yM (Norris et a l . , 1983) as well as around 50 pM (Nachshen and Blaustein, 1979) from the frog neuromuscular junction. The agent, therefore, appears to be a good tool to distinguish between pre- and postsynaptic C a + + channels. In the present studies, doses of 0.33-4 pM verapamil were used with the hope that only C a + + blocking properties of the drug and not the other nonspecific effects would be seen. It would, however, be important that studies similar to those of Dingledine (1983) and Gr i f f i th and Brown (1982) on CA-^  neurones be conducted using these low concentrations of verapamil to confirm that C a + + channels are indeed blocked. Tetanus-induced LLP and homo- and heterosynaptic depressions It is clear from the results that LLP of the CA^ population spike is only masked, but not permanently reversed, by heterosynaptic depression. It also appears that heterosynaptic depression is brought about by a CA-^  ++ neuronal accumulation of Ca produced by the tetamzed input. Indeed, ++ the studies u t i l i z ing Ca -sensit ive electrodes showed that the largest ++ drop in extracellular Ca during low frequency tetanic stimulation of inputs occurred around the CA-^  cel l body area (Benninger et a l . , 1980). In addition, intracel lular measurements of C a + + revealed that low f r e -quency tetani produced much more C a + + influx than high frequency tetani (Chirwa et a l . , 1983). It is proposed that a CA-^  neuronal accumulati on ++ of Ca leads to a generalized depression of the neuronal responsiveness to inputs. How such a depression is brought about by the C a + + accumu-lation is at present unclear. Receptor desensitization has been reported to be fac i l i ta ted by C a + + (Magazanik and Vyskodil, 1970; Manthey, 1966; Mi ledi , 1980; Nastuk and Parsons, 1970). Perhaps, the CA1 neuronal accumulation of C a + + leads to the desensitization of glutamate - 78 -receptors (presuming that glutamate is the transmitter [Storm-Mathisen, 1977a]). In this connection, i t is interesting that Lynch and associates reported that during LLP the responsiveness of CAj neurones to applied glutamate was depressed (Lynch et a l . , 1976). The uncovering of new glutamate binding sites observed by Baudry and Lynch (1980) during LLP may be related to a compensation produced by the cel l to the lost functional receptors. A swelling of CA-^  dendritic spines was reported to occur following a 30 Hz tetanus (Van Harreveld and Fifkova, 1975). Perhaps, the CA^  neuronal C a + + accumulation as well as water accumulation leads to such a swelling. Since the dendritic total surface area would be in -creased by this growth, i t would be logical to expect the exc i tabi l i ty of the dendrites to be decreased and not increased. Consistent with this idea, during spreading depression a swelling of dendrites was reported (Van Harreveld and Khattab, 1967). It is interesting that the dendritic swelling occurs following a 30 Hz tetanus (Van Harreveld and Fifkova, 1975) that could induce a depression, but not a 100 Hz tetanus (Lee et  a l . , 1980) that induces LLP. It has, however, been reported that the var iab i l i ty of dendritic spines decreases and the extent of synaptic contact on dendritic shafts increases during LLP (Lee et a l . , 1980). This morphological change has been interpreted to improve synaptic transmission through a postsynaptic change (Horwitz, 1981; Lee et a l . , 1980). The co-occurrence of homo- and heterosynaptic depressions induced by tetanic stimulation of an input , the depression of CA-^  population spike that follows iontophoresis of C a + + on CA^ neurones, the depression produced by glutamate or APV during tetanic stimulations and the late depression produced by NMA application (to be discussed in detail in - 79 -following sections) al l point to the CA-^  neurone as the locus for these depressions. Whether homo- and heterosynaptic depressions are a conse-quence of altered synaptic transmission by a change at the subsynaptic regions or due to a change in the electr ical properties of the dendritic and/or the somatic membrane of the CA^ neurone is unclear. An exami-nation of antidromic CA-^  f i e l d potential by the stimulation of alveus during homo- and heterosynaptic depressions could be useful to determine i f the electr ical properties of the ce l ls are changed. It i s , however, unlikely that a pure antidromic f i e ld can be evoked by alvear stimula-t ion . Studies should be conducted with intracel lular recordings to deter-mine i f the electr ical properties of CA-^  neuronal dendrites and somata are changed during homo- and heterosynaptic depressions. What appears to be clear in the present studies is that homo- and heterosynaptic depres-sions are postsynaptic. The development of LLP has been reported to be dependent on the ++ presence of extracellular Ca (Dunwiddie and Lynch, 1979; Wigstrom et a l . , 1979). If this were the case, i t is puzzling as to why verapamil, ++ which blocks Ca entry through soma-dendritic membranes, did not ++ counteract LLP. Perhaps, LLP is Ca -dependent but is presynaptic and i t would be worth pursuing this poss ib i l i ty . On the other hand, verapamil may not block Ca entry through the subsynaptic membrane that is res-ponsible for LLP. If this were true, one has to postulate that specific subsynaptic C a + + channels rather than a general accumulation of CA-^  neuronal Ca are involved in events leading to LLP. It i s , however, interesting that verapamil counteracts the depression of CA^ population spike produced by applied glutamate to the CA-^  ce l l body layer (dis-- 80 -++ cussed in the following section), indicating that the Ca entry into CA^  cel ls induced by the transmitter can also be prevented by vera-pamil. The depolarization of CA-^  neurones produced by N-methyl-DL-aspartate (NMA) (Dingledine, 1983) and applied intracel lular current pulses in the presence of tetrodotoxin (Gr i f f i th and Brown, 1982) reportedly involve a verapamil sensitive Ca + conductance. Nicoll and Alger (1981) reported that synaptic activation as well as applied ++ glutamate induce a Ca conductance in the CA-^  neurones. It is ++ possible that such a Ca accumulation can lead to changes triggering the long-lasting homosynaptic and heterosynaptic depressions. A C a + + -dependent K + conductance induced by both synaptic excitation and amino acid application has been reported (Nicoll and Alger, 1981). In the present studies, the 20 Hz-induced depression and the depression produced by the application of Ca are much more long lasting than the depres-sion that would be expected to be generated by a Ca -dependent K conductance. The action of iontophoretically applied C a + + to produce the observed depression may be thought to be due to a "charge screening" effect of the ion. This poss ib i l i t y is unlikely since the ce l l body application of C a + + depressed the population "EPSP" recorded near the dendrites, where presumably the divalent ion was not reaching. Moreover, a direct application of Ca on the dendrites did not produce a depres-sion. Depression and LLP may be due to di f ferent ial quantal accumulation of C a + + into CA^ neurones, low levels inducing LLP while high levels producing a depression. This idea is supported by the observation that during verapamil application a 20 Hz tetanus induces LLP (the total C a + + accumulation may be less because of verapamil's presence). The dependence - 81 -++ of LLP on c r i t i ca l Ca levels, however, has to be specif ic for the tetanized input because during verapamil application heterosynaptic depression is blocked and not converted into LLP. Tetanic stimulation of a specif ic hippocampal input induces LLP while producing a generalized decrease in the synaptic responsiveness of the postsynaptic neurones, although there seems to be an inverse relationship between tetanic frequency and the latter effect. Therefore, during LLP, transmission across the potentiated synapse is not only more ef f ic ient , but the postsynaptic cel l would respond less favourably to other non-tetanized synapses. Perhaps a suppression of a nonspecific responsiveness of CA^  neurones to various inputs during the early stages of LLP is necessary to consolidate the information carried through the potentiated synapse and, hence, may have an implication in the establishment of synap-t i c p last ic i ty . Since i t appears that both LLP and the co-occurring depression are Ca + +-dependent, i t would be d i f f i cu l t to analyze the mechanism(s) of one without dealing with the other. In this regard, use of drugs l ike vera-pamil that selectively interfere with the depression should prove useful. To distinguish between a presynaptic and a postsynaptic involvement of ++ ++ Ca , a selective Ca blocker that interferes with one, but not the other is needed. As is shown in the present studies, verapamil, while not ++ interfering with transmitter release, could counteract a presumably Ca dependent postsynaptic depression. L-glutamate The observations on the f i r ing rate of CA-^  ce l l s during drug app l i -cation via a push-pull cannula to both the CA-^  and CAg areas indicate - 82 -that LLP of the CA-^  population spike is associated with minimal act iva-tion of postsynaptic cel ls to discharge whereas the depression is asso-ciated with an increase in CA-^  neuronal f i r i n g . Although this suggests a presynaptic mechanism for LLP, a change in the postsynaptic membrane caused by asynchronous transmitter release from the presynaptic terminals is also possible. This postsynaptic change, however, should have been greater when glutamate was applied in the CAj area but, as has- been demonstrated, i f a postsynaptic change did occur, i t only seemed to contribute to a depression rather than to LLP. With this method of application, however, i t was not possible to separate out somatic vs den-dr i t i c changes induced by the drug and this has to be done before conclu-ding that the agent has similar effects at both s i tes . One might argue that the f i r s t application of glutamate in the CAg region e l ic i ted a maximal LLP and, therefore, there could have been no more enhancement of the response by the second application of the agent in the CA^ area. As can be seen from the results , even if the CA^ application did not induce LLP in 5 cases, the application of the amino acid in the CA-^  area fa i led to in i t iate LLP. Moreover, in 2 experiments in which glutamate was applied only to the CA^  area, a post-application depression rather than LLP was observed. Collingridge et a l . (1983b) also reported that in the CA-^  area, iontophoretically applied glutamate produced a short lasting depression and not LLP. Various laboratories have reported the presence of a Ca+ +-dependent K+ conductance in hippocampal neurones following repetitive f i r i ng (Alger and N ico l l , 1980; Hotson and Prince, 1980) as well as activation of an electrogenic Na , K -pump following excita-tion by glutamate (Segal, 1981). The hyperpolarization observed as a - 83 -result of Ca + -act ivated K+ conductance or the Na + , K+-pump could account for the in i t i a l (1-3 min) depression following increased act iv i ty of CA-l neurones by glutamate excitation, but the later depression seen ( i . e . , beyond 5 min) cannot be accounted for by either of these two events. Since the above application of glutamate in the CA^ region produced a depression of the population spike and i t was not possible to separate somatic vs synaptic effects of the amino acid due to the large area of application, i t was decided to examine the actions of ionto-phoretically applied glutamate at either the cel l body layer or the dendritic/synaptic area of CA^. The results obtained for somatic application clearly indicate that the ++ drug induces a Ca dependent depression of the CA^ population spike which is enhanced by tetanic stimulation of Sch (which presumably further activates the CA^ cel l during glutamate administration and hence allows increased C a + + inf lux) . Zanotto and Heinemann (1983) have demonstrated that glutamate applications on CA^ neurones induce reductions in extra-ce l lu lar free C a + + concentrations in the pyramidal cel l body layer. In addition, tetanic stimulations of Sch also deplete extracellular C a + + in the CA^  cel l body layer (Benninger et a l . , 1980) and this C a + + seems to be entering into the soma of the cel ls as evidenced by intracel lular ion sensitive recordings (Chirwa et a l . , 1983; Morris et a l . , 1983). It is clear that entry of C a + + into CA-^  ce l l s via the soma is not res -ponsible for LLP. If LLP was masked by the depression, verapamil, which interferes with the development of homo- and heterosynaptic depressions but not LLP, should have revealed the potentiation but i t did not. Furthermore, the powerful depression induced by glutamate and tetanus was - 84 -absent when verapamil was instituted during the conditioning and the tetanus-induced LLP was unmasked. Glutamate applied near the synaptic zone of CA-^  c e l l s , in the absence or presence of verapamil, in the majority of cases produced LLP following termination of the application. However, in a few cases, a depression was observed. Since somatic glutamate applications on CAj cel ls induce a depression, i t is logical to think that the same effect is e l i c i ted on the dendrites of these c e l l s . The drug, when applied at the synaptic zone wil l probably exert effects on both the presynaptic ter -minals of Sch and the postsynaptic dendrites of GA-^  neurones. It is possible that LLP is generated presynaptically and depression post-synaptically so that both phenomena are induced by glutamate application but only one or the other is observed depending on which is predominant. Exci tabi l i ty testing on the Sch presynaptic terminal during the amino acid application revealed a decrease in threshold, probably ref lect ing a depolarization, which in turn allows voltage-dependent C a + + entry. Since LLP is Ca+ +-dependent (Dunwiddie and Lynch, 1979; Wigstrom et  a l . , 1979), the Ca influx following depolarization may trigger events in the presynaptic terminal leading to LLP. It has previously been reported that LLP is associated with a decrease in presynaptic terminal exc i tabi l i ty (Sastry, 1982). In fact , following glutamate application, a similar observation results in Sch terminals and i t may be due to a hyperpolarization which would be consistent with an enhancement in synap-t i c transmission, although other poss ib i l i t ies such as N a + + - i n a c t i -vation, an increase in membrane capacitance and an alteration in resting membrane conductance cannot be ruled out. - 85 -The above observations indicate that neuronal changes occurring to produce LLP are confined to the synaptic zone and Ca movement into CA-^  somata is at least partly involved in producing depression but not LLP. Although the results suggest that LLP is generated presynaptically, i t cannot be ruled out that Ca channels located on the postsynaptic dendritic membrane which are insensitive to verapamil are responsible. This would suggest that synaptically located receptors and/or C a + + channels are dist inct from extrasynaptic ones and, in fact , there is a report in l i terature (Fagni et a l . , 1983) which demonstrates that there is a difference between synaptic transmitter receptors and extrasynaptic glutamate receptors in that the latter ones desensitize and the former ones do not. N-Methyl-DL-aspartate It has been suggested that NMA receptor activation increases a voltage-++ sensitive Ca conductance in hippocampal pyramidal cel ls (Dingledine, 1983). The depression of CA^ neurones appears to be dependent on intracel lu lar C a + + (Chirwa et a l . , 1983). There is also evidence for Ca+ +-dependence of LLP (Dunwiddie and Lynch, 1979, Wigstrom et a l . , 1979). The results obtained with NMA application to the whole s l ice indicate that the drug has two effects on the CA-^  population spike: an induction of an early potentiation as well as a late depression. It is probable that both the potentiation and depression are co-occurring at any time but i t seems that the former is predominant at the i n i t i a l (about 30 min) period post-NMA but the latter is more powerful and hence takes over at the later stages. The mechanism of this depression is as yet unclear (except that verapamil appears to minimize i ts presence) and should be - 86 -investigated further. There is a decrease in the exci tabi l i ty of Sch terminal regions that is present throughout the potentiation as well as the depression. It has previously been reported that there is a similar exc i tabi l i ty change in presynaptic terminals that is associated with tetanus-induced LLP (Sastry, 1982). The nature of this alteration and its involvement in LLP is as yet undetermined. Verapamil blocks only the late depression of the population spike and has no effect on the potentiation of the response or the presynaptic threshold change induced by NMA. Presuming that verapamil blocks CA^ Ca channels, this depression appears to be due to a postsynaptic change. Moreover, Mn + + also blocked this depression, supporting the idea that Ca is involved. Mn ++ which is known to block pre- and postsynaptic Ca channels counteracted potentiation of the population spike as well as the presynaptic exci ta -b i l i t y change associated with i t , while verapamil had no effect on either „ ++ of these. It i s , therefore, logical to think that a presynaptic Ca dependent change can account for this potentiation. The observations following iontophoretic NMA application at the CA^ synaptic zone suggest that the effects of the amino acid can be explained at least partly by an action on Sch-CA^ synapses. As evidenced by the different NMA doses (iontophoretic) to e l i c i t depression vs potentiation, ++ i t seems that the former can be produced more easi ly . Perhaps, the Ca influx into presynaptic terminals to induce the potentiation (presuming that i t is generated presynaptically) has to be greater than that into the subsynaptic CA-^  neurones to induce depression (since both LLP and depression are Ca + + -dependent). Alternatively, there may be a fewer number of NMA preferring receptors present on terminals than on CA^ - 87 -neurones so higher doses of NMA may be required to activate a suff icient number at terminals to trigger events leading to potentiation. These results indicate that the depression and potentiation of the ++ population spike as well as the presynaptic exc i tabi l i ty change are Ca -dependent. Verapamil, however, appears to block the depression only suggesting a selective action of the drug on the postsynaptic neurone. Amino acid "antagonists" and LLP Collingridge et a l . (1983b) reported that 2-amino-5-phosphonovalerate (APV) interfered with LLP because they found that a tetanic stimulation during the drug application did not induce LLP. They therefore suggested that an activation of NMA receptors, presumably during the tetanic stimu-lat ion, leads to the development of LLP. Although the lack of appearance of LLP if the tetanus is given during APV application is supported by the present resul ts , i t is quite clear that LLP development was not "anta-gonized" by APV. It appears that during tetanic stimulations if APV is administered the CA-^  population spike is depressed (for a prolonged period of time after the termination of the drug application) and that this post-drug depression is co-occurring with LLP. LLP is therefore masked, rather than blocked by APV. Verapamil, which had no signif icant effect on the control CA-^  popu-lation spike, counteracted the depression of the population spike produced by low frequency tetani given to the Schaffer col lateral input. It is ++ suggested that a CA-^  neuronal accumulation of Ca which occurs during such tetanic stimuli (Chirwa et a l . , 1983) is at least partly responsible for the depression. Perhaps, APV (as in the case of glutamate) increases the C a + + influx into CA-^  neurones during the tetanic stimulation - 88 -leading to a buildup of the ions in the c e l l s , thereby inducing a depres-sion. Verapamil, by interfering with this C a + + accumulation, could diminish the development of the APV-induced depression. It is interesting that APV induces a depression, i f applied during a tetanus, but not the population spike evoked at 0.1 Hz. Perhaps, the agent fac i l i ta tes the influx of C a + + that is dependent on CA-^  neuronal act iv i ty . The above results with APV indicate that NMA receptors are not involved in the induction of LLP by tetanic stimulation of the input. It has recently been reported that y-D-glutamylglycine, another NMA antagonist used by Collingridge et a l . (1983b), did not block but only masked LLP in the dentate gyrus (Dolphin, 1983). It has also been claimed that glutamic acid diethyl ester (GDEE) blocked LLP (Krug et a l . , 1982). These authors, however, showed that the agent had a direct depressant effect that lasted for two hours. Their finding that GDEE blocked LLP may, therefore, be due to a masking rather than blockade of LLP. In fact , i t has been shown in this laboratory that GDEE does not block LLP in the dentate gyrus (Sastry et a l . , 1982). DL-Aminophosphonobutyric acid (APB) was reported to block LLP (Dunwiddie et a l . , 1978). This agent, however, does not block the effect of applied glutamate in the hippocampus (Dunwiddie et a l . , 1978) and perhaps blocked LLP not through a blockade of the amino acid receptors. D- or DL-Aminoadipic acid ( a A A ) , another amino acid antagonist (Collingridge et a l . , 1983a; Davies and Watkins, 1979), did not prevent the development of LLP and in fact this agent when applied to the whole s l i ce produced LLP of the CA^ population spike presumably by an activation of the input neurones. One should, therefore, be extremely cautious in interpreting results from the studies ut i l i z ing - 89 -these amino acid antagonists. It is clear that these "antagonists" have effects unrelated to amino acid antagonism. CONCLUSIONS 1. Long-lasting potentiation (LLP) of synaptic responses in the CA^ region of hippocampus is probably generated presynaptically (but a verapamil insensitive subsynaptic dendritic alteration cannot be ruled out). 2. Tetanus-induced homo- and heterosynaptic depressions as well as amino acid-induced depressions of synaptic transmission are localized to the postsynaptic CA-^  neurone. 3. Verapamil, a C a + + antagonist, appears to preferential ly block the ++ actions of Ca postsynaptically. The induction of both LLP and the ++ above depressions are Ca -dependent, but verapamil selectively counteracts the depressions and not LLP. ++ 4. There is a Ca -dependent, verapamil insensitive, decrease in exci -tab i l i t y at the terminal regions of Schaffer col laterals which is associated with and has a similar time course to LLP. 5. N-Methyl-DL-aspartate preferring amino acid receptors are not involved in the induction of LLP. - 90 -NEURONE A ( 1 ) DEPRESSION: -OBSERVATION OF HOMO- AND HETERO-SYNAPTIC DEPRESSIONS FAVOURED BY LOW RATHER THAN HIGH TETANIC FREQUENCIES -FACILITATED BY GLUTAMATE, NMA AND APV -DEPENDENT ON CALCIUM -SENSITIVE TO VERAPAMIL DEPRESSION (1) (2) LLP: -OBSERVATION OF LLP FAVOURED BY HIGH RATHER THAN LOW TETANIC FREQUENCIES -ASSOCIATED WITH DECREASED TERMINAL EXCITABILITY -DEPENDENT ON CALCIUM -NOT SENSITIVE TO VERAPAMIL DEPRESSION ? FIG 21 Schematic diagram of a CA^ neurone and Schaffer col lateral input + t o i l lust rate proposed sites of Ca + +-dependent depression and Ca -dependent LLP. - 91 -REFERENCES ABRAHAM, W. C. and GODDARD, G. V. (1983). Asymmetric relationships bet-ween homosynaptic long-term potentiation and heterosynaptic long-term depression. Nature (Lond.) 305: 717-719. ALGER, B. E . , MEGELA, A. L. and TEYLER, T. J . (1978). 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Effects of localized applications of L-glutamate on the population spike in the hippocampal s l ice preparation. Li fe Sc i . 33: 1673-1678 (1983). Goh, J . W. and Sastry, B. R. Mutual inhibition of nociceptive pathways in fel ine spinal cord. Can. Fed. B io l . Soc. (Abstract) 25: 72 (1982). Goh, 0. W. and Sastry, B. R. Mutual inhibition of pain pathways in cat spinal cord. Pacif ic Cascade Soc. Neurosci. (Abstract) (1982). Maretic, H., Chirwa, S. S . , Goh, J . W. and Sastry, B. R. Actions of calcium antagonists and glutamic acid diethylester on presynaptic terminal exc i tabi l i ty and on long-term potentiation in dentate gyrus. Paci f ic Cascade Soc. Neurosci. (Abstract) (1982). List of Publications con't J . W. GOH Maretic, H., Murali Mohan, P., Chirwa, S . , Goh, J . W. and Sastry, B. R. Verapamil counteracts depression rather than long-lasting potentiation (LLP) of rat hippocampal CA^ population spike. Fed. Proc. 43: 924 (1984). Sastry, B. R., Chirwa, S. S . , Goh, J. W., Maretic, H. and Pandanaboina, M. M. Verapamil counteracts depression but not long-lasting potentiation of the hippocampal population spike. Life  Sc i . 34: 1075-1086 (1984). Sastry, B. R., Chirwa, S. S . , Goh, J . W. and Maretic, H. Calcium, long-term potentiation (LTP) and depression of hippocampal population spike. Soc. Neurosci. (Abstract) 9: 480 (1983). Sastry, B. R., Chirwa, S. S . , Goh, J . W. and Maretic, H. Is long-term potentiation in the dentate gyrus dependent on an alteration in the presynaptic terminal act iv i ty Soc. Neurosci. (Abstract) 8: 146 (1982). Sastry, B. R. and Goh, J. w. Long-lasting potentiation in hippocampus is not due to an increase in glutamate receptors. Life Sc i . 34: 1497-1501 (1984). ~~ Sastry, B. R. and Goh, J . W. Actions of morphine and met-enkephalin-amide on nociceptor driven neurones in substantia gelatinosa and deeper dorsal horn. Neuropharmacology 22: 119-122 (1983). Sastry, B. R. and Goh, J . W. Actions of morphine (M) and met-enkephalin-amide (MEA) on nociceptor driven neurones (NDN) in substantia gelatinosa (SG) and deeper dorsal horn (DH). Can. Fed. B io l .  Soc. (Abstract) 25: 96 (1982). Sastry, B. R., Goh, J. w. and Pandanaboina, M. M. Verapamil counteracts the masking of long-lasting potentiation of hippocampal population spike produced by 2-amino-5-phosphonovalerate. Li fe Sc i . 34: 323-329 (1984). 

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