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Studies on the induction of short- and long-term synaptic potentiation in the hippocampus May, Patrick B. Y. 1987

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Studies on the Induction of Short- and Long-Term Synaptic Potentiation in the Hippocampus by Patrick Bing Yee May B.Sc.(Pharm.), The University of British Columbia, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PHARMACOLOGY AND THERAPEUTICS We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1987 © Patrick Bing Yee May, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pharmacology & Therapeutics The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date October 14, 1987 DE-6(3/81) - i i -ABSTRACT High frequency repet i t i ve st imulat ion of an exci tatory input in the hippocampus resul ts in a post- tetanic potent iat ion (PTP) of short duration (about 3 min) that can be followed by a long-term synaptic potent iat ion (LTP) of the same exci tatory input (Schwartzkroin and Wester, 1975; Andersen et a l . , 1977). It has been reported that th is tetanus-induced LTP cannot be 2+ 2 + e l i c i t e d in a Ca - f ree medium and i s therefore a Ca -dependent process (Dunwiddie et a l . , 1978; Dunwiddie and Lynch, 1979; Wigstrbm et a l . , 1979). 2+ Whether the induction of LTP is d i r ec t l y dependent upon Ca , or whether „ 2+ . Ca is required because synaptic transmission i s needed to i n i t i a t e cer ta in postsynaptic process(es) (a postsynaptic depolar izat ion, fo r instance) leading to LTP, i s unknown. Recent studies from th is laboratory showed that both short-term potent iat ion (STP; with a duration resembling PTP) and LTP can be assoc ia t ive ly induced i f ac t iva t ion of a test input co-occured with e i ther a tetanic st imulat ion of separate exci tatory inputs or a su f f i c ien t depolar izat ion of the postsynaptic neurone (Sastry et a l . , 1985). In th i s study, experiments were performed to invest igate (1) whether associat ive STP could be induced when act iva t ion of the test input preceded or followed the onset of the condit ioning t ra in and (2) whether LTP could be 2+ induced in the absence of Ca in the ex t race l l u la r medium i f s u f f i c i e n t depolar izat ions of the presynaptic terminals and postsynaptic neurones were provided. A l l experiments were performed using the transversely sectioned hippocampal s l i c e preparat ion. Test s t imul i ' were del ivered v ia an electrode located in the stratum radiatum while the condi t ioning tetani (100 Hz, 10 pulses per t ra in) were del ivered v ia another electrode located in the stratum or iens. Population exci tatory postsynaptic potent ia ls (EPSPs) were recorded from the apical dendr i t ic area of CA^ neurones. A f te r the i n i t i a l control st imulat ion per iod, 5 condit ioning tetani were given at a frequency of 0.2 Hz. The test s t imul i e i ther preceded (-) or fol lowed ( + ) the onset of each condi t ioning t r a i n by 0 to 100 ms. When the test stimulus followed the onset of each condi t ioning t r a i n , there was s i gn i f i can t STP of the test EPSP up to a condi t ion ing- test in terva l of +80 ms. When the test stimulus preceded the onset of each condit ioning t r a i n , there was s ign i f i can t STP of the test EPSP up to a condi t ion ing- test in terva l of -50 ms. Conditioning tetani that were given without co-ac t iva t ion of the test input resulted in a subsequent depression of the test EPSP. It i s suggested that e i ther the test or the condit ioning input can i n i t i a t e some postsynaptic process(es) which can in turn af fect the act ivated presynaptic terminals to increase t ransmit ter release or a l te r the subsynaptic dendr i t i c propert ies. For studying the p o s s i b i l i t y of the induction of LTP in the absence of Ca in the ex t race l l u la r medium, population EPSPs were recorded from apical dendr i t ic area of CA^ neurones in response to stratum radiatum st imulat ion. Af ter the control st imulat ion per iod, s l i c e s were exposed e i ther to Ca -contain ing or Ca - f ree (with Mn and Mg replacing Ca ) medium, with the concentration of KC1 at 10 to 80 mM. Long-term potent iat ion of the population EPSPs was observed fol lowing the exposure to + 2+ high K in Ca - f ree media. Following a br ie f period of potent iat ion i n i t i a l l y , population EPSPs often exhibi ted a tendency toward depression + 2+ a f ter exposure to high K in Ca -containing media. LTP induced by high + 2 + K in Ca - f ree medium could also be observed when a f ixed number of axons were being ac t iva ted, ind icat ing that a recruitment of presynaptic f ib res cannot en t i re l y account for the potent ia t ion. LTP of the i n t r a c e l l u l a r EPSPs in CA^ neurones could be induced when postsynaptic d e p o l a r i z i n g commands were paired with a c t i v a t i o n of the stratum radiatum 2+ while the s l i c e s were exposed to Ca - f r e e medium (normal co n c e n t r a t i o n of 2+ K C 1 ) . These r e s u l t s suggest that e x t r a c e l l u l a r Ca , s y n a p t i c transmission and thus subsynaptic receptor a c t i v a t i o n are not necessary f o r the induction of LTP as long as s u f f i c i e n t d e p o l a r i z a t i o n s of the presynaptic terminals and postsynaptic neurones are provided. Bhagavatula R. S a s t r y , Ph.D. (Supervisor) - V -TABLE OF CONTENTS Page Abstract i i Table of Contents v L i s t of Tables v i i L i s t of Figures v i i i Acknowledgements ix Abbreviations x CHAPTER 1. INTRODUCTION 1 2. REVIEW.OF THE LITERATURE 4 2.1 Anatomy of the hippocampal formation 4 2.2 Major afferents to the hippocampal formation 7 2.2.1 Afferents to the dentate area. 7 2.2.2 Afferents to area CA 3. 8 2.2.3 Afferents to area CA 1. 8 2.3 Inhibi tory interneurones 9 2.4 Efferents from the hippocampal formation 11 2.5 Short-term potent iat ion 11 2.5.1 F a c i l i t a t i o n . 13 2.5.2 Augmentation. 15 2.5.3 Potent ia t ion. 16 2.5.4 Locus and possible mechanisms for STP. 18 2.6 Long-term potent iat ion 31 3. METHODS 54 3.1 Preparation of s l i ces 54 3.2 S l i ce chamber 57 3.3 Physio logical medium 57 3.4 Instrumentation 59 3.4.1 Stimulat ion systems. 59 3.4.2 Recording systems. 59 3.5 Temporal requirements of assoc ia t ive ly induced short-term 60 potent iat ion - vi -CHAPTER 3.6 Induction of long-term potent iat ion in the absence of ex t race l l u la r calcium 3.6.1 Effects of increasing ex t race l l u la r potassium concentration in both the absence and presence of calcium in the perfusing media. 3.6.2 Effects of increasing concentration of ex t race l l u l a r potassium ( in calc ium-free medium) in the presence and absence of p ic ro tox in . 3.6.3 Effects of increasing concentration of ex t race l l u la r potassium ( in calcium-free medium) when a f ixed number of axons were being act ivated. 3.6.4 Effects of pai r ing postsynaptic depolar izat ions with presynaptic action potent ia ls in calcium-free medium. 4. RESULTS 69 4.1 Temporal requirements of assoc ia t ive ly induced short-term 69 potent iat ion 4.2 Induction of long-term potent iat ion in the absence of 69 ex t race l l u la r calcium 4.2.1 Effects of increasing ex t race l l u la r potassium 69 concentrations in both the absence and presence of calcium in the perfusing media. 4.2.2 Effects of increasing ex t race l l u la r potassium 73 concentration ( in calcium-free medium) in the absence and presence of p ic ro tox in . 4.2.3 Effects of increasing concentration of ex t race l l u l a r 73 potassium ( in calcium-free medium) when a f ixed number of axons were being act ivated. 4.2.4 Effects of pai r ing postsynaptic depolar izat ions 75 with presynaptic act ion potent ia ls in calcium-free medium. Page 62 • 62 65 6 5 66 5. DISCUSSION 78 5.1 Temporal requirements of assoc ia t ive ly induced short-term 78 potent iat ion 5.2 Induction of long-term potent iat ion in the absence of 83 ex t race l l u la r calcium 6. CONCLUSIONS 93 - V l l -LIST OF TABLES Effects of increasing concentrations of ex t r ace l l u l a r potassium on the CAj population EPSP magnitude 15 minutes fo l lowing termination of exposure to elevated ex t race l l u la r potassium • - v i n -LIST OF FIGURES Figures 1 Anatomical arrangement of a transversely sectioned rat hippocampal s i i ce . 2 S l i ce chamber and perfusion system for the maintenance of and e lect rophys io log ica l recordings from in v i t r o rat hippocampal s l i c e s . 3 Experimental arrangements fo r the associat ive induction of STP. 4 A schematic diagram to fur ther i l l u s t r a t e the experimental arrangements in the invest igat ion of the temporal l im i t s for the induction of associat ive STP. 5 General experimental arrangements for sections 3.6.1 to 3 .6 .3 . 6 • Diagram i l l u s t r a t i n g the experimental arrangements fo r the pair ing of postsynaptic depolar izat ions with presynaptic action po ten t ia ls . 7 Potent iat ion of the test population EPSPs at d i f fe rent condi t ion ing- test in terva ls (CTl) . 8 Magnitudes of population EPSPs fo l lowing appl icat ion of elevated ex t race l l u l a r potassium ( in calcium-free medium) in the absence and presence of p ic ro tox in . 9 Magnitudes of population EPSPs fol lowing appl icat ion of elevated ex t race l l u la r potassium ( in calcium-free medium) when a f ixed number of axons were being ac t iva ted. 10 EPSP magnitudes p r io r to and fo l lowing the ac t iva t ion of presynaptic f i b res concurrently with depolar izat ions of a CAj neurone during perfusion with calcium-free medium. - ix -ACKNOWLEDGEMENTS I am grateful to Dr. B. R. Sastry, my supervisor, fo r his i ns igh ts , guidance and encouragement during the course of t h i s study. I thank Mr. Anthony Auyeung, Mr. Sanika Chirwa and Dr. Joanne Goh fo r t he i r f r iendship and help. Thanks are also due to Mr. Chr is t ian Cari tey for developing the s l i c e chamber used fo r our experiments. Financial support of the Medical Research Council of Canada, the B r i t i s h Columbia Health Care Research Foundation, the Faculty of Medicine (Universi ty of B r i t i s h Columbia) Summer Research Studentship, and the B r i t i s h Columbia Post Secondary Scholarship, is great ly appreciated. - X -LIST OF ABBREVIATIONS Ach Acetylcho 1ine AP7 2-amino-7-phosphonoheptanoate APV 2-amino-5-phosphonovalerate (2-amino-5-phosphonopentanoic acid) CA Cornu ammonis cAMP Cyclic adenosine monophosphate CNS Central nervous system CT Conditioning tetanus (tetani) CTI Conditioning-test interval DGG y-D-glutamylglycine DLH DL-homocysteate EDTA Ethylenediaminetetraacetic acid EGTA Ethyleneglycol-bis-(B-aminoethyl ether)-N, N, N', N'-tetraacetic acid epp End plate potential EPSP Excitatory postsynaptic potential GABA Y-aminobutyric acid GAD Glutamic acid decarboxylase IPSP Inhibitory postsynaptic potential LTP Long-term potentiation mepp Miniature end plate potential NMDA N-methyl-D-aspartate PTP Post-tetanic potentiation STP Short-term potentiation TS Test stimulus (stimuli) TTX Tetrodotoxin - 1 -1. INTRODUCTION In the mammalian hippocampus, high frequency repet i t i ve st imulat ion of the exc i ta tory af ferents can lead to a post- tetanic potent iat ion of the synaptic response that i s evoked by the same input (B l i ss and L/zfmo, 1973). This synaptic potent iat ion can be of very short durat ion, having a duration of only mi l l iseconds to minutes, and i s therefore referred to as short-term potent iat ion (STP; McNaughton, 1982). Depending on various factors that w i l l be discussed l a t e r , th is synaptic potent iat ion can also be of a more persistent nature, las t ing fo r a period of 30 min or much longer, and i s referred to as long-term potent iat ion (LTP; B l i s s and Gardner-Medwin, 1973; B l i s s and Ltfmo, 1973; Douglas and Goddard, 1975). It was la te r found that both STP (Creager et a l . , 1980) and LTP (Schwartzkroin and Wester, 1975) can be e l i c i t e d in the in v i t ro hippocampal s l i c e preparation (Skrede and Westgaard, 1971), thus fur ther f a c i l i t a t i n g the study of synaptic potent iat ion in the hippocampus. LTP has espec ia l l y attracted the attention of invest igators because an enhancement of synaptic potent ia ls i s thought by many to have a ro le in learning and memory (Goddard, 1980; Morris et a l . , 1986). It has recently been shown that both STP and LTP can be induced when act ivat ion of a test input co-occured with e i ther a tetanic st imulat ion of separate exci tatory inputs or su f f i c i en t depolar izat ion of the postsynaptic neurone (Sastry et a l . , 1986). In other words, potent iat ion of the synaptic response can be e l i c i t e d without d i rec t tetanic act ivat ion of the afferents evoking the response providing that the above condit ions are met. The present study was performed using the rat transverse hippocampal s l i c e preparation in v i t r o . The aim of the f i r s t part of th is study was to invest igate whether assoc iat ive STP could be induced when a temporal - 2 -separation existed between the act ivat ion of the test stimulus and the onset of the condit ioning tetanus. Stimulation of the test input was given before as well as af ter the onset of the condit ioning tetanus and the response to the test stimulus was monitored af ter such "paired" st imulat ions. The induction of hippocampal LTP has been thought to be a 2+ , Ca -dependent process (Dunwiddie et a l . , 1978; Dunwiddie and Lynch, 1979; Wigstrom et a l . , 1979). A high frequency stimulus given in the 2+ absence of Ca in the ex t race l lu la r medium f a i l e d to e l i c i t LTP (Dunwiddie and Lynch, 1979; Wigstrom et a l . , 1979). It i s unknown whether 2+ 2+ Ca is d i r ec t l y required for the induction of LTP, or whether Ca has to be present because synaptic transmission i s required to i n i t i a t e cer ta in postsynaptic process(es) leading to LTP. Based on the f indings of Sastry et a l . (1986), who indicated that both pre- and postsynaptic a c t i v i t y seem to be required fo r the induction of LTP, experiments were performed to 2+ invest igate whether LTP could be e l i c i t e d in a Ca - f ree medium when both the presynaptic terminals and the postsynaptic neurones were depolar ized. This was achieved by exposing the s l i ces to elevat ing concentrations of + 2+ ex t race l l u la r K in a Ca - f ree medium and monitoring the synapt ic response a f te r the above treatment. A l te rna te ly , depolar izat ion of the postsynaptic neurone by d i rect current in jec t ion was paired with ac t i va t ion 2+ of the input f ib res while the s l i ces were being exposed to Ca - f ree medium. To ensure that any increase in synaptic e f f i cacy subsequent to high ex t race l l u la r K + was not merely a resul t of a recruitment in the number of f i b r e s , experiments were performed to observe the ef fect of elevated ex t race l l u la r K ( in Ca - f ree medium) on the synaptic response when a f ixed number of axons were being act ivated. This was being done to ensure that any ef fect of h igh-K + on the synaptic response was not en t i r e l y due to any change in the e x c i t a b i l i t y and therefore in the number of axons that were being act ivated. Many Ca -mediated events .have been implicated in the induction of LTP (Finn et a l . , 1980; Turner et a l . , 1982; Ecc les , 1983; Mody et a l . , 1984; Lynch and Baudry, 1984). It i s hoped that resul ts from these experiments w i l l provide some ins ights into the role ( i f any) Ca plays in the induction of LTP. - 4 -2. REVIEW OF THE LITERATURE 2.1 Anatomy of the hippocampal formation It has been pointed out that the hippocampus is the most conspicuous of a l l the cytoarchi tectonic structures in the mammalian brain (Schwerdtfeger, 1984). Because of i t s c lear lamination into several d i s t i nc t layers, the hippocampus has been considered a simple model of the neuronal cortex (Ramon y Ca ja l , 1893) and d i f ferent aspects of hippocampal morphology have been extensively studied. The hippocampal formation i s considered to be part of - the l imbic system, a term that i s used to include a l l of the l imbic lobe as well as associated subcort ical nuclei (Truex and Carpenter, 1969). The hippocampal formation is made up of two types of cortex-the a l locor tex and the per ia l locor tex (Chronister and White, 1975). The al locortex can be fur ther divided into palaeocortex and archicortex (Schwerdtfeger, 1984). The archicortex comprises the subiculum, Ammon's horn, fasc ia dentata, precommissural hippocampus as well as supracommissural hippocampus (Schwerdtfeger, 1984). The terms "arch icor tex" and "hippocampus" are often used as synonyms (Schwerdtfeger, 1984). The per ia l locor tex consists of the presubiculum, the area re t rosp len ia l i s e, the parasubiculum and the entorhinal region (Chronister and White, 1975). When one refers to the hippocampal formation, one is r ea l l y re fer r ing to the hippocampus proper (Ammon's horn), the dentate gyrus, and the subiculum (Chronister and White, 1975). The hippocampus i s s t ruc tu ra l l y b i l a t e r a l l y symmetrical and i s shaped somewhat l i ke a cashew nut (Teyler and DiScenna, 1984). Both the hippocampus proper and the dentate gyrus are folded into a shape somewhat l i ke the l e t t e r " C " , and the two " C ' s " i n te rd ig i ta te as shown in F i g . 1 (Teyler and DiScenna, 1984). The dentate gyrus consists of a s ingle layer - 5 -B A S I L A R D E N D R I T E S S O M A - A P I C A L D E N D R I T E S S. polymorph* F i g . 1. Anatomical arrangement of a t ransversely sectioned rat hippocampal  s l i c e . The major a f ferent , efferents and i n t r i n s i c pathways are shown. Alv-alveus B-Basket c e l l s Comm-commissural inputs Ento-entorhinal cortex Fim-fimbria HF-hippocampal f i ssure mf-mossy f ib res pp-perforant path Sch-Schaffer co l l a te ra l s - 6 -of granule c e l l s (Teyler and DiScenna, 1984) with the apical dendrites oriented towards the p ia l surface within the hippocampal f i ssure (or away from the center of the "C") (Chronister and White, 197 5; Teyler and DiScenna, 1984). Next to the granule c e l l s are the polymorphic ce l l s (Ram5n y Ca ja l , 1893; Lorente de N5, 1934) which, together with the t rans i t i ona l pyramidal c e l l s of f i e l d CA^ of Ammon's horn, made up the region known as the hi 1 us of the dentate gyrus (Chronister and White, 1975). The t rans i t i ona l pyramidal c e l l s at t r ibuted to f i e l d CA^ of Ammon's horn have been a source of some controversy, since i t has been d i f f i c u l t to d i f fe ren t ia te whether they are r ea l l y part of Ammon's horn or whether they are a part of the dentate gyrus (Teyler and DiScenna, 1984). Blackstad (1955) c l a s s i f i e d them as part of the dentate gyrus, even though these c e l l s do appear to form a s t ructura l t r ans i t i on into the hippocampus proper. The hippocampus proper (Ammon's horn) and the subiculum comprises predominantly of pyramidal c e l l s (Lorente de No, 1934; Teyler and DiScenna, 1984). Ammon's horn can be divided into four main areas, or zones: C A ^ a k cy Ch^, ^ 3 ( a ^ c ) and CA^ (F ig . 1). The pyramidal c e l l s in f i e l d CA^ (and part of CA£) are what Lorente de No (1934) referred to as the "giant pyramids". F ie ld CA^ consists of smaller pyramidal neurones which extend from those of CA^ as one layer of c e l l s (Chronister and White, 1975). F ie ld CA,, i s considered to be the t rans i t i ona l f i e l d between CA^ and CA^ the i r pyramidal c e l l s being somewhat smaller than those of f i e l d CA^ (Lorente de No, 1934). At the border between CA^ and the subiculum, the pyramidal c e l l s appear to lose the i r c l us te r i ng , the region between the pyramidal c e l l s and the alveus gradual ly diminishes and then becomes a c e l l layer (Chronister and White, 1975). Besides the pyramidal and granule c e l l s , which predominate the hippocampal formation, a number of basket c e l l s (general ly interpreted as inh ib i tory interneurones) - 7 -can be found to contact numerous granule as well as pyramidal neurones (Ramon y Ca ja l , 1893). As shown by the transverse sect ion in F i g . 1, the layers of the hippocampal formation (Ammon's horn and dentate gyrus) are: stratum or iens, stratum pyramidale, stratum radiatum, stratum lacunosum, stratum moleculare, stratum granulosum and stratum polymorphe (Ramon y Ca ja l , 1893). Dorsal to stratum oriens i s a zone of myelinated f i b r e s , the alveus (Schwerdtfeger, 1984). 2.2 Major afferents to the hippocampal formation 2.2.1 Afferents to the dentate area. The entorhinal cortex gives r ise to the major exc i ta tory input to the hippocampal formation-the perforant path (Ramon y Ca ja l , 1893; Lorente de No, 1934). E l e c t r i c a l st imulat ion of the perforant path f i b res or the entorhinal area can evoke a large negative going population spike in the dentate granule c e l l layer , ind icat ing the synchronous discharge of many such granule c e l l s (Andersen, 1975). The input from the perforant path to the dentate area is organized in a ser ies of para l le l l ines (L0mo, 1971). The perforant path, which ar ises from the la te ra l part of the entorhinal cor tex, crosses the subiculum and the hippocampal f i ssure and innervates the dentate gyrus (Lorente de No, 1934). It has been noted that the ventral part of the dentate gyrus receives many more branches from the perforant path than the dorsal or ven t r i cu la r part (Lorente de No, 1934). Lorente de No (1934) observed that the perforant path also innervates the prosubiculum and f i e l d s CA^, Zk^ and CAg of Ammon's horn. The entorhinal cortex also gives r i se to the alvear path, which gives numerous co l l a t e ra l s to the prosubiculum and to f i e l d CA.^ (Lorente de No, 1934). A second major source of afferents to the dentate gyrus i s the commissural pathway (Andersen, 1975), which enters the hippocampus through the f imbr ia (Lorente de No, 1934). - 8 -2.2.2 Afferents to area CA.,. The major afferent pathway to the CA^ pyramidal neurones is probably the mossy f ib res (Andersen, 1975), the axons of the dentate granule c e l l s which course through the hi lus of the dentate and form en passant synapse with the dendr i t ic spines of the proximal port ion of the apical dendrites (Andersen et a l . , 197lb). The mossy f ib res have large synaptic swel l ing at 2 50 to 450 urn in tervals and course along the whole of f i e l d CA^ (Blackstad and Kjaerheim, 1951). The branched dendrites are completely engulfed by these synaptic swel l ings mentioned above (Hamlyn, 1961). It was also found that both the mossy f ibres and the perforant path f ib res run s t r i c t l y pa ra l le l and that both sets of f i b res are oriented d i r ec t l y transverse to the longi tudinal axis of the hippocampal formation (Andersen et a l . , 1971b). The commissural pathway to area CA^ ar ises from the homotopic point of the contra la tera l hippocampus (Andersen, 1975) and enters the hippocampal formation through the f imbr ia (Lorente de No, 1934). H i s to l og i ca l l y , the commissural f ib res terminate in the basal dendrites of the pyramidal neurones in f i e l d CA^ (Blackstad, 1955) (F ig . 1). 2.2.3 Afferents to area CAp The commissural pathway mentioned in the previous subsection also crosses through the stratum pyramidale of areas CA^ and CA^ to form en passant synapses with the apical dendrites of the pyramidal neurones of area CAj (Lorente de No, 1934; Blackstad, 1955) (F ig . 1). In area CAp the commissural pathway also t ravels by way of the alveus and synapses with the basal dendrites of the pyramidal neurones (Blackstad, 1955; Andersen, 197 5) (F ig . 1). The most prominent exc i ta t ion of area CA^ neurones can be observed fo l lowing act ivat ion of CA^ pyramidal neurones or the i r Schaffer co l l a t e r a l s (Andersen et a l . , 1971b). The Schaffer c o l l a t e r a l s are the coarse, myelinated co l l a t e ra l s of the axons of the CA_ pyramidal neurones (Andersen et a l . , 1971b). These c o l l a t e r a l s - 9 -curve back through the CA^ stratum pyramidale towards area CA^ and then form en passant synapses with d i s ta l portions of the apical dendr i t ic trees' of CA^ pyramidal neurones (Andersen et a l . , 1971b) (F ig . 1). As was mentioned e a r l i e r , the alvear path from the entorhinal cortex gives off co l l a te ra l s to the prosubiculum and to f i e l d CA^ a (Lorente de No, 1934). To conclude, the hippocampal formation contains a four membered exci tatory neuronal chain consis t ing of entorhinal c e l l s , dentate granule c e l l s , "CAg and CA^ pyramidal neurones (Ramon y Ca ja l , 1893; Lorente de No, 1934). Axons from these neurones form a ser ies of sequent ia l ly activated pathways consis t ing of the perforant path, the mossy f i b r e s , the Schaffer co l l a te ra l s and the alveus (Skrede and Westgaard, 1971). Tr isynapt ic exc i ta t ion of CA^ neurones by perforant path st imulat ion has been demonstrated, suggesting that a l l the members of the aforementioned exci tatory neuronal chain can be found in a t ransversely sectioned hippocampal s l i c e (Skrede and Westgaard, 1971). In addi t ion, there ex is t the f ib res const i tu t ing the intrahippocampal longi tudinal associat ion pathways (Lorente de No, 1934; Swanson et a l . , 1978) which, although incompletly understood, is thought to be involved in in te r lamel la r communication within the hippocampus (Teyler and DiScenna, 1984). 2.3 Inhibi tory interneurones Inhibi tory interneurones, or basket c e l l s , can be found d is t r ibu ted in stratum oriens close to the pyramidal neurones as well as in stratum granulosum close to the dentate granule c e l l s (Ramon y Ca ja l , 1893; Lorente de No, 1934; Andersen et a l . , 196 3, 1964). A var iety of basket c e l l types can be located in the areas mentioned above (Ramon y Ca ja l , 1893; Lorente de N5, 1934), although the u l t ras t ruc tu ra l charac te r i s t i cs of these interneurones appear to be quite s im i l a r (Buzsaki, 1984). A l l of the basket c e l l s , however, have axonal terminals that make up a dense network of - 10 -f i b res -a "basket l ike entanglemenf'-around the somata of pyramidal as well as granule c e l l s (Ramon y Ca ja l , 1893; Lorente de No, 1934; Andersen et a l . , 1953, 1964). The axonal terminals form synapses on the somata and the proximal dendrites of granule c e l l s (Buzsaki , 1984). Basket c e l l axons have been observed to extend for up to 1 mm in the longi tudinal axis of the rat hippocampal formation and synapse with up to about 500 granule c e l l s (Strube et a l . , 1978). With the pyramidal c e l l s , the synapses formed by the axonal terminals of the basket c e l l s are almost a l l concentrated on the somatic region (Ramon y Ca ja l , 1893; Lorente de No, 1934). From the drawings of Lorente de No (1934), Andersen et a l . (1964) estimated that one basket c e l l makes about 200 to 500 synaptic contacts with pyramidal neurones. Using i n t r a c e l l u l a r recording of hippocampal pyramidal c e l l s , a hyperpolar izat ion associated with c e l l discharge from d i f ferent afferent sources has been observed (Kandel and Spencer, 1961; Andersen et a l . , 1963, 1964). Andersen et a l . (1964) put forward the model of recurrent or feed-back i nh ib i t i on . Namely, inh ib i to ry interneurones receive exci tatory afferents from neighbouring p r inc ipa l c e l l s and then send inh ib i to ry processes back to the pr inc ipa l c e l l somata to complete the inh ib i to ry c i r c u i t (Andersen et a l . , 1964). Therefore, the f i r i n g of the inh ib i to ry interneurone depends on the f i r i n g of the p r inc ipa l neurones. Buzsaki and Eidelberg (1981) thought that recurrent i nh ib i t i on alone could not account for the ef fect of commissural path st imulat ion on the dentate granule c e l l s , since act ivat ion of the input has been reported to suppress spontaneous as well as evoked c e l l discharges of the granule c e l l s without previous exc i ta t ion of a large number of the p r inc ipa l c e l l s (McNaughton et a l . , 1978). Buzsaki and Eidelberg (1981, 1982) provided evidence for a feed-forward in addit ion to the recurrent type i n h i b i t i o n . These authors observed that interneurones in both the CA, and dentate area responded to - l i -very low in tens i ty commissural vol leys (Buzsaki and Eidelberg, 1981, 1982). Moreover, the f i r i n g of some of these interneurones preceded or coincided with the onset of the synaptic potent ia l recorded ex t race l1u lar ly (Buzsaki and Eidelberg, 1981, 1982). Buzsaki (1984) also reported that the same interneurone can par t i c ipa te in both feed-forward and recurrent i n h i b i t i o n . Using spec i f i c ant isera to glutamic acid decarboxylase (GAD-the enzyme catalyzing the formation of y-aminobutyric acid [GABA]), Ribak et a l . (1978) loca l ized a dense network of GABAergic neurones around the pyramidal and granule c e l l somata which corresponds to the d i s t r i bu t ion of basket c e l l axon terminals. Douglas et a l . (1983) observed that b icucu l l i ne had no ef fect on the f i e l d potent ial evoked by commissural input st imulus, but prevented the commissural stimulus from suppressing the population spike evoked by perforant path s t imulat ion. 2.4 Efferents from the hippocampal formation One of the major outputs of the hippocampal formation is probably the axons from the CA^ pyramidal neurones, which jo in the axons of the CA^ pyramidal neurones running in the alveus and t rave l out through the f imbr ia to the septum, hypothalamus and the contra la tera l hippocampus (Andersen et a l . , 1971b; Andersen, 197 5). The other major efferent system seems to be the project ions by the axons of the CA^ neurones to the subiculum by way of the alveus (Swanson et a l . , 1978). 2.5 Short-term potent iat ion Short-term potent iat ion (STP) can perhaps be general ly defined as a shor t - las t ing (mi l l iseconds to minutes) increase in the magnitude of postsynaptic response as a resu l t of a preceeding repet i t i ve as well as single pulse ac t iva t ion of the presynaptic nerve f ib re (s ) (Ecc les, 1957; Mal lart and Mart in, 1967; Creager et a l . , 1980). Based on the durat ion of the observed ef fects and t he i r time course of decay, STP can be subdivided - 12 -into three components: (1) f a c i l i t a t i o n , which i t s e l f can be subdivided into two components based on the i r decay time constants (Mallart and Mart in, 1967; Magleby, 1973a,b; Younkin, 1974; Zengel et a l . , 1980); (2) augmentation (Magleby and Zengel, 1976a,b,c; Zengel and Magleby, 1982) and (3) potentiat ion (Hubbard, 1963; Gage and Hubbard, 1966; Rosenthal, 1969; Magleby, 1973b; Magleby and. Zengel, 1976a,b,c). These d i f ferent components of STP w i l l be fur ther discussed in l a te r sect ions. H i s t o r i c a l l y , the study of STP can be traced back to the work of Sch i f f in 1858 using the gastrocnemius-sciat ic preparation of the f rog . Schi f f (1858) observed that fol lowing tetanic st imulat ion of the s c i a t i c nerve, single test pulses produced larger responses (greater twitch tension) than before the tetanus. In 1894, Boehm reported a temporary decurarizing ef fect on the response of the muscle to test shocks a f te r te tan iz ing a curar ized, previously unresponsive s c i a t i c gastrocnemius preparat ion. Hughes (1958) suggested that th i s decurar izat ion ef fect observed by Boehm was probably d i rec t l y related to post- tetanic potent iat ion (PTP). Like, the studies discussed above, STP in other ear ly studies were reported mostly in the neuromuscular junct ion (Guttman et a l . , 1937; Feng, 1937; Feng et a l . , 1938; Brown and von Euler , 1938; Feng et a l . , 1939; Feng and L i , 1941; Feng, 1941), although there have been ear ly reports of STP in the peripheral nervous system as well (Ecc les, 1935; Larrabee and Bronk, 1938, 1947). In 1949, Lloyd showed post- te tanic STP in central neurones by demonstrating PTP in the monosynaptic ref lex of the spinal cord. In 1955, Gloor reported STP in the hippocampus subsequent to repe t i t i ve st imulat ion of the amygdala. Addit ional evidence suggesting STP to be a general ized phenomenon has been found in the v isual system (Grani t , 1955; Hughes and Evarts, 1955; Hughes et a l . , 1955 ), o l fac tory system (MacLean, 1957) and auditory system (Hughes, 1954), as well as in iso la ted cardiac t issues (Rosin and Farrah, 1955; - 13 -Katzung and Farah, 1955). Various components of STP were also observed in the rat fasc ia • dentata . (McNaughton, 1982). In 1983, Racine and Mil gram found that a l l three components of STP can be observed in the rat l imbic forebra in. In the studies discussed above, the terms f a c i l i t a t i o n , augmentation and potent iat ion were often used interchangeably. It was not un t i l the studies of Mall art and Martin (195 7) and la te r Magleby and Zengel (Zengel and Magleby, 1982) that STP was c l a s s i f i e d according to the durat ion and time course of decay of the various components. In the fo l lowing sect ions , the three components of STP w i l l be discussed in. more de ta i l with references to the factors inf luencing them and some possible mechanisms of ac t ion . 2.5.1 Fac i1 i t a t i on . Mal la r t and Martin (1967) observed that when one or more condit ioning st imul i were applied to the nerve supplying a motor end-plate, the end-plate potent ia ls (epps) produced by subsequent test s t imul i were increased in amplitude. These and other authors demonstrated that there are two components to th i s " f a c i l i t a t i o n " fo l lowing a s ing le condit ioning stimulus based on the time course of t he i r decay (Mal lar t and Mar t in , 1967; Magleby, 1973a; Younkin, 1974). The f i r s t component i s maximal immediately fo l lowing the impulse and decays exponent ia l ly with a time constant of about 35 ms while the second component peaks 120 ms fol lowing the- impulse and decays exponential ly with a time constant of about 2+ 250 ms (Mal lart and Mar t in , 1967). It was reported that Sr increased the magnitude and the time constant of decay of the second component of f a c i l i t a t i o n while having l i t t l e e f fect on the other components of STP (Zengel and Magleby, 1980; Zengel et a l . , 1980). The decay of f a c i l i t a t i o n was also found to proceed at a slower rate as the temperature was lowered (Charlton and B i t tner , 1978). Tetanic st imulat ion i s not necessar i l y required for f a c i l i t a t i o n , since f a c i l i t a t i o n can be observed when the same - 14 -input is stimulated twice in rapid succession ( th is is commonly referred to as paired-pulse or twin-pulse f a c i l i t a t i o n ) (Creager et_a_L, 1980). Feng (1940) reported that at the frog neuromuscular junct ion, two shocks given close together (2.0 to 68 ms in terva ls) resulted in the second response being much greater in amplitude than that of the response to a s ing le shock. S imi lar observations were made at the frog neuromuscular junct ion (Eccles et a l . , 1941; Braun et a l . , 1966), at the c ray f ish neuromuscular junct ion (Dudel and Ku f f l e r , 1961; Dudel, 196 5), at the mammalian neuromuscular junct ion (L i l ey and North, 1953; Lundberg and Qu i l i s ch , 1953; Hubbard, 1963; Hubbard and Schmidt, 1963), in cat sympathetic ganglion (Job and Lundberg, 1953), rabbit superior cerv ica l ganglion (Zengel et a l . , 1980), squid synapses (Charlton and B i t tne r , 1978), and in the hippocampal s l i c e preparation (Creager et a l . , 1980). Hubbard (1963) reported a potent iat ion of the epp amplitude which could be separated into two components - primary and secondary potent ia t ion. "Primary potent ia t ion" corresponded to the two components of f a c i l i t a t i o n discussed in th is sect ion while "secondary potent ia t ion" corresponded to the longer last ing PTP which w i l l be discussed in la te r sections (Mallart and Mart in, 1967). The f a c i l i t a t i o n that i s observed during a short t r a i n of st imulat ion (frequency f a c i l i t a t i o n ) can perhaps be regarded as an extension of paired-pulse f a c i l i t a t i o n (Racine and Milgram, 1983). Mal lar t and Martin (1967) found that the increase in the amplitude of the epp during short t ra ins of repet i t i ve st imulat ion at frequencies up to 100 Hz and i t s decay af ter the t ra in could be predicted by assuming that (1) the magnitude and time course of both components of f a c i l i t a t i o n are the same af ter every stimulus in the t ra in and (2) the f a c i l i t a t o r y ef fects for every stimulus in the t r a i n sums l i n e a r l y . Magleby (1973a) found that the above l i near f a c i l i t a t i o n model predicted the growth of the epp amplitude only during the - 15 -f i r s t several hundred ms of repet i t i ve s t imula t ion, a f ter which the amplitudes of the epps were found to be f a c i l i t a t e d more than predicted by the l inear model. Studying f a c i l i t a t i o n at squid synapses, Charlton and B i t tner (1978) agreed that the l inear f a c i l i t a t i o n model applied only to very short t ra ins of s t i m u l i . At longer stimulus t r a i n , the second component of f a c i l i t a t i o n as well as other components of STP would have added s i gn i f i can t l y to the summed f i r s t component of f a c i l i t a t i o n (Magleby, 1973a,b; Charlton and B i t tner , 1978; Creager et a l . , 1980). 2.5.2 Augmentation. Augmentation i s a component of STP that resu l ts from repet i t i ve s t imulat ion, i s intermediate in duration between f a c i l i t a t i o n and potent ia t ion, and decays approximately exponent ial ly over most of i t s time course with a mean time constant of about 7 sec (Magleby and Zengel, 197 5, 1976a). Augmentation has been observed at the f rog and toad neuromuscular junctions (Magleby and Zengel, 1976a,b,c; Eru lkar and Rahamimoff, 1978) as well as the superior cerv ica l ganglion of the rabbit (Zengel et a l . , 1980). McNaughton (1982) observed STP in rat f asc ia dentata which decayed to control levels with double exponential time course, one of which was comparable to that of augmentation at neuromuscular synapses. It is d i f f i c u l t to evaluate whether augmentation was observed in other studies because th is component could eas i l y have been masked by the longer las t ing PTP. Magleby and Zengel (1976a) argued that augmentation was evident but had gone unnoticed in the e a r l i e r studies of Larrabee and Bronk (1947), L i l ey (1955a) and Landau et a l . (1973). It was found that test s t imulat ions (given once every 5 to 10 sec) applied immediately af ter the condi t ioning t ra in to test for augmentation did not s i g n i f i c a n t l y change the estimates of i t s magnitude and.time course (Magleby and Zengel, 1976a). Therefore un l ike f a c i l i t a t i o n , augmentation cannot seem to be generated by one or a few impulses. - 16 -Magleby and Zengel (1976a) observed that the decay time constant for augmentation remained re la t i ve l y constant as the duration of the condit ioning st imulat ion was increased. The magnitude of augmentation, on the other hand, was found to increase with the duration and the frequency of st imulat ion (Zengel and Magleby, 1982). Whereas the magnitude of each increment remains constant during a t ra in of stimulus for f a c i l i t a t i o n , the magnitude of each increment appears to increase during the t ra in fo r 2 + augmentation (Zengel and Magleby, 1982). Ba reportedly increases the magnitude but not the time constant of decay of augmentation (Zengel and Magleby, 1980; Zengel et a l . , 1980). Despite the e f fo r ts of Magleby and Zengel in attempting to estab l ish augmentation as a d i s t i nc t component of STP (Magleby and Zengel, 1976a,b,c; Zengel et a l . , 1980; Zengel and Magleby, 1982), augmentation has not been extensively studied by other invest igators . Further studies w i l l have to be performed before the exact nature of th is postulated component of STP can be more thoroughly understood. 2.5.3 Potent ia t ion . Post- tetanic potent iat ion i s perhaps the most thoroughly studied of a l l the components of STP (see Hughes, 1958, fo r review). As was mentioned before, ear ly studies of PTP were done using neuromuscular preparations - an enhancement of muscle contract ion or a temporary decurar izat ion of a previously unresponsive neuromuscular preparation af ter tetanus (Guttman et a l . , 1937; Feng, 1937; Feng et a 1., 1938; Brown and von Euler , 1938; Feng et a l . , 1939; Feng, 1941; Feng and L i , 1941; Walker, 1947; Hutter, 1952; L i l e y and North, 1953). Larrabee and Bronk (1938, 1947) reported on f a c i l i t a t i o n as well as PTP in the s te l l a te ganglion of cats . Af ter a period of repe t i t i ve a c t i v i t y , the amplitude of the postganglionic spike potent ia l was severa l - fo ld greater than the response to a s imi la r preganglionic vo l ley before repe t i t i ve st imulat ion (Larrabee and Bronk, 1947). The same invest igators also - 17 -reported that the degree of potent iat ion increased in d i rect proport ion to the number of condit ioning st imul i un t i l i t reached a maximum of about three- fo ld greater than control a f ter about 40 s t i m u l i , although more prolonged te tan izat ion continued to produce longer las t ing PTP (with, however, a longer latency to the peak of potent iat ion) (Larrabee and Bronk, 1947). Post- tetanic potent iat ion has also been observed in the central nervous system (CNS), f i r s t in the spinal cord, and la te r in other central neurones as we l l . Part of the reason for the in terest in PTP in the CNS i s that th is form of synaptic potent iat ion has been thought to be part of the basis underlying learning and memory (Eccles and Mclntyre, 1951; Ecc les , 1953; Goddard, 1980; Kandel, 1981; Kandel and Schwartz, 1982), although the focus has now been shi f ted to the study of Jong-term potent iat ion (LTP) (see Tey ler and DiScenna, 1987, for review). Lloyd (1949) reported PTP of a monosynaptic ref lex pathway fo l lowing tetanic afferent s t imula t ion. Like Larrabee and Bronk (1947), Lloyd also observed increasing potent iat ion with increasing st imulat ion frequencies of up to about 300 Hz, beyond which the duration (and latency to peak) of potent iat ion continued to increase. S imi lar observations were made by Eccles and Rai l (1951). PTP of polysynaptic ref lexes were not found by Lloyd (1949), although Eccles (1953) argued that no fundamental di f ferences should ex is t between polysynaptic and monosynaptic re f lexes , but merely less opportunity for reveal ing potent iat ion in the former case. PTP of a monosynaptic ref lex in the cat spinal cord was also reported by Wall and Johnson (1958). Post- tetanic potent iat ion has been extensively studied in the marine sna i l Aplys ia (Waziri et a l • , 1959; Kretz et a l . , 1982; Walters and Byrne, 1984). Af ter tetanic st imulat ion of a presynaptic neurone (L10) in Ap l ys i a , Kretz et a l . (1981) observed PTP of 200 to 800% that lasted up to 5 min. - 18 -Walters and Byrne (1984) reported PTP ( las t ing 2 to 3 min) produced by high frequency i n t r a c e l l u l a r act ivat ion of mechanosensory neurones. These authors suggested a possible connection between PTP and other types of synaptic p l a s t i c i t y which have been observed in Aplys ia sensory neurones (Kandel, 1981; Kandel and Schwartz, 1982). Gloor (1955, 1954) observed PTP in the hippocampus of the cat subsequent to tetanic st imulat ion of the amygdala as well as of the perforant path. More recent ly , McNaughton (1982) reported PTP in rat f a s c i a dentata which decayed with a time constant of about 90 sec. PTP in various l imbic forebrain pathways were examined by Racine and Milgram (1983). These authors observed two components of PTP - potent iat ion 1, which decayed with a time constant of about 70 sec, and potent iat ion 2, which decayed with a time constant of about 6 .5 min. The l a t t e r component was found to be present in nearly a l l pathways, while the former was often missing ( i t was suggested that potent iat ion 1 was present but was masked by a post- te tanic depression ef fect) (Racine and Milgram, 1983). 2.5.4 Locus and possible mechanisms for STP. At the neuromuscular junct ion, i t has been suggested that changes responsible for PTP occurs in the muscle con t rac t i l e mechanism (Feng et a l . , 1939; Walker, 1947). Guttman et a l . (1937) argued that the muscle f i b re cannot be the locus of enhancement since experiments with d i r ec t l y stimulated curarized muscle showed no enhancement. These authors concluded that the locus of the potent iat ion was at the neuromuscular junct ion. L i l ey and North (1953) suggested the s i te of PTP to be at the motor nerve terminals and that a f ter repet i t i ve a c t i v i t y an increased f rac t ion of the acety lchol ine (Ach) present at the motor nerve terminals is released with each impulse. Hutter (1952) found no changes in the s e n s i t i v i t y of the motor end plate to Ach fo l lowing - i y -repet i t i ve st imulat ion of motor nerve and also suggested that more Ach was released per nerve vol ley fo l lowing repet i t i ve s t imulat ion. S t a t i s t i c a l analysis of f a c i l i t a t i o n and potent iat ion has also been conducted. A nerve impulse a r r i v ing at a motor end plate causes Ach to be released at certa in s i tes in d iscrete quanta (Fatt and Katz, 1952). The release of each quanta i s an a l l -or -none event and the p robab i l i t y of any 0 + one unit responding to a nerve impulse can be reduced by lowering the Ca 2+ and/or ra is ing the Mg concentration in the physio logica l medium (del C a s t i l l o and Katz, 1953; del C a s t i l l o and Engbaek, 1954; del C a s t i l l o and Katz, 1954b). Under these condi t ions, the quantal nature of the neuromuscular transmission process can be more e f f ec t i ve l y demonstrated (del C a s t i l l o and Katz, 1953). del C a s t i l l o and Katz (1953, 1954c) found (using i n t r a c e l l u l a r recording of the miniature end-plate potent ia ls [mepps]) that PTP or paired-pulse f a c i l i t a t i o n of the epp was mainly due to a s t a t i s t i c a l recruitment of quantal components at each neuromuscular junc t ion . In s t a t i s t i c a l terms, del C a s t i l l o and Katz (1954c) stated that i f each synapse contains a population of n uni ts whose mean probab i l i t y of responding is expressed by the term p, then the average number of quanta responding to one impulse, or m, can be expressed as: m = n x p The probab i l i t y of any quantal unit responding to a nerve impulse appeared to be increased by a preceeding impulse (or impulses). Martin (1955) confirmed the f indings of del C a s t i l l o and Katz (1954b) using larger values of m. L i l ey (1955 ) also f e l t that the post -act ivat ion potent iat ion of an epp of low quantal content was a resul t of recruitment of more quanta and that the amplitude and duration of th is ef fect were determined by the number of condit ioning impulses. L i l e y (1956) fur ther observed a post -ac t iva t ion - 20 -increase in the frequency of the mepps whose time course para l le led that of the post-act ivat ion potent iat ion of the epps. Hubbard and Schmidt (1962) and Hubbard (1963) found that both f a c i l i t a t i o n af ter one or a few impulses ("primary potent iat ion") and PTP ("secondary potent iat ion") were caused by an increased release of Ach from nerve terminals. Hubbard (1963) and Braun et a l . (1966) stated that th is increased release could be at t r ibuted to a "mobi l i za t ion" of t ransmit ter , presumably leading to an increase in the amount of avai lab le t ransmit ter . However, Hubbard et a l . (1971) provided evidence showing that f a c i l i t a t i o n and PTP are so le ly a t t r ibutab le to changes in p instead of to an increase in n. Magleby (1973a,b) agreed that changes in p were responsible for the increase in transmitter re lease, but went fur ther to suggest that f a c i l i t a t i o n and potent iat ion represented increases in two independent factors which act j o i n t l y to increase the probab i l i t y of t ransmit ter re lease. Augmentation at the neuromuscular junct ion was also found to be a resul t of an increase in the number of quanta of t ransmitter released from the nerve terminal (Magleby and Zengel, 1976a). The d i f f e ren t i a l e f fects of successive condi t ion ing- test ing t r i a l s on augmentation and potent iat ion suggest that at least some of the factors involved in increasing t ransmit ter release by these processes may be d i f ferent for the two processes (Magleby and Zengel, 1976b). Studies in other systems also seem to indicate a presynaptic locus for the d i f ferent forms of STP. In the sympathetic gangl ion, Larrabee and Bronk (1947) found that potent iat ion was not caused by a long-term a l te ra t ion in the " i r r i t a b i l i t y " of the ganglion c e l l s . A f ter repet i t i ve e l e c t r i c a l st imulat ion of e i ther the preganglionic or the postganglionic nerve, the discharge of the ganglion c e l l s in response to Ach appl icat ion was reduced, rather than augmented, at a time when prolonged f a c i l i t a t i o n can be - 21 -demonstrated by the appropriate tes ts . Job and Lundberg1s (1953) study on the sympathetic ganglion of the cat also favoured a presynaptic locus for f a c i l i t a t i o n . In the c i l i a r y ganglion of the ch ick , Martin and P i l a r (1964a) observed an increase in the mepp frequency fo l lowing a b r ie f tetanus applied to the presynaptic nerve. F a c i l i t a t i o n , augmentation and potent iat ion observed at the superior cerv ica l ganglion of the rabbit were also found to be a resul t of increased transmit ter re lease, although the mechanisms underlying these increases were not speculated upon (Zengel et a l . , 1980). In the spinal cord, Lloyd (1949) suggested that PTP could be explained in terms of a hyperpolar izat ion of the afferent presynaptic structures which was ref lected by a pos i t ive a f te r -po ten t ia l . Indeed, there i s considerable evidence re la t ing PTP in the spinal cord with the pos i t i ve a f te r -po ten t ia l in afferent presynaptic st ructures. In the bu l l f rog splanchnic nerve, Gasser (1935) and Richards and Gasser (1935) observed a decreased e x c i t a b i l i t y associated with the pos i t i ve a f ter -potent ia l while the s ize of the response was supernormal. In the saphenous or the phrenic nerve of the cat , Gasser and Grundfest (1936) also observed an associat ion between a subnormality in threshold and the pos i t ive a f t e r -po ten t i a l . With increasing te tan iza t ion , there was an increased in amplitude and then in duration of the po ten t ia l . Woolsey and Larrabee (1940) found that te tan izat ion of a spec i f i c dorsal spinal root resulted in a prolonged p o s i t i v i t y in the tetanized but not the untetanized adjacent roots. This prolonged p o s i t i v i t y lasted for more than 1 min and increased in amplitude with increases in frequency and duration of tetanus. Furthermore, Graham (1942) observed that in frog s c i a t i c or cat saphenous nerve, anodal po la r i za t ion of the nerve caused an increase in the spike height of the action po ten t ia l . S i m i l a r l y , del C a s t i l l o and Katz (1954d) observed an increase in the amplitude of the - 22 -epp during weak or moderate anodic po lar iza t ion of frog motor nerve endings. The pos t - te tan ica l l y hyperpolarized nerves of the a f ferents , according to Lloyd (1949), would respond to afferent impulses with an "enhanced in tens i ty " that i s proportional to the potent ia t ion. Like L loyd, Eccles and Ra i l (1951) observed a potent iat ion which lasted "some minutes" a f ter tetanus (90 vol leys at 300 Hz). These authors, however, reported a decrease in the amplitude of the presynaptic spike with the potent iat ion that was observed af ter less than 300 vo l leys . Eccles and Ra i l (1951) stated that only a f ter very prolonged tetani was there an increase in the s ize of the presynaptic spike associated with PTP as reported by Lloyd (1949). Although Eccles and Ra i l (1951) agreed with Lloyd (1949) that whatever underlying change(s) causing the potent iat ion i s probably presynaptic in o r i g i n , these authors maintained that PTP can occur with or without being associated with an increased presynaptic sp ike. Eccles and Ra i l (1951) raised the p o s s i b i l i t y that repet i t i ve st imulat ion might a l t e r the spat ia l re la t ionship of the synaptic knobs to the post-synaptic membrane temporarily and thus lead to potent ia t ion. - In 1958, Wall and Johnson re-examined the f indings of Lloyd (1949) and tested the e x c i t a b i l i t y changes in afferent f ib res associated with PTP of a monosynaptic ref lex by antidromic s t imulat ion. A decrease in the e x c i t a b i l i t y of the afferent f i b re was indeed observed to para l le l the increase in the monosynaptic re f l ex . When the nerves to the two heads of the gastrocnemius muscle were separated, te tan izat ion of the nerve from one head produced no change in the s ize of the re f lex evoked by st imulat ing the nerve from the other head (Wall and Johnson, 1958), strong evidence suggesting a presynaptic locus for PTP. These authors, however, observed a delayed onset of the f u l l potent iat ion of the monosynaptic ref lex whereas the decrease in e x c i t a b i l i t y of the f ib res reached a maximum immediately - 23 -af ter the end of the tetanus. They at t r ibuted th is delay also to presynaptic events, namely a hyperpolar izat ion of the presynaptic f ib res resu l t ing in a possible anodal block in some of the branches (Wall and Johnson, 1958). Using i n t r ace l l u l a r recording techniques, Eccles and Krnjevic (1959a,b,c) reported more evidence suggesting a presynaptic locus fo r PTP in the spinal cord. Eccles and Krnjevic (1959a,b,c) reported that a tetanus (400 Hz for 10 sec) usual ly led to a phase of hyperpolar izat ion of the membrane rest ing potent ia l that can las t fo r up to 50 sec. This post- tetanic hyperpolarizat ion of the afferent f i b res was associated with an increase in spike height and duration (Eccles and Krn jev ic , 1959a, c ) . Based on evidence provided by del C a s t i l l o and Katz (19 54d) at the frog neuromuscular junct ion and by Hariwara and Tasaki (1958) at the squid giant synapse, Eccles and Krnjevic (1959c) suggested that a larger spike may release larger amounts of the synaptic t ransmit ter fo r a given nerve impulse. As was mentioned before, del C a s t i l l o and Katz (1954d) reported an increase in the epp amplitude during anodic po la r iza t ion of the nerve. Hagiwara and Tasaki (1958) observed that small changes in the presynaptic spike can lead to large changes in the postsynaptic po tent ia ls . Eccles and Krnjevic (1959c) suggested that larger presynaptic spikes might be at least p a r t i a l l y responsible for PTP in the spinal cord. In other systems, there had been s im i l a r attempts to cor re la te potent ia l changes in the presynaptic (or prejunct ional) nerve f i b re with potent iat ion of the postsynaptic (or post junct ional) response. In the mammalian neuromuscular junct ion, Hubbard and W i l l i s (1962) reported large and progressive increases in the amplitude of the epp produced by hyperpolar iz ing currents applied very close to the end-plate. They raised the p o s s i b i l i t y that instead of causing the release of t ransmit ter in - 24 -greater amounts as suggested by Eccles and Krnjevic (1959c), the hyperpolarizing current might be increasing the amount of t ransmitter avai lable for release by nerve impulses. Hubbard and Schmidt (1962, 1963) correlated the ex t race l l u la r epp and the diphasic (posi t ive-negat ive) spike potent ial preceding i t . They found that a f te r repe t i t i ve s t imula t ion, both the epp and the diphasic spike were increased in amplitudes. Moreover, both the epp and the diphasic spike returned to control s ize with a s im i la r time course. Paired-pulse f a c i l i t a t i o n was, however, associated with a decrease of the spike potent ia l amplitude (Hubbard and Schmidt, 1963). It was concluded that only a f ter repet i t i ve st imulat ion would there be an increased presynaptic spike amplitude due to hyperpolar izat ion of nerve terminals. It was also suggested that f a c i l i t a t i o n of the epp fo l lowing a s ing le antecedent pulse was brought about by a d i f fe rent mechanism (Hubbard and Schmidt, 196 2, 196 3). Contrary to the f indings of Hubbard and Schmidt (1962, 1963) in the mammalian neuromuscular junct ion, Takeuchi and Takeuchi (1962) reported that paired-pulse f a c i l i t a t i o n in the squid giant synapse (recorded i n t r a c e l l u l a r l y ) was accompanied by an increase in the amplitude of the presynaptic action po ten t ia l . Changes in the synaptic current caused by 2+ 2+ increasing or decreasing the Ca or Mg concentration was not accompanied by any appreciable changes in the presynaptic action potent ia l (Takeuchi and Takeuchi, 1962), ind i rec t evidence suggesting that t ransmit ter release may not be determined only by e l e c t r i c a l changes in the presynaptic axon but also through some other intermediate process(es). In the avian c i l i a r y gangl ion, Martin and P i l a r (1964b) reported that both f a c i l i t a t i o n and PTP can occur without any change in the presynaptic spike or in the rest ing membrane potent ia l of the nerve terminal . . - 25 -Transient changes in the concentrations of various ions have also been postulated to be responsible for the d i f ferent components of STP. In the squid giant axon, Baker et a l . (1967) demonstrated that ra is ing internal +' + Na or lowering external Na concentration increased the in f lux of 2+ Ca through the membrane. Banks et a l . (196 5) observed that the secret ion of catecholamines by ' the adrenal medulla in response to carbamylcholine and elevated K concentration was enhanced when the internal Na + concentration was high. Birks and Cohen (196 5) reported that a frog neuromuscular preparation treated with the cardiac glycoside digoxin showed a progressive increase in the amplitude of the epp. The increased internal Na + concentration presumably led to an increase in internal f ree Ca because of the competition between these ions for binding s i tes at the nerve terminal membrane (Birks and Cohen, 196 5). The increase in the epp amplitude was due to an increase in transmitter release which was in 2 + turn a resul t of the increase in the amount of in ternal f ree Ca (Birks and Cohen, 196 5). Sherman and Atwood (1971) reported a r e l a t i v e l y long-term f a c i l i t a t i o n in the c ray f i sh neuromuscular junct ion a f te r a 20 to 30 min period of st imulat ion at 5 to 10 Hz. Addit ion of ouabain to the normal bathing solut ion was found to enhance the rate and magnitude of th is "long-term f a c i l i t a t i o n " while lowering NaCl concentration in the medium v i r t u a l l y el iminated i t s occurence. Sherman and Atwood (1971) suggested that th is "long-term f a c i l i t a t i o n " was a resul t of an accumulation of Na + within the nerve terminals - ouabain increased the internal Na + concentration while lowering ex t race l l u l a r Na + concentration reduced the entry of Na + ions into the nerve terminal during spike a c t i v i t y . Gage and Hubbard (1966),' on the other hand, showed that procedures which presumably cause Na + accumulation in nerve terminals e i ther blocked or reduced the magnitude of PTP. S i m i l a r l y , Weinreich (1971) showed that PTP can be - 26 -+ obtained in a Na - f ree medium or in the presence of tetrodotoxin (TTX), where t ransmit ter release evoked by e lec t ro ton ic depolar izat ion of the motor nerve terminal was potentiated subsequent to presynaptic st imulat ion by a t r a i n of depolar iz ing pulses. More recent ly , there has been evidence suggesting that the post- tetanic hyperpolar izat ion thought to be responsible fo r PTP was (at least par t ly ) a resu l t of an increased ac t i v i t y of the sodium pump (Ri tch ie and Straub, 197 5; McDougal and Osborn, 1976). The various components of STP have also been suggested to be a resu l t of changes in the external K + concentrat ion. Brown and Feldberg (1936) demonstrated that the administrat ion of excess KC1 into the perfusing medium enhanced paired-pulse f a c i l i t a t i o n at the superior cerv ica l ganglion of the cat . Feldberg and Vart iainen (1934) found that small doses of KC1 favor the transmission of nerve impulses at the sympathetic ganglion. Rosenblueth's (1950) opinion was that K + was the only possible agent involved with transmission that was stable enough to explain the r e l a t i ve l y long l as t i ng changes associated with PTP. At the mammalian neuromuscular junct ion, L i l e y and North (1953) found that cer ta in of the post- tetanic ef fects could be reproduced by increasing the external K + concentrat ion. They explained that a c t i v i t y resulted in a loss of K + from the nerve terminals and thus a reduction in the K + concentration gradient across the nerve membrane. According to these invest igators , t h i s might have had an ef fect on the release of Ach that was s im i la r to the e f fec t when the K + concentration in the perfusing medium was increased. In the hippocampal s l i c e preparat ion, Creager et a l . (1980) suggested that an increase in the ex t race l l u l a r concentration of K might be one of the underlying factors of frequency + f a c i l i t a t i o n . An increase in the K concentration in the perfusing medium produced ef fec ts mimicking those of frequency f a c i l i t a t i o n . - 27 -There have also been evidence which argued against the involvement of K + in PTP. Feng et a l . (1939) reported that PTP decayed more rapid ly and to a greater extent in the presence of excess K + . Feng and Li (1941) argued that whatever s i m i l a r i t i e s existed between the post- te tanic effects and the K +- induced ef fects were rea l l y quite s u p e r f i c i a l . They added that a high excess of K + at the neuromuscular junct ion would l i k e l y be associated with some depression. Takeuchi and Takeuchi (1961) found that the increase in the frequency of miniature discharge caused by elevated-K + medium could be markedly decreased by the passage of a hyperpolarizing current through the muscle membrane at the end-plate region. They suggested that the post- tetanic increase in discharge frequency (as observed by L i ley [1955]) might be decreased by the passage of a hyperpol a r iz ing current i f the ef fect was mediated by increased K concentration around nerve terminals. The decay of the post- te tanic increase in mepp frequency was, however, not changed even when the membrane was hyperpolarized by about 50 mV (Takeuchi and Takeuchi, 1961). Like Feng et a l . (1939), Gage and Hubbard (1966) found that exposure of a neuromuscular preparation to an el.evated-K + medium resulted in reduced PTP ( in both the extent and duration of increase of epp amplitude) and a fas te r decay. L i l ey and North's (1953) hypothesis that PTP was due to a depletion of i n t r a c e l l u l a r K + was tested by exposing the preparation to K + - f ree medium, which abolished PTP. These f indings were thought to be inconsistent with the hypothesis that PTP was due to a reduction of K + concentration in nerve terminals (Takeuchi and Takeuchi, 1961, Gage and Hubbard, 1966). Calcium is the ion that has most often been implicated to play a role in STP. At the neuromuscular junct ion, del C a s t i l l o and Katz (1954a) stated 2+ ?+ that Ca and Mg inf luence in opposite manners the release of Ach by a motor nerve impulse. They explained that " . . . t h e nerve impulse acts in the - 23 -f i r s t instance by releasing such ca r r i e r molecules (X') from an inact ive calcium compound (CaX) giving r ise to an epp which is a s t a t i s t i c a l fus ion of miniature potent ia ls" (del C a s t i l l o and Katz , 1954a). Takeuchi and Takeuchi (1962) agreed that, the release of t ransmit ter may be p a r t i a l l y 2+ determined by the combination of Ca with some ca r r i e r molecule. Katz and Miledi (1967) observed f a c i l i t a t i o n of t ransmit ter release i f a b r i e f 2+ iontophoretic appl icat ion of Ca was given before a depolar iz ing pulse was applied to the motor nerve ending. Dodge and Rahamimoff (1967) observed 2+ a highly non- l inear re la t ionship between the Ca concentration in the medium and the epp amplitude. They suggested that the number of packets of Ach released is proport ional to the fourth power of the concentration of 2+ CaX, that i s , a co-operative action of about four Ca ions is required for the release of one quantal packet of t ransmit ter by the nerve impulse. Based on some of the above f ind ings , i t has been suggested that PTP and 2+ f a c i l i t a t i o n are due to some change in residual Ca at a membrane s i t e which is important fo r the potent iat ion of t ransmit ter release (Gage and Hubbard, 1956; Rahamimoff, 1968). Rahamimoff (1968) showed that C a 2 + affects both the magnitude and the time course of paired-pulse f a c i l i t a t i o n . Katz and Mi ledi (1968) proposed the hypothesis that when two pulses are given within a short i n t e r va l , the degree of f a c i l i t a t i o n of t ransmitter release depends on the accumulation of "act ive calcium" a f te r 2+ the f i r s t pulse, which in turn depend on the decl ine of the in f lux of Ca af ter the f i r s t pulse and the removal of the "act ive calcium" from the c r i t i c a l s i tes in the nerve terminal . When the in terva l between the two pulses i s so short that the second one i s given before the rate of in f lux of 2+ Ca returns to basel ine, t ransmit ter released a f ter the second pulse w i l l be greater than that released a f ter the f i r s t because of an increased concentration of "act ive calcium" during the second pulse. This concept i s - 29 -usual ly referred to as the residual CaX hypothesis (Younkin, 1974). S im i l a r l y , PTP has been suggested to be associated with an increased 2+ i n t r a c e l l u l a r accumulation of Ca during but not a f ter the high frequency tetanus (Rosenthal, 1969). A very shor t - las t ing PTP was observed when the 2+ Ca concentration in the medium was lowered only during the tetanic st imulat ion whereas PTP las t ing 4 to 5 min was obtained when the tetanus was 2+ given in normal Ca medium. Weinreich (1971) agreed that PTP is at least 2+ par t l y a resul t of Ca accumulation during the tetanus. It was also shown that the magnitude and time course of PTP was dependent upon the 2+ number of tetanic s t i m u l i , presumably because of an increasing Ca accumulation with an increasing number of tetanic s t i m u l i . In support of 2+ the Ca accumulation hypothesis, Stinnakre and Tauc (1973) demonstrated 2+ an increase in Ca in f lux in Aplysia neurones during an evoked t ra in of action potent ia ls using the calcium act ivated photoprotein aequorin. 2+ In ject ion of Ca into Aplysia nerve c e l l s (Meech, 1972) and spinal motoneurones (Krnjevic and L i s iew icz , 1972) have been shown to hyperpolarize these neurones through an increase in K permeabi l i ty . S im i l a r l y , the post- tetanic hyperpolar izat ion in Aplys ia neurones was not observed when the 2+ . . ex t race l l u la r Ca concentration was lowered (Meech, 1974). Furthermore, 2+ + PTP has been corre lated with a slow outward Ca -dependent K current, 2+ ind i rect evidence supporting the residual Ca hypothesis fo r PTP (Kretz 2+ et a l . , 1981). Sastry (1979) demonstrated a Ca -dependent post- tetanic hyperpolar izat ion of primary afferent terminals of cat which was found to be reduced by the appl icat ion of Ca antagonists and enhanced by the Ca 2+ "agonist" Sr . It was also observed that af ter tetanic st imulat ion the absolute refractory period in the afferent terminal regions was prolonged, presumably re f lec t ing an increase in the duration of the action potent ia l during the post- tetanic period (Sastry, 1979). This post- tetanic increase - 30 -in the absolute ref ractory period was also found to be sensi t ive to the 2 + 2 + 2 + various Ca antagonists and the Ca "agonist" Sr (Sastry, 1979). Hubbard et al.(1971) agreed that f a c i l i t a t i o n and PTP have a common underlying cause. These authors, however, added that the rate of removal of 2+ Ca is at least as important a determining factor of the magnitude and 2+ duration of PTP as is the accumulation of Ca during the tetanus. Younkin (1974) found that the quant i tat ive behaviour of f a c i l i t a t i o n can be adequately predicted by the residual CaX hypothesis (Katz and M i l e d i , 1968) when n i s assigned a value of 4 in the re la t ionsh ip : epp = K[CaX] n This above re la t ionsh ip appeared to have been an outgrowth of the independent observations of Dodge and Rahamimoff (1967) that the number of packets of Ach released i s proportional to the fourth power of the concentration of CaX. Erulkar and Rahamimoff (1978) provided evidence suggesting the existence of a second process which causes an increase in transmit ter release during and a f ter the tetanus, possib ly a release of 2+ + Ca from i n t r a c e l l u l a r stores coupled with Na which entered the nerve terminal during the impulse. In the c ray f ish neuromuscular junct ion, Zucker (1974) found that no s ingle value of n adequately predicted the r i se of f a c i l i t a t i o n during the tetanus. Other invest igators have also expressed 2+ doubts about the adequacy of the residual Ca hypothesis in accounting for the quant i tat ive behaviour of f a c i l i t a t i o n af ter one or more nerve impulses (B i t tner and Schatz, 1981; Quastel, 1984), even though some maintained that the hypothesis i s compatible with the f a c i l i t a t i o n of t ransmit ter release (Stockbridge and Hines, 1982). Short-term sens i t i za t i on of the g i l l -wi thdrawal ref lex in Ap l ys ia , a simple form of learn ing, has been extensively invest igated and i s bel ieved to be caused by a f a c i l i t a t i o n of synaptic transmission from the presynaptic - 31 -terminal of the sensory neurones to various target c e l l s (Caste l lucc i and Kandel, 1976; Carew et a l . , 1979). Serotonin is believed to be the putative transmit ter that i s responsible for th is f a c i l i t a t o r y process (Brunel l i et a l . , 1976). K le in and Kandel (1980) provided evidence suggesting that the increase in t ransmit ter is mediated by an increase in the concentration of c y c l i c adenosine monophosphate (cAMP) in the sensory neurones. Serotonin released from modulatory neurones increases the cAMP level in the terminals of the sensory neurones (Klein and Kandel, 1980). This increase in cAMP level d issoc ia tes the regulatory subunit(s) from the ca ta l y t i c subunit of a cAMP-dependent protein k inase, which then phosphorylates a " . . . nove l species of the K + channel protein or a regulatory protein that is associated with i t " (Kandel and Schwartz, 1982). Phosphorylation inact ivates the channel, slows repo lar iza t ion of and therefore broadens the action po ten t ia l , thus allowing greater in f lux of 2+ Ca into the presynaptic terminals, enhancing transmit ter release (Klein and Kandel, 1980). In a l a te r study, the theory was complicated by the 2+ f inding of a component of serotonin-sensi t ive enhancement of Ca accumulation that is not secondary to changes in K + current but is an 2+ i n t r i n s i c a l te ra t ion in Ca handling by the c e l l (Boyle et a l . , 1984). More recent ly, Hochner et a l . (1986a,b) confirmed that the broadening of the action potent ia l was indeed a fac tor causing the f a c i l i t a t i o n of t ransmit ter re lease. 2.6 Long-term potent iat ion In contrast to STP, long-term potent iat ion (LTP) can be defined as a pers is tent , long- las t ing (greater than 30 min) increase in the magnitude of the postsynaptic response to a constant afferent vo l ley subsequent to a br ie f tetanic st imulat ion of the same afferent pathway (B l i ss and L^mo, 1973; Douglas and Goddard, 197 5; Byrne, 1987; Teyler and DiScenna, - 32 -1987). I n i t i a l studies on LTP were performed in vivo using both anaesthetized (B l i ss and L0mo, 1973) and unanaesthetized (B l i ss and Gardner-Medwin, 1973; Douglas and Goddard, 1975) animals. In a l a t e r study, Schwartzkroin and Wester (1975) observed LTP in area CA^ using an in v i t ro hippocampal s l i c e preparat ion. Many subsequent studies were performed using the in v i t ro hippocampal s l i c e preparation where LTP was observed in areas CA^ (Andersen et a l . , 1977; Lynch et a l . , 1977; Buzsaki , 1980), CA3 (Alger and Teyler, 1976) as well as in the dentate gyrus (Alger and Teyler, 1976). Since the ear ly studies on hippocampal LTP (B l i ss and L#mo, 1973; B l i s s and Gardner-Medwin, 1973), long- las t ing increases in synaptic e f f i cacy resu l t ing from previous high-frequency a c t i v i t y have subsequently been observed in other systems, both centra l and peripheral (Dolphin, 1985; Teyler and DiScenna, 1987). LTP has, fo r example, been observed in the neocortex (Lee, 1982; Racine et a l . , 1983; Voronin, 1985) and in deep cerebel lar nuclei (Racine et a l . , 1986). At peripheral synapses, LTP has been observed in the superior cerv ica l ganglion of rat (Brown and McAfee, 1982) as well as in bu l l f rog sympathetic ganglion (Koyano et a l . , 1985). In Ap lys ia , LTP has been observed at the synapses mediating the defensive g i l l and siphon withdrawal ref lex (Walters and Byrne, 1985). Wojtowicz and Atwood (1986) observed a post- tetanic long-term f a c i l i t a t i o n of t ransmit ter release at the c ray f ish neuromuscular junct ion that pers is ts for many hours. Most recent ly , M i l l e r et a l . (1987) reported LTP in the c ray f ish la te ra l giant escape reaction c i r c u i t which shares many propert ies with hippocampal LTP. This survey of the l i t e ra tu re w i l l , however, be mostly l imi ted to LTP of the hippocampus, i t s propert ies and some possible mechanism(s) of ac t ion . - 33 -The enhancement of synaptic e f f icacy during LTP can be characterized by changes in several parameters. There can be an increase in the amplitude of the ex t race l l u l a r l y recorded population exc i ta tory postsynaptic potential (EPSP), ind icat ing an increase in the inward current at the synapse of the tetanized inputs (B l i ss and L^mo, 1973; Andersen and Hvalby, 1986). There can also be an increase in the peak-to-peak amplitude and a reduction in the latency of the population spike (B l i ss and L0mo, 1973), a re f lec t ion of the number of discharging neurones as well as the synchrony with which they f i r e (Andersen et a l . , 1971a). Andersen et a l . (1977) observed a post- tetanic long-term enhancement of the probab i l i t y of discharge of s ingle CA^ neurones and a reduced discharge latency of the neurones to a constant afferent input. Long-term potent ia t ion in the hippocampus has been shown, at least in area CAp to be input spec i f i c - only the inputs that are te tan ica l l y activated show LTP (Andersen et a l . , 1977, 1980; Lynch et a l . , 1977). Moreover, Lynch et a l . (1977) found in area CA-^  that the potentiat ion of one set of af ferents tended to depress the target neurones' response to a second converging but independent set of inputs. In dentate granule c e l l s , i t was shown that perforant path te tanizat ion can depress the synaptic function of converging afferents for periods exceeding 3 hours (Abraham and Goddard, 1983). Analogous resu l ts were observed by Chirwa et a l . (1983), who reported that establ ished LTP in CA^ neurones could be masked by a transient depression induced by a low frequency tetanus. Voronin (1983) suggested that th is depression of the responses in control pathways can be explained by a superimposit ion of generalized post- tetanic depression, possibly a resul t of a post- tetanic decrease in t ransmit ter s e n s i t i v i t y (Lynch et a l . , 1976). In area CA^, on the other hand, heterosynaptic as well as homosynaptic LTP have been reported (Yamamoto and Chujo, 1978; - 3 4 -Misgeld et a l . , 1979). Yamamoto and Chujo (1978) suggested that the heterosynaptic LTP was at least par t ly caused by a reduction in inh ib i to ry inf luences, as was suggested by a decrease in the amplitude of i nh ib i to ry postsynaptic potent ia ls (IPSPs) during LTP (also see Haas and Rose, 1984). Wigstrom and Gustafsson (1983b, c , 1985a) also observed a f a c i l i t a t i o n of LTP induction during blockade of i n h i b i t i o n . Misgeld et a l . (1979), however, observed increases as well as decreases in the IPSP amplitudes during LTP. G r i f f i t h et a l . (1986) also provided evidence that decreased inh ib i t i on plays l i t t l e or no role in LTP observed in CA^ neurones. Hagashima and Yamamoto (1985) d i f fe ren t ia ted between ear ly and la te responses in f i e l d potent ia ls induced in area CA^ by mossy f i b r e s t imulat ion. These authors reported that LTP of the late response (which could be mediated heterosynapt ical ly) was not merely a consequence of potent iat ion of the ear ly response (which could only be e l i c i t e d homosynaptically) but was the resul t of an independent mechanism, poss ib ly an enhanced in teract ion among CA^ neurones. Abraham et a l . (1985) observed homosynaptic as well as heterosynaptic ef fects between two converging but independent perforant path inputs to the dentate granule c e l l s . Tetanizat ion of the la te ra l perforant path resulted in a homosynaptic LTP of the conditioned pathway, a long-term heterosynaptic depression of the responses evoked by the untetanized medial perforant path (as described by Abraham and Goddard in 1983), and a long-term sh i f t to the le f t of the population spike (S) versus f i e l d EPSP (E) curve. This "E-S potent ia t ion" , which r e f l e c t s , according to the authors, an increased coupling between spike, generation and synaptic input, i s presumably a resu l t of an increase in coupling between granule c e l l s (Abraham et a l . , 1985). -35 -Unlike STP (paired-pulse and frequency potent iat ion as well as PTP), which can be induced in low Ca and/or high Mg medium (Dunwiddie and Lynch, 1979), the induction of LTP is widely believed to be a Ca -dependent process (Dunwiddie et a l . , 1978; Dunwiddie and Lynch, 1979; Wigstrom et a l . , 1979). When hippocampal s l i c e s were exposed to 2+ 2+ medium in which Ca was subst i tuted by e i ther Mg (Dunwiddie and Lynch, 1979; Izumi et a l . , 1987) or M n 2 + (Wigstrom et a l . , 1979), a high frequency stimulus del ivered to the synaptic input f a i l e d to induce LTP. 2+ When the same s l i c e s were re-perfused with normal Ca -medium, tetanic st imulat ion of the same input resulted in LTP (Dunwiddie and Lynch, 1979; 2+ Wigstrom et a l . , 1979). On the other hand, when Ca in the medium was 2 + 2 + replaced with Sr , a Ca "agonis t " , LTP very s im i l a r to that observed in a "normal" s l i c e could be readi ly induced by te tan ic st imulat ion of the synaptic input (Wigstrom and Swann, 1980). It has also been shown that 2-chloroadenosine, an adenosine agonist which has been suggested to block 2+ Ca in f lux (Proctor and Dunwiddie, 1983), blocks the induction of LTP (Dolphin, 1983a). Following the induction of LTP in CA^ pyramidal neurones, Baimbridge and M i l l e r (1981) observed an increase in the uptake and retention of ^ C a which para l le led the potent iat ion of the CA^ population spike. Kuhnt 2+ et a l . (1985) also demonstrated an increased Ca content in tetanized as compared to non-tetanized hippocampal s l i c e s . Furthermore, i t was reported 2+ that a t ransient increase in the ex t race l l u l a r Ca concentration (from 2 2+ 2+ to 4 mM; Mg was removed when Ca concentration was elevated to 4 mM) resulted in an " L T P - l i k e " increase of the stratum radiatum evoked CA^ population spike as well as the population EPSP (Turner et a l . , 1982; Reymann et a l . , 1986). Turner et a l . (1982) suggested that calmodulin, a 2+ Ca -binding protein which i s believed to play an important role in - 36 -i n t r ace l l u l a r regulatory functions (Cheung, 1980; Klee et a l . , 1980), i s involved in LTP. Tr i f luoperaz ine as well as pimozide, drugs which are believed to be inh ib i to rs of calmodulin-mediated events, have been shown to block the induction of LTP (Finn et a l . , 1980; Mody et a l . , 1984). Most recent ly, Gamble and Koch (1987) proposed a model for pred ic t ing the 2+ increase in i n t r a c e l l u l a r Ca in response to repet i t i ve synaptic input , 2+ which par t ly depends on the binding of Ca to calmodulin. In 1978, McNaughton et a l . reported that a threshold stimulus in tens i t y during high-frequency act iva t ion was required before LTP could be observed. This threshold in tens i ty suggests presumably that a minimum number of afferent f ib res needs to be act ivated during high frequency s t imulat ion before potent iat ion can occur, since the threshold was considerably greater than the stimulus threshold needed to observe a minimum synaptic response (McNaughton et a l . , 1978). Concurrent te tanizat ion of medial and l a te ra l perforant path f ib res produced potent iat ion at stimulus i n tens i t i es where ident ica l st imulat ion of e i ther pathways was less e f fec t ive in producing LTP (McNaughton, 1978). Based on the c e l l assembly theory postulated by Hebb (1949), McNaughton et a l . (1978) suggested a possible cor re la t ion between the high frequency tetanus and the discharge of the postsynaptic neurones. This "cooperat iv i ty" or " assoc i a t i v i t y " of afferent f ib res was confirmed and further investigated by Wigstrom and Gustafsson (1983a) and Lee (1983), who found that concurrent high frequency act ivat ion of the Schaffer c o l l a t e r a l s and commissural pathways from the CA^ and subicular aspects of the s l i c e produced a greater degree of LTP in the CA^ region than ac t i va t ion of f ib res from the CA^ aspect alone. Aside from the studies discussed above, there have been many other studies demonstrating that a concurrent te tanizat ion of independent, converging "weak" and "strong" inputs resul ts in LTP of the weak input - 37 -whereas tetanizat ion of the weak input alone does not produce LTP (see Teyler and DiScenna, 1987 for review). The crossed entorhinal c o r t i c a l pathway to the dentate gyrus, which did not show LTP when a high frequency condit ioning stimulus was presented, exhibi ted potent iat ion only when concurrent condit ioning st imulat ions were presented to the i p s i l a t e r a l and the contra latera l entorhinal project ions (Levy and Steward, 1979, 1983). Associat ive LTP was also demonstrated with the septal and entorhinal project ions to the dentate gyrus (Robinson and Racine, 1982; Robinson, 1986), as well as with two independent Schaffer co l l a te ra l inputs to CA^ neurones (Barrionuevo and Brown, 1983). Kelso and Brown (1986) observed that associat ive LTP occured when the tetanus to the weak input overlapped the tetanus to the strong input. Winson and Dahl (1986) observed that substant ial LTP can be produced when lower frequency s t imul i were given asynchronously to the medial and la te ra l perforant pathways so that the composite stimulus frequency to the granule c e l l dendrites was high. Less or no potent iat ion was observed when the lower frequency st imulat ion was given synchronously or separately (Winson and Dahl, 1986). In 1986, Sastry et al • observed that LTP as well as STP (with a duration s im i la r to PTP) could be assoc ia te ly induced in CA^ neurones without tetanic st imulat ion of the weak ( test ) input, but when i t was act ivated during e i ther a tetanic st imulat ion of other exc i ta tory (condit ioning) inputs that presumably terminated in the same region or a depolar izat ion of the postsynaptic neurone. S imi la r observations were made by other invest igators (Gustafsson et a l . , 1986; Gustafsson and Wigstrom, 1986; Kelso et a l . , 1986; Wigstrom et a l • , 1986; Wigstrom and Gustafsson, 1986; also see: Larson and Lynch, 1986). Furthermore, Auyeung (1986) and Ito et a l . (1986) found that LTP could be induced when act ivat ion of the stratum radiatum afferents to the CA, neurones was paired with concurrent - 38 -high frequency (antidromic) st imulat ion of the alveus. This was in contrast to the f indings of Lee (1983), who observed no substant ia l ly greater potent iat ion with concurrent tetanic st imulat ion of the stratum radiatum and the alveus as compared to that observed with tetanic st imulat ion of the stratum radiatum only. These authors seem to agree that ac t iva t ion of both the presynaptic terminals and the postsynaptic neurones is important fo r the induction of LTP. These studies also indicate that postsynaptic depolar izat ion seems to be one of the necessary requirements fo r the induction of LTP - the te tanizat ion of the other exci tatory inputs that terminate in the same region as the test input (Gustafsson and Wigstrom, 1986; Sastry et a l . , 1986), te tan izat ion of the alveus (Auyeung, 1986; Ito et a l . , 1986), as wel l as d i rec t i n t r a c e l l u l a r depolar izat ion of the postsynaptic neurone (Kelso et a l . , 1986; Sastry et a l . , 1986 ), could a l l induce a c r i t i c a l level of depolar izat ion at the postsynaptic s i t e s . In support of the above idea, Kelso et a l . (1986) found that no LTP could be i. ————— induced when high frequency st imulat ion was given while the soma of the postsynaptic neurone was being voltage clamped at a hyperpolarized l e v e l . Furthermore, i t has been observed that the induction of LTP can be blocked by i n t r ace l l u l a r in jec t ion of hyperpolar iz ing current in the postsynaptic neurone during the high frequency stimulus (Malinow and M i l l e r , 1986). Wigstrom and Gustafsson (1986) suggested a role fo r the N-methyl-D-aspartate (NMDA) receptors in the associat ive induction of LTP. This was based on the hypothesis (Co l l ingr idge, 1985; Col l ingr idge and B l i s s , 1987) that NMDA receptors are responsible for the induction of LTP while the kainate and/or quisqualate subtypes of glutamate receptors are responsible for generating the EPSP responsible for exci tatory synaptic transmission. Tetanic st imulat ion of the exc i ta tory afferents leads to a 2+ . temporary unblocking of the NMDA receptors by Mg (a resul t of the c e l l s - 39 -becoming more depolarized during tetanic st imulat ion) and allows a transient opening of the NMDA receptor-mediated channels; the NMDA channels allow a further depolar izat ion of the c e l l , i n i t i a t i n g the processes that lead to a long-term enhancement of synaptic responses (Col 1ingridge, 1985). Wigstrom and Gustafsson (1986) reported that 2-amino-5-phosphonovalerate (APV), a drug that is thought to be an NMDA receptor blocker (Col 1 ingridge et a l • , 1983b), blocked the potent iat ion induced by conjunction between s ingle vol leys and br ie f tetani to a separate input or depolar iz ing current pulses to the postsynaptic neurone. Although there seems to have been some agreement towards both a pre- as well as postsynaptic involvement in the induction of LTP, there is s t i l l controversy as to the locus of modulation as well as the mechanism(s) underlying these modulatory processes that is involved in the maintenance of LTP. There have been much evidence supporting both presynaptic and postsynaptic involvement in LTP (Goh and Sast ry , 1985a; Andersen and Hvalby, 1986), some of which w i l l be presented and discussed here. Results from many e lect rophys io log ica l as well as biochemical studies have suggested a presynaptic involvement in the maintenance of LTP. In the c ray f ish opener neuromuscular junc t ion , quantal analys is has demonstrated that LTP can be en t i re l y accounted for by an increase in the average number of quanta of t ransmi t ter released by the presynaptic terminal (Baxter et a l . , 1985). In the superior cerv ica l ganglion of the ra t , LTP has been shown to be due to an increase in the amount of evoked Ach released. In Ap lys ia , presynaptic f a c i l i t a t i o n leading to increased transmitter release has been shown to be responsible for sens i t i za t i on of sensory neurone terminals (Kandel and Schwartz, 1982). In the hippocampus, quantal analys is of exci tatory postsynaptic potent ia ls has always been performed in the synapse between the granule c e l l mossy f ib res and the pyramidal neurones of - 4 0 -the CA^ region (Brown et a l . , 1979; Yamamoto, 1982; Voronin, 1983; Brown and Johnston, 1983). Large (about 2 mV) miniature EPSPs, which f a c i l i t a t e quantal ana lys is , have been observed in the CA^ neurones, mainly because of the high input resistance of these neurones and the close proximity of the mossy f i b re synapse to the pyramidal c e l l soma (Brown et a l . , 1979). Voronin (1983) demonstrated an increase in the number of quanta of released transmit ter but no s ign i f i can t change in the quantal s ize during LTP. Voronin's quantal analysis have, however, been c r i t i c i z e d for i t s high noise l e v e l , an inadequate ca lcu la t ion of the noise cont r ibu t ion , and an apparently great ly var iab le number of f ib res involved (Andersen and Hvalby, 1985). Recently, miniature EPSPs have been observed in CA^ neurones (Chirwa and Sastry , 1987b; Malenka et a l . , 1987). Chirwa and Sastry (1987b) reported that LTP induced in CA^ neurones by te tan ic st imulat ion of stratum radiatum is associated with an increase in the number of miniature EPSPs. There is also more d i rect evidence suggesting an increased release of neurotransmitter during LTP. Glutamate and aspartate have both been suggested to be possible exci tatory neurotransmitters in hippocampal afferents (Nadler et a l . , 1976; di Lauro et a l . , 1981). Skrede and Malthe-S^frenssen (1981) preloaded hippocampal s l i ces with [ H]D-aspartate and studied i t s release before and af ter the induction of LTP. These authors observed an increased rest ing and evoked release of 3 [ H]D-aspartate fol lowing tetanic st imulat ion of the Schaffer c o l l a t e r a l s (Skrede and Malthe-S^frenssen, 1981). In a l a te r study, Dolphin et a l . (1982) demonstrated an increase in the release of [ Hjglutamate (converted 3 from preloaded [ H]glutamine by the enzyme glutaminase) from perforant path terminals fol lowing the induction of LTP in the pathway. Release of 3 preloaded L-[ H]glutamate has also been shown to increase in s l i c e s - 4 1 -prepared a f te r LTP has been induced in the dentate gyrus (Lynch et a l . , 1985). More recent ly , B l i s s et a l . (1986) demonstrated a cor re la t ion between LTP induced in the dentate gyrus and an increased level of aspartate as well as glutamate in the physio logical medium perfusing the molecular layer of the dentate gyrus by means of a push-pull cannula. In support of an increased level of neurotransmitter released during LTP, Agoston and 45 Kuhnt (1986) demonstrated an increased Ca uptake of r e l a t i ve l y pure synaptosomes - pinched off nerve terminals - from the CA^ area of hippocampal s l i c e s a f te r LTP induction as compared to synaptosomes prepared from control s l i c e s in which LTP was not induced. Furthermore, e lectron micrograph studies provided evidence suggesting a migration of synaptic ves ic les towards the synaptic act ive zone during LTP (Applegate et a l . , 1987). The f rac t i on of local synaptic ves ic les (ves ic les located close to the act ive zones) attached to the act ive zone membrane was found to have s i gn i f i can t l y increased in t issues where LTP have been induced (Applegate et a l . , 1987). Evidence supporting a presynaptic role for the maintenance of LTP was also provided by Sastry (1982), who observed with LTP a concomitant decrease in the presynaptic terminal e x c i t a b i l i t y . In analogous to the post - te tan ic hyperpolarizat ion that has been observed in the primary afferent terminals of the spinal cord, Sastry (1982) suggested that th is decrease in presynaptic terminal e x c i t a b i l i t y may re f l ec t a hyperpolar izat ion of the terminal membrane po ten t ia l , which may in turn lead to an increased release of evoked t ransmit ter (Wall and Johnson, 1958). A s im i la r decrease in presynaptic terminal e x c i t a b i l i t y was also observed to pa ra l l e l the associat ive STP as well as LTP induced by pai r ing act ivat ion of a weak input to the CAj neurones with a tetanic st imulat ion of other exci tatory inputs (Sastry et a l . , 1986 ). Perhaps a cor re la t ion can also be drawn to an - 42 -ea r l i e r study by Sastry et a l . (1985), who demonstrated that a transient increase in the f i r i n g rate of CA^ neurones produced by the iontophoretic appl icat ion of DL-homocysteate (DLH) on the pyramidal c e l l soma resulted in a prolonged increase of the threshold fo r antidromic ac t iva t ion in the CA^ area. A loca l ized appl icat ion of glutamate in area CA^ induced LTP in CAj neurones evoked by Schaffer co l l a te ra l st imulat ion while the same appl icat ion of glutamate in area CA-^  resulted in a prolonged depression of the response (Goh and Sastry, 1983). An increase in the a c t i v i t y of the Schaffer co l l a te ra l s from CA^ neurones (caused by glutamate appl icat ion in area CA^) thus induces LTP in CA^ neurones while an increased postsynaptic ac t iva t ion (appl icat ion of glutamate in area CA^) seems to resul t in depression. A decrease in the uptake of neurotransmitter has also been suggested to be par t ly responsible for the maintenance of hippocampal LTP (Wieraszko, 1983), although the phys io log ica l s ign i f icance of t h i s , uptake change has been questioned (Goh and Sast ry , 1984; Goh et al • , 1986 ). An a l ternat ive p o s s i b i l i t y causing increased transmit ter release has been put forward by Benjamin et a l . (1985), who suggested that an altered membrane f l u i d i t y resul t ing from an increased methylation of presynaptic components may account for LTP. Recent ly, Andersen and Hvalby (1986) suggested that synapsin I, a protein that has been suggested to a l t e r the mobi l i ty of synaptic ves ic les by i t s degree of phosphorylation (deCamil l i and Greengard, 1986), may play a role in LTP. Further studies appear to be required before such a corre la t ion can be confirmed. There has also been some evidence for the existence of presynaptic autoreceptors (McBean and Roberts, 1981), in which a change in number or a f f i n i t y may play a role in LTP. In a recent current- and voltage-clamp study of the mossy f ibre-CA^ system of the guinea pig hippocampal s l i c e , no change in input res is tance, membrane time - 43 -constant, postsynaptic e x c i t a b i l i t y , or the ionic s e l e c t i v i t y property of subsynaptic receptors that mediate the EPSP, was found to be associated with LTP (Barrionuevo et a l . , 1985). An increase in the synaptic conductance was, however, observed during LTP (Barrionuevo et a l . , - 1986). The underlying cause(s) of th is conductance change, which could involve a presynaptic or postsynaptic locus, was not fur ther invest igated. The cooperat iv i ty among input f ib res that seems to be required fo r the induction of LTP has often been interpreted to indicate a postsynaptic locus fo r LTP (McNaughton, 1978; Wigstrom and Gustafsson, 1983a). However, Goh and Sastry (1985b) demonstrated an in teract ion among presynaptic terminals , possibly through an accumulation of ex t race l l u la r K + or secondary to the increased transmit ter released during high frequency a c t i v i t y . An increased ex t race l l u la r K released during high frequency ac t i v i t y can act on nearby presynaptic terminals to reinforce the ef fects of the tetanus in causing LTP (Goh and Sastry, 1985a). Increased amount of t ransmitter released during the tetanus can also depolarize other presynaptic terminals to increase the 2+ voltage-dependent inward Ca current (Andersen and Hvalby, 1986). 2+ An in f lux of Ca across the granule or pyramidal c e l l membranes have been suggested to be the i n i t i a l step which resul ts in whatever secondary changes that leads to LTP (Eccles, 1983; Kudo et a l . , 1987; Smith, 1987). It was, however, shown that a low frequency (1 to 20 Hz) te tan ic st imulat ion, which favours the observation of depression rather than LTP, 2+ produces a marked increase in the i n t r a c e l l u l a r Ca leve ls (Chirwa et a l . , 1983). A high frequency st imulat ion (100 to 500 Hz), which favours the observation of LTP, resu l ts in no substant ial increase in the 2+ i n t r a c e l l u l a r levels of Ca (Chirwa et a l . , 1983). Moreover, i t has been 2+ shown that verapamil as well as other organic Ca -channel blockers are inef fec t ive in blocking the induction of LTP (Sastry et a l . , 1984a; Taube - 44 -and Schwartzkroin, 1986). Verapamil has, however, been observed to counteract the depression induced by low frequency tetanic st imulat ion (Sastry et a l . , 1984a). It was therefore suggested that a postsynaptic 2+ neuronal accumulation of Ca due to in f lux resul ts in generalized depression rather than LTP (Sastry et a l . , 1984a). It i s p laus ib le that an 2+ in f lux of Ca into hippocampal CA^ neurones leads to a reduction in the responsiveness of these neurones to the neurotransmitter, poss ib ly through receptor desensi t izat ion (Murali Mohan and Sastry , 1985). It i s poss ib le , 2+ however, that an i n t r a c e l l u l a r mobi l izat ion of Ca might be required to cause the secondary change(s) leading to LTP. In 1980, Baudry and Lynch (1980a) proposed the hypothesis that tetanic 2+ st imulat ion resul ts in a Ca -mediated act ivat ion of a membrane-associated protease which in turn exposes, or unmasks, addi t ional glutamate receptors (also see: Baudry and Lynch, 1980b, 1981a,b). The level of Na+-independent binding was presumed to re f lec t the binding of glutamate to subsynaptic receptors while the level of Na+-dependent binding was thought to be an ind ica t ion of h igh -a f f i n i t y glutamate uptake in the hippocampus (Baudry and Lynch, 1981a,b). Using synaptic membrane prepared from s l i ces which have been t e tan i ca l l y st imulated, Baudry et a l . (1980) reported an increase in the maximal number of Na -independent h igh-a f f in i t y binding s i tes for [ H]glutamate without any changes in the i r a f f i n i t y . This increase in the number of glutamate binding s i t es has also 2+ been observed af ter exposure of hippocampal membranes to elevated Ca (Baudry and Lynch, 1979). Ethyleneglycol-bis-(e-aminoethyl e ther) -N, N, N 1 , N ' - te t raacet ic acid (EGTA), as well as the proteinase i nh ib i t o r leupeptin 2+ reportedly blocked th i s Ca -induced increase in glutamate binding s i t es (Baudry et a l . , 1983). A more " s p e c i f i c " hypothesis for LTP was put forward by Lynch and Baudry in 1984. Namely, high frequency a c t i v i t y causes a - 45 -2+ t ransient elevat ion of Ca in the dendr i t ic spines which then act ivates a 2+ / membrane-associated ca lpa in , a form of Ca -act ivated proteinase (Lynch and Baudry, 1984). Activated calpain breaks up a loca l i zed portion of a fodr in network ( fodr in is suggested by the authors to par t ic ipate in the "capping" of c e l l surface receptors) , causing s t ructura l and chemical changes in the region of the subsynaptic membrane and exposing prev iously occluded glutamate receptors (Lynch and Baudry, 1984). The hypothesis proposed by Baudry and Lynch (1980; Lynch and Baudry, 1984) has subsequently been challenged by other invest igators . Sastry and Goh (1984) reported that an increase in the number of glutamate binding s i tes is not necessary for LTP in the hippocampus. The invest igators did observe a s l i gh t increase in the number of Na+-independent glutamate binding s i tes with low frequency tetanic s t imulat ion, which favours depression rather than LTP (Sastry and Goh, 1984; Goh et a l . , 1986 ). Goh et a l . (1986) suggested that the increase in glutamate binding observed by Baudry et a l . (1980) might have been associated with depression and not potent ia t ion. Verapamil, which counteracted the depression induced by low frequency tetanic st imulat ion as mentioned e a r l i e r (Sastry et a l . , 1984a), was observed to also counteract the increase in glutamate binding associated with the depression (Sastry and Goh, 1984). S imi la r f indings were l a t e r reported in a study by Lynch et a l . (1985). Furthermore, e i the r ex t race l l u la r appl icat ion or i n t r a c e l l u l a r in jec t ion of leupept in, which has been suggested to be an i nh ib i t o r of the a c t i v i t y of calpain (Lynch and Baudry, 1984), has been shown not to in ter fere with the induction of LTP in hippocampal s l i ces (Sastry, 1985). Following tetanic s t imulat ion, long- las t ing morphological changes have been observed in the dendr i t ic spines of the dentate granule c e l l s as wel l as that of the pyramidal neurones in area CA, (Fifkova and van Harreveld, - 46. -1977; Lee et a l . , 1980; Applegate et a l . , 1987). Fi fkova and van Harreveld (1977 ) and Applegate et a l . (1987) reported an increase in the area of the dendr i t ic spines fol lowing tetanic st imulat ion of the af ferents. Theoretical analysis using mathematical as well as computer generated models of hippocampal neurones and the i r dendr i t ic spines also suggest a role for changes in dendr i t ic morphology in modulating synaptic e f f i cacy (Horwitz, 1981; Brown et a l . , 1984). Lee et a l . (1980) did not observe any large changes in the s ize or shape of the dendr i t ic spine. What these authors did observe was a decrease in the v a r i a b i l i t y of the spine population, which they interpreted as e i ther a change in some population of spines with atypical shapes so that they assume a more "normal" configurat ion or an a l te ra t ion in a s i gn i f i can t percentage of the spines such that the i r shape becomes more uniform (Lee et a l . , 1980). Lee et a l . (1980) also pointed out that the study of Fi fkova and van Harreveld (1977) did not make use of e lect rophysio log ica l techniques to ascertain that LTP had indeed been establ ished in the regions that were selected for analys is . An increase in the density of dendr i t ic shaft synapses was also observed af ter the induction of LTP (Lee et a l . , 1980). Desmond and Levy (1983, 1986a,b) did not observe any increase in the density of shaft synapses with the induction of LTP, but an increase in the density of concave spine synapses which reportedly provides a . la rger postsynaptic area leading to an enhanced e f f i cacy for synaptic transmission. As was mentioned e a r l i e r , NMDA receptors have been implicated in the induction of LTP (Col 1ingridge, 1985; Wigstrom and Gustafsson, 1986; Col l ingr idge and B l i s s , 1987). 2-amino-7-phosphonoheptanoate (±AP7) and APV, presumably NMDA receptor antagonists, have been reported to block the induction of LTP in area Ck^ of hippocampal s l i ces as well as in the dentate gyrus (Harris et a l . , 1984; Errington et a l . , 1987). However, - 47 -Dolphin (1983b) and Sastry et a l . (1984b) reported, respect ive ly , that Y-D-glutamylglycine•(DGG) as well as APV merely mask rather than block the induction of LTP. Sastry et a l . (1984b) suggested the p o s s i b i l i t y that APV 2+ increases Ca in f lux into CA^ neurones during the tetanic s t imula t ion, causing a depression and thus masking any potent iat ion that might have been induced. More recent ly , Harris and Cotman (1986) and Goh (1986) reported that LTP of the mossy f i b re pathways to the area CA^ pyramidal neurones i s not blocked by NMDA antagonists. It seems that more studies are needed before the exact r o l e , i f any, of the NMDA subtype of glutamate receptors can be e luc idated. It also seems at th is stage that the locus of change underlying LTP w i l l remain e lus ive . It i s hoped that more sens i t i ve techniques, possib ly a vo l tage-sensi t ive dye (Saggau et a l . , 1985 ), w i l l become avai lab le so that the c e l l u l a r s i te at which LTP occurs can eventually be located. The synthesis and the release of proteins has been suggested to be associated with LTP (Duffy et a l . , 1981). Duffy et a l . (1981) demonstrated an increase in the incorporat ion of radiolabeled val ine into and the secretion of labeled proteins from areas in the hippocampal s l i c e in which LTP has been induced. Control s l i c e s or s l i ces which were given the te tan ic st imulat ion but did not exhib i t LTP did not show enhanced secret ion of proteins into the ex t race l l u la r environment (Duffy et a l . , 1981). The idea of new protein synthesis with LTP can also account for the long- las t ing morphological changes of the dendr i t ic spines that have been observed fol lowing tetanic st imulat ion (Fifkova and van Harreveld, 1977). Eccles (1983) suggested the p o s s i b i l i t y of a postsynaptic release of a second messenger that can af fect the presynaptic terminals leading to an increase output of t ransmit ter . Stanton and Sarvey (1984) demonstrated the a b i l i t y of a ser ies of protein synthesis inh ib i to rs to block the induction of LTP in - 48 -the CA^ region of the rat hippocampal s l i c e . Moreover, the blockade of LTP induction was found to be well correlated with the a b i l i t y of the protein synthesis inh ib i to rs to i nh ib i t incorporat ion of [ H]valine into proteins (Stanton and Sarvey, 1984). Stanton et a l . (1987) reported that one of the monoclonal antibodies generated to dentate gyri of postnatal rats blocked the induction as well as the maintenance of LTP in both area CA^ and the dentate gyrus. The authors suggested that the antibody binds to a spec i f i c c e l l surface protein that i s important to both the hippocampus and the dentate gyrus in t he i r a b i l i t y to produce and maintain LTP (Stanton et a l . , 1987). Chirwa and Sastry (1987a) demonstrated the release of a heat-sensi t ive chemical, presumably a peptide (or pept ides), into the perfusing f l u i ds of the guinea pig hippocampus af ter tetanic s t imula t ion. When these co l lected samples were applied onto hippocampal s l i c e s , LTP of the CAj population spike was observed (Chirwa and Sastry, 1987a). Most recent ly , there i s some evidence that a peptide s im i la r to the mast c e l l degranulating peptide from bee venom may play an important ro le in LTP (Cherubini et a l . , 1987). There i s some evidence that the ac t iva t ion of protein kinase C (a calcium-dependent phosphol ip id-sensi t ive kinase) may be involved in the induction of LTP (Routtenberg and Lovinger, 1985; Linden et a l . , 1986; Lovinger et a l . , 1986; Malenka et a l . , 1986; Routtenberg et a l . , 1986). Protein kinase C, which can be loca l i zed in various brain structures including the hippocampus (Mochly-Rosen et a l . , 1987), is believed to have an important role in the regulat ion of neuronal e x c i t a b i l i t y ( M i l l e r , 1986; Schwartz and Greenberg, 1987). Af ter the induction of LTP, Lovinger et a l . (1986) and Routtenberg and Lovinger (1985) reported an increase in the phosphorylation of protein F^, reportedly a substrate for protein kinase C. Iontophoretic appl icat ion of 12-o-tetradecanoylphorbal-13-acetate - 4 9 -(Routtenberg et a l . , 1985 ) or oleate (Linden et a l . , 1985 ), agents that reportedly act ivate protein kinase C, was observed to enhance the durat ion of LTP induced by tetanic s t imulat ion. Malenka et a l . (1986) applied a var iety of analogues of phorbal esters (known to act ivate protein kinase C) by superfusion over hippocampal s l i ces and observed a very long-term enhancement of the population spike recorded in area CA^. The act ivat ion of protein kinase C has been observed to increase the release of t ransmit ter from nerve terminals (Haimann et a l . , 1987; Malenka et a l . , 1987; Shapira et a l . , 1987). Storm (1987) observed a broadening of the action potent ia l recorded from CA^ neurones of hippocampal s l i c e s subsequent to bath appl icat ion of phorbal es te rs . Af ter exposure of agents that act ivate protein kinase C, Aplys ia bag c e l l neurones were observed to 2+ express a second c lass of Ca• channels with a higher unitary conductance that was never observed in untreated c e l l s (Strong et a l . , 1987). In hippocampal s l i c e s , phorbal esters were found to markedly increase the frequency of spontaneous miniature EPSPs recorded from CA^ pyramidal 2+ neurones (Malenka et a l . , 1987). When Ca was removed from the bathing medium, appl icat ion of the phorbal ester was s t i l l capable of e l i c i t i n g the spontaneously occuring miniature EPSPs (Malenka et a l . , 1987). It has been suggested that catecholamines play a modulatory role in LTP production. Peripheral in ject ions of amphetamine and epinephrine have been shown to enhance the development of LTP in the dentate gyrus (Gold et a l . , 1984). On the other hand, Morimoto et a l . (1987) found that an acute in ject ion of amphetamine s l i g h t l y reduced LTP of the population spike in rats previously unexposed to amphetamine and s i g n i f i c a n t l y reduced LTP of the population spike in rats that have been chron ica l l y treated with amphetamine. LTP of the EPSP was, however, not affected by e i ther acute or chronic in ject ions of amphetamine (Morimoto et a l . , 1987). Dunwiddie et a l . - 50 -(1982) observed no ef fect of norepinephrine, amphetamine, reserpine, or adrenergic antagonists on the production of LTP in CA^ neurones. In area CAg, however, Hopkins and Johnston (1984) reported a modulatory e f fec t of noradrenaline on LTP induced by tetanic st imulat ion of the mossy f i b r e s . It has been general ly assumed that the mechanisms underlying the induction of LTP and PTP in the hippocampus are not the same (Dunwiddie and Lynch, 1979; McNaughton, 1982). As was mentioned e a r l i e r , both STP (resembling PTP in i t s duration) and LTP can be induced by concurrent act ivat ion of a weak test input with a te tanic st imulat ion of other exci tatory inputs or a su f f i c i en t depolar izat ion of the postsynaptic neurone (Sastry et a l . , 1986). Moreover, a decrease in the presynaptic terminal e x c i t a b i l i t y was found to be c lose ly correlated with both assoc ia t i ve ly induced STP and LTP (Sastry et a l . , 1986). Whether STP or LTP is induced depends on the number of such "pa i r ings" (Sastry et a l . , 1986). On the other hand, i t i s not known whether LTP can be gradual ly developed as an extension of STP with an increasing number of "pa i r i ngs " , or whether a c r i t i c a l number of such paired st imulat ions has to be given before whatever c r i t i c a l changes causing LTP can take place. In Ap lys ia , experimental resul ts suggest an ident ica l synaptic locus and ion ic mechanism fo r both long- and short-term forms of sens i t i za t ion (Kandel and Schwartz, 1982). Kandel and Schwartz (1982) suggest that the only di f ference between the two forms of sens i t i za t ion response may be that long-term sens i t i za t ion involves some morphological a l tera t ions which require the synthesis of new macromolecules such that the conversion from the short- to the long-term process may require the synthesis of new genes (also see: Goelet and Kandel, 1986). It i s therefore possible that PTP and LTP in the hippocampus do not involve en t i re l y d i f fe rent mechanisms. Perhaps a s i tua t ion analogous to that of short- and long-term forms of sens i t i za t i on in Aplys ia could - 51 -explain the di f ference between the induction of PTP and LTP in the hippocampus. The hippocampus has long been thought to be in some manner involved in the storage and/or re t r ieva l of information in the mammalian brain (see Deadwyler [1985] and Green [1964] for reviews). Studies revealed impairment of various learned behaviour subsequent to lesions of the hippocampus-from disrupt ion of r e l a t i v e l y simple conditioned ref lexes (Weikart and Berger, 1986) to that of r e l a t i ve l y more complicated spat ia l memory tasks that were previously accquired (Morris et a l . , 1982; Barnes and McNaughton, 1985; Whishaw, 1987). LTP of the hippocampus has been seen as a very promising model for learning and memory (Chung, 1977; Ecc les , 1986; Racine and K a i r i s s , 1987). Indeed, LTP can be a convenient model for explaining the mechanisms of learning and memory, mainly because of i t s persistence fo r days and even weeks af ter r e l a t i ve l y br ie f periods of intense a c t i v i t y (B l i ss and Gardner-Medwin, 1973; Douglas and Goddard, 1975). In the hippocampal s l i c e preparat ion, i t has been pointed out that the duration of LTP is l imi ted mainly by the surv ival of the preparation (Reymann et a l . , 1985). Landfield et a l . (1978) observed an impairment of synaptic potent iat ion in the hippocampus of aged rats that also exhib i t memory def ic iency. It i s not known, however, whether the def ic iency in memory i s due to a general deter iorat ion of a l l brain structures with age or to some spec i f i c deter iorat ion of the hippocampus only. Berger (1984) demonstrated that the induction of LTP in the hippocampus of the animals before t ra in ing increased the rate at which the animals subsequently learn a simple condit ioning task. The author suggests that LTP may occur in an animal during associat ive learning (Berger, 1984). On the contrary, McNaughton et a l . (1986) observed that high-frequency, LTP-inducing st imulat ion of the perforant pathway in chron ica l l y prepared animals resulted in a profound and - 52 -persistent def ic iency in the acquis i t ion of new spat ia l information but had no ef fect on wel l -estab l ished spat ia l memory. Protein synthesis, which has been shown to occur in various regions of the hippocampus during learning (Yanagihara and Hyden, 1971; Lossner et a l . , 1982), i s also thought to be associated with hippocampal LTP (Duffy et a l . , 1981). Recently, antibodies to S-100, a g l i a l protein which has been shown to be involved in learning and memory, has been observed to block the induction of LTP (Lewis and Teyler, 1986). Fi fkova and van Harreveld (1977), who observed a swel l ing of the dendr i t ic spines of the dentate granule c e l l s a f te r te tanic s t imula t ion, also reported a s im i l a r change of the dendr i t ic spines fo l lowing the acquis i t ion of a conditioned ref lex in mice (Fifkova and van Harreveld, 1978). APV, which reportedly blocks the induction of LTP by blockade of NMDA receptors (Errington et a l . , 1987), has also been observed to impair spat ia l learning in rats when given in the same concentration (Morris et a l . , 1986). Despite a l l of the above f ind ings, the use of hippocampal LTP as a model for learning and memory i s not without problems. As was pointed out by Racine and K a i r i s s (1987), the dose of APV used by Morris et a l . (1986) can often lead to ataxia as well as other behavioural abnormal i t ies. The reported impairment of spat ia l learning by APV may, therefore, involve more than just a d isrupt ion of the storage process. Laroche (1985) monitored the dentate f i e l d potent ia ls e l i c i t e d by perforant path st imulat ion during instrumental learning ( lever-pressing for food reward) as well as c l ass i ca l condit ioning (tone-shock associat ion) in ra ts . No s ign i f i can t changes in the amplitude of the population spike was noted during various phases of instrumental learning or during c l a s s i c a l condit ioning experiments (Laroche, 1985). Racine's group was also unable to detect any changes in the responses evoked by perforant path st imulat ion during various types of - 53 -learning (Racine and K a i r i s s , 1987). Racine and Ka i r i s s (1987) also pointed out the fact that an animal is constantly acquir ing new information about t he i r environments-the animal is constantly " l ea rn ing " . If LTP is indeed involved in " l ea rn ing " , synaptic responses should be constantly changing and not merely change a f te r the animal has acquired a spec i f i c task presented to i t by the invest igator . Moreover, since LTP can be induced in other centra l as well as peripheral synapses (Teyler and DiScenna, 1987), one wonders whether LTP might merely be a mean by which cen t ra l . as well as peripheral structures improve t he i r general funct ional e f f i cacy . It appears that the re la t ionsh ip , i f any, between LTP and memory and learning i s more complex than any present funct ional or mathematical model can account fo r . In the foregoing sect ion, some of the propert ies of both short- and long-term potent iat ion are given. Before concluding, the purpose of the fol lowing set of experiments should perhaps be i t e ra ted . The object ives of the fol lowing study are (1) to observe whether STP can be induced when act ivat ion of the test input preceded as well as followed the onset of the condit ioning tetanus and (2) to observe whether LTP can be induced in the 2+ absence of ex t race l l u la r Ca i f su f f i c i en t depolar izat ions of the presynaptic terminals and the postsynaptic neurones are given. These experiments are based on the f indings of Sastry et a l . (1986) that LTP can be induced in the hippocampus when ac t iva t ion of a test input i s simultaneously paired with e i ther a te tanic st imulat ion of separate exci tatory inputs or su f f i c ien t depolar izat ion of the postsynaptic neurone. It i s hoped that resul ts from the fo l lowing set of experiments w i l l contribute to a fur ther understanding of the induction of both short - and long-term forms of synaptic potent ia t ion. - 54 -3. METHODS 3,1 Preparation of s l i c e s A l l experiments were performed using the in v i t ro transverse hippocampal s l i c e preparat ion. Hippocampal s l i ces were prepared from male Wistar rats weighing 75 to 100 g obtained from the un i ve rs i t y ' s animal care un i t . The animal was placed inside a glass dessicator j a r on top of a polyethylene bag containing i ce . Cooling of the animal decreased i t s metabolic rate and thus minimized the p o s s i b i l i t y of metabolic shock when the brain was subsequently removed fo r preparation of s l i ces (Pandanaboina and Sastry, 1984). A p l as t i c tube was introduced into the dessicator j a r through a hole that was made in the l i d of the j a r . The animal was anaesthesized with a mixture of halothane (2 to 2.5%) and carbogen (95% 0-p and 5% CO2) fo r about 25 to 35 min. At the end of the per iod, the rec ta l temperature of the animal was usual ly about 31 to 32 degrees ce l c i us . The animal was then removed from the j a r and an inc i s ion of the skin was made sag i t a l l y to expose the s k u l l . The sku l l plate and the dura mater were then removed, a f ter which the brain was drenched with pre-cooled and oxygenated (about 4 degrees ce lc ius) standard physio logical medium (refer to sect ion 3.3 for composition of medium). The brain was dissected free from the lower brain stem at the pontine l e v e l , and the o l factory t racts as well as the opt ic nerve were severed. The ent i re brain was then removed from the crania l vault and again drenched with pre-cooled and oxygenated physiological medium. The hippocampus (hippocampi) from one or both s ide(s) of the brain was then located and exc ised. The hippocampus was placed on a layer of moistened f i l t e r paper attached to the chopping block of a Mcllwain t issue chopper, the chopping block was adjusted to obtain the desired or ientat ion for s l i c i n g (Teyler, 1980), and transverse s l i ces (500 pm) were made. 1 - 55 -S l i ces were then gently transferred into a petr i dish containing oxygenated (95% 0^ and 5%C02) physio logical medium maintained at 4 degrees ce lc ius to decrease the metabolic rate of the s l i c e s . Using a spatula, s l i ces were being arranged on a nylon mesh (F ig . 2, IN). The arranged s l i ces were then sandwiched between the nylon mesh they were rest ing on and a second nylon mesh (F ig . 2, ON) in order to minimize movement during recording. The " f i xed" s l i ces were then transferred to the s l i c e chamber and were allowed to equi l ib ra te fo r 1 hour before recordings begun. Inside the s l i c e chamber, s l i c e s were submerged underneath about 0 .5 mm of oxygenated physio logical medium. Temperature of the chamber was maintained at 32 ± 0.5 degrees ce lc ius throughout the equ i l ib ra t ion period as well as the experiments. An ^extra carbogen l ine (F ig . 2, Co) was introduced into the s l i c e chamber to ensure that the s l i c e s were adequately oxygenated. The inf low rate of the physio logical medium was maintained at approximately 2 to 3 ml per min and the bathing medium was continuously removed by the suct ion l ine connected to a vacumn pump so that the level of medium was kept constant. In the process of arranging the s l i ces on the nylon mesh, an i n i t i a l v isual inspection was often carr ied out in order to el iminate obviously damaged s l i c e s . "Healthy" s l i c e s have smooth, well formed borders and a f i rm consistency (Alger et a l . , 1984). Moreover, both the pyramidal c e l l layer and the dentate granule c e l l layer can be c l ea r l y seen under the microscope. It has been shown that hippocampal s l i c e s have l i fe t imes of 6 to 19 hours (Schurr et a l . , 1984). Experiments described in th is study have durations of 2 to 3 hours each. Furthermore, only one s l i c e per animal was used for the experiment in order to ensure optimal condit ions of s l i c e s fo r experiments. The ent i re procedure from the i n i t i a l i nc i s ion made on the - 55 -F i g . 2. S l i c e chamber and p e r f u s i o n system f o r the maintenance, of and  e l e c t r o p h y s i o l o g i c a l recordings from i n v i t r o r a t hippocampal s l i c e s . AB-aluminium block Co-extra carbogen l i n e GW-ground wire HE-heating element I L - i n f l o w l i n e IN-inner nylon mesh LM-1ines f o r medium Mf-manifold Mn-manipulator 01-outlet f o r medium ON-outer nylon mesh S C - s l i c e chamber SL-suction l i n e SS-securing screw - 57 -animal to the t rans fer ra l of the s l i ces into cooled, oxygenated physiological medium took less than 3 min. 3.2 SI ice chamber F i g . 2 shows an i l l u s t r a t i o n of the s l i c e chamber and the perfusion system. The s l i c e chamber (SC) a'llows the pos i t ion ing of the two nylon meshes f ixed onto inner (IN) and outer (ON) p lex ig lass r ings. The s l i c e chamber s i t s above an aluminium block (AB) which contains a temperature sensing device. The aluminium block i s in d i rec t contact with a heating element (HE) which warms the aluminium block and thus the s l i c e chamber to the desired temperature. The perfusion system for the s l i ces consists of separate storage barrels (50 ml each) for the standard as well as the any drug-containing media. The storage barrels are connected to the l ines for the medium (LM) by s ta in less steel hypodermic syringe needles which f i t the bar re ls . A l l the l ines for the medium are connected to the s ingle inf low l ine by way of a manifold. At any given time, only one of the l ines for medium is opened and a l l the other l ines are clamped shut. The out let (01) of the suction l i ne (SL) is adjusted depending on the desired depth of the perfusing medium. The diameter of the l ines for the medium (LM), the s ingle inflow l ine (IL) and the suction l ine (SL) are ident ica l in diameter so that there are no s ign i f i can t di f ferences between the inflow and outflow rate. A more detai led descr ipt ion of the s l i c e chamber can be found in Pandanaboina and Sastry (1984). 3.3 Physiological medium The standard medium was used during the preparation of the s l i c e s . A l l of the media used were constantly aerated with carbogen, the pH being maintained at 7.4. - 5 3 -Standard medium: 120 mM NaCl, 3.1 mM KC1, 1.3 mM NaH 2P0 4 , 25 mM NaHC03, 2 mM C a C l 2 , 2 mM MgCl 2 > 10 mM dextrose. Picrotoxin medium: 120 mM NaCl, 3.1 mM KC1, 25 mM NaHC03, 4 mM CaClr,, 4 mM M g C ^ , 10 mM dextrose, 10 yM p ic ro tox in . P ic ro tox in was used in some of the experiments to f a c i l i t a t e the induction of associat ive STP as well as LTP (Wigstrom and Gustafsson, 1983b, 1985). 2 + 2 + The level of Mg and Ca was increased from 2 to 4 mM in order to minimize epi lept i form a c t i v i t y that could be induced when p ic ro tox in was added (Wigstrom and Gustafsson, 1983b, 1985a). The buffer ing capacity of the p icrotox in-conta in ing medium was maintained even with the omission of NaH^PO^ as long as i t was aerated with carbogen. EDTA medium: same as standard medium except that 200 yM ethylenediaminetetraacetic acid (EDTA; added from stock solut ion which was adjusted to pH 7 . 4 ) was added. EDTA-picrotoxin medium: same as p icrotox in medium except that 200 uM EDTA was added to the medium. 2+ 2 + Ca - f ree (Mn ) picrotoxin-EDTA medium: same as EDTA-picrotoxin medium except that CaCl^ was omitted, 1 mM MnC^ was added and the concentration of MgCl^ was increased to 7 mM to compensate fo r the divalent ion concentrat ion. Ca ' - f ree (Mnc ) EDTA medi urn: same as EDTA medium except that CaClg was omitted, 1 mM MnC^ was added and the concentrat ion of MgCl2 was increased to 7 mM. 2+ ?+ ?+ Ca - f ree (Co ) picrotoxin-EDTA medium: same as Ca - f ree 2+ (Mn ) picrotoxin-EDTA medium except that CoC^ (1 mM) was used instead of MnCl 0 . - 59 -Elevated K + medium: when the concentration of KC1 was increased in any of the media described above, the concentration of NaCl was reduced by the appropriate amount in order to maintain a comparable osmolar i ty. 3.4 Instrumentation 3.4.1 Stimulat ion systems. The st imulat ion electrodes used for a l l experiments were concentric b ipolar metal electrodes (SNEX 100, Rhodes E lec t ron ics , resistance 1 to 2 Mfl ). Current pulses (negative square waves) were del ivered using a Grass S88 2-channel st imulator and passed through a Grass PSIU6 constant current stimulus i so la t i on unit before reaching the st imulat ion electrodes. 3.4.2 Recording systems. For ex t race l l u la r recordings, recording microelectrodes were prepared from f i b r e - f i l l e d c a p i l l a r y tubing (borosi 11 icate g lass , O.D. 1.5 mm, I.D. 1.0 mm, Frederick Haer and Company) using a Narishige PE-2 microelectrode pu l le r and were f i l l e d with 4 M NaCl ( f i l t e r e d ) . Tip diameter of these recording electrodes was about 1 pm "and resistance about 1 to 2 Mo,. In t race l lu la r recording electrodes were pul led from the same cap i l l a r y tubing with a Brown-Flaming pu l le r and were f i l l e d with a 10:1 mixture of 1 M KC1 and 1.6 M K c i t r a t e . I n t race l l u la r electrodes served the dual purpose of recording and in ject ing current fo r i n t r ace l l u l a r depo lar iza t ion. Ex t race l lu la r s ignals were ampli f ied by e i ther a World Prec is ion Instruments DAM-5A d i f f e ren t i a l preampl i f ier or a Medical Systems Neurolog AC-preampli f ier and AC-DC ampl i f ie r . Responses were then stored and averaged using a DATA 6000 (Data Precis ion) waveform analyser and plot ted on paper using a Hewlett-Packard 7470A graphics p lo t te r . In t race l lu la r s ignals were amplif ied using a World Precis ion Instruments M-707 i n t r a c e l l u l a r ampl i f ie r . I n t race l lu la r responses were monitored using a Tektronix type 5113 dual beam storage osc i l loscope which could also simultaneously monitor - 60 -the injected current pulses. Records could e i ther be captured on polaroid f i lm or stored on audio tape using a Hewlett-Packard 3968A instrumentation recorder. Records from the tape could then be plotted on paper using the Hewlett-Packard 7404A chart recorder and then analysed. 3.5 Temporal requirements of assoc ia t ive ly induced short-term potent iat ion Experiments were carr ied out using p icrotox in medium. Two st imulat ing electrodes were placed in two separate convergent inputs (stratum oriens and stratum radiatum) (F ig . 3) to a population of CA^ pyramidal neurones. The condit ioning tetani (SI) were del ivered through the electrode located in the stratum oriens while the test s t imul i (S2) were del ivered v ia the electrode located in the stratum radiatum. Stimulus in tens i ty was adjusted to produce a weak population EPSP (200 to 600 yV; F i g . 3 inset) from the test input and a strong population EPSP from the condit ioning input. Population EPSPs were recorded from the apical dendr i t ic area of CA^ neurones. Except during paired condi t ion ing- test s t imulat ions, both the test and condit ioning inputs were stimulated at 0.1 Hz, a l ternat ing every 5 sec (pulse durat ion: 0.2 ms). When a paired st imulat ion (50 ms delay) was given to the weak input, the response to the second stimulus was increased in s ize compared to that to the f i r s t . On the other hand, when the condit ioning input was stimulated 50 ms p r io r to the test input, no such f a c i l i t a t i o n of the second response was observed, an ind icat ion that the two sets of f ib res were indeed independent of each other. Both test and condit ioning inputs were stimulated with 0.2 ms pulses at 0.1 Hz (a l ternat ing every 5 sec) . After a control s t imulat ion period of at least 30 min, the condi t ioning input was te tan i ca l l y stimulated with a ser ies of 5 t ra ins (each t r a i n was made up of 10 pulses at 100 Hz) at 5 sec in te rva ls . The test s t imul i (TS) e i ther preceded (-) or followed (+) the onset of each condit ioning t ra in (CT) by 0 to 100 ms (F ig . 4) . After each ser ies of 5 pair ings of test - 61 -F i g . 3. Experimental arrangements for the associat ive induction of STP. Two bipo1ar st imulat ing electrodes were placed in separate convergent inputs to a population of CA} neurones. The condit ioning tetani (5 t r a i ns , each t ra in consist ing of 10 pulses at 100 Hz, one t ra in every 5 sec) were del ivered v ia the electrode located in the stratum oriens (SI) while the test s t imul i were del ivered v ia the electrode located in the stratum radiatum (S2: a representative response is shown in the inse t ; ca l i b ra t i on bars represent 0.3 mV and 10 ms). Population EPSPs were recorded from the apical dendr i t ic area of CA^ neurones by a glass microelectrode (R). Comm-commissural inputs Sch-Schaffer co l l a t e ra l s - 6 2 -s t imul i and condit ioning te tan i , the test population EPSP was monitored fo r 10 to 15 min to ensure that the response returned to the control l e v e l . If STP was followed by LTP, no fur ther paired condit ionings were given and the experiment was discontinued. Otherwise, each experiment consisted of 5 to 10 ser ies of pair ings at d i f ferent condi t ion ing- test in terva ls (CTl) with test stimulus e i ther preceding (negative CTl) or fo l lowing (posi t ive CTl) the condit ioning tetanus. 3.6 Induction of long-term potent iat ion in the absence of ex t race l l u l a r  calcium 3.6.1 Effects of increasing ex t race l l u la r potassium concentration in both the absence and presence of calcium in the perfusing media. Except during the period in which s l i c e s were exposed to elevated ex t race l l u l a r K + medium, s l i ces were perfused with p icrotox in medium. Population EPSPs were recorded from apical dendrites of CA^ neurones in response to stratum radiatum st imulat ion at 0.2 Hz (F i g . 5 and inset) and stable responses were obtained fo r at least 30 min before the experiment began. Regardless of „ 2+ 2+ whether the s l i ces were exposed to Ca - f ree or Ca -containing medium, 2 + 2 + they were f i r s t perfused with Ca - f ree (Mn ) picrotoxin-EDTA (3.1 mM KC1) medium (4 min appl icat ion period) before each elevated K + app l ica t ion 2+ to completly block the synaptic responses and to remove the Ca contained in the normal medium. The response was usual ly completly blocked in under 2 2+ + min. The addi t ional 2 min of exposure to Ca - f ree (normal K ) medium 2+ was to ensure the complete removal of Ca and that an equi l ibr ium with the new medium has been at ta ined. S l i ces were then exposed to elevated ex t race l l u l a r K concentrations given in e i ther Ca - f ree (Mn ) 2+ picrotoxin-EDTA medium or control (Ca -containing medium) for a period of 3 min. The concentration of KC1 was varied from 10 to 80 mM. The 2+ 2+ Ca - f ree (Mn ) picrotoxin-EDTA (3.1 mM KC1) medium was applied fo r an - 53 -100 m s C T I - 1 0 0 C T I 0 C T I +100 C T ( S , ) T S ( S 2 ) F i g . 4 . A schematic diagram to fur ther i l l u s t r a t e the experimental  arrangements in the invest igat ion of the temporal l im i t s for the induction  of associat ive STP. One test stimulus (TS; given v ia S2 Tn F i g . 3) was paired with each of the 5 condit ioning t ra ins (CT; given v ia SI in F i g . 3) at each of the condi t ion ing- test in terva ls (CTI) that was examined. The condi t ioning-test in terva l was varied between -100 ms and +100 ms. As shown in the diagram, a negative CTI indicates that the test stimulus precedes the onset of each condit ioning t r a i n . A pos i t ive CTI indicates that the test stimulus was act ivated fo l lowing the onset of the condit ioning t r a i n . - 64 -Comm Sch F i g . 5. General experimental arrangements for sections 3.6.1 to 3 .6 .3 . A b ipo lar st imulat ing electrode (S) was posit ioned in the stratum radiatum and a glass recording microelectrode (R) was posit ioned in the apical dendr i t i c area of CA} neurones to monitor the population EPSP evoked at 0.2 Hz. Stimulat ion strength was adjusted to e l i c i t a response of approximately 1.0 to 1.5 mV. A representative response is shown in the inset ( ca l i b ra t i on bars represent 10 ms and 0.5 mV). Comm-commissural inputs Sch-Schaffer co l l a t e ra l s - 6 5 -+ " ' addit ional 3 min fol lowing elevated K appl icat ion to wash out any excess + K that might not have been immediately removed before re-exposure to 2+ Ca -containing (3.1 mM KC1) medium. As cont ro ls , a number of s l i ces were 2+ 2+ exposed only to Ca - f ree (Mn ) picrotoxin-EDTA (3.1 mM KC1) medium for a period of 10 min. The population EPSP was monitored fo r a period of at 2+ least 30 min af ter perfusion with the Ca -containing medium resumed. 3.6.2 Ef fects of increasing concentration of ex t race l l u la r potassium  ( in calcium-free medium) in the presence and absence of p ic ro tox in . Stimulating and recording s i tes for th is set of experiments were the same as that of the experiments described in sect ion 3 .6 .1 ' (see also F i g . 5 and 7+ i nse t ) . The sequence and duration of exposure of s l i ces to Ca - f ree media were also s im i l a r to that described in the previous sect ion. B r i e f l y , 2+ 2 + s l i ces were f i r s t exposed to Ca - f ree (Mn ) EDTA (3.1 mM KC 1) medium fo r a period of 4 min, followed by a 3 min exposure to Ca - f ree (Mn ) EDTA (elevated K +) medium and an addit ional 3 min period exposure to C a 2 + - f r e e (Mn 2 +) EDTA (3.1 mM KC1) medium. Instead of a. range of + • 2+ ex t race l l u la r K concentrat ions, s l i ces were exposed to Ca - f ree medium with an elevated K + concentration of 40 mM. 10 experiments each were performed using p icrotox in-conta in ing and p ic ro tox in- f ree media. For the experiments using p ic ro tox in , s l i c e s were exposed to media containing picrotoxin throughout the experiment-from the i n i t i a l incubation period to the end of the experiment. Again, responses were monitored for a period of 2+ at least 30 min af ter treatment with Ca media. 3.6.3 Ef fects of increasing concentration of ex t race l l u la r potassium  ( in calcium-free medium) when a f i xed number of axons were being act ivated. This series of experiments was performed to observed the ef fects of elevated ex t race l l u la r K + when a f ixed number of axons were being maximally act ivated. Stimulation and recording s i tes were essen t i a l l y s im i l a r to that - 66 -of previous experiments. Experiments were performed using p ic ro tox in - f ree media. Before the experiments, a cut was made in between the s t imulat ing and recording electrodes from the alveus to part of the stratum radiatum and from the lower part of the stratum radiatum towards the hippocampal f i s s u r e such that only a very small band of stratum radiatum input to CA^ neurones remained. The stimulus strength was then increased to twice that i s required to produce a maximal population EPSP. The sequence and duration of 2+ exposure of s l i c e s to Ca - f ree media were also the same as that described in the las t sect ion (3 .6 .2) . Again, the concentration of elevated K + used in these experiments was 40 mM. 3.6.4 Ef fects of pai r ing postsynaptic depolar izat ions with presynaptic action potent ia ls in calcium-free medium. It has previously been shown that LTP can be induced when act ivat ion of a weak test input occurs during a depolar izat ion of the postsynaptic neurone (Sastry et a l . , 1986). The purpose of th i s ser ies of experiments was to examine i f LTP can be induced with the. same parameters when ex t race l l u l a r Ca has been removed. Experiments were performed using p icrotox in-conta in ing media. I n t r ace l l u l a r EPSPs were recorded from CA^ neurones in response to stratum radiatum st imulat ion at 0.2 Hz (F ig . 6 and i nse t ) . Only neurones that had membrane potent ia ls greater than -60 mV and showed stable membrane potent ia ls fo r extended periods of time were used fo r these experiments. After obtaining 2+ stable controls fo r about 10 to 15 min, s l i ces were exposed to Ca - f ree 2+ (Mn ) picrotoxin-EDTA (3.1 mM KC1) medium for a period of 5 min. At the 2+ fourth minute of exposure to Ca - f ree medium, depolar iz ing commands (150 ms, 2 to 3 nA, 10 commands at 0.2 Hz) paired with a near simultaneous act ivat ion of the input f ib res were given with the i n t r a c e l l u l a r - 67 -F i g . 6. Diagram i l l u s t r a t i n g the experimental arrangements fo r the pa i r ing  of postsynaptic depolarizations^ with presynaptic action po ten t ia ls . A b ipo lar st imulat ing electrode (S) was posit ioned in the stratum radiatum in order to evoke EPSPs in CA^ neurones. An i n t r a c e l l u l a r microelectrode (R/D) in a CAi neurone both applied the depolar iz ing commands (150 ms, 2 to 3 nA, 10 commands at 0.2 Hz; inse t , l e f t : the ca l i b ra t i on bars represent 150 ms and 40 mV) and recorded the i n t r a c e l l u l a r EPSP (a representative response is shown in inse t , r i gh t : the ca l i b ra t i on bars represent 10 ms and 5mV). The st imulat ion strength was adjusted such that the s ize of the EPSPs would be about 30% of maximum. Comm-commissural inputs Sch-Schaffer co l l a t e ra l s - 68 -microelectrode-the stratum radiatum-induced EPSP was evoked 1 ms a f ter the onset of each depolar iz ing command. As cont ro ls , the same depolar iz ing commands were given without pair ing them with act ivat ion of the input f i b res . The i n t r a c e l l u l a r EPSP was monitored for a period of 25 to 30 min af ter s l i ces were re-exposed to control medium. - 69 -4. RESULTS 4.1 Temporal requirements of assoc ia t ive ly induced short-term potent iat ion In experiments where the test stimulus (S2) followed the onset of the condit ioning tetanic t r a i n (SI ) , there was s ign i f i can t STP of the test population EPSP up to CTI +80 ms (F ig . 7) . When S2 preceded the onset of the condit ioning t r a i n (SI) , there was s ign i f i can t potent iat ion up to a CTI of -50 ms (F ig . 7 ) . The greatest degree of STP was induced when the tes t input was act ivated simultaneously with the onset of the condit ioning t r a i n (CTI 0 ms; F i g . 7) . When the condit ioning tetani (SI) were given without coact ivat ion of the test EPSP, a s ign i f i can t heterosynaptic depression of the test EPSP ensued (F ig . 7, f i l l e d diamond; n = 23). The duration of the associat ive STP did not usual ly exceed 3 min. The degree of potent iat ion was quantif ied 1 min af ter the end of the condit ioning te tan i - tes t s t imu l i pa i r ings. In some instances, STP with a duration of considerably less than 1 min has been observed. Whether or not these observed potent iat ion represent the shorter components reported in the l i t e ra tu re (Mallart and Mart in, 1967; Magleby and Zengel, 1976a,b,c) remain to be determined. 4.2 Induction of long-term potent iat ion in the absence of ex t race l l u l a r  calcium 4.2.1 Ef fects of increasing ex t race l l u la r potassium concentrations in both the absence and presence of calcium in the perfusing media. In the 2+ absence. ..of Ca in the ex t race l l u la r perfusing medium, increasing + ex t race l lu la r K (10 to 80 mM) led to a dose-dependent post-treatment potent iat ion of the population EPSP. This was quantitated 15 min a f te r termination of exposure to elevated ex t race l l u l a r K + (Table 1). S l i c e s exposed to Ca - f ree medium containing normal (3.1 mM) level of ex t race l l u la r K + also exhibi ted a s l i gh t potent iat ion of the EPSP - 70 -120r-o . Z O o u. o 110 < if) < CL V) a. Ui o 100 •< a. o a. f-UJ r" 9 0 L -100 - 8 0 —60 —40 —20 0 20 40 60 80 100 CONDITIONING-TEST INTERVAL (ms) F i g . 7. Potent iat ion of the test population EPSPs at d i f fe rent cond i t ion-ing- test in tervals (CTl) . Each point on the curve ( f i l l e d cTrcTes) indicates the mean ± SEM of the test population EPSP represented as a percent of cont ro l . The population EPSP magnitudes were measured 1 min fo l lowing the 5 paired t r a i ns . Each point on the curve was compared to i t s own preconditioned magnitude using the paired Student's t - t es t . The f i l l e d diamond represents the magnitude of the test population EPSP 1 min fo l lowing 5 unpaired condit ioning t r a i n s . The number below each point on the curve represents the number of recorded observations (n) fo r each condi t ioning-test i n t e r va l . 2+ (population EPSP as a A of control 15 min a f ter exposure to Ca - f r e e , 3.1 mM K medium: 111 ± 3 SEM, n = 8). However, i f one compares th i s value to those presented in the top row of Table 1, one could observe that increasing concentrations of ex t race l l u la r K + during exposure to 2+ Ca - f ree medium produced increasingly greater potent iat ion of the population EPSP. Therefore, the potent iat ion of the population EPSP „ 2+ + fol lowing exposure to Ca - f r e e , elevated K media appears to be a + consequence of the elevat ion of ex t race l l u l a r K and not merely an 2+ 2 + ar t i f ac t resul t ing from exposure to Ca - f ree medium or to the Mn contained in i t . Appl icat ion of elevated ex t race l l u la r K + in „ 2+ Ca -containing medium also led to a br ie f potent iat ion of the EPSP fol lowing termination of app l i ca t ion . This br ie f potent iat ion was fol lowed in the majority of cases by a prolonged depression of the population EPSP. This tendency towards depression was espec ia l l y apparent with the higher concentrations of ex t race l l u la r K + (Table 1). In order to fur ther es tab l ish that the potent iat ion produced in _ 2+ 2+ Ca - f ree medium was not merely caused by some property unique to Mn , 2+ 2 + v the experiments were repeated using Ca - f ree (Co ) medium instead of 2+ 2+ Mn in the Ca - f ree medium. A comparable potent iat ion of the + 2 + population EPSP was observed with 80 mM K in a Ca - f ree medium 2+ ? + / containing Co instead of Mn (population EPSP as a % of control 15 min af ter termination of exposure to 80 mM K + : 142 ± 14 SEM, n = 5). 2+ 2+ Exposure of s l i ces to the same Ca - f ree (Co ) medium produced no such potent iat ion of the EPSP (population EPSP as a % of control a f te r 2+ 2 + + termination of exposure to Ca free [Co ] , 3.1 mM K medium: 92 ••* 3 SEM, n = 4 ) , fur ther suggesting that the potent iat ion of the population EPSP + was indeed an ef fect of elevated ex t race l l u l a r K . The recovery of the 2+ responses from Mn -contain ing medium seemed to have been s l i g h t l y f as te r Table 1. Effects of increasing concentrations of ex t race l l u la r potassium on the CA-^  population EPSP magnitude 15 min fol lowing termination of exposure to elevated  ex t race l lu la r potassium. Population EPSP as a % of control 15 minutes af ter exposure to media with [K + ] in mM 10 20 40 80 2+ 2+ Mn -Mg Range 106-128 109-129 120-155 122-197.5 medium Mean ± SEM 116.5 ± 4.79 120.4 ± 3.28 136.3 ± 4.6 153.7 ± 9.9 n 4 6 7 7 2+ 2+ Ca -Mg Range 103-111 94-115 86-109 61-114 medium Mean ± SEM 106.3 ± 1.7 104.4 ± 3.39 101 ± 3.27 90.1 ± 7.63 n 4 5 6 7 Af ter obtaining stable controls for at least 30 min, s l i c e s were f i r s t exposed to Ca^ + - f ree (Mn^+) picrotoxin-EDTA (normal K +) medium for a period of 4 min. This was followed by the appl icat ion of elevated-K + medium given in e i ther Ca2 + - f ree (Mn2+-Mg2+) or Ca2 +-containing (Ca2+-Mg2+) medium for a period of 3 min. S l i ces were exposed to Ca^ + - f ree (Mn^+) picrotoxin-EDTA (normal K +) medium for an addit ional 3 min before perfusion with Ca2 +-containing (normal K +) medium resumed. The magnitudes of the EPSP given in the table were recorded 15 min fo l lowing the termination of the elevated-K + treatment and expressed as a percent of t he i r own con t ro l . The range of magnitudes fol lowing each treatment, as well as the mean±SEM and the number of recorded observations, are given. - 73 -2+ than that from Co -containing medium, possib ly because of a greater a f f i n i t y of the EDTA towards M n 2 + . 4.3.1 Effects of increasing ex t race l l u l a r potassium concentration ( in calcium-free medium) in the absence and the presence of p ic ro tox in . This ser ies of experiments was performed to examine the necessity of p icro tox in + fo r the elevated K -induced potent iat ion of the EPSP observed in the previous sect ion. Regardless of whether p ic ro tox in was added to the medium, appl icat ion of elevated ex t race l l u la r K (40 mM) in Ca - f ree medium resulted in a s ign i f i can t potent iat ion of the population EPSP (F ig . 8 ) . In p ic ro tox in- f ree medium, appl icat ion of elevated K + did produce a s ign i f i can t post-treatment potent iat ion of the population EPSP (population EPSP as a % of control 15 min af ter termination of exposure to elevated K : 130 ± 7 SEM, n = 10) (F ig . 8) . In p icrotox in-conta in ing media, appl icat ion of elevated K + resulted in a s l i g h t l y greater post-treatment potent iat ion of the population EPSP (population EPSP as a % of control 15 min a f ter termination of exposure to elevated K + : 143 ± 10 SEM, n = 10) (F ig . 8). Comparison of the above two values did not y i e l d s t a t i s t i c a l l y s ign i f i can t dif ferences (P > 0.1 using the unpaired Student's t - t e s t ) . 4.3.2 Ef fects of increasing concentration of ex t race l lu la r potassium  ( in calcium-free medium) when a f ixed number of axons were being ac t iva ted . This set of experiments was performed to es tab l i sh that the elevated K -induced potent iat ion was not merely a resul t of an increase in the number of axons act ivated because of increased e x c i t a b i l i t y . The procedures described in Methods (sect ion 3.5.3) ensured that a f ixed number of axons were being act ivated even i f there was an increase in the e x c i t a b i l i t y . + 2+ Elevation of ex t race l l u la r K (40 mM) during perfusion with Ca - f ree medium led to a post -appl icat ion potent iat ion of the population EPSP - 74 -16CK C o u ««-» c 0) E 0) CL (/) CL UJ C o a o Q. 140-120-100- -8 0 J ** r 0 10 I 15 20 — i — 25 i 30 time after exposure to 40 mM K + (minutes) F i g . 8. Magnitudes of ex t race l l u la r potassium population EPSPs (in calcium-free mediumT fol lowing appl icat ion of  — " ' the absence and elevated in presence Of p ic ro tox in . S l i ces "were f i r s t exposed to Ca^ + - f ree (Mn2+) EDTA (normal \C) medium fo r a 4-min per iod, followed by a 3 min exposure to C a 2 + - f r e e (Mn 2 +) EDTA (40 mM K + ) medium and an addi t ional 3-min period of exposure to C a 2 + - f r e e (Mn 2 +) EDTA (normal K + ) medium. Magnitudes of population EPSPs were expressed as a percent of and compared with t he i r own controls using the paired Student's t - t es t ( * * = P < 0.01; * = P < 0.05). F i l l e d c i r c l e s represent mangitudes of the population EPSPs (mean±SEM) fol lowing the exposure of s l i c e s to Ca2 + - f ree medium containing 40 mM K + in the presence of 10 yM p ic ro tox in . F i l l e d squares represent magnitudes of the population EPSPs (mean ± SEM) fo l lowing the exposure of s l i c e s to the same Ca2 + - f ree (elevated K + ) medium in the absence of p ic ro tox in . - 7 5 -(population EPSP as a % of control 15 min af ter termination of app l ica t ion of elevated K + : 1 2 1 * 2 SEM, n = 15) (F ig . 9) , ind icat ing that the potent iat ion was not merely a resul t of an increase in the number of f i b res act ivated. 4.2.4 Effects of pair ing postsynaptic depolar izat ions with presynaptic action potent ia ls in calcium-free medium. As in the previous experiments where elevated ex t race l l u l a r K + was exposed to the ent i re s l i c e , act ivat ion of presynaptic f ib res concurrently with depolar izat ions of the postsynaptic CA^ neurone produced a s ign i f i can t potent iat ion of the the i n t r a c e l l u l a r ^ recorded EPSP (EPSP as a % of control 15 min fo l lowing termination of exposure to Ca - f ree medium: 139 * 5 SEM, n = 9) (Figure 10). Postsynaptic depolar izat ion without concurrent ac t iva t ion of the presynaptic f ib res did not produce a s ign i f i can t potent iat ion of the i n t r a c e l l u l a r EPSP in the same neurones. Results from present experiments further strengthen the f indings of the previous sections (3.6.1 to 3.6.3) 2+ that LTP can be induced in the absence of ex t race l l u l a r Ca as long as concurrent postsynaptic depolar izat ions and act ivat ion of the presynaptic terminals are provided. - 76 -140-1 O c o u c time after exposure to 40 mM K + (minutes) Figure 9. Magnitudes of population EPSPs fo l lowing appl icat ion of elevated  ex t race l l u la r potassium (in calcium-free medium) when a f ixed number of"  axons were being act ivated. Fn between the stimul ating and recording electrodes, a cut was made from the alveus to part of the stratum radiatum and from the lower part of the stratum radiatum towards the hippocampal f i ssure such that only a small f rac t ion of the stratum radiatum input to CAi neurones remained. The sequence and duration of exposure to control and C a 2 + - f r e e media were essen t ia l l y the same as that described in the legends to F i g . 8 and in Section 3.6.2. No p icro tox in was included in any of the media used. Magnitudes of the population EPSPs were expressed as a percent of and compared with the i r own controls using the paired Student's t tes t ( • •= P < 0.01; • = P < 0.05). - 77 -— i I I i i i — I i I I 0 5 10 15 20 25 30 35 40 45 50 Time (minutes) F i g . 10. EPSP magnitudes p r io r to and fo l lowing the act ivat ion of  presynaptic f ib res concurrently with depolar izat ions of a CAj neurone  during perfusion with calcium-free medium. THe bar indicates the time period during which the s l i c e was perfused with Ca 2 + - f r ee (normal K + ) medium. The arrow represents the time at which depolar iz ing commands (150 ms, 2 to 3 nA, 10 commands at 0.2 Hz) were given concurrently with act ivat ion of the presynaptic f i b res . The membrane potent ial was -57 mV at the beginning and -64 mV at the end of the experiment. EPSP sizes were expressed as a percent of the f i r s t recorded con t ro l . This is a t yp ica l experiment. - 78 -5. DISCUSSION 5.1 Temporal requirements of assoc ia t ive ly induced short-term potent iat ion Depending upon the duration of the observed ef fect and i t s time course of decay, short-term potent iat ion has been general ly subdivided into three major components - f a c i l i t a t i o n (Mallart and Mart in , 1967), augmentation (Magleby and Zengel, 1976a,b,c) and potent iat ion (Magleby and Zengel, 1976a,b,c). Based on the above c l a s s i f i c a t i o n , the various components of STP have been observed in many central as well as peripheral systems, including the hippocampus (Creager et a l . , 1980; McNaughton, 1982; Racine and Milgram, 1983). Although there seems to have been some agreement towards a presynaptic locus for the d i f fe rent forms of STP (Larrabee and Bronk, 1947; L loyd, 1949; del C a s t i l l o and Katz, 1954a,b,c, d; Erulkar and Rahamimoff, 1978), there has not been any concensus as to the exact mechanism(s) underlying STP. Recently, Sastry et a l . (1986) found that STP can be induced in area CA^ of the hippocampus without tetanic s t imulat ion of the input, but when act ivat ion of the input i s paired with the simultaneous te tan izat ion of other converging exc i ta tory inputs. The aim of the present experiments i s to fur ther examine the temporal requirements of assoc ia t ive ly induced STP using the same experimental arrangement as that of Sastry et a l . (1986). Present resul ts fur ther confirm e a r l i e r f indings by Sastry et a l . (1986 ) that in area CA^ of the hippocampus, STP can be induced with concurrent act ivat ion of a test input and tetanic st imulat ion of other converging exci tatory inputs. It has now been fur ther observed that associat ive STP can be induced even when a temporal separation occurs between the act ivat ion of the test input and the onset of the condi t ioning tetanus. The greatest amount of associat ive STP was observed when the onset - 79 -of the condit ioning t ra in occured simultaneously with the act ivat ion of the test input (Figure 7). The amount of potent iat ion appears to decrease roughly symmetrically about CTI ± 0 ms for both pos i t i ve ( + ) and negative (-) CTI up to CTI ± 40 ms (F ig . 7) . From a CTI of +50 ms to a CTI of +70 ms there appears to be a re la t i ve increase in the degree of potent iat ion even though the condi t ion ing- test in terva l i s increasing (F ig . 7) . After a CTI of +70 ms, the amount of potent iat ion decreases to i ns ign i f i can t values for longer pos i t i ve CTI. For negative CTI, the amount of potentiat ion continuously decreases for longer in terva ls un t i l i ns ign i f i can t potentiat ion was observed for a CTI greater than -50 ms (F ig . 7 ) . STP observed in the present study as well as that observed by Sastry et a l . (1986) resembled PTP in i t s durat ion, although whether the two forms of potent iat ion share common underlying mechanism(s) remains to be determined. For each experiment, the degree of potent iat ion was quanti f ied 1 min a f ter the pair ing of the condit ioning te tan i - tes t s t i m u l i . There were experiments where potentiat ion that lasted less than 1 min was observed. Whether these very short last ing potent iat ion represent the shorter components of STP ( f a c i l i t a t i o n or augmentation), or whether they merely represent potent ia t ion that i s masked by a subsequent depression (Abraham and Goddard, 1983; Chirwa et a l . , 1983; Abraham et a l . , 1985), cannot be determined from the present resu l t s . With fur ther studies i t may be possible to determine whether other shorter forms of STP can indeed be induced with the methods used in th is study. Presently avai lab le resul ts make i t d i f f i c u l t to ascerta in the cause of the apparent increase in the degree of potent iat ion with a CTI longer than +40 ms. In hippocampal CA^ pyramidal neurones, i t has been reported that a hyperpolarizing IPSP occurs fo l lowing the EPSP (Newberry and N i c o l l , 1984). The IPSP has been reported to resul t from an increase in the - 80 -chlor ide conductance of the neurone and be sens i t i ve to GABA antagonists (Nico l l and Alger, 1981). The IPSP has also been reported to peak at a latency of about 50 ms (Newberry and N i c o l l , 1984). The trough on the potentiat ion curve at a CTl of + 40 ms may be due to an incomplete blockade of the GABA-mediated inh ib i t i on mentioned above. The concentration of picrotoxin used in these experiments (10 uM) was r e l a t i v e l y low compared to the 100 uM used in some studies from other laborator ies (Wigstrom and Gustafsson, 1983b, 1985), although the concentration had been found to be adequate in blocking GABA-mediated inh ib i t i on (Sastry et a l . , 1986). The trough on the curve at a CTl of +40 ms could also be merely due to s t a t i s t i c a l v a r i a b i l i t y of the resu l t s . Further experiments, perhaps u t i l i z i n g a higher concentration of p ic ro tox in , may be required before the issue can be c l a r i f i e d . When the condit ioning tetani were given without concurrent act ivat ion of the test input, there was a s ign i f i can t short-term depression of the response to the test input (F ig . 7) . This may perhaps be analogous to the f indings of Chirwa et a l . (1983) in CA^ neurones, and that of Abraham and Goddard (1983) and Abraham et a l . (1985), who observed that tetanizat ion of e i ther the la te ra l or medial perforant path (which e l i c i t e d LTP of the tetanized pathway) resulted in a long-term heterosynaptic depression of the other pathway. The observations made in these experiments for associat ive STP are s imi la r to those made for associat ive LTP (Gustafsson and Wigstrom, 1986). In order of assoc iat ive LTP to be induced, Gustafsson and Wigstrom (1986 ) also reported that the test input had to be activated during the condit ioning tetanus (pos i t ive CTl) or less than 40 ms before the onset of the condit ioning t r a i n (CTl -40 ms). Wigstrom and Gustafsson (1985b) proposed a mechanism for associat ive 2+ LTP involving a purported Ca -current resul t ing from act ivat ion of NMDA - 81 -receptors. Wigstrom et a l . (1985b) reported that this NMDA receptor-act ivated current can be observed even af ter a single afferent st imulat ion and has a half-decay time of 40 to 50 ms. An overlapping of th is current would exaggerate the Ca in f lux through these purported channels because of larger levels of subsynaptic depolar izat ion and thus induce LTP (Gustafsson and Wigstrom, 1986). This would also be a convenient theory to explain the associat ive induction of both STP and LTP since i t would explain the induction of potent iat ion when the EPSP preceded the condit ioning tetanus by up to 50 ms (CTI -50 ms). No s ign i f i can t potent iat ion was observed with condi t ion ing- test in terva ls of greater than +80 ms despite some overlapping between the EPSP and the las t pulses of the condit ioning t ra in (F ig . 7) . This can perhaps be explained by the f ind ing that the las t pulses of each t ra in are not as e f fec t i ve in sett ing up the potent iat ion as the f i r s t pulses (Gustafsson and Wigstrom, 1986). Although the "NMDA receptor theory" discussed above is a very a t t rac t ive one in explaining the induction of associat ive STP, other p o s s i b i l i t i e s should not be ruled out. Col l ingr idge and B l i s s (1987), fo r instance, suggested that the same voltage-dependent presynaptic NMDA receptors may be involved in the induction of LTP; i t i s p lausible that these same receptors may also be involved in the associat ive induction of STP. The involvement of presynaptic NMDA receptors in the induction of associat ive STP would also account for the decrease in presynaptic terminal e x c i t a b i l i t y that was observed to be associated with both associat ive STP and LTP (Sastry et a l . , 1986). Sastry et a l . (1986) suggest the p o s s i b i l i t y of ephaptic in teract ions (Richardson et a l . , 1984) between the postsynaptic neurones and the act ivated presynaptic terminals. A release of potassium ions from the dendrites which in turn af fect the presynaptic terminals has also been proposed as a possible mechanism (Sastry et a l . , 1986). Although the presence of a high level of ex t race l l u la r K suggests an increase in presynaptic terminal e x c i t a b i l i t y , i t i s possible that K have cer ta in spec i f i c membrane ef fects (not related to i t s ef fects on e x c i t a b i l i t y ) on the act ivated presynaptic terminals that can in turn af fect t ransmi t ter re lease. Furthermore, i t i s possib le that a high level of ex t r ace l l u l a r K produce changes that resu l t in a decrease rather than an increase in presynaptic terminal e x c i t a b i l i t y . High concentrations of ex t r ace l l u l a r K can cause a decrease in presynaptic terminal e x c i t a b i l i t y by inact iva t ion of Na channels or by a hyperpolar izat ion due to an enhanced a c t i v i t y of the Na + pump (Goh and Sastry, 1985b). It i s , however, d i f f i c u l t for th is hypothesis to account for the induction of assoc ia t ive potent iat ion when the test EPSP precedes the condit ioning tetanus. I t i s also possible that changes in presynaptic terminal e x c i t a b i l i t y may not be d i r ec t l y related to associat ive STP or LTP. Although a decrease in presynaptic terminal e x c i t a b i l i t y has been found to pa ra l l e l the time course of both associat ive STP and LTP, th is reported e x c i t a b i l i t y change may not necessar i ly be playing a causative role in the associat ive induction of STP or LTP. Since i t i s now observed that associat ive STP can occur regardless of whether the test stimulus precedes or fol lows the onset of the condi t ion ing t r a i n , i t appears reasonable to suggest that some postsynaptic process - possibly the release of some chemical(s) - can be act ivated by e i ther the test or the condi t ioning input which in turn a f fec ts the act ivated presynaptic terminals. This seems to be in agreement with the suggestion of Sastry et a l . (1986) that a "subl iminal f a c i l i t o r y process" can be activated even a f ter one action potent ial and can lead to potent iat ion when overlapped with the condit ioning t r a i n , presumably due to an increase in t ransmit ter re lease. Presuming that associat ive STP i s same - 83 -as tetanus-induced STP, i t seems reasonable to think that the presynaptic terminal is the actual locus of potent iat ion and that the e x c i t a b i l i t y changes associated with associat ive STP and LTP re f lec t a presynaptic hyperpolar izat ion, which has been suggested to be the cause of increased transmitter release in some other systems (L loyd, 1949; Eccles and Krn jev ic , 1959c). On the other hand, assoc ia t ive STP observed in the present study may not share the same underlying mechanism(s) as tetanus-induced STP. Present resul ts cannot rule out the p o s s i b i l i t y of a postsynaptic locus of change being at least par t ly responsible for the associat ive induction of STP, although the change(s) w i l l have to be loca l ized to the subsynaptic areas act ivated by the test input. The "NMDA receptor theory" (Wigstrom and Gustafsson, 1985b; Gustafsson and Wigstrom, 1985) discussed e a r l i e r can very well account fo r the associat ive induction of STP. A short-term change in the s e n s i t i v i t y of the subsynaptic receptors to released t ransmit ter that may be induced by e i ther the test or the condit ioning input, can also account for present resu l t s . Morphological changes in the act ivated dendr i t ic spines (Fi fkova and van Harreveld, 1977) as well as the synthesis of new proteins (Duffy et a l . , 1981) may also play a role in the associat ive induction of STP, although such changes are more l i k e l y associated with long- rather than short-term forms of potent iat ion. Further studies w i l l have to be performed before any of the above p o s s i b i l i t i e s can be el iminated. 5.2 Induction of long-term potent iat ion in the absence of ex t race l lu la r  calcium The induction of hippocampal LTP is often thought to be a 2+ Ca -dependent process (Dunwiddie et a l . , 1978; Dunwiddie and Lynch, 2 + 1979; Wigstrom et a l . , 1979). When Ca in the ex t race l l u la r medium was 2 + 2 + replaced by Mg or Mn , tetanus-induced LTP cannot be e l i c i t e d - 84 -9 + (Dunwiddie and Lynch, 1979; Wigstrom et a l . , 1979). Whether Ca is d i r ec t l y required - presynapt ica l ly to affect t ransmit ter release or 2+ postsynapt ical ly to i n i t i a t e cer ta in Ca -dependent changes - or i nd i rec t l y required because synaptic transmission during the tetanus is needed to induce a postsynaptic depolar izat ion that may be required for the induction of LTP, i s at present unknown. It has been reported that LTP can be induced when act iva t ion of a test input i s coupled with su f f i c i en t depolar izat ion of CA^ neurones (Gustafsson et a l . , 1986; Kelso et a l . , 1986; Sastry et a l . , 1986). The present ser ies of experiments aim to invest igate whether Ca would be required fo r LTP induction i f one d i rec t l y provides a concurrent ac t iva t ion of the presynaptic f i b res and depolar izat ion of the postsynaptic neurones. An af f i rmat ive answer would suggest the p o s s i b i l i t y that the induction of LTP is d i r ec t l y dependent upon ?+ 2+ Ca . Present resul ts suggest that ex t race l l u la r Ca i s not absolutely required for the induction of LTP as long as a su f f i c i en t depolar izat ion of the presynaptic terminals and postsynaptic neurones co-occur. In studies from other laborator ies in which synaptic transmission was 2+ blocked during tetanic st imulat ion by the removal of ex t r ace l l u l a r Ca (Dunwiddie et a l . , 1978; Dunwiddie and Lynch, 1979; Wigstrom et a l . , 1979), induction of LTP might have been prevented because postsynaptic depolar izat ion did not take place during act ivat ion of the presynaptic terminals. When depolar izat ion of the postsynaptic neurone co-occurs with act ivat ion (or depolar izat ion) of the presynaptic terminals, LTP can be 2+ induced in the absence of ex t race l l u l a r Ca . During tetanic st imulat ion of the presynaptic terminals, synaptic transmission and thus subsynaptic receptor act ivat ion may therefore be required to induce a su f f i c i en t 2+ postsynaptic depolar izat ion. Even in experiments where ex t race l l u l a r Ca was merely reduced and not en t i re l y el iminated (Dunwiddie and Lynch, 1979), - 85 -the postsynaptic depolar izat ion during the tetanic st imulat ion may not have been su f f i c ien t to e l i c i t LTP-inducing changes since synaptic transmission was considerably reduced by the treatment. Therefore, the "requirement1' of 2+ LTP-induction for ex t race l l u la r Ca may rea l l y be a dependence on synaptic transmission and thus postsynaptic depolar izat ion during te tan ic 2+ st imulat ion and not a d i rect dependence on ex t race l l u l a r Ca per-se. As was mentioned before, the induction of LTP requires the concurrent depolar izat ion of the presynaptic terminals and the postsynaptic neurones. The appl icat ion of elevated K + depolarizes the presynaptic terminals as well as the postsynaptic neurones and thus meets the requirement fo r the induction of LTP. Furthermore, present resul ts seem to indicate that appl icat ion of elevated ex t race l l u la r K in the absence of Ca resu l t s in potent iat ion while the same treatment in the presence of ex t race l l u l a r 2+ Ca resul ts in a tendency towards depression instead of greater potent iat ion. Chirwa et a l . (1983) and Sastry et a l . (1984a) suggest that homosynaptic as well as heterosynaptic depression involve a postsynaptic 2+ locus and are associated with an in f lux of Ca into CA^ neurones. On the other hand, high frequency tetanic st imulat ion that induce LTP was found 2+ not to be associated with any measurable increase in i n t r a c e l l u l a r Ca levels (Chirwa et a l . , 1983). The inef fect iveness of verapamil as well as 2+ other organic Ca -channel blockers in blocking the induction of LTP also argues against an induction process that requires a general postsynptic 2+ in f lux of Ca . Some caution should perhaps be exercised in the 2+ in terpretat ion of experiments involv ing Ca channel blockers because there has been some controversy as to the i r true effect iveness in blocking 2+ Ca entry into various neuronal t issues (Augustine et a l . , 1987). It i s 2+ possible that in the presence of ex t race l l u l a r Ca , elevat ing the concentration of ex t race l l u la r K + induces a depression (s imi la r to that - 86 -induced by low-frequency tetanic st imulat ion as described by Chi'rwa et a l . , ' 1983) which co-occurs with the potent iat ion of the population EPSP and thus mask the expression of the potent ia t ion. 2+ It was reported in sect ion 4.2.1 that s l i c e s exposed to Ca -free medium containing normal (3.1 mM) level of ex t race l l u l a r K + exhibi ted a s l igh t potent iat ion of the CA^ population EPSP. This observation is perhaps analogous to that of Goh (1986), who reported a potent iat ion of the population EPSP a f ter a 10 min period where input st imulat ion was interrupted. Goh (1986) also observed a s l igh t potent iat ion of the EPSP 2 + subsequent to a 10 min exposure of s l i c e s to Ca - f ree medium. Therefore, a mere interrupt ion of synaptic a c t i v i t y can produce a cer ta in degree of synaptic potent ia t ion. However, i t can be seen (Table 1) that increasing + 2 + concentrations of ex t race l l u la r K ( in Ca - f ree medium) did produce subs tan t ia l l y greater potent iat ion of the EPSP, ind icat ing that the potent iat ion was not merely a consequence of the in terrupt ion of synaptic transmi ss ion. Turner et a l . (1982) reported a LTP- l ike potent iat ion of the population spike and population EPSP responses of CA^ pyramidal neurones fo l l ow ing .a t ransient elevat ion of ex t race l l u l a r Ca levels (from 2 to 4 mM). 2+ Moreover, an increase in the " i n t r a c e l l u l a r " Ca content reportedly pa ra l l e l s the above LTP- l ike state (Turner et a l . , 1982). It i s , however, 2+ possible that increasing ex t race l l u l a r Ca resulted in increased membrane 2 + binding of Ca and that th is "potent ia t ion" was a resu l t of the bound 2+ 2 + 2 + Ca and not an increase in i n t r a c e l l u l a r Ca . Ca cart bind to or screen the negative charges on the presynaptic membrane surface to cause an increase in transmitter release (Bl ioch et a l . , 1968). The slowness in the 2+ removal of th is membrane bound Ca could also account for the prolonged 2+ increase in tota l Ca content. As was mentioned before, app l icat ion of - 87 -+ 2+ elevated K in the presence of ex t race l l u la r Ca led to prolonged depression rather than greater potent ia t ion. It i s conceivable that depolar izat ion of the postsynaptic neurone leads to a greater in f lux of 2+ Ca into CA^ neurones and thus resul ts in prolonged depression. As was also discussed e a r l i e r , neuroleptics have been reported to block the induction of LTP in hippocampal s l i c e s (Finn et a l . , 1980; Mody et a l . , 1984). These authors suggested that neuroleptics exert the i r LTP-blocking ef fects through an antagonism of calmodulin-act ivated events (Finn et a l . , 1980; Mody et a l . , 1984). However, the s e l e c t i v i t y of t r i f l uoperaz ine or other neuroleptics as a calmodulin i nh ib i to r has often been questioned (Augustine et a l . , 1987). Because of i t s highly hydrophobic nature, t r i f l uoperaz ine , fo r instance, has been shown to have a wide var ie ty of actions upon various other molecules with hydrophobic components (Augustine et a l . , 1987). Among i t s many other e f f ec t s , t r i f l uoperaz ine has been shown to block the actions of protein kinase C (Wise et a l . , 1982), a substance that has been implicated to have more than a minor role in the induction of LTP (Malenka et a l . , 1985, 1987). The phenothiazines have also been shown to i nh ib i t the Ca -uptake of the mitochondria (Hirata et a l . , 1982), 2+ which may in turn af fect the mobi l izat ion of i n t r a c e l l u l a r Ca and possibly the induction of LTP (Stanton and Schanne, 1986). Moreover, Augustine et a l . (1987) pointed out that because of the mult ip le ro les of calmodulin in c e l l funct ion, the in terpretat ion of experiments using even very highly se lect ive inh ib i to rs of calmodulin would not be a simple matter. A popular hypothesis for the induction of LTP is that te tan ic st imulat ion of exc i ta tory pathways produces a temporary unblocking of the 2+ NMDA receptors by Mg and allows a transient expression of the NMDA receptor system, thus i n i t i a t i n g the process(es) leading to LTP (Col 1ingridge, 1985). Since transmit ter release and thus receptor T 8 8 -act ivat ion appear not to be absolutely required to induce LTP as long as a coincident depolar izat ion of the presynaptic terminals and postsynaptic neurones i s provided, resul ts from th is study are not in support of the above hypothesis. APV, a purported NMDA receptor antagonist, has been reported to block tetanus-induced (Col 1ingridge et a l . , 1983b) as well as associat ively- induced LTP (Wigstrom and Gustafsson, 1985). Whether APV rea l l y prevents the induction of LTP by blocking NMDA receptors has never been c lea r l y es tab l ished. Moreover, the propert ies of APV (as well as those of other amino acid "antagonists") have not been rea l l y c l ea r l y e luc idated. It has been suggested that APV masks rather than blocks LTP induction and 2+ that APV increases the postsynaptic Ca in f lux into CA^ neurones during the tetanic st imulat ion leading to depression (Sastry et a l . , 1984b). APV may not be as se lec t ive an antagonist as was claimed (Col 1 ingridge et a l . , 1983b) and may have some other propert ies that are unrelated to i t s action as a NMDA receptor "antagonist" . King and Dingledine (1985) reported that APV can act ivate before blocking NMDA receptors in rat hippocampal s l i c e s . It appears that fur ther studies are required before these p o s s i b i l i t i e s can be c l a r i f i e d . Based on the present resu l t s , i t seem possible that co-ac t iva t ion (or a depolar izat ion) of the presynaptic terminals and postsynaptic neurones 2+ induces some vo l tage-sens i t i ve , ex t race l l u la r Ca -independent process whose components have yet to be e luc idated. Perhaps depolar izat ion of the postsynaptic neurone causes a release of some second messanger that may in turn act on the act ivated presynaptic terminals to induce cer ta in long-term changes (as was f i r s t suggested by Eccles in 1983). This i s supported by a recent study by Chirwa and Sastry (1987a) which demonstrated the tetanus-induced release of a heat-sensi t ive chemical that causes LTP of the CAj population spike when applied onto hippocampal s l i c e s . There i s also - 89 -the p o s s i b i l i t y that a coupling of postsynaptic depolar izat ion with presynaptic terminal act ivat ion leads to some e l e c t r i c a l interact ions that modify the presynaptic release process resu l t ing in LTP. Results from the present study do not rule out the p o s s i b i l i t y that 9 + i n t r a c e l l u l a r mobi l izat ion (or red is t r ibu t ion) of Ca may be responsible fo r the induction of LTP. Lynch et a l . (1983) reported that i n t r a c e l l u l a r in jec t ion of EGTA into postsynaptic neurones block the induction of 2+ tetanus-induced LTP. The mobi l izat ion of i n t r a c e l l u l a r Ca may very wel l be t r igger ing the release of the second messenger discussed above. If LTP 9+ involves only a postsynaptic locus, the mobi l izat ion of i n t r a c e l l u l a r Ca may also be the cause of the subsequent events which leads to changes in the shape of the dendrites or any other changes that resul ts in the enhancement of synaptic e f f i cacy (Lynch and Baudry, 1984). If postsynaptic changes are indeed responsible fo r the induction of LTP, these changes w i l l l i k e l y be loca l ized to the act ivated subsynaptic s i t e s , since presynaptic ac t i va t ion i s also a prerequsite condit ion for i t s induct ion. It has been observed that caf fe ine (Auyeung and Sastry, unpublished observations) and 3- isobutyl- l -methylxanthine (IBMX; Mody and M i l l e r , 1984) induce LTP of the CA^ neuronal EPSP when appl ied, even when synaptic transmission was s i g n i f i c a n t l y reduced. Mody and M i l l e r (1984) suggested 9+ i n t r a c e l l u l a r Ca release from storage s i tes to be the cause of the IBMX-induced LTP. Whether or not th i s IBMX- or caffeine-induced LTP can be blocked by APV (and i f so, whether the blockade i s mediated through NMDA receptor or through some other mechanisms) needs to be invest igated. MacVicar and Baker (1985) demonstrated that in a low Ca -cadmium medium caffeine e l i c i t s a slow prolonged depolar izat ion of the CA^ neurone caused by a slow inward Na + current that i s sens i t i ve to tetrodotoxin (TTX). In t race l lu la r EGTA in jec t ion was found to block th i s caffeine-induced slow - 90 -• + depolar izat ion, suggesting that the slow inward Na current may be 2 + regulated by i n t r a c e l l u l a r Ca mobi l izat ion (MacVicar and Baker, 1985). This raises the p o s s i b i l i t y that LTP can be induced when th is caf fe ine- or IBMX-induced slow depolar izat ion co-occured with ac t iva t ion of the input f i b r e s . A l te rna te ly , caf fe ine or IBMX could induce the slow inward current in presynaptic terminals, thereby depolar iz ing them in associat ion with a s im i la r postsynaptic depolar izat ion leading to LTP. Results from section 4.2.2 show that the induction of LTP in 2+ Ca - f ree medium can occur in the absence of p ic ro tox in in the perfusing media, suggesting that p icrotoxin f a c i l i t a t e s rather than i s mandatory for the induction of potent iat ion of the EPSP caused by depolar izat ion of the presynaptic terminals and postsynaptic neurones. Wigstrom and Gustafsson (1984) suggested that besides causing a reduction of i n h i b i t i o n , p icrotoxin also act ivates an addit ional dendr i t ic depolar iz ing process during tetanic act ivat ion that enhances the production of LTP. Present resul ts also seem to suggest that GABA receptor blockade (by picrotoxin) i s not mandatory fo r , but perhaps plays a modulatory role in the induction of LTP. Based on present ly avai lab le resul ts from th is and other laborator ies, many d i f ferent models for the induction of hippocampal LTP can be put forward. Protein kinase C is a promising candidate whose act ivat ion may at least be par t l y responsible for the induction of LTP in the hippocampus (Routtenberg and Lovinger, 1985; Linden et a l . , 1985; Lovinger et a l . , 1986; Malenka et a l . , 1986; Routtenberg et a l . , 1986 ). Girard et a l . (1985) provided evidence suggesting that protein kinase C can be found in presynaptic terminals. Phorbal esters, agents that act ivate protein kinase C by mimicking the actions of the second messenger d iacy lg lycero l (Castagna et a l . , 1982), have been observed to cause LTP of the CA^ population spike and population EPSP (Malenka et a l . , 1985). Phorbal esters did not, when - 91 -appl ied, have any ef fect on the f i e l d responses of postsynaptic neurones in hippocampus e l i c i t e d by pressure appl icat ion of glutamate (Malenka et a l . , 1985). This perhaps suggest that the phorbal ester is probably not inducing LTP by any a l te ra t ions in the postsynaptic parameters (Malenka et a l . , 1985). It has been suggested e a r l i e r that a su f f i c ien t postsynaptic depolar izat ion act ivates a vo l tage-sens i t ive release of a chemical(s) that may act on the act ivated presynaptic terminals (Sastry et a l . , 1985). Perhaps th is (these) chemical(s) released can act ivate the presynapt ical ly located protein kinase C and thus lead to LTP. In support of the above hypothesis, Malenka et a l . (1987) demonstrated an increase in the frequency of spontaneous miniature EPSPs recorded from CA^ neurones with the 2+ appl icat ion of phorbal esters in Ca - f ree medium. The exact mechanism(s) by which the act ivat ion of protein kinase C is at present unknown. Some of the p o s s i b i l i t i e s may involve a broadening of the presynaptic act ion potent ial (Kle in et a l . , 1980; Storm, 1987), or a recruitment of new Ca -channels on the presynaptic terminals (Strong et a l . , 1987), e i ther of which can lead to an enhanced release of t ransmit ter substances. Very recent ly, Hu et a l . (1987) reported that i n t r a c e l l u l a r in ject ions of prote in kinase C in some CA-^  pyramidal neurones induced a LTP- l ike enhancement of synaptic transmission. This raises the p o s s i b i l i t y of the involvement of a postsynaptic ac t iva t ion of protein kinase C in the induction of LTP. Act ivat ion of protein kinase C postsynapt ica l ly may induce changes in the act ivated subsynaptic s i tes that can lead to LTP. A l te rna te l y , a postsynaptic ac t iva t ion of protein kinase C can cause the release of the chemical(s) that may act on the act ivated presynaptic terminals to increase transmitter release (Sastry et a l . , 1986). Spec i f ic inh ib i to rs of prote in kinase C (Augustine et a l . , 1987) could probably be used in future experiments in order to c l a r i f y these p o s s i b i l i t i e s . - 92 -The present observation that synaptic potent iat ion can be induced in 2+ 2+ the absence of Ca leads one to re-examine the role Ca plays in the induction of synaptic potent iat ion and perhaps opens up some new perspectives toward the understanding of synaptic modulatory processes in the central nervous system. There is of course the question as to whether the LTP observed in the present work (with minimal ex t race l l u la r Ca ) is the same as the "normal" LTP induced by te tan izat ion of exci tatory inputs (Schwartzkroin and Wester, 197 5; Andersen et a l . , 1977; McNaughton, 1982). LTP observed in th is study i s comparable to that e l i c i t e d by high frequency tetanic st imulat ion of the input (Andersen et a l . , 1977). As have been discussed before, perhaps during a tetanic st imulat ion of the input , synaptic transmission and therefore subsynaptic receptor ac t iva t ion is involved in inducing th is postsynaptic depolar izat ion which appears to be needed for the induction of LTP (Sastry et a l . , 1986). When one can d i rec t l y provide th is postsynaptic depolar izat ion concurrently with act ivat ion of the presynaptic f i b r e s , act ivat ion of any subsynaptic receptors does not appear to be required to cause th is process of induct ion. Therefore, the induction of LTP does not seem to be d i r e c t l y 2+ 2 + dependent on Ca or any spec i f i c ex t race l l u l a r Ca -mediated event. - 93 -6. CONCLUSIONS Ever since the ear ly works of Schi f f (1858) and Boehm (1894) in the neuromuscular junc t ion , STP has been extensively studied in many di f ferent systems (Hughes, 1958). STP in central neurones, espec ia l l y PTP, has pa r t i cu la r l y attracted the attent ion of invest igators because i t has been used as a model fo r learning and memory. At least a large part of that interest has been shi f ted towards LTP ever since i t was f i r s t reported (B l i ss and Gardner-Medwin, 1973; B l i s s and L0mo, 1973). For various reasons discussed e a r l i e r , LTP i s at present seen as the most promising model for explaining the processes involved in memory and learning (Chung, 1977; Ecc les , 1985; Racine and K a i r i s s , 1987). The induction of LTP is widely believed to be a Ca -dependent process (Dunwiddie et a l . , 1978; Dunwiddie and Lynch, 1979; Wigstrom et a l . , 1979), although various 2+ 2+ components of STP have been shown to be inducible in low Ca /high Mg medium (Dunwiddie and Lynch, 1979). In 1986, Sastry et a l . reported that LTP as well as STP could be assoc ia t ive ly induced in CA^ neurones without d i rec t tetanic st imulat ion of the (test) input f i b res , but when i t was act ivated during e i ther a tetanic st imulat ion of other converging but independent exc i ta tory inputs or a depolar izat ion of the postsynaptic neurone. In the f i r s t part of th is study, i t was observed that STP could be induced when the act iva t ion of the test input preceded or followed the onset of the condit ioning tetanic t ra in by a cer ta in temporal i n t e r va l . Some of the possible mechanism underlying th is potent iat ion were discussed. In the second part of th is study, i t was reported that LTP could be 2+ induced in the absence of Ca in the ex t race l l u la r medium i f su f f i c ien t depolar izat ions of the presynaptic terminals and postsynaptic neurones - 94 -co-occur. LTP of the CA^ population EPSP was observed fol lowing the + 2+ appl icat ion of elevated ex t race l l u l a r K in Ca - f ree medium. Potent iat ion of the i n t r a c e l l u l a r l y recorded EPSP was also observed when the presynaptic f ib res was concurrently act ivated with depolar izat ions of the CA^ neurone by d i rect current i n jec t i on . Because of th is f ind ing , i t was suggested that ex t race l l u la r Ca and thus synaptic transmission are not required for the induction of LTP as long as act ivat ion of the presynaptic terminals and a su f f i c i en t depolar izat ion of the postsynaptic neurones co-occured. 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