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Differential effects of calcium and tetanic stimulation frequencies on hippocampal synaptic potentiation… Chirwa, Sanika Samuel 1985

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DIFFERENTIAL EFFECTS OF CALCIUM AND TETANIC STIMULATION FREQUENCIES ON HIPPOCAMPAL SYNAPTIC POTENTIATION AND DEPRESSION. By SANIKA SAMUEL CHIRWA B. Sc (Pharm.)., The University of Brit ish Columbia, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology & Therapeutics, Faculty of Medicine, The University of Brit ish Columbia) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1985 © S a n i k a Samuel Chirwa, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements fo r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publi c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of fH-A^t^ACOUOW ( c f e f ^ P f c l C n C ^ The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 - i i -ABSTRACT In the hippocampus, tetanic stimulation of an input results in a long lasting potentiation (LLP) of synaptic transmission involving that input. While high frequency tetanic stimulations are preferred to e l i c i t LLP, low frequency tetanus induces homosynaptic and heterosynaptic depressions. The present investigations were conducted to (1) analyse the characteristics of pulses in orthodromic and antidromic tetanic stimulations and relate them to post-tetanic changes in evoked potentials (2) determine i f potentiation and depression co-occur and (3) determine whether an established LLP in one input is subsequently modified by the in i t ia t ion of LLP in another input (to the same CAlb neurons) or whether LLP can be reversed by homosynaptic and heterosynaptic depressions and last ly (4) determine how interference or enhancements of calcium and potassium fluxes with pharmacological substances related to potentiation and depression. Experiments were conducted on transversely sectioned rat hippocampal s l ices . Evoked potentials in subfield CAlb were e l i c i ted with stimulations of CAlb axons, commissural (Com), or Schaffer col laterals (Sch). Sch ter-minal exc i tabi l i ty was tested with a stimulating electrode placed in the Sch/CAlb synaptic regions. Recordings were made with microelectrodes posi-tioned in the CAlb ce l l bodies and/or dendritic regions, and in f i e ld CA3. It was found that potentiation and depressions co-occur. Presynaptic volleys accompanied a l l tested tetanic tra ins . Similarly , antidromic trains discharged CAlb neurons continuously but did not cause LLP. Low frequency tetanic trains caused fac i l i ta ted synchronous discharges of CAlb neurons during significant portions of these trains . In contrast, few i f any syn-chronous discharges followed high frequency tetanus. Yet high frequency tetanus e l i c i t e d LLP and low frequency tetanus caused homo- and heterosynap-t i c depressions. An establ ished LLP could be masked but not reversed by homo- and heterosynaptic depressions but t h i s LLP was not interrupted by subsequently induced LLP of a separate input. Iontophoretic L-glutamate on CAlb c e l l bodies caused depression which was more pronounced i f a tetanus was evoked during L-glutamate e jec t ions . The depressions to low frequency tetanus and L-glutamate were counteracted by verapamil. L a s t l y , barium and 4-aminopyridine potentiations were reversed with washing. Appl icat ions of these drugs d id not a l t e r Sch terminal e x c i t a b i l i t y . Tetanus induced during the presence of 4-aminopyridine s t i l l e l i c i t e d LLP. It i s concluded that homo- and heterosynaptic depressions are par t l y due to the accumulation of calcium into the CAlb neurons. The magnitude of c a l -cium entry into presynaptic and postsynaptic regions is governed by the t e t -anic frequencies evoked. The r e s u l t s are consistent with a presynaptic mediated LLP. B. R. Sastry - iv -TABLE OF CONTENTS Chapter Ti t l e Page No. A TITLE PAGE i B ABSTRACT • i i C TABLE OF CONTENTS iv D LIST OF TABLES ix E LIST OF FIGURES x F ABBREVIATIONS xi i G ACKNOWLEDGEMENTS , xi i i H DEDICATION xiv 1 INTRODUCTION , 1 2 BASIC MORPHOLOGY OF THE HIPPOCAMPAL FORMATION 3 2.1 Introduction 3 2.2 The hippocampal region 4 2.3 The hippocampus 4 2.4 Dentate gyrus 6 2.5 Hilus and CA4 region 6 2.6 Cornu ammonis 7 2.7 Fields and subfields of the cornu ammonis 8 2.8 Interneurons 9 3 ENTORHINAL CORTEX, DG AND CA MAJOR AFFERE.NTS 10 3.1 The perforant path 10 3.2 The DG mossy fibers 12 3.3 The associational and commissural projections from hilar/CA4, CA3 and CA2 f ields 12 - V -Chapter Ti t le Page No. 3.4 Interneurons 14 3.5 Summary 15 4 FURTHER MORPHOLOGICAL CHARACTERISTICS OF FIELD CA1 15 5 CA1 FIELD ELECTROPHYSIOLOGY 17 5.1 Electr ical properties of CA1 pyramids 17 5.2 CA1 pyramids and interneuron discharges 17 5.3 Basic features of evoked potentials 18 6 INTRINSIC NONSYNAPTIC IONIC CONDUCTANCES 20 6.1 Basic features 20 6.2 Sodium conductances 20 6.3 Calcium conductances 21 6.4 Potassium conductances 21 6.5 Functional role of ionic conductances 22 7 ANTIDROMIC, ORTHODROMIC AND OTHER EVOKED POTENTIALS 24 7.1 Antidromic f ie ld potentials 24 7.2 Orthodromic responses 24 7.3 Inhibitory postsynaptic potentials 26 7.4 Electrotonic coupling and ephaptic interactions 27 8 SYNAPTIC INTERACTIONS AND POSSIBLE NEUROTRANSMITTER CANDIDATES ' 28 8.1 Recurrent and feed-forward inhibition 28 8.2 Extrinsic modulatory pathways 28 8.3 Neuroactive substances 29 8.4 Excitatory amino acids in commissural and Schaffer afferents 30 - vi -Chapter Ti t l e Page No. 9 CENTRAL NERVOUS SYSTEM SYNAPTIC PLASTICITY 33 9.1 Simple model of brain function 33 9.2 Features of long-lasting potentiation and depression 34 9.3 Summary 38 10 CONSIDERATION OF POSSIBLE MECHANISMS MEDIATING LLP 40 10.1 Post-tetanic potentiation 40 10.2 Increases in afferent volley 40 10.3 Increased transmitter release 41 10.4 Spine morphology changes 42 10.5 Increased synaptic receptors 43 10.6 Miscellaneous changes 44 10.7 Homo- and heterosynaptic depression 45 11 SOME INHIBITORS OF POTASSIUM AND CALCIUM FLUXES 46 11.1 Barium 46 11.2 4-Aminopyridine 47 11.3 Verapamil 48 12 METHODS 50 12.1 Animals 50 12.2 Slice preparations 51 12.3 Slice selection and v iab i l i t y 52 12.4 Slice chamber and perfusion method 53 12.5 Preparation of standard and test media 55 12.6 Stimulating and recording electrodes 56 12.7 Positioning of electrodes 58 12.8 Laboratory e lectr ical instruments 60 - v i i -Chapter Ti t l e Page No. 13 EXPERIMENT SCHEMES 61 13.1 Induction of evoked responses 61 13.2 Tetanic frequencies 62 13.3 4-AP dose-response curves 62 13.4 Effects of 4-AP 63 13.5 Verapamil studies 63 13.6 Iontophoretic L-glutamate 64 13.7 Effects of B a + + 64 ++ + 13.8 Monitoring extracellular Ca and K changes 64 13.9 Schaffer boutons exci tabi l i ty testing 65 13.10 Homo- and heterosynaptic p las t ic i ty 65 14 RESULTS 65 14.1 Evoked potentials 65 14.2 4-AP dose-response curves 66 14.3 Verapamil 70 14.4 LLP 70 14.5 Homo- and heterosynaptic p las t ic i ty 72 14.6 Characteristics of tetanic trains 75 14.7 Extracellular C a + + and K + 80 14.8 B a + + and evoked potentials 84 14.9 Effects of 4-AP 84 ,14.10 Iontophoretic L-glutamate 91 14.11 Exci tabi l i ty of Schaffer collateral terminals 94 - vi i i -Chapter Ti t le Page No. 15 DISCUSSION 94 15.1 LLP 94 15.2 Tetanic stimulations 96 15.3 Homo- and heterosynaptic depression 97 15.4 Barium 98 15.5 4-Aminopyridine 101 15.6 Miscellaneous 103 16 CONCLUSIONS 104 17 REFERENCES 107 - ix -LIST OF TABLES Table T i t l e Page No. I Ef fects of verapamil on long las t ing potent ia t ion . 76 II Ef fects of verapamil on homosynaptic and heterosynaptic depression 77 III Drug induced enhancements associated with 4-aminopyridine perfusions during s t imulat ion , no st imulat ion and mag-nesium-mediated synaptic blockade 89 - X -LIST OF FIGURES Figure Ti t l e Page No. 1 General morphology of the hippocampal formation 5 2 The major afferent systems in the hippocampus 11 3 Evoked population spike 19 4 Antidromic and orthodromic potentials recorded in CA1 f i e ld 25 5 Slice chamber 54 6 Positioning of stimulating and recording electrodes 59 7 Population spike amplitude and latency measurements 67 8 Stimulus strength and evoked potentials 68 9 Dose response curves to the cumulative addition of 4-aminopyridine showing increases in the amplitude of evoked population spikes 69 10 Representative enhancements in evoked population spikes of CAlb pyramidal cel ls to cumulative addition of 4-aminopyri-dine during the determination of the dose response curves 71 11 Verapamil and evoked potentials 73 12 Il lustration of hippocampal long lasting potentiation 74 13 Records of individual pulses during antidromic low and high frequency tetanic stimulations 79 14 Records of individual pulses during orthodromic low and high frequency tetanic stimulations 81 - xi -Figure Ti t l e Page No. 15 Records of individual presynaptic volleys during low and high frequency tetanic stimulation of Schaffer col laterals 82 16 Effects of raised potassium concentrations on evoked population spike 85 17 Effects of barium on evoked population spike 86 Changes in evoked population spike following 18 3, 10 and 15 min exposures to barium medium. 87 19 Linear regression plots of decay times of evoked population spike following barium applications. 87 20 Plot of 4-aminopyridine perfused during synaptic transmission blockade with raised magnesium medium 90 21 4-aminopyridine induced bursting act ivity in CAlb subfield 92 22 Maintained enhancements of evoked population spike following 4-aminopyridine and 100Hz treatment 93 23 Il lustration of an antidromic single spike evoked in CA3 93 - x i i -ABBREVIATIONS AP Action potential . Asp L-aspartate. CA Cornu ammonis. CNS Central nervous system Com Commissural afferents DG Dentate gyrus EPSP Excitatory postsynaptic potential GABA Gamma-aminobutyric acid GAD Glutamic acid decarboxylase IPSP Inhibitory, postsynaptic potential NMA N-methyl-DL-aspartate NMDA N-methyl-D-aspartate PP Perforant pathway PS Population spike PW Positive wave Sch Schaffer col laterals - x i i i -ACKNOWLEDGEMENTS I am deeply indebted to my supervisor Dr. Bhagavatula R. Sastry for h is encouragement, emotional support and academic guidance. His patience and be l ie f in me afforded me the unique opportunity to make something out of my l i f e . My hear t fe l t thanks are due to Joanne W. Goh, Dr. Hermina Maretic and Dr. P. Murali Mohan for the i r f r iendship and co l laborat ion in some of the experiments in t h i s t h e s i s . I thank Elaine L. Jan and L isa Heiduschka for the i r expert assistance in the ed i t ing and format l i n e set t ing of the manu-s c r i p t , Chr is t ian Caritey for developing the s l i c e chambers used in the laboratory. The f inanc ia l supports of Graduate Student Research Assistantship from the Medical Research Council and Staff Development Fellowship from the Univer-s i t y of Zambia are greatly appreciated. - xi v -Dedicated to my father and mother brothers and s i s te rs to my f i r s t loves T i i se tso and Sanika Jnr . Thank you for your patience and s a c r i f i c e s - 1 -1. INTRODUCTION Once neuroblasts d i f f e r e n t i a t e into nerve c e l l s , they never div ide again to give r i s e to daughter nerve c e l l s (Gazen, 1970; Jacobson, 1970). In maturi ty , the only nerve c e l l development that occurs is in completing gene-t i c a l l y determined st ructural and funct ional spec ia l i za t ions whose pheno-typ ic expressions are depended on the i r regional l o c a t i o n . Hence the ner-vous system is f u l l y constructed in i t s nerve c e l l numbers and connections before i t is used (Eccles, 1977). C lear ly changes in the performance of the brain during l i f e are not due to the addit ion of new nerve c e l l s . There must e x i s t , therefore, other mechanisms that mediate processes such as learning and memory. Most neurobiologists are presently engaged in invest igat ions that are aimed at determining the subtle changes in the chemistry or microstructure and microfunction that mediate such complex funct ions . In p a r t i c u l a r , the phenomena of long las t ing potent iat ion (LLP) in the hippocampus has been receiv ing close scrut iny , for i t is thought to be involved in learning and memory (Swanson, Teyler and Thompson, 1983 review). The f i r s t detai led account of LLP was given in the early seventies ( in v ivo: B l i s s and L/mo, 1973; B l i s s and Gardner-Medwin, 1973; in v i t r o : Schwartzkroin and wester, 1975; Alger and Teyler , 1976) when i t was shown that repet i t i ve tetanic st imulations of s p e c i f i c hippocampal inputs led to the development of post -tetanic LLP. The increase in synaptic e f f i cacy could be maintained for days or weeks in v ivo . LLP presented i t s e l f as a decrease in onset latencies and/or increases in amplitudes of the evoked synaptic potent ia l s . At the i n t r a c e l l u l a r l e v e l , LLP was seen as an increased probab i l i t y in c e l l d i s -charge. - 2 -Over the last decade, i t became apparent that LLP could not be e l i c i t e d with antidromic tetanic st imulations or d i rect soma ( i n t r a c e l l u l a r ) depolar-i z a t i o n s . While orthodromic tetanic st imulations could e l i c i t LLP, such t ra ins had to be evoked in the presence of e x t r a c e l l u l a r calcium. More importantly, only the tetanised input subsequently exhibited LLP. These studies led to the conclusion that changes associated with LLP development were loca l i zed to the synaptic regions (Swanson, Teyler and Thompson, 1983 revi ew). A re lat ionsh ip between LLP development and tetanic frequencies became apparent when i t was noted that (a) high frequency tetanic st imulations most r e l i a b l y induced LLP of immediate onset (Schwartzkroin and Wester, 1975) whereas (b) low frequency tetanic st imulations usual ly caused a post - tetanic depression, even of non-tetanized inputs to the same output neurons, and t h i s depression was sometimes followed by a gradually developing LLP in the tetanized input (Dunwiddie and Lynch, 1978; Chirwa, Goh, Maretic and Sastry , 1983). The charac te r i s t i cs of the depression were consistent with a gener-a l i sed change to the postsynaptic neurons. While many features of LLP and depression have now been e luc idated, the exact mechanisms mediating LLP (or depression) have yet to be determined. Despite the concerted e f fo r ts of many invest igators , i t i s s t i l l not known whether the changes in LLP are loca l i sed to presynaptic or postsynaptic com-ponents or involve both regions. The hypothesis of Baudry and Lynch (1980) contended that there was an increase in subsynaptic receptors during LLP. Yet evidence is avai lable that correlates presynaptic a l te rat ions with LLP (Skrede and Malthe-Sorenssen, 1981; Sastry, 1982). - 3 -Prel iminary reports from our laboratory have given resu l ts that are con-s is tent with a presynaptic mediated LLP (Chirwa, Goh, Maretic and Sastry, 1983). On the other hand, the resu l t s on depression were in accordance with a postsynaptic change. The studies in t h i s thesis further examine the d i f -f e r e n t i a l e f fects of calcium and tetanic frequencies on presynaptic and postsynaptic regions and re late these ef fects to LLP and/or depression. The studies attempted to show the fo l lowing . 1. The net in f lux of calcium into presynaptic and postsynaptic regions was a function of the tetanic st imulat ion frequency. 2. Calcium entry into presynaptic regions mediated LLP development. 3 . Calcium entry into postsynaptic regions mediated depressions. 4. Presynaptic and postsynaptic ef fects of calcium could s e l e c t i v e l y be dissected out with pharmacological substances. A l l the studies in t h i s thes is were conducted on the rat hippocampal s l i c e in v i t r o . 2 . BASIC MORPHOLOGY OF THE HIPPOCAMPAL FORMATION 2.1 Introduction The bulk of the information on the hippocampal formation presented here comes from the c l a s s i c studies of Cajal (1911) and Lorente De No (1934). Hence the descr ipt ion and naming of parts of the hippocampal formation used in th i s thesis are consistent with the schemes employed by Cajal (1911) and Lorente De No (1934). However, some sections have been updated and/or c l a r -i f i e d based on recent detai led studies such as those of Blackstad (1959), White (1959) and Swanson, Wyss and Cowan (1978). - 4 -2.2 The hippocampal region The cortex is often divided into the a l locor tex or the isocortex . Dur-ing ontogenic development, the isocortex separates from the c o r t i c a l mantle. The a l locortex however does not cleave but remains as an S-shaped in fo ld ing of the c o r t i c a l mantle (F i l imonoff , 1947). This mantle layer i s further folded into a C-shape along the i n f e r i o r horn of the l a t e r a l vent r i c le and l i e s ventral and medial to the rh inal f i s s u r e . The cortex immediate to the al locortex is denoted as the peri a l locor tex . The hippocampal formation is comprised of the a l locortex and the per ia l locor tex (Chronister and White, 1975; Teyler and Discenna, 1984). The a l locor tex is subdivided into the hippocampus proper, the dentate gyrus (which together are denoted as the 'hippocampus') and much of the sub-iculum (Lorente De No, 1934; Swanson, Wyss and Cowan, 1978; Teyler and DiScenna, 1984). Parts of the subiculum ly ing adjacent to the presubiculum are usual ly assigned to the per ia l locor tex (Lorente De No, 1934). The p e r i -a l locortex i t s e l f i s largely composed of the presubiculum (area 27), the area re t rosp lena l i s e (area 29e), the parasubiculum (area 49) and the entorhinal region (area 28) (Brodman, 1909; Lorente De No, 1934; Blackstad, 1956; Chronister and White, 1975). 2.3 The hippocampus The hippocampus is a b i l a t e r a l l y symmetrical s t ructure , each consist ing of two i n t e r d i g i t a t i n g a r c h i c o r t i c a l f i e l d s termed cornu ammonis and the dentate gyrus (see f i g . 1) . The cornu ammonis and the dentate gyrus con-t a i n densely packed sheets of c e l l s , the pyramids and the granule c e l l s , which are the pr inc ipa l c e l l type of the i r respective f i e l d s . In each f i e l d are a var iety of interneurons, with the i r dendrites intermingled among those of the pr inc ipa l c e l l types. - 5 -Layers 1. Alveus.-2. Stratum o r i e n s . 3. Stratum pyramidale. 4. Stratum radiatum. 5. CA1 Stratum moleculare. 6. D6 Stratum moleculare. 7. Stratum granulosum. F i g . 1. General morphology of the hippocampal formation. The dots along the v e r t i c a l l i n e give the approximate posi t ions of the layers above. Abbreviat ions: CA, cornu ammonis; DG. dentate gyrus; Hf, hippocampal f i s s u r e ; Sub. , subiculum. - 6 -2.4 Dentate gyrus In the dentate gyrus, granule c e l l s ex is t in a single layer termed the stratum granulosum. Between the stratum granulosum and the hippocampal f i s -sure i s the second dentate gyrus layer denoted as the stratum moleculare. The granular c e l l s have the i r apical dendrites oriented toward the hippocam-pal f i ssure but with in the stratum moleculare. The dentate gyrus curves into a V- and/or U-shape around the la t te r part of the cornu ammonis f i e l d . In th i s dentate gyrus curvature, the blade or side which is adjacent to the subiculum and the i n i t i a l parts of the cornu ammonis is denoted as the suprapyramidal region. The other dentate gyrus blade, which i s i n t r a v e n t r i c u l a r , i s termed as the infrapyramidal region (Swanson, Wyss and Cowan, 1978). 2.5 Hilus and CA4 region Within the dentate gyrus concavity and close to i t s apex is the h i lus region. Some authors have considered the h i lus as the t h i r d layer of the dentate gyrus (Ca ja l , 1911; Blackstad, 1956). But within the h i l u s , the boundaries between the dentate gyrus and the cornu ammonis are not readi ly d iscern ib le (Amaral, 1978; Swanson, Wyss and Cowan, 1978). In fact th i s region is extremely var iable in appearance across species. According to Lorente De No (1934) the cornu ammonis on approaching the h i lus bends on i t s e l f , f i r s t upwards ( f i r s t blade) and then downwards (second blade) . These def lect ions are least developed in rodents and increase in complexity in the rabb i t , monkey and man, respect ive ly . A s i g -n i f i can t number of c e l l s between the f i r s t blade and the cornu ammonis pre-sumably belong to the cornu ammonis f i e l d . Yet the c e l l s between the second blade and the granular layer send the i r axons to the dentate gyrus (see later sect ions ) . Parts of the h i lus region, therefore , seem to belong - 7 -to the dentate gyrus and yet other sections are associated with the cornu ammonis. The h i l a r region therefore can be viewed as being a s t ructura l t r a n s i t i o n zone from the dentate gyrus into the cornu arrmonis. There are pyramidal c e l l s that stream out of th i s h i l a r region, and t h i s l a t t e r part comprises the area CA4 of Lorente De No, (1934). 2.6 Cornu ammonis The pyramids of the cornu ammonis f i e l d are arranged in the stratum pyramidale layer . The other layers of the cornu ammonis f i e l d include the alveus, which i s next to the epithelium of the l a t e r a l v e n t r i c l e . Between the alveus and the stratum pyramidale l i e s the stratum or iens . Next to the stratum pyramidale, but on the opposite side to the stratum or iens , are found the stratum lucidum, stratum radiatum, stratum lacunosum and stratum moleculare, in that order. The extent of development of the d i f ferent layers in the cornu ammonis varies among species. In rodents, for example, the d i v i s i o n between stratum radiatum and stratum lacunosum is somewhat a r t i f i c i a l . Consequently, in rodents, the stratum radiatum and stratum lacunosum are often described together (Lorente De No, 1934). S i m i l a r l y the stratum pyramidale and the stratum lucidum are described together. The boundaries of the cornu ammonis i t s e l f fo l low the length of the stratum radiatum. Using methods . such as reduced s i l v e r impregnation, the stratum radiatum is revealed as a dense f iberplexus above the pyramidal c e l l layer (Lorente De No, 1934). At the dentate gyrus, the cornu ammonis ends where t h i s dense f iberplexus (which i s the stratum radiatum) abruptly ceases within the hilus/CA4 regions. The boundary between the cornu ammonis and the subiculum is r e l a t i v e l y well defined and occurs where the stratum radiatum disappears. In th i s - 8 -region, the pyramidal c e l l s are not clustered together but ex is t in a s ingle layer . The main regions of the hippocampal formation are i l l u s t r a t e d in f igure 1. 2.7 F ie lds and subf ie lds of the cornu ammonis The f i e l d s in the cornu ammonis (CA) are delineated on the basis of st ructural features of the primary c e l l types in the region. For instance, two c e l l s with s i m i l a r axonal apparatus but d i f f e r i n g in dendr i t ic d i s t r i b u -t ions are taken to represent two d i s t i n c t c e l l types (Lorente De No, 1934). A l t e r n a t i v e l y , i f the dendrites are s i m i l a r but the axonal ramif icat ions are d i s i m i l a r , then such c e l l s are not equivalent . In the CA, the pyramids are the primary c e l l types. Hence the various f i e l d s and subf ie ld c l a s s i f i c a t i o n s are based on the general morphology of the pyramids in each area. Other st ructura l features such as s i z e , presence and/or absence of dendr i t i c spines are also used to describe the subf ie lds . The cornu ammonis is divided into four main f i e l d s namely CA1, CA2, CA3 and CA4 (see f i g . 1) . F ie ld CA4 has been described in section 2.5 above. The CA3 pyramids are large with ascending shafts (apical dendrites) that do not have side branches in the stratum radiatum but branch out upon reach-ing the stratum moleculare (Lorente De No, 1934). The i n i t i a l parts of these apical dendrites possess thick spines, which are in contact with the terminals of mossy f ibers from the dentate gyrus granule c e l l s . F i e l d CA3 is further subdivided into CA3a, CA3b and CA3c. Subf ield CA3c i s adjacent to CA4 and i t s pyramidal c e l l s have thick spines on both apical and basal dendr i tes . Presumably only CA3c pyramids are simultaneously i n -nervated by mossy f ibers of the infrapyramidal and suprapyramidal DG (gran-ule c e l l s ) blades (Lorente De No, 1934). - 9 -Subfield CA3b consists of mixed pyramids; and some 5C% of these have Schaffer c o l l a t e r a l s (see l a t e r sect ions ) . In contrast , the pyramids in CA3a (unlike CA3b and CA3c) do not give Schaffer c o l l a t e r a l s according to Lorente De No (1934). Most of the CA3a pyramids send out myelinated axons that give out one or two myelinated c o l l a t e r a l s that ascend to the stratum radiatum where they form an associat ional pathway running within the stratum radiatum of CA3 to CAlb. This powerful associat ion pathway has yet to be f u l l y character ized. Next to subf ie ld CA3a is the f i e l d CA2. Some authors consider CA2 as a smal l , t r a n s i t i o n a l zone between CA3 and CA1 (Swanson, Wyss and Cowan, 1978). Though the CA2 pyramids are large ( i . e . , s i m i l a r to those of CA3), the i r dendrites lack thick spines and are without mossy f ibe r inputs (Lorente De No, 1934; Haug, 1974; Swanson, Wyss and Cowan, 1978). Unlike CA3 pyramids, the apical dendrites of CA2 pyramids begin to div ide immediately after leav-ing the c e l l body layer . CA2 pyramids do not give Schaffer c o l l a t e r a l s . Even though CA1 is subdivided into CAla, CAlb and CAlc , these d i v i s ions seem to be more c lear in primates than in rodents (Lorente De No, 1934). The onset of CAlc from CA2 area is characterized by the presence of smaller pyramidal c e l l s . Their dendrites are f i n e , with numerous side branches. Calb has the smallest pyramids in the whole cornu arrmonis. The Schaffer c o l l a t e r a l s of CA3 and CA4 cease at the l i m i t of CAlb and CAla. Subfield CAla i t s e l f has a mixed primary c e l l population that comprises, in par t , of subiculum c e l l s . 2.8 Interneurons The pyramidal c e l l s in the CA and the granule c e l l s in the DG make up 96-98% of the neuropil of the hippocampus (Buzsaki , 1984). However, other types of neurons are d is t r ibuted within these regions. - 1 0 -In the stratum granulosum layer can be found a var iety of interneurons ( i . e . , the basket c e l l s of Lorente De No, 1934; C a j a l , 1911). On the basis of the i r shape, the c l a s s i f i c a t i o n of these interneurons include pyramidal, ho r i zonta l , fus i form, inverted fusiform and mult ipolar (Ribak and Seress, 1934; Buzsaki, 1984). The dendrites of these interneurons have l i t t l e or no spines (Amaral, 1978; Ribak and Seress, 1983; Buzsaki , 1984). Their axons form an extensive plexus in the granule c e l l s and inner stratum mole-culare layers that subsequently form synapses on the c e l l body and/or p r o x i -mal dendrites of granule c e l l s . S i m i l a r l y , interneurons c l a s s i f i e d as fus i form, s t e l l a t e , spheroid or unipolar are d is t r ibuted in the hilar/CA4 regions (Amaral, 1978). Their dendrites lack spines and these interneurons possess l o c a l l y arboriz ing axons (Buzsaki , 1984). Within the CA f i e l d s are found large interneurons of the fol lowing va r ie ty ; b ipo la r , fus i form, t r i angu la r , polygonal, hor izontal and Golgi type II (Ribak, Vaugh and S a i t o , 1978). In add i t ion , small f u s i -form and ovoid interneurons have been i d e n t i f i e d in the CA f i e l d s . 3 . ENTORHINAL CORTEX, DG AND CA MAJOR AFFERENTS 3.1 The perforant path Figure 2 i l l u s t r a t e s the major afferents of the hippocampus. The medial and la te ra l parts of the i p s i l a t e r a l entorhinal cortex send out f ibers term-ed perforant path (PP) that form synapses on the spines of the dentate gyrus granule c e l l dendrites in the middle and outer th i rds of the DG molecular layer (Hjorth-Simonsen, 1973; Matthews, Cotman and Lynch, 1976). Even the contra latera l entorhinal cortex sends out a small f rac t ion of i t s perforant path f ibers to each hippocampal DG area (Zimmer and Hjorth-Simonsen, 1975). F i g . 2. The major afferent systems in the hippocampus. Abbreviations denote: PP, perforant pathway; Mf, mossy f i b e r s ; Com, commissural a f fe rents ; Sch, Schaffer c o l l a t e r a l s . The arrows in diagram show the branches of the CA1 axons in the alveus. - 12 -In rodents, the PP projection is perpendicular to the main axis of the hippocampal formation. Some divergence has been observed in the l a t e r a l PP pro jec t ion , where a small zone in the entorhinal cortex actual ly innervates a much wider zone within the DG (Wyss, 1981). Though not completely character ized, evidence is ava i lab le that has demonstrated a d i rec t PP projection to the CA f i e l d s (Gott l ieb and Cowan, 1972; Steward, 1976). It i s also known that some DG interneurons project the i r dendrites into the termination zone of the PP (Ribak and Seress, 1983). It is probable that some PP f ibers make contact with the dendrites of i n t e r -neurons . 3.2 The DG mossy f ibers The DG granule c e l l s give out narrow bands of axons termed mossy f ibers that extend transversely across f i e l d CA3 (Blackstad, Brink, Hem and Jeune, 1970). There is l i t t l e overlap of the mossy f ibers from granule c e l l s at adjoining l e v e l s . Mossy f ibe rs are highly laminated, and form synapses on the spines of proximal dendrites of CA3 pyramids. The mossy f ibers of gran-ule c e l l s in the infrapyramidal DG blade innervate CA3c. Granule c e l l s in the suprapyramidal DG blade send out mossy f ibers across the ent i re CA3 f i e l d (Lorente De No, 1934; Haug, 1974; Swanson, Wyss and Cowan, 1978). A small f rac t ion of mossy f ibers synapse with c e l l s in the h i l a r region (Blackstad and Kjaerheim, 1961) and interneurons in f i e l d CA3 (Tombol, Babosa, Hajdu and Somogyi, 1979; Misgeld , Sarvey and Klee, 1979). 3.3 The associat ional and commissural projections from hilar/CA4, CA3 and  CA2 f i e l d s H i l a r c e l l s send out associat ional and commissural f ibers which termin-ate on the inner t h i r d of the DG molecular layer . As is true for most of the major afferents in the hippocampus, i t i s not known exactly whether h i l -- 13 -ar projections terminate on DG granule c e l l s and/or just make contact with the DG interneurons (Swanson, Sawchenko and Cowan, 1981). CA4 and some CA3 pyramids send out axons to the i p s i l a t e r a l and cont ra -l a t e r a l CA3 f i e l d s (Gottl ieb and Cowan, 1973). These f ibe rs establ ish syn-apt ic contacts with CA3 c e l l dendrites in the stratum oriens and the stratum radiatum layers (Swanson, 1973). CA3 and CA4 axons also const i tute a com-missural projection to the contra latera l CA1 f i e l d which synapse with CA1 c e l l dendrites mostly in the stratum oriens (Schaffer , 1892; Laurberg and Sorensen, 1981). L a s t l y , from the CA3 and CA4 pyramids th ick axons, branch out several c o l l a t e r a l s . Some of these c o l l a t e r a l s are short and end within the CA3 stratum oriens and/or between the loca l CA3 pyramids. There are also th ick and longer c o l l a t e r a l s from axons of CA4, CA3c and some CA3b pyramids (Lorente De No, 1934) that cross the stratum pyramidale and the stratum radiatum layers and then enter the stratum lacunosum ( s t i l l considered as stratum radiatum in rodents) where they const i tute myelinated horizontal f i be rs termed Schaffer c o l l a t e r a l s (Sch). The Schaffer c o l l a t e r a l s inner -vate the apical dendrites of CA1 pyramids. According to Lorente De No, (1934) CA2 pyramids and most of CA3c do not give out Schaffer c o l l a t e r a l s . Instead, CA2 pyramids send out axons from which branch out thick c o l l a t e r a l s which cross through the stratum pyramid-ale and enter the stratum radiatum, where they const i tute a strong l o n g i t u -dinal (ax ia l ) associat ion pathway (up to CAlb) . In add i t ion , the CA2 pyra-midal c e l l axons form horizontal c o l l a t e r a l s and t ravel within the stratum oriens towards the subiculum and entorhinal cortex. - 14 -CA1 pyramids send out axons that (a) branch out within the stratum oriens (b) ascend and ramify in the stratum radiatum and/or (c) have c o l l a -te ra ls that reach and travel in the alveus. These CA1 axonal c o l l a t e r a l s in the alveus ex i t out v i a the f imbr ia , and some go to the subiculum and areas beyond the entorhinal cortex. Some CA1 axon c o l l a t e r a l s d i s t r ibu te back to CA1 and CA2 pyramidal layers . But these CA1 axons are not in contact with the pyramids of CA3 or CA4, nor the DG granule c e l l s . CA1 axons project out of the hippocampus to other brain regions such as the la te ra l septal nuclei and prefrontal cortex (Swanson and Cowan, 1977; Swanson, 1981). 3.4 Interneurons Evidence in the l i t e r a t u r e indicates that most of the major inputs to the hippocampus simultaneously innervate interneurons and pr inc ipa l c e l l s . The primary physiological function of these interneurons is to cause i n h i b i -t ion of the pr inc ipa l c e l l s in each hippocampal f i e l d (Kandel, Spencer and Br in ley , 1961; Andersen, Eccles and L i n i n g , 1963; Storm-Mathisen, 1977; Ribak, Vaugh and S a i t o , 1978; Seress and Ribak, 1983). A var iety of other putative transmitters and neuromodulators are contained in some interneurons (Buzsaki , 1984 review). Though i t i s l i k e l y that some interneurons in fact exc i te the p r inc ipa l c e l l s , the overwhelming evidence presently implicates interneurons as medi-ating i n h i b i t i o n (Andersen, Eccles and Ljrfyning, 1964; Andersen,1975; Turner and Schwartzkroin, 1981; Fox and Ranck, 1981; F inch, Nowlin and Babb, 1983). Both recurrent and feed-forward i n h i b i t i o n have been demonstrated (Buzsaki, 1984 review). - 15 -3.5 Summary The hippocampal formation can be viewed as consist ing of adjoining cor -t i c a l bands (DG, CA3, CA1 and subiculum) that are folded along the i r l o n g i -tudinal a x i s . Axons from one c o r t i c a l band ( e . g . , DG) are in p a r a l l e l arrangement and cross the border to innervate the next s t r i p at r ight angles (Andersen, 1983). Each major pathway ends on a l imited part of the dendr i t -i c t ree of the rec ip ient c e l l s in a par t i cu la r f i e l d ( t h i s is afferent lam-i n a t i o n ) . In general, the segregation of d i f ferent synapses are t y p i c a l l y arranged as fol lows (a) exc i tatory c e l l s are confined to dendr i t ic spines whereas (b) inh ib i to ry interneurons terminate on the soma, i n i t i a l axon and/or i n i t i a l parts of dendrites (Andersen, Blackstad and Ltfmo, 1966). Other than the inputs discussed in the preceding sect ions , there ex is ts a multitude of pathways that project to the hippocampal formation (Buzsaki, 1984). These have not been discussed here since detai led information i s presently not a v a i l a b l e . L a s t l y , much i s known about the organization of the longer intrahippocampal pathways, e . g . , mossy f i b e r s , Schaffer c o l l a t e r -a l s , e tc . However, de ta i l s on (a) the i r s p e c i f i c connections on indiv idual neurons and/or (b.) the exact connections with the many classes of interneur -ons remain to be e luc idated. 4. FURTHER MORPHOLOGICAL CHARACTERISTICS OF FIELD CA1 The hippocampus has been well defined anatomically and phys io log ica l l y and therefore serves as a convenient model for the e luc idat ion of central nervous function and structure . Most of the experimental data presented in t h i s thes is were obtained from subf ie ld CAlb of the rat hippocampus. For that reason much of the discussion in the fo l lowing sections pertain to the CA1 pyramids. - 16 -Each CA1 pyramidal soma i s t y p i c a l l y oval and oriented such that the long axis i s v e r t i c a l to the alvear surface. On average the soma is about 40 by 20 microns. Its basal dendrites ex i t from the soma in a bush- l ike fashion with i r regu lar branches that div ide repeatedly. The apical den-d r i tes extend outward, in p a r a l l e l , towards the hippocampal f i s s u r e . Then at i n t e r v a l s , thinner secondary dendrites emerge out of the apical dendr i t ic shaf ts . These smaller dendr i t ic branches are inundated with numerous spines. Re lat i ve ly fewer spines are located on the primary dendr i t ic shaf t . Andersen, S i l f v e n i u s , Sundberg and Sveen (1980) used electron micro-graphs prepared from u l t r a t h i n sections of guinea-pig hippocampus to further analyse the f ibe r or ientat ion on pyramids in f i e l d CA1 that golgi impregna-t ion studies had previously shown to be in p a r a l l e l with the pyramidal layer (Golgi , 1886; Schaffer , 1892; C a j a l , 1911). In addi t ion , the electron microscopic studies of westrum and Blackstad (1962) had revealed that at in terva ls these p a r a l l e l afferents in the s t rata oriens/radiatum sent out en  passage boutons that made contact with the CA1 dendri tes. In t h i s scheme each f ibe r makes a series of synaptic contacts on the dendrites of numerous c e l l s as the axon procedes along i t s transverse t ra jec to ry . Andersen, S i l f v e n i u s , Sundberg and Sveen (1980) found that the number of synapses possessing spec ia l ised contacts with the dendr i t ic spines was u n i -form (about 42 per 100 urn) across the main parts of the st rata oriens and radiatum. In these regions, close to 95% of a l l synaptic contacts termin-ated on asymmetric dendr i t i c spines and the rest were in d i rec t contact with the dendrites. Towards the ends of the dendrites, however, the number of spine-contact ing synapses decreased and there was an increase of about 30% in the number of synapses in d i rec t contact with the dendrites (Ander-- 17 -sen, S i l f v e n i u s , Sundburg and Sveen, 1980). S i m i l a r l y a high d i s t r i b u t i o n of d i rec t contact spines was observed in the soma area, i . e . , stratum pyra-mid ale and the i r immediate regions. 5. CA1 FIELD ELECTROPHYSIOLOGY 5.1 E l e c t r i c a l properties of CA1 pyramids Schwartzkroin (1975; 1977) used guinea pig hippocampus in v i t r o to character ize the passive membrane properties of CA1 pyramidal neurons and found them to be s i m i l a r to those of the pyramidal neurons described in intact animal preparations (Kandel and Spencer, 1961; Spencer and Br in ley , 1961; Spencer and Kandel, 1961). The average spike amplitudes obtained were 64.9 ± 10 mV in v i t r o . The c e l l res is tances , calculated from the slope of the current -voltage (I-v) curves yielded values of 16.3 ± 5.3 Mn. The c e l l time constant, i . e . , the latency from onset of the pulse to l - ( l / e ) of the peak voltage d e f l e c t i o n , was 9.8 ± 3 . 4 msec (Schwartzkroin, 1977). Schwartzkroin (1977) att r ibuted the considerable v a r i a b i l i t y of resistance and time constant measurements as being due to real di f ferences among the sampled neurons. In any case, recent studies that have used advanced tech -niques (Turner and Schwartzkroin, 1980; Brown, F r i ck le and Perke l , 1981; Turner, 1982) have generated data on CA1 neuronal input res is tance, time constant, time constant ra t ios and e lect rotonic length which are cons is tent -ly s i m i l a r among d i f fe rent invest igators and are in agreement with values reported by Schwartzkroin in 1977. 5.2 CA1 pyramid and interneuron discharges CA1 pyramidal neurons often generate a burst of 2-10 action potent ials (Ranck, 1973) of decreasing amplitude and increasing durat ion, i . e . , complex - 18 -sp ike. I n t race l lu la r records (Schwartzkroin, 1975) have shown that the com-plex-spike is comprised of long duration action potent ia ls and depolar iz ing a f t e r - p o t e n t i a l s . However, orthodromic and antidromic act ivat ion tend to e l i c i t a s ingle action potential due to the act ivat ion of recurrent i n h i b i -t ion (Andersen, Eccles and Ltfyning, 1964). Unlike CA1 pyramids, the interneurons mediating synaptic i n h i b i t i o n give c h a r a c t e r i s t i c short -duration action p o t e n t i a l s . Activated interneur-ons exhib i t repet i t i ve and high frequency f i r i n g (Andersen, Eccles and L i n i n g , 1964). 5.3 Basic features of evoked potent ia ls Andersen, Eccles and Loyning (1964) showed that large i n t r a c e l l u l a r hyperpolarizations in CA1 neurons could be evoked by st imulat ion of the com-missural and the Schaffer c o l l a t e r a l s . Concurrent with these large i n t r a -c e l l u l a r hyperpolar izat ions, termed inh ib i to ry postsynaptic potent ia ls (IPSP), were the laminated e x t r a c e l l u l a r pos i t ive potent ia ls termed posi t ive waves (PW). S imi lar times were demonstrated between the onset of the IPSP and of the PW. When there was an exc i tatory postsynaptic potent ia l (EPSP) or action potent ia l superimposed on the IPSP response, the latency of the IPSP was always longer by 0.8 to 2.3 msec. If such an EPSP and/or action potent ia l preceded the IPSP, the PW exhibited an e a r l i e r onset than the i n t r a c e l l u l a r l y recorded IPSP. It was concluded that the PW was the net f i e l d potent ia l produced by hyperpolariz ing current sources at or near the somata of CA1 neurons and by sinks in the dendri tes. To a lesser extent and accounting for the e a r l i e r onset of the PW, r e l a t i v e to i n t r a c e l l u l a r IPSP, was the addit ion of p o s i -t i ve potent ia ls produced by the action of depolar iz ing synapses on the - 19 -apical/basal dendr i tes . The soma region was the passive source to act ive sinks (Andersen, Eccles and Loyning, 1964). It i s now well establ ished that with increasing st imulat ion strength the population PW can be interrupted by a negative-going potent ia l which is the population spike (PS). Andersen, B l i s s and Skrede (1971) demonstrated that the PS i s the potent ia l produced at the s i t e of the ind iv idua l action poten-t i a l s of many neighbouring, synchronously discharging neurons. These inves-t igators also showed a temporal cor re la t ion between PS and unit spikes in the same region. The PS amplitude introduced two peaks in the PW; and the second posi t ive peak corresponded to the maximal i n t r a c e l l u l a r IPSP. These features of the PS/PW are shown in f igure 3. F i g . 3 . Evoked population sp ike. The stimulus a r t i f a c t i s i d e n t i f i e d by the s t a r . Abbreviations denote: 1PW, f i r s t pos i t i ve wave component; PS, population sp ike ; 2PW, second pos i t i ve wave component. Negat iv i ty i s downwards. - 20 -It should be noted that in CA1 neurons potent ia l s h i f t s can be caused by transient s h i f t s in the ionic gradients. These ionic s h i f t s and associated potent ia l changes a l te r the magnitude of evoked EPSP, Pw and PS. For instance, calcium activated K + conductances fo l lowing neuronal a c t i v i t y (Hotson and Pr ince , 1980) probably enhances CA1 c e l l hyperpolar izat ions. Indeed CA1 pyramidal neurons possess a d i s t r i b u t i o n of nonsynaptic ionic conductances (see section 6) that can modify the net signal t ransfer from input s i t e s to the recording s i t e . 6. INTRINSIC NONSYNAPTIC IONIC CONDUCTANCES 6.1 Basic features A var iety of ionic conductances are known to occur in the hippocampal and other central nervous system (CNS) neurons. These conductances have a loca l i sed d i s t r i b u t i o n on the soma and/or dendr i t ic regions. They exhibi t ion ic s p e c i f i c i t y , are voltage-dependent and can be modulated by neurotrans-mitters and neuropeptides ( L l i n a s , 1984). I n t r i n s i c ion ic conductances par t i c ipate in the neuronal integrat ion of s igna ls . In fac t much of the electrophysiology observed in mammalian neurons derives from the i n t r i n s i c e l e c t r i c a l properties of the c e l l s , i . e . , interplay of ion ic conductances that co-occur with c l a s s i c synaptic in teract ions . 6.2 Sodium conductances At least two types of sodium (Na+) conductances are known to operate + in the CA1 pyramidal neurons. There is the c l a s s i c inac t i va t ing Na con-ductance (Terzuolo and A r a k i , 1961; L l inas and Sugimori, 1980) which is implicated in the generation of fas t action po ten t ia l s . A noninactivating Na+ conductance has also been ident i f i ed and is thought to mediate graded plateau potent ia ls las t ing up to 15 seconds (L l inas and Sugimori, 1980; Strafstrom, Schwindt and C r i l l , 1982). 6.3 Calcium conductances A high threshold inact i vat ing calcium (Ca + + ) conductance located in the soma and possibly in the dendrites (Sugimori and L l i n a s , 1982) i s pres-ent in the hippocampus (Schwartzkroin and Slawsky, 1977). This inact i vat ing ++ ++ Ca conductance is involved in the generation of Ca -dependent action potent ia ls termed C a + + spikes. There ex is ts a second type of C a + + con-ductance which does not inact ivate but i s able to induce C a + + spikes. ++ This somatic Ca conductance has a low threshold of act ivat ion since i t i s activated by hyperpolarizations from rest level (L l inas and Jahnsen, 1982). 6.4 Potassium conductances Hi therto , the largest number of ionic conductances known are those of + + potassium (K ). At least seven types of K conductances are known and probably a l l of them occur in the hippocampus (Segal and Barker, 1984). F i r s t , there is the c l a s s i c Hodgkin-Huxley type (Hodgkin and Huxley, 1952) delayed r e c t i f i e r K+ current which generates the f a l l i n g phase of the fast action p o t e n t i a l . Another K + current , denoted as the M-current, has been observed in the hippocampus. The M-current does not inac t i va te , is a low threshold K + current and is e l i c i t e d by depolarizations and modulated by synaptic (chol inergic ) neurotransmitter substances (Adams, Brown and H a l l i w e l l , 1981). The M-current increases input resistance and t h i s e f fec t could conceivably f a c i l i t a t e dendr i t i c to soma communications. Another type of inact i vat ing delayed r e c t i f y i n g K + current d i f f e r s from the M-current in that the former can be blocked by caesium and shows a dependency on C a + + (L l inas and Yaro, 1980). Two and possibly three types - 22 -of anomalous inward r e c t i f y i n g K + conductances operate in the hippocampus (Holston, Prince and Schwartzkroin, 1979). Type one is the instantaneous r e c t i f i e r whose charac te r i s t i cs were i n i t i a l l y given by Katz in 1949 and Adrian in 1969. The second type i s denoted as delayed or time-dependent r e c t i f i e r K + current (Wilson and Goldner, 1975). Anomalous r e c t i f i c a t i o n s seem to modulate input resistance and invar iab ly improve coupling between dendrite and soma (L l inas , 1984). L a s t l y , a fast transient K + conductance has been noted in the hippo-campus (Gustaffsson, Gal van, Graffe and Wigstrom, 1982) which presumably serves to prevent the rapid return of membrane potent ia l to basel ine , f o l -lowing hyperpolar izat ions. A possible function of t h i s K + conductance would be in the prevention of rebound exc i ta t ion as the c e l l membrane poten-t i a l returned to basel ine. 6.5 Functional ro le of ionic conductances As stated previously , the hippocampal ionic conductances contribute s i g -n i f i c a n t l y toward neuronal signal in tegrat ion . For instance, the pyramidal c e l l s ' dendr i t ic electrophysiology is in part generated by changes in Na + , + ++ K and Ca conductances (cf . Schwartzkroin and Slawsky, 1977). Spencer and Kandel (1961) postulated the existence of a fas t and ear ly p repotent ia l , + + which is now thought to be mediated by Na conductance, i . e . , Na spike. Wong, Prince and Basbaum (1979) have since confirmed the in t radendr i t i c Na + spike and proposed that these Na + spikes served as e lectrotonic couplers between the dendrites and the soma. However, the exact function of the potent ia ls i s not known. A l l or some of the bursting behavior seen in pyramidal neurons seem to be caused by changes in i n t r i n s i c ion ic conductance changes. Dentate gyrus granule c e l l s do not f i r e bursts but CA3 c e l l s can burst spontaneously. Both somata and dendrites of CA3 c e l l s burst read i l y (Wong, Prince and Basbaum, 1979). In contrast , CAl pyramidal c e l l s can burst but do not do so o r d i n a r i l y (Masukawa, Bernado and Pr ince , 1982; A lger , 1984). CAl pyramidal c e l l dendrites can be induced to burst ( i . e . discharge) d i r e c t l y but CAl somata cannot. The differences in bursting behaviour between soma and dendr i te , or indiv idual cornu ammonis f i e l d s and the dentate gyrus, may be due to differences in d i s t r i b u t i o n and/or a c t i v i t y of recurrent or feed-forward i n h i b i t i o n (Alger, 1984). Anyhow, in t radendr i t i c and intrasomatic recordings from a pyramidal neuron seem to implicate both the soma and the dendrites as the most l i k e l y s i tes for generation of bursting a c t i v i t y (Wong and Pr ince, 1978; Wong, 1982). Wong (1982) postulated that sodium spikes i n i t i a t e d by membrane potent ia l f luctuat ions in the hippocampal pyramidal neurons act ivate a C a + + conductance. Upon membrane r e p o l a r i z a t i o n , the C a + + conductance decays slowly and may induce secondary depolar izat ions . Whatever the mechanisms, tetrodotoxin res is tant spikes that are thought to be mediated by C a + + have been demonstrated in the mammalian CNS (Kandel and Spencer, 1961; Barrett and Bar re t t , 1976; Schwartzkroin and Slawsky, 1977; Wong and Pr ince , 1978). In summary, the preceding discussion points out a var iety of i n t r i n s i c ion ic conductances that are present in the hippocampal pyramidal neurons. Among other funct ions, these ionic conductances par t ic ipate in the genera-t ion of action po ten t ia l s , soma-dendritic e l e c t r i c a l coupl ing, modi f i ca -t ions/control of synaptic s ignals and termination of neuronal a c t i v i t i e s . Some of the other known ion ic conductances in the hippocampus have not been discussed since they have s t i l l to be properly character ized. Subsequent studies are l i k e l y to ident i fy 'nove l ' i on ic conductances. C lear ly post -- 24 -synaptic neurons do not simply act as a slave to presynaptic commands. At least in the hippocampal pyramidal neurons, there ex i s t s a battery of ionic conductances that d ic tate the ult imate global response emanating from these postsynaptic neurons, fo l lowing synaptic a c t i v a t i o n . 7. ANTIDROMIC, ORTHODROMIC AND OTHER EVOKED POTENTIALS 7.1 Antidromic f i e l d potent ials Synaptic potent ia ls in CA1 pyramids cannot be evoked by st imulat ion of f ibers in the alveus. Instead, a short latency spike (see f igure 4A) show-ing l i t t l e latency var ia t ion and capable of fol lowing high frequency ( e . g . , 100. Hz) i s evoked. Thus alvear st imulation (where CA1 pyramidal axons traverse) e l i c i t s spikes which f u l f i l the c r i t e r i a for antidromic invasion of the pyramidal neuron. These spikes are not abolished by high magnesium or manganese containing media ( i . e . , Ringer's solut ions without C a + + ) . 7.2 Orthodromic responses Typ ica l ly st imulat ion of the afferents in the stratum oriens or the stratum radiatum cause a presynaptic potent ia l in a s t r i p - l i k e region at the stimulated level (Andersen, S i l f v e n i u s , Sundberg, Sveen and Wigstrom, 1978) This e x t r a c e l l u l a r l y recorded negative d e f l e c t i o n , as shown by the arrow in f igure 4D, i s termed the presynaptic vo l ley (PV) and i t s magnitude i s an index of the number of activated f i b e r s . Evoked commissural or Schaffer c o l l a t e r a l s e l i c i t postsynaptic poten-t i a l s in the CA1 pyramids. The negative f i e l d synaptic potent ia ls have the i r maximum in the region where the activated f ibe rs terminate and show reversal ( i . e . , to p o s i t i v i t y ) when recorded from distant posit ions along the dendr i t i c axis (Andersen, S i l f v e n i u s , Sundberg and Sveen, 1980). Depending on the magnitude of the orthodromic exci tatory p o t e n t i a l s , i . e . , F i g . 4. Antidromic and orthodromic potent ia ls  recorded in CAl f i e l d . Dot corresponds to 2Pw and arrow points at the presynaptic v o l l e y . Note the ear ly onset of PS in (A) r e l a t i v e to orthodromic PS in (B) and (C). Negat iv i ty i s downwards. - 26 -reaching threshold , numerous action potent ia ls (AP) are discharged in the pyramidal neurons. The magnitude of the summated APs generated is maximum, when recorded in the stratum pyramidale layer and show p o l a r i t y reversal on both sides of the pyramidal layer . Andersen, S i l f v e n i u s , Sundberg and Sveen (1980) caused se lect ive a c t i v a -t ion of a small group of afferent f i be rs to e l i c i t f i e l d potent ia ls in the CA1 neurons. These investigators demonstrated that the proximal and d i s t a l synapses in CA1 were largely equipotent in evoking f i e l d po ten t ia l s . The input across a s i m i l a r number of proximal and d i s t a l synapses gave the same high p robab i l i t y of discharging s ingle neurons. 7 . 3 . Inhibi tory postsynaptic potent ia ls Orthodromic, but not antidromic act ivat ion of CA1 pyramidal neurons is more e f fec t i ve in producing IPSPs (NB: same s ize f i e l d potent ia ls and asso-ciated IPSP measured concurrently ; see Alger and N i c o l l , 1982). Pharmaco-log ical evidence supports the d i s t i n c t i o n between or tho - and antidromical ly evoked IPSPs (Alger and N i c o l l , 1979). The morphology of IPSPs caused by antidromic st imulat ion d i f f e r from those evoked during orthodromic s t imula -t i o n . Besides, pyramidal c e l l IPSPs are inducible fol lowing stratum r a d i -atum st imulat ion even in the absence of recordable population spikes. C lear ly recurrent i nh ib i t i on (which operates only af ter CA1 discharges or after antidromic exc i tat ion of CA1 axons) alone cannot account for the d i s -crepancies in antidromic versus orthodromic mediated IPSPs. The above e v i -dence (cf . A lger , 1984) renders s i g n i f i c a n t e lect rophys io log ica l support for the existence of feed-forward i n h i b i t i o n . - 27 -7.4 E lectrotonic coupling and ephaptic interact ions Though avai lable evidence is incomplete, two types of nonsynaptic i n t e r -actions have been implicated in the hippocampus. MacVicar and Dudek (1981) have proposed the operation of e lect rotonic coupl ing, where a c t i v i t y in one neuron is presumed to be transmitted d i r e c t l y to other neurons v ia anatomic-a l l y i d e n t i f i a b l e junct ions . The dye Luci fer Yellow, which is very sparsely taken up from the ex t race l lu la r space and does not cross chemical synaptic junct ions , has been used to reveal the gap junct ions . In these studies, however, the p o s s i b i l i t y of mechanical coupling being introduced by the electrode i t s e l f p a r t i a l l y impaling both c e l l s (cf . A lger , McCarren and F isher , 1983) has not been ruled out e n t i r e l y . It should be noted that pyramidal neurons are t i g h t l y packed together (Lorente De No, 1934). Ephaptic interact ions are thought to be the influence on a neuron caused by e x t r a c e l l u l a r current flow v i a e x t r a c e l l u l a r resistances (Jefferys and Haas, 1982; Taylor and Dudek, 1982). The demonstration of ephaptic t rans -mission requires that in v i t r o hippocampal s l i c e s be bathed for prolonged periods in a low C a + + medium. Apparently there is complete chemical syn-aptic blockade. Thereafter rhythmic bursts las t ing for many seconds and which can be blocked by high (6 mM) but not low (less than 4 mM) M g + + are recorded (Taylor and Dudek, 1982; Alger , 1984). These bursts can be spon-taneous or they can be evoked by e l e c t r i c a l s t imulat ion . Taylor and Dudek (1982) analysed d i f f e r e n t i a l recordings of e x t r a c e l l u -lar and i n t r a c e l l u l a r potent ia ls simultaneously and reported that the ex t ra -c e l l u l a r f i e l d potent ia ls had ef fects on the membrane po ten t ia l s . It is not c lear why these ephaptic interact ions take long to develop; and neither are the mechanisms for the synchronous bursting understood. If i t is assumed that e lect roton ic and ephaptic interact ions have a funct ional ro le in hippo-campal physiology, these could be some of the means by which synchronization of c e l l f i r i n g occurs. - 28 -8 . SYNAPTIC INTERACTIONS AND POSSIBLE NEUROTRANSMITTER CANDIDATES. 8 .1 Recurrent and feed-forward i n h i b i t i o n Inhib i tory inf luences, both recurrent and feed-forward, probably use gamma-aminobutyric acid (GABA) as the i r p r inc ip le neurotransmitter (Storm-Mathisen, 1977; Frotscher, Leranth, Lubbers and Oer te l , 1984). GABA synthe-t i c enzyme glutamic acid decarboxylase (GAD) as well as GABA-ergic receptor subtypes, are d is t r ibuted in a l l layers of f i e l d CA1 (Storm-Mathisen, 1977; Andersen, Dingledine, Gjerstad, Langmoen and Mosfeldt -Laursen, 1980). Evidence indicates that synapt ica l ly released GABA on c e l l bodies and around the i n i t i a l parts of axons and/or dendr i tes, causes a conductance change that induces a net in f lux of ch lor ide (Cl~) ions . This in f lux of Cl~ causes hyperpolarizations in c e l l s and thereby prevents them from d i s -charging when, synapt ica l ly dr iven. In add i t ion , inh ib i to ry interneurons terminate on the primary and secondary parts of CA1 dendrites (Lorente De No, 1934) and release GABA. Here GABA presumably produces a conductance increase, involving sodium and chlor ide ions , and resu l t ing in a depolar izat ion action (Andersen, 1983). Andersen, Dingledine, Gjerstad, Lanmoen and Mosfeldt-Laursen (1980) proposed that t h i s GABA-mediated depolar izat ion in the dendr i t i c regions created i n -tense conductance changes which subsequently shunted the ef fects of e x c i t a -tory synapses in the v i c i n i t y (c f . Alger and N i c o l l , 1979; 1982). 8.2 Ex t r ins i c modulatory pathways A var iety of ex t r ins i c modulatory pathways in the hippocampus have been i d e n t i f i e d . In most cases, the exact target c e l l s for these ex t r ins ic inputs and/or the i r source of o r ig in are not f u l l y known. Some of these inputs that seem to innervate parts of CA1 inc lude; the medial septum and - 29 -diagonal chol inerg ic input (Storm-Mathisen, 1977: Lynch, Rose and G a l l , 1978), noradrenergic outflow from the locus coeruleus (L indval l and Bjork land, 1974) and the serotonergic projection from the medial and dorsal raphe nuclei (Azmitia and Segal, 1979). The probable modulatory ro le of these e x t r i n s i c pathways is i l l u s t r a t e d by acetylchol ine actions (ACh). Acetylchol ine is known to cause a reduction in the M-current which is active over the -70 mV to -40 mV range (Dodd, Dingledine and K e l l y , 1981; Bernado and Pr ince, 1982). The actions of ACh are accompanied by slow depolar izat ions in the c e l l s and a raised input res is tance. These actions can conceivably predispose the CAl pyramids to bursting (Dingledine, 1984) espec ia l l y since ACh also i n h i b i t s the release of inh ib i to ry and/or exci tatory afferents to CAl pyramidal neurons (Yamamoto and Kawai, 1967; Ben -Ar i , Krn jev ic , Rei f fenste in and Ropert, 1981). 8.3 Neuroactive substances Recent f indings have revealed a diverse d i s t r i b u t i o n of neuroactive sub-stances in the hippocampus (reviewed by Dingledine, 1984). It remains to be establ ished whether these neuroactive substances form separate pathways and/or co -ex i s t with other neurotranmitters. It can be speculated that neuroactive substances might even reside and/or be released from the CAl neurons dendr i t ic spines and influence synaptic in te rac t ions . But the experimental evidence for these p o s s i b i l t i e s are presently lack ing . The main neuroactive substances that have been characterized so far in the CAl f i e l d include enkephalin-1ike substances ( G a l l , Brecha, Karten and Chang, 1981), cholecystokinin and somatostatin (Greenwood, Godar, Reaves and Hayward, 1981), vasoactive i n t e s t i n a l polypeptides (Loren, Emson, Fahren-krug, Alumets, Hakanson and Sundler, 1979), substance-P (Vincent, Kimura and - 30 -McGeer, 1981) and angiotensin- I I (Haas, F e l i x , Ce l io and Inagami, 1980). The l i s t of neuroactive substances presented here is by no means exhaustive. A more detai led discussion of neuroactive substances and the i r mechanism of actions has been presented by Dingledine (1984). 8.4 Exci tatory amino acids in commissural and Schaffer afferents Several of the hippocampal exc i tatory pathways seem to u t i l i s e ac id ic amino acids as neurotransmitters. The evidence for a neurotransmitter role of glutamate and/or aspartate in the hippocampal commissural and Schaffer afferents are based on the biochemical and autoradiographic l o c a l i z a t i o n of high a f f i n i t y uptake s i tes (Storm-Mathisen and Iversen, 1979; Fonnum, Lund-Karl sen, Malthe-Sorensen, Skrede and Walaas, 1979), induction of changes in the endogenous leve ls of amino acids a f ter se lect ive lesions (Fonnum and Walaas, 1978) and the demonstration of C a + + mediated release fol lowing K + or e l e c t r i c a l st imulat ion (Nadler, White, Vaca, Perry and Cotman, 1978; Wieraszko and Lynch, 1979; Malthe-Sorensesen, Skrede and Fonnum, 1979). It needs emphasizing that the abundant data in the l i t e r a t u r e ( e . g . , Nadler, Vaca, White, Lynch and Cotman, 1976; White, Nadler and Cotman, 1979; Koerner and Cotman, 1982) only implicates L-glutamate and/or L-aspartate or the i r analogues, as l i k e l y neurotransmitter candidates. In fac t the methods used to implicate L-glutamate (Glu) or L-aspartate (Asp) as neurotransmit-ters are not without c r i t i c i s m . For example, many antagonists of acidic amino acid mediated exc i tat ion have been character ized, but the i r antagonism was to exogenously applied ac id ic amino acids . F i r s t l y , these studies em-ploy iontophoretic and/or other means of ac id ic amino acid appl icat ions , which is not necessar i ly the same as d i r e c t l y st imulat ing the f ibers thought to contain ac id ic amino acids as neurotransmitters. There is lack of e v i -dence for the se lect ive/spec ic ic antagonism of endogenous ac id ic amino acid - 31 -transmission (reviewed by P u i l , 1981). Secondly, binding studies and e l e c -t rophys io log ica l manipulations have been used to character ize ac id ic amino acid subtypes. The v a l i d i t y of any l igand binding technique i s dependent on the demonstration that the radioact ive l igand se lec t i ve l y labels the physio-log ica l or pharmacological receptors under study. This requirement is often not f u l f i l l e d (Foster and Fagg, 1984). Th i rd ly , even though a Ca and voltage dependency are demonstrated in the release s tud ies , i t i s not known for cer ta in whether the released t r i t i a t e d transmitter comes from the same i n t r a c e l l u l a r compartment as the endogenous transmitter i t s e l f (Laduron, 1984). In other systems, i t has been shown that t r i t i a t e d ligands can be trapped in d i f ferent i n t r a c e l l u l a r compartments of in tact c e l l s (Maloteaux, Gossuin, Waterkeyn and Laduron, 1983). These ligands can be displaced by unlabel led compounds. S t i l l , Glu or Asp are probably the neurotransmitter substances in the commissural and Schaffer afferents and are treated as such here. In spinal neurons, microiontophoresed Glu causes a fast onset exc i ta t ion followed by a rapid termination of ac t ion . This excitant action is mediated by a d i rect G lu - induced depolar izat ion of the spinal neurons ( reviewed by P u i l , 1981). The e lect rophys io log ica l studies indicate that Glu induces an inward move-ment of cat ions , mostly Na + and/or perhaps C a + + . Pu i l (1981) summar-ised that the i n i t i a t i n g ionic event in Glu-induced depolarizat ions could be an increase in C a + + conductance or even release of membrane-bound C a + + . ++ The subsequent inward movement of Ca could cause s i g n i f i c a n t depolar-++ i z a t i o n s , since Ca has a steep dr iv ing force and car r ies twice the amount of charge of Na + . Whatever the i n i t i a l mechanisms of ac t i va t ion , G l u - dependent depolar izat ions subsequently cause an i n t r a c e l l u l a r accumu-l a t i o n of Na + (which probably act ivates electrogenic Na + extrusion) and also an outward K + f l u x . Both these processes probably contribute towards r e p o l a r i z a t i o n . Most of the act ions/effects of Glu on spinal neurons have now been demonstrated in the hippocampal pyramidal neurones (Dingledine, 1983a; Co l l ing r idge , Kehl and McLennan, 1983). According to Habl i t z and Langmoen (1982) the reversal potent ia l for the Glu-mediated depolar izat ion in the hippocampus was comparable to that of the EPSP. Both sh i f ted in a negative d i rec t ion in low Na + medium. In CA1 pyramids, Dingledine (1983b) found that N-methyl-DL-aspartate (NMA) evoked C a + + spikes accompanied by an apparent increase in input res is tances . But Glu caused depolarizat ions with an associated decrease in res is tance. Another observed dif ference was the se lect ive blockade of NMA-induced responses by C a + + antagonists whereas Glu actions remained (Dingledine, 1983b). It was concluded that NMA activated C a + + conduc-tances but Glu activated Na + and possibly K + conductances (Dingledine, 1983b; 1984; c f . Westbrook and Mayer, 1984). Many of the ac id ic amino ac ids , notably aspartate, quisqualate and k a i n -ic ac id , exhib i t s i m i l a r actions to Glu. Their differences in potencies, 'antagonis t ic ' p ro f i l es and/or ion ic conductances activated have led to the impl icat ion of ac id ic amino acid receptor subtypes. At least two types of ac id ic amino acid receptors have been impl icated; the NMA receptor and the non-NMA receptor(s).(McDonald and Wojtowicz, 1982; Westbrook and Mayer,1984; Watkins, 1984; Foster and Fagg, 1984; Dingledine,1984). N-methyl-D-aspar-tate (NMDA) is se lect ive for NMA receptors which, when act ivated , increase a voltage-dependent Ca conductance. Both quisqualate and kainate are - 33 -preferred at non-NMA receptors, whose act ivat ion e l i c i t s Na + and possibly K + conductances. Glu is active at both NMA and non-NMA receptors. Con-ceivably both receptor subtypes may be d is t r ibuted in the same subsynaptic regions of the hippocampus. 9. CENTRAL NERVOUS SYSTEM SYNAPTIC PLASTICITY 9.1 Simple model of brain function Once pr imit ive neuroblasts d i f f e r e n t i a t e into nerve c e l l s , they loose the i r mi to t i c competency and never div ide again (Gazen 1970; Oacobson, 1970). Subsequent nerve c e l l development is in completing genet ica l ly determined st ructural and funct ional spec ia l i za t ions whose phenotypic expression is governed by t h e i r regional locat ion , i . e . , environmental inf luences. While microgrowths s t i l l occur that replace components such as synapses, none of the nerve c e l l s d iv ide to give r i s e to daughter nerve c e l l s . The nervous system therefore is f u l l y constructed in i t s c e l l numbers and connections before i t i s used (Eccles, 1977). In very simple terms, the nervous system uses i t s own language to per-form i t s integrat ive funct ions . Hence any external s ignals presented to i t must f i r s t be converted into th i s unique nervous system language before pro-cess ing. Eccles (1977) has stated that signal in tens i ty i s t ranslated into increased frequency of action potential propagation in some peripheral a f -fe rents . In t h i s scheme, then, t ransient changes in action potential ampl i -tude and duration or frequency of action potent ial propagation or post -signal potent iat ion then represent some of the elements comprising the ner-vous system alphabet. It then becomes conceivable how any s ignal can be modified and encoded to const i tute a s p e c i f i c message to the nervous system - 34 -for in tegrat ion . If changes in the performance of the brain during l i f e are not due to the addit ion of new daughter nerve c e l l s then there must ex is t other mechanisms that could mediate, for instance, learning and memory func-t i o n s . It could be argued that these complex processes involve subtle changes in the chemistry or microstructure and,microfunction of the ex is t ing neuronal populations. Let us suppose that u l t imately memory functions are encoded as biochemi-cal changes within selected neuron populations in s p e c i f i c regions of the brain then conceivably preferent ia l mechanisms ex is t that mediate signal t ransfer or re t r ieva l from neuronal memory banks. At any one time arrays of memory and non-memory s ignals c r i s s - c r o s s in the same neuronal networks. If the fore-going summation is accurate then how can the preferent ia l t ransfer of information be achieved? Most inves-t igators in neurobiology implicate the phenomena of long - las t ing potent ia -t ion as the l i k e l y mechanism for the se lect ive and e f f i c i e n t t ransfer of learning and memory signals (review Swanson,Teyler and Thompson, 1982). During long-lasting potent iat ion , synaptic e f f i c a c i e s of s p e c i f i c inputs are greatly augmented for very prolonged periods (cf . Hebb, 1949). 9.2 Features of long - las t ing potentiat ion and depression B l i s s and his co-workers (B l i ss and Lfrfmo, 1973; B l i s s and GardnerMedwin, 1973) gave the f i r s t detai led account of post - te tan ic long - las t ing potent ia -t ion (also ca l led long-term potent iat ion) of evoked f i e l d potent ia ls in the dentate gyrus. In anaesthetized rabb i t s , condit ioning st imul i o f . 1 0 - 2 0 Hz for one or more seconds given to the perforant path caused longlast ing potent iat ion (LLP) of DG f i e l d responses evoked by post - te tan ic test pulses ( 0.5 Hz frequency ) to that input (B l i ss and Lomo, 1973). LLP was of - 35 -several hours duration and was manifested as a decrease in PS latency and/or an increase in amplitude of the PS or f i e l d EPSP. LLP was found to be asso-c iated with a decrease in PS v a r i a b i l i t y to the same test pulses. During the low frequency (10-20 Hz) t r a i n a rapid bui ld up of PS was observed, a phenomena termed as frequency potentiat ion (Andersen, Holmqvist and Voorhoeve,1966). On cessation of th i s condit ioning s t i m u l i , a decaying PS potent iat ion to test pulses f i r s t occurred which was quickly followed by a phase of PS depression las t ing from seconds to minutes. Thereafter, LLP became apparent. In contrast , high frequency ( i . e . , 100 Hz) condit ioning st imul i caused a rapid diminution of the synchronouusly discharging PS dur-ing the t ra ins but t h i s led to a post - tetanic development of LLP without the i n i t i a l phase of depression. The LLP e l i c i t e d by high and low frequency tetanic t ra ins exhibited s i m i l a r c h a r a c t e r i s t i c s . Once LLP was established i t only began to diminish when the qual i ty of recordings in the experimental and the control inputs were poor. Whenever there were changes r e f l e c t i v e of LLP, these were confined to the tetanized and not the non-tetanized per for -ant path sect ions . In the same tetanized perforant path, subsequent condi -t ion ing st imul i caused an augmentation of the established LLP u n t i l an asym-ptote was reached. LLP could not be accounted by changes in st imulat ion electrode after tetanic st imulations since a second electrode (along the same tetanized PP) that was not used to de l iver the condit ioning t ra ins to t h i s PP, could s t i l l evoke LLP in the DG. Furthermore, condit ioning st imul i caused the stimulus versus response ( i . e . , EPSP or PS) curves to s h i f t to the l e f t . However, potent iat ion of the PS and EPSP did not always co-occur (B l i ss and L$no, 1973). It was argued that LLP was not a simple s h i f t up the stimulus versus - 36 -response curves since a post-conditionong dendr i t ic EPSP, though matched in amplitude with a pre-condit ioning dendr i t ic EPSP, e l i c i t e d a bigger PS. The above studies were subsequently reproduced in unanaesthetized rab -b i ts (B l i ss and Gardner-Medwin, 1973) where i t also became c lear that LLP could be maintained for up to three days. Presently i t i s known that LLP can be maintained for weeks in in tact animals (review, Swanson, Teyler and Thompson, 1982). A more s i g n i f i c a n t development, however, was the demon-st rat ion of LLP in the CA1 region of the guinea-pig transverse hippocampal s l i c e in v i t ro (Schwartzkroin and Wester, 1975). Alger and Teyler (1976) were later to show that LLP could be e l i c i t e d in the CA1, CA3 and DG sub-regions of the rat hippocampus in v i t r o . Schwartzkroin and Wester (1975) noted that a st imulat ing electrode placed in the stratum radiatum could evoke an orthodromic potential in CA1 and an antidromic potent ial in CA3. Yet tetanizat ion with t h i s radiatum placed st imulat ion electrode only poten-t ia ted the orthodromic po ten t ia l s . In add i t ion , the antidromic t e t a n i z a -t ion of CA1 axons in the alveus could not e l i c i t LLP in the CA1 region. Two separate inputs, one in the stratum oriens and the second in the stratum radiatum , presumably to the same CA1 output neurons were used to test for the input s p e c i f i c i t y of LLP (Andersen, Sundberg, Sveen and Wigstrom, 1977; Andersen, Sundberg, Sveen, Swann and Wigstrom, 1980). Immediately af ter the condit ioning st imulat ion to the stratum radiatum afferents there was a generalised post - tetanic b r ie f depression to both inputs. Af ter t h i s depressive phase, only the tetanised afferents showed LLP. Schwartzkroin and Wester (1975) had previously found that condit ioning s t imul i caused an increase in the p robab i l i t y of discharge in s ingle CA1 units (cf . review Voronin, 1983). Subsequent studies in indiv idual c e l l s indicated that membrane resistance and rest ing membrane potent ia l were - 37 -apparently not changed during LLP (Andersen, Sundberg,Sveen, Swann and WigstrSm, 1980). Dunwiddie and Lynch (1979) reported that frequency potent iat ion , pa i red -pulse f a c i l i t a t i o n and short-term post - te tan ic potentiat ion could read i l y be e l i c i t e d in low C a + + Ringer's so lut ion whereas LLP was only occassional ly induced in these condit ions. Hence LLP seemed to d i f f e r from these other forms of neuronal p l a s t i c i t y since LLP induction was most sensi t ive to e x t r a c e l l u l a r C a + + l e v e l s . The dependence of LLP i n i t i a t i o n on e x t r a c e l -++ ++ l u l a r Ca was further investigated as fo l lows . Ca - f r e e Ringer's and Ringer's solut ions containing increased manganese (Wigstrom, Swann and Andersen, 1979) or increased magnesium (Dunwiddie and Lynch, 1979) were used to block synaptic t ransmission. The presynaptic vo l ley amplitude in normal Ringer's and during the synaptic blockade was e s s e n t i a l l y the same (wigs-trom, Swann and Andersen, 1979). It was found that tetanic st imulations given during the synaptic b lock -ade did not e l i c i t LLP when post - te tan ic responses were monitored in normal R inger 's . Yet after th i s return to normal R inger 's , i f the same input was s i m i l a r l y tetanised, t h i s caused the development of LLP (Dunwiddie and Lynch, 1979; Wigstrom, Swann and Andersen, 1979). In terest ing ly , the i n s t i -tut ion of a b r ie f synaptic blockade with high magnesium Ringer 's , after LLP was i n i t i a t e d , did not abolish LLP (Dunwiddie, Madison and Lynch, 1978). This indicated that only the i n i t i a t i o n of LLP was dependent on the ex t ra -++ ++ c e l l u l a r Ca but i t s maintenance seemed to be independent of t h i s Ca . Most studies that have used low frequency tetany ( i . e . , 10-30 Hz) to induce LLP have usual ly reported an i n i t i a l phase of PS depression pr ior to LLP formation. In fact under these condi t ions , LLP has been reported as - 38 -'developing gradually ' (Alger and Teyler , 1976). In contrast , high frequen-cy tetanic stimulations i . e . , greater than 100 Hz) cause the ' r a p i d ' deve l -opment of LLP without an associated depressive phase (Schwartzkroin and Wester, 1975; Swanson, Teyler and Thompson, 1982). Dunwiddie and Lynch (1978) examined the ef fects of st imulat ion t ra ins of 100 pulses at rates of 5-100 Hz, on LLP development. The magnitude of the e l i c i t e d LLP was shown to be greatest with the highest frequencies. It was confirmed that the low-er frequencies evoked a post - te tan ic depression pr ior to any other p o s t - t e t -anic changes (Barrionuevo, Schott ler and Lynch, 1980; Chirwa, Maretic and Sastry , 1983). The duration of th i s depression varied from minutes to hours. P r io r to tetanus, a constant depolar iz ing current of 0.56 nA was used to e l i c i t action potent ia ls in a CA1 pyramid (Andersen, Sundberg, Sveen, Swann and Wigstrom, 1980). The latency for action potent ial discharge, which was the time from the onset of the depolar iz ing pulse u n t i l the f i r i n g of an action potent ial was then determined. This latency was termed as the depol -a r i z i n g current pulse (abbreviated as DPP). Immediately after the tetanic s t imulat ion , and only during the time when the transient depression was present, the DPP was found to be increased. It was suggested that when the post-tetanus depression was present, th i s was associated with a generalized but t ransient decrease in CA1 neuronal e x c i t a b i l i t y . 9.3 Summary In conclusion i t can be stated that LLP presents i t s e l f as an increase in synaptic e f f i c a c y , fo l lowing appropriate condit ioning of spec i f i c inputs. During LLP, previously subthreshold neurons are recrui ted and even neurons that were previously reaching threshold have an increased probabi l i t y to discharge. With e l e c t r i c a l manipulations, only Ca + + -dependent orthodromic - 39 -condit ioning caused the development of LLP. Under these condi t ions , LLP w i l l s t i l l develop even i f the postsynaptic neurons are kept ' s i l e n t ' with i n t r a c e l l u l a r l y injected hyperpolariz ing currents. In fac t there i s no e v i -dence in the l i t e r a t u r e that has shown the induction of LLP by in jec t ing depolar iz ing currents into the CAl neurons. It is presumed that antidromic influences or hyperpolarizing (and even depolar iz ing currents) in the soma do not 'reach' the dendrit ic/subsynaptic regions or the terminal boutons, where the changes mediating LLP seem to occur. Recently, a variant to LLP in DG and CA3, termed associat ive LLP,has been described. Associat ive LLP is an enhanced synaptic e f f i cacy that occurs in one (weak) synaptic input only i f i t is t e t a n i c a l l y stimulated in conjunction with nearly concurrent st imulat ion of a second (strong) synaptic input (summary of Johnston and Brown, 1984). Furthermore, potentiat ion phenomena in other brain regions have been described ( e . g . , Racine, Milgram and Hafner, 1983) though the i r charac te r i s t i cs are not exactly l i k e those of hippocampal LLP. L a s t l y , the wide range of frequencies that induce LLP also cover the frequencies operative in normal physiology. LLP should therefore be induc-ib le in a behaving animal. As stated previously , LLP is an at t ract ive can-didate for mediating some memory and learning funct ions . The role, of the depressions that can be induced fol lowing repet i t i ve st imulat ion is unclear. More importantly the actual mechanisms mediating LLP ( and depression) or the loc i for the change remain to be conclusively e luc idated . The next sec -t ion examines some of the mechanisms thought to account fo r LLP development. The possible loc i for these changes are discussed. - 40 -10 CONSIDERATION OF POSSIBLE POSTSYNAPTIC MECHANISMS MEDIATING LLP 10.1 Post - te tan ic potentiat ion Post - te tan ic potentiat ion (PTP) i s a short duration (2-5 min) increase in synaptic e f f i cacy which occurs after repet i t i ve st imulat ion of an input and has been shown to be mediated by an increase in neurotransmitter r e -leased. The basic change in PTP is an -afferent terminal hyperpolarization which leads to augmented action potent ial amplitudes (Eccles and Krn jev ic , 1959). It is un l ike l y that LLP is an extension of PTP per se, since the l a t t e r can be evoked in low C a + + (Dunwiddie and Lynch, 1979) and i s inde-pendent of tetanic st imulation i n t e n s i t i e s (McNaughton, Douglas and Goddard, 1978; McNaughton, 1982). PTP can be evoked in a s ingle f i b e r (Eccles and Krn jev ic , 1959) whereas LLP has yet to be demonstrated in one hippocampal af ferent . McNaughton, Douglas and Goddard (1978) contended that there ex-isted a threshold stimulus in tens i t y below which LLP could not be e l i c i t e d . The avai lable evidence in the l i t e r a t u r e rules out a common process between PTP and LLP. 10.2 Increases in afferent vo l ley B l i s s and Ltfmo (1973) proposed that LLP could be explained in terms of increases in presynaptic vol ley (PV), i . e . , increase in number of recruited f i b e r s . Andersen, Sundberg, Sveen and Wigstrom (1977) f a i l e d to detect any changes in the afferent f ibers f i e l d potent ia ls after tetanus, ind icat ing that the number of afferents being activated was unaltered. The use of con-stant current stimulators or biphasic s t imul i has shown that st imulation electrode resistances are not changed by tetanus. L a s t l y , i t has been r e -ported that even when an i p s i l a t e r a l input to a subf ie ld in the hippocampus i s potentiated, i t s c o l l a t e r a l s to the contra latera l hippocampus do not ex-- 41 -hi b i t LLP (Voronin, 1983 review). Such an e f fec t would be unattainable, i f indeed there was an increase in the presynaptic f i b e r s rec ru i ted . S t i l l none of the above objections conclusively ru le out an increase in PV in LLP. The ear ly component which is often taken to be the PV merges into the dendr i t ic EPSP without giving a c lear demarcation (see f i g . 4 ) . The ear ly onset of a potentiated dendr i t ic EPSP tends to d i s t o r t t h i s PV and complicates ana lys is . The absence of LLP in a cont ra latera l component of a tetanised i p s i l a t e r a l input could be due to the inexact pos i t ioning of r e -cording electrodes in the target areas of the cont ra latera l hippocampal sub-f i e l d s . Besides, i t is known that each s ingle afferent f i be r gives out a ser ies of en passage boutons along i t s t ra jec to ry . It i s not known for cer -ta in whether a l l the boutons belonging to the same afferent f ibe r are a c t i -vated, each time an action potent ial i s generated in that f i b e r . While there may not be any discernable changes in PV amplitude, i t is l i k e l y that in LLP more inact ive boutons are rec ru i ted . Such a p o s s i b i l i t y i s funct ion -a l l y equivalent to an increase in PV and has yet to be tested. 10.3 Increased transmitter release Voronin (1983) summarised that LLP was mediated by an increase in the number of transmitter quanta released per presynaptic spike. LLP presents i t s e l f as an increase in amplitudes and/or a decrease in r i s e time of evoked p o t e n t i a l s . More importantly, various invest igators have reported an i n -crease in the i n t r a c e l l u l a r l y recorded EPSP during LLP (Yamamoto and Chujo, 1978; Andersen, Sundberg, Sveen, Swann and Wigstrom, 1980). These f indings are consistent with an increase in neurotransmitter released (for a l te rna -t i v e explanations, see later sect ions ) . A presynaptic change was indicated when i t was found that terminal e x c i t a b i l i t y of tetanised inputs decreased - 42 -af ter LLP i n i t i a t i o n (Sastry, 1982). L a s t l y , an increase in glutamate r e -lease after the i n i t i a t i o n of LLP has been demonstrated (Skrede and Malthe-Sorenssen, 1981; Dolphin, Errington and B l i s s , 1982). However, these last studies quoted here have not const i tuted the d e f i n i t i v e proof that LLP is mediated by an increase in t ransmitter released. Some objections w i l l be c i ted here. It i s not known for certa in whether label led ligands actual ly occupy the same s i tes as the endogenous neurotransmitter (Laduron, 1984). Besides, L-glutamate has been implicated but not conclus ively shown to be the neurotransmitter in Schaffer c o l l a t e r a l s (see section 8.4 in th i s t h e s i s ) . 10.4 Spine morphology Van Harreveld and Fifkova (1975) observed swel l ings of spines fol lowing tetanic stimulations and l inked th is to LLP. It was assumed that the evoked tetanus caused LLP even though th is potentiat ion was not actual ly checked. Mixed resu l ts on spine changes and LLP have since been reported. Desmond and Levy (1981) reported increases in spine s ize and synaptic densi t ies and associated these changes with LLP. Yet i t was found by Lee, Schot t le r , Ol iver and Lynch (1980) that LLP was not associated with s i g n i f i c a n t changes in spine area, spine number, spine neck diameter or length of the postsynap-t i c density . S imi lar f indings have been reported by Chang and Greenough (1984) who further noted that the only consistent morphological change seen during LLP was an increase in the number of s e s s i l e spines and d i rect con-tact synapses. It should be noted that the funct ional s ign i f icance of dendr i t ic spines has yet to be e luc idated. I t has been proposed that spines might serve to attenuate synaptic s ignals (Chang, 1952) or permit the 'weighting' of s i g -- 43 -nals from di f ferent afferents impinging on the same dendrite ( R a i l , 1970). Spines could be a structural mechanism for the separation of synaptic appa-ratus and thereby del imit synaptic c r o s s - t a l k . Few studies have elaborated on the above p o s s i b i l i t i e s . Computer modelling studies that have used dimensions obtained from hippocampal h i s t o l o g i c a l studies indicate that spine signal t ransients are only attenuated by less than 2% in conduction across the spine neck (Turner, 1984; Kawato and Tsukahara, 1984). Therefore, the dendrites essent ia l l y 'see' the to ta l s ignal transduced at the subsynaptic membrane. C lear ly no advantage would be offered by increased spine s i z e , even i f there i s a concomittant reduction in axial resistance ( i f spines increase in volume), making such a change of less importance in LLP. It is apparent from the above discussion that i f the i n t r a c e l l u l a r dendr i t ic EPSP increases after LLP (Yamamoto and Chujo, 1978) only two processes could account for the increase namely (a) increase in neurotransmitter depolariz ing the subsynaptic membrane or (b) an increase in the conducting a b i l i t y of the subsynaptic membrane. Point (a) can be incorporated in mechanisms discussed in section 1 0 . 3 . Point (b) w i l l be considered in section 10.5. 10.5 Increased synaptic receptors Baudry and Lynch (1980) proposed the fol lowing scheme for LLP i n i t i a t i o n . Tetanic st imulations caused C a + + in f lux into postsynaptic dendrites. The increased i n t r a - d e n d r i t i c C a + + tr iggered a biochemical change which i n -volved phosphorylation of Y -pyruvate dehydrogenase. Then a membrane bound proteinase was activated which in turn caused proteolys is of some membrane associated component(s) thereby e f fect ing re -organizat ion of the dendr i t ic membranes. Ult imately these changes caused the uncovering of glutamate receptors previously not accessible in synaptic transmission (Baudry - 44 -and Lynch, 1980; Eccles,1983 review) Since glutamate receptors are assumed to be coupled to ionic channels, c l e a r l y the above changes would lead to an increased conducting c a p a b i l i t y ( i . e . , more channels now avai lab le) of the subsynaptic membrane. Such an e f fec t would s a t i s f y point (b) in section 10.4 above. Much of the evidence in support of the receptor increase hypothesis was obtained from binding studies that showed an increase in spec i f i c glutamate binding s i tes fol lowing the induction of LLP (Baudry and Lynch, 1979; Baudry, O l i ver , Creager and Wieraszko, 1980). However, these studies can be faulted for the fo l lowing reasons, Sastry and Goh (1984) ca re fu l l y repro -duced these binding studies but found that the increase in glutamate binding correlated better with depression rather than LLP. Results from the studies conducted in t h i s thesis w i l l further demonstrate some inconsistencies of the receptor hypothesis as a mechanism for LLP. 10.6 Miscellaneous changes. I n t r a c e l l u l a r recordings have so fa r f a i l e d to reveal long - las t ing changes in rest ing membrane p o t e n t i a l , spike threshold or input resistances (Andersen, Sundberg, Sveen, Swann and Wigstrom, 1980). In any case, the s p e c i f i c i t y of CAlb LLP seem to ru le out the p o s s i b i l i t y of a generalised postsynaptic increase in e x c i t a b i l i t y (Andersen, Sundberg, Sveen and Wigs-trom, 1977). In fac t immediately after tetanic s t imulat ions , post - tetanic hyperpolarizations occur which invar iab ly decrease c e l l e x c i t a b i l i t y (Yama-moto and Chujo, 1978). Other studies have shown a decrease in L-glutamate s e n s i t i v i t y af ter tetanus (Lynch, Gribkoff and Deadwyler, 1976). These changes do not support the notion that generalised postsynaptic changes can account for LLP. In addi t ion , d is inh ib t ion of interneurons and/or net i n -- 45 -creases in synaptic exci tatory to inh ib i to ry influences (Schwartzkroin and Wester, 1975, Haas and Rose, 1982) have been examined and found to be un-l i k e l y candidates fo r LLP changes (Swanson, Teyler and Thompson, 1983 review). However, one postsynaptic change offers a possible contr ibut ing mechan-ism to LLP. It was shown in section 6 that CAlb neurons are endowed with a multitude of ionic conductances, which par t ic ipate in neuronal integrat ion of synaptic s igna ls . These conductances have been shown to contribute towards dendr i t ic spike generation (Wong,1982) and may be involved in the soma-dendr.it i c e lect rotonic coupling (Spencer and Kandel, 1961; Wong, Prince and Basbaum, 1979). It i s feas ib le that dendr i t ic changes in e f f i cacy of some ion ic conductances, which may not be read i l y detected with i n t r a c e l l u -la r recordings, improve e lect rotonic coupling during LLP. L a s t l y , another possible mechanism for LLP that is presently incompletely characterised is the involvement of neuromodulators and related substances. For instance, i t has been reported that depletion of noradrenaline reduces LLP ( B l i s s , God-dard, Robertson and Sutherland, 1981). Future studies w i l l elaborate on these mechanisms. 10.7 Homo- and heterosynaptic depression Homo- and heterosynaptic depression have yet to be f u l l y character ised. It i s known that lengthy low frequency tetanus induce homo- and heterosynap-t i c depressions (Dunwiddie and Lynch, 1978). Unlike LLP, depressions can be induced by nonsynaptic ac t i va t ion . Lynch, Gr ibkoff and Deadwyler (1976) reported a ce l l -w ide reduction in responsiveness to glutamate iontophoresis. Andersen, Sunberg, Swann and Wigstrom (1980) used i n t r a c e l l u l a r methods and detected a decrease in c e l l e x c i t a b i l i t y . However, th i s tes t ing was only - 46 -done for in terva ls immediately af ter tetanus and not when a f u l l y developed homo- and heterosynaptic depression was present. It s t i l l has to be deter -mined what i n t r a c e l l u l a r changes are associated with the depressions. Recently i t became evident that both depression and potent iat ion could c o - e x i s t . A balance is established between potentiat ion and depression (Sastry , Chirwa, Goh, Maretic and Pandanaboina, 1984). As best as can be determined, the locus for homo- and heterosynaptic depression i s postsynap-t i c . The exact mechanisms mediating these changes are s t i l l under i n v e s t i -gation . 11 SOME INHIBITORS OF POTASSIUM AND CALCIUM FLUXES 11.1 Bari urn , ++. ++ Barium (Ba ) competit ively hinders Ca entry into c e l l s , and when ++ + inside the c e l l , Ba diminishes K ef f lux (Werman and Grundfest, . . . ++ 1961). Sastry (1979) demonstrated a Ba -mediated increase in presynaptic terminal action potent ial (AP) ref ractory per iod, ind icat ive of a widened AP caused by the blockade of the delayed K r e c t i f i e r current . In the hippo-campus, Hotson and Prince (1981) found that bath appl icat ions of B a + + augmented the C a + + potent ia ls but attenuated the K +-dependent hyperpol -++ a r i z a t i o n s . It was concluded that Ba effects were caused by the in f lux ++ ++ + of Ba through Ca channels followed by the ions ' reduction of K conductance. ++ ++ Ba i s c lose ly re lated to Ca in atomic number, valence and chemi-cal propert ies . The hydrated radius of B a + + i s smaller than that of Ca (Stokes, 1964) and may account for the favoured entry of Ba than ++ ++ Ca through physiological Ca channels (Potreau and Raymond, 1980). Extensive studies are lacking that have explored a l l the C a + + functions - 47 -that B a + + can mimic. B a + + w i l l bind to calmodulin though with a lower a f f i n i t y than C a + + (Wolff, Hubner and S iege l , 1972). It was of interest ++ ++ to f ind out whether Ba can subst i tute for Ca during the i n i t i a t i o n of LLP. 11.2 4-Aminopyridine There are r e l a t i v e l y few studies that have been concerned with the possible cor re la t ion between pharmacologically induced enhancement of f i e l d responses (Haas and R y a l l , 1980; Linsemann and C o r r i g a l l , 1981; Turner, Baimbridge and M i l l e r , 1982; Goh and Sastry, 1983) and the e l e c t r i c a l l y evoked LLP (B l i ss and L0mo, 1973). Pharmacological agents such as 4-Amino-pyridine (4-AP) enhance inh ib i to ry and exci tatory influences in the hippo-campus (Buckle and Haas, 1982). 4-AP blocks voltage s e n s i t i v e , fas t K + channels in a var iety of excitable t issues (Lechat, Thesleff and Bowman, 1981) . Consequently 4-AP w i l l enhance most processes that are dependent on action potent ia ls whose dec l in ing phase is par t l y due to fas t K + channel conductances. In para l le l f ibers of the rat cerebel lar cortex, 4-AP caused the widening of the presynaptic vol ley (Kocsis, Malenka and Waxman, 1982). In presynaptic terminals , K + blockade invar iably leads to prolongation of the action potent ia l and t h i s causes vo l tage-sens i t i ve C a + + channels to open for a longer period (Yeh, Oxford, Klu and Narahashi, 1976; Tokunaga, Sandri and Akeset, 1979; Sastry , 1979; Theslef f , 1980; Letchat, 1981). Hence C a + + in f lux i s enhanced and t h i s f a c i l i t a t e s coupling between terminal depolar izat ions and transmitter release at chemical, synapses. The actions of 4-AP therefore lead to an increase in impulse-evoked transmitter release (Thesleff , 1980). An in terest ing feature of 4-AP interact ions is in i t s enhancement of C a + + entry into presynaptic terminals (Buckle and Haas, 1982) , which is a possible l o c i for LLP (Sastry, 1982). This property of - 48 -4-AP was u t i l i z e d in an attempt to i n i t i a t e LLP via pharmacological methods. 11.3 Verapami1 . Verapamil i s a synthetic der ivat ive of papaverine, an a lka lo id found in opium poppy. In cardiac pharmacology, verapamil blocks activated and ++ inact ivated Ca -channels . I ts e f fec ts are more marked in t issues that discharge frequently or are less polar ized at rest and where act ivat ion is dependent on the C a + + current (Henry, 1980; Katzung and Chatterjee, 1982). The s i n o a t r i a l and a t r iovent r i cu la r nodes exhib i t both these c h a r a c t e r i s t i c s . Verapamil a c t i v i t y i s s tereospec i f ic ( i . e . , possible receptor interact ion) with the S(-) isomer presenting a much greater potency than the R(+) isomer. Presumably verapamil acts from the inner side of the membrane and binds more e f f e c t i v e l y to channels in depolarized membranes. This block i s p a r t i a l l y revers ib le by elevat ing e x t r a c e l l u l a r C a + + or by drugs that increase the transmembrane f lux of Ca , e . g . , adrenergic stimulants (Katzung and Chatterjee, 1982). The actions of verapamil stated above have made t h i s substance useful in c l i n i c a l therapeutics Katzung and Chatterjee, 1982) where i t i s indicated for the fo l lowing condit ions; (a) a t r i a l f i b r i l l a t i o n o r , a t r i a l f l u t t e r of recent onset, with rapid vent r icu lar response (especia l ly i f not cont ro l lab le with d i g i t a l i s preparations) (b) paroxysmal supraventricular tachycardia (c) chronic stable angina of e f fo r t (d) angina resu l t ing from coronary artery spasm and (e) non-surgical obstruct ive hypertrophic cardiomyopathy (Canadian Pharma-ceut ica l Assoc iat ion , 1984). The therapeutic e f fec ts correspond to verapamil plasma levels of 60-70 ng/mL., i . e . , 0 .13-0.15 uM, t y p i c a l l y less than 1 yM. At these concentrations, verapamil exerts i t s i n h i b i t i o n of C a + + entry into cardiac t i s s u e . Data from cardiac and other excitable t issues indicate that low verapam-++ i l concentrations do not prevent the Ca -dependent release of t ransmitter - 49 -substances. Annunziato, DiRenzo, Amoroso and Quattrone (1984) found that the K +-evoked release of endogenous dopamine from tuberoinfundibular neurons in v i t r o was enhanced by lpM verapamil but depressed by verapamil concentrations greater than 10 uM. S i m i l a r l y low verapamil ( less than 10 uM) interfered with the Ca + +-dependent con t rac t i l e responses of iso lated canine saphenous veins caused by transmural nerve st imulation and yet t h i s same verapamil concentration did not reduce release of t r i t i a t e d norepinephrine from the adrenergic nerve endings in t h i s system (Takata and Kato, 1984). Only verapamil concentrations greater than 30 uM could reduce the release of t r i t i a t e d norepinephrine fo l lowing transmural adrenergic nerve s t imulat ion . In rat cerebrocort ical synaptosomes, Norr is , Dha l iwa l l , Druce and Bradford (1983) demonstrated that while low verapamil concentrations (equal to or less than 1 uM) could not prevent transmitter 45 + + re lease, i t was e f fec t i ve in blocking Ca entry . In excised rabbit , . ++ nodose ganglia C somata, Ca spikes could be e l i c i t e d which were ++ maintained by further st imulat ion of Ca in f lux ( I to , Sakanashi, Kawamura and N i s h i , 1984). In t h i s t i s s u e , i t was reported that in Na + free s o l u -t ions i n t r a c e l l u l a r ^ recorded C a + + spikes were reduced in amplitude by 10 uM verapamil. In normal physiological so lu t ions , 1 yM verapamil caused reductions in maximal rate of r i s e and maximal rate of f a l l . I to, Sakanashi, Kawamura and Nishi (1984) concluded that these low concentrations of verapamil depressed C a + + i n f lux and possibly could in ter fere with Na + and K + f l u x e s . The preceding studies demonstrate that low verapamil concentrations in ter fere with C a + + entry into neurons and/or other exci table c e l l s but do not prevent the Ca + +-dependent neurotransmitter re lease. It is well docu-- 50 -merited that the i n i t i a t i o n of hippocampal p l a s t i c i t y are Ca -dependent. However, i t remains to be resolved whether t h i s C a + + requirement i s needed to the pre - and/or postsynaptic elements. The ef fects of low verapamil concentration was thought to be a potent ia l method that could be used to determine which C a + + currents ( i . e . , presynaptic versus post-synaptic) were important for the development of LLP. For that reason, low verapamil concentrations were selected and used in some of the studies reported in t h i s t h e s i s . 12. METHODS 12.1 Animals Male Sprague-Dawley ra ts obtained from Charles River in Ontario (Canada) were used for some of the ear ly experiments reported in the present t h e s i s , i . e . , the demonstration and character izat ion of LLP and depression and the assessment of 4-AP ef fects in the hippocampus. But to minimise costs as well as f a c i l i t a t e rapid del ivery of animals, male Wistar rats obtained from the univers i ty of B r i t i s h Columbia (Canada) animal unit were subsequently used. The animal unit uses standard animal care procedures for the maintenance of laboratory animals. Their Wistar rats are weaned after 21 days. Once a week, t y p i c a l l y on Mondays, 8-12 male Wistar ra ts were received from the animal unit and used for studies in that same week. Upon a r r i v a l and pr ior to experimentation, Wistar rats (about 30 days o l d ; 75-100g ) were kept in wire cages in the modern departmental animal room. These rats had free access to food (rat chow) and water. The animal room had control led temperature (22-23°C) and humidity (50-55%) with set 12-hourly day and night peri ods. - 51 -Firs t of a l l , studies on LLP and depression as well as effects of 4-AP in Sprague-Dawley rats could be duplicated in Wistar rats in v i t ro . In fact no significant differences in pharmacological responsiveness and/or electro-physiological behavior could be detected between slices from Sprague-Dawley rats and Wistar rats (cf. Chirwa, Goh, Maretic and Satry, 1983 to Sastry, Chirwa, Goh, Maretic and Pandanaboina, 1984). Hence whenever applicable the overall experimental data from Sprague-Dawley rats and Wistar rats have been assessed together without c r i t i c a l considerations of the rat subtypes used. 12.2 Slice preparations Hippocampal sl ices were prepared from male Wistar rats as follows. Animals were i n i t i a l l y cooled (30-40 min) on an ice pack in a dessicator to a rectal temperature of 30-31° C and maintained on a mixture of halothane and oxygen. The dessicator was pre-equilibrated with this halothane/oxygen mixture (in concentrations sufficient for general anaesthesia) before introducing the animal. To obtain s l ices , the skin was cut and an insertion made under the base of the skul l . A pair of small scissors was used to cut through the skull along the sagittal suture line and the sides pulled apart to expose the brain. The brain was carefully removed and hippocampus dissected free on a cooled glass surface. The dorsal surface of the hippocampus was then sectioned transversely to the septotemporal axis at a thickness of 500 u on a Mcllwain tissue chopper. Serial sections were separated with a fine brush and a stainless steel spatula in a plate containing previously cooled (5° C) a r t i f i c i a l cerebrospinal f lu id (termed 'standard medium' or 'standard solution' in this thesis) and equilibrated with 5% carbon dioxide and 95% oxygen (carbogen). The procedure from surgery to s l ice preparetion was completed within 3 minutes. In i t ia l - 52 -cool ing of the animal s i g n i f i c a n t l y increased the proportion of v iable s l i c e s obtained from each hippocampus. F i n a l l y s l i c e s were transferred to the s l i c e chamber. S l i ces were positioned between two nylon nets to minimise movement as well as permit submersion. The chambers were perfused at a rate of 2-3 mL/min with the standard medium containing in mM: NaCl, 120; KC1, 3 . 1 ; NaHC033, 26; NaH 2P0 4 , 1 .3; C a C l 2 > 2 .0 ; MgCl 2 , 2.0 and glucose, 10.0. The standard medium was pre-gassed with carbogen (pH of medium, ca . 7.4) and maintained at 32 ± 0.5° C. In add i t ion , the carbogen flowed over the top of the s l i c e s in the s l i c e chamber. S l i ces were allowed to equ i l ib ra te with the standard medium in the s l i c e chamber for a minimum of 60 minutes pr ior to recording. 12.3 S l i ce se lect ion and v i a b i l i t y S ign i f i cant damage occurs along the cut surfaces of the s l i c e (Schurr, Reid, Tseng and Edmonds, 1984). So e lect rophys io log ica l recordings were usually obtained from a depth of about 100-300 u from the surface of a transversely cut hippocampus sect ion . It i s known that immediately after s l i c i n g , the metabolic status of brain s l i c e s is s i g n i f i c a n t l y altered r e l a -t i v e to in vivo a c t i v i t y . For instance, the levels of to ta l adenylates and creatines or peak cAMP and cGMP leve ls are decreased (Whittingham, Lust , Chr is tak is and Passonneau, 1984). In fac t red i s t r ibu t ion of cations occurs, i . e . , lowered i n t r a c e l l u l a r K + /Na + r a t i o . Evidence indicates that some 1-2 hours are needed for the s l i c e s to return back to a steady metabolic state (Whittingham, Lust, Chr is tak is . and Passonneau, 1984). This steady metabolic state in s l i c e s is presumably maintained for 6-8 hours of incuba-t i o n . Perez-saad, Valouskova and Bures (1984) found that immediately af ter s l i c e cutt ing and submerging, the ex t race l lu la r K + p r o f i l e s were very high. After 1-2 hours these invest igators found that the ex t race l lu la r K + p r o f i l e s decreased and approached the K + concentrations of the a r t i f i c i a l cerebrospinal f l u i d used. It i s for reasons as presented above that in the present s tud ies , s l i c e s were incubated for at least one hour to permit recovery from the mechanical and metabolic trauma. To begin w i th , selected s l i c e s (4-6 out of a possible 8-10 obtained in s l i c i n g ) had the fo l lowing c h a r a c t e r i s t i c s . They were i n -tact with well defined and unmashed borders, i . e . , s l i c e edges. Only s l i c e s with a c l e a r , translucent CA and DG c e l l u l a r layers and in add i t ion , a V- and/or U-shaped DG were chosen. Furthermore, the selected s l i c e s had clean and even (smooth) surfaces, i . e . , not 'mashy' or ' f l a k y ' . Evoked responses could be obtained af ter one-hour incubations and main-tained for up to 12 hours (Sastry, Chirwa, Goh, Maretic and Pandanaboina, 1984). Each experiment was t y p i c a l l y of 2-4 hours durat ion. Hence 2-3 of the selected s l i c e s could have been used concurrently after the incubation per iod. But to minimise v a r i a b i l i t y between s l i c e s from the same animal (and those of other animals) caused by d i f fe rent exposure times to the standard medium, only one s l i c e per animal was u l t imately used. Consequently, the to ta l times of exposures to bathing medium and experimentation for d i f ferent s l i c e s were comparable. 12.4 S l i c e chamber and perfusion method Detailed descript ions of the s l i c e chamber used were reported in a publ icat ion from t h i s laboratory (Pandanaboina and Sastry , 1984). Figure 5 i s a simple sketch of the s l i c e chamber. The main components of the chamber were (a) a raised stage constructed of p lex ig lass and (b) with a c i r c u l a r chamber of diameter 7.5 cm and depth 0.7 cm, bored into the top surface and (c) a special temperature-regulating aluminum bar that was stuck beneath the c i r c u l a r chamber. - 54 -F i g . 5. SI ice chamber. A. Mani fo ld . B. Suction l i n e . C. Raised p lex ig lass stage. D. Temperature regulat ing aluminum bar. E. The s l i c e chamber. F. Medium i n l e t . - 55 -The standard and test media were contained in separate 50-mL po lye thy l -ene b a r r e l s . Carbogen l ines for medium oxygenation terminated in each of these b a r r e l s . The barrel containing standard medium was in turn connected to an elevated feeding tank (volume, ca . 2 L) which was the source of stand-ard medium. From each 50-mL barrel came out a connecting tube (that could be opened or closed with a c l i p ) that led to a common 'manifo ld ' on one end and which in turn , gave out a s ingle out let on the opposite s i d e . This s ingle o u t l e t , i . e , v ia connecting polyethylene tubes, eventually fed the selected medium to the s l i c e chamber. B r i e f l y , both standard and/or test solut ions were i n -troduced into the s l i c e chamber from above. Continuous drainage out of the s l i c e chamber was v ia a side suction out let created fo r that purpose. A balanced inflow and outflow of solut ions ensured the maintenance of constant solut ion leve ls in the s l i c e chamber. At a l l t imes, the s l i c e s were sub-merged in the medium. Among other propert ies , the whole perfusion set-up permitted (a) the rapid exchange of standard and test solut ions (b) complete oxygenation of solutions (c) removal of dead spaces within the system and (d) the regu la -t ion of solut ion temperature. 12.5 Preparation of standard and test media The standard medium was f reshly prepared on each experimental day, to the f i n a l constituent concentrations described in sect ion 12 .2 . Test media of B a + + , M n + + and M g + + were prepared by omitting 2 mM C a + + in the standard medium and replacing i t with 2 mM of B a + + or M n + + or M g + + respect ive ly . Stock solutions of L-glutamate (the monosodium sa l t ) and 4-Aminopyridine, were prepared by d isso lv ing appropriate amounts of each of - 56 -these substances in de- ionised water to make f i n a l concentrations of 100 mM. The L-glutamate solut ion was used to f i l l the iontophoretic microelectrode. The 4-AP solut ions were d i lu ted to the appropriate f i n a l concentrations by adding measured amounts to the standard medium or the other test media. In a l l cases, the pH of the f i n a l 4-AP containing media was found to be ca . 7.4 while being bubbled with carbogen. The stock solut ions were ref r igerated when not being used. The remain-ing standard media at the end of the (experiment) day was discarded. The laboratory chemicals used in the preparation of solut ions were obtained from the fo l lowing sources; (a) B a C l 2 , 4-Aminopyridine and L-glucose came from Sigma, (b) NaHC03, , NaCl, NaH2P04, MgCl 2 and C a C l 2 , came from Fisher and (c) KC1 and MnCl 2 came from Baker. 12.6 Stimulating and recording electrodes Concentric b ipolar st imulat ing electrodes, model SNEX-100 with shaft lengths of 50 mm (Rhodes Medical Instruments) were used. These electrodes had resistances of 1.0 'Mo,. Subsequently each st imulat ing electrode was replaced, whenever i t s resistance s i g n i f i c a n t l y increased, i . e . , greater than 3 Ki (occurred after 5-7 weeks of continuous use) and i f t h i s r e s i s t -ance could not be lowered by basic techniques of cleaning the e lectrode. Standard f i b e r f i l l e d glass micropipettes ( internal diameter, 1.02 ± 0 .15 ; outside diameter, 1.5 ± 0.1 mm: WPI) were used to prepare recording e lectrodes. Most of these micropipettes were pul led to f ine t i p s ( t ip diameter, 1-3 micrometers) on the Narishige v e r t i c a l pu l le r type PE-2 . Single barrel recording micropipettes were f i l l e d with 4M NaCl and had resistances of 0 . 5 - 1 . 5 Mo,. These micropipettes were used to monitor or record spontaneous and evoked potent ia ls in the hippocampus in v i t r o . For - 57 -iontophoretic appl icat ions and recordings, seven-barrel micropipettes were assembled, then pul led ( t ip diameter, 2-3 micrometers) and f i l l e d as fo l lows . The central b a r r e l , which was usual ly used to record potent ia ls was f i l l e d with 4M NaCl. Three side barrels also contained 4M NaCl, and were used to check for current e f fects and to i n i t i a t e current balancing when necessary. The remaining three side barrels were f i l l e d with 0.1M L-glutamate ( r e s i s t -ance t y p i c a l l y 30-50 Mfi). A t h i r d type of recording micropipette was assembled and used to monitor C a + + and K + l e v e l s . A double-barrel ion se lect ive micropipette was pul led to f ine t i p s izes (diameter 0 ; 5 - l micrometers) on a model P—77, Brown-Flaming micropipette p u l l e r . Then a very f ine hypodermic needle was inserted down to the taper of one b a r r e l . Using a double-switch system, t h i s hypodermic needle could be used to pass e i ther nitrogen (N2) or vapors of dichloromethylsi lane (si lane) caused by f i r s t passing N 2 over s i l a n e . I n i t i a l l y , the taper of the micropipette was heated to 200°C in a c o i l . During t h i s heating, one barrel was dried by passing N 2 for one minute. This was followed by passing s i lane vapors down the taper for 5 minutes. This process led to the baking of s i lane onto the inner surfaces of the treated micropipette barrel thereby making i t hydrophobic. Each si laned t i p + was s y r i n g e - f i l l e d with either ion-exchange resin IE-190 for K or i o n -++ exchange res in IE-202 for Ca (both resins obtained from Narco). Then the barrels were b a c k - f i l l e d with 0.1M KC1 or 0.1M CaCl 2 respect ive ly . The non-si laned barrel in the double-barrel assembly was used as a reference electrode and f i l l e d with 0.1M NaCl (for K + -e lec t rode) and 0.1M KC1 (for the C a + + - e l e c t r o d e ) . - 58 -The K + electrode was cal ibrated by measuring the mV def lect ions on a vibronmeter, after exposure to graded [K + ] so lu t ions . In the range 10-150 mM K + , a l inear re lat ionsh ip was observed. Typ ica l l y a 40-45 mV d e f l e c -+ ++ t ion was seen with every 10 mM increase in [K ] . S i m i l a r l y , the Ca -electrode was ca l ib rated using graded [Ca + + ] so lu t ions . In the l inear range, (within physiological C a + + ranges), increases of 10 mM [Ca + + ] caused approximately 25 mV def lec t ions . These ca l ib ra ted values for K + and C a + + were used as references for comparison when recording K + and C a + + changes in the hippocampus, in v i t r o . 12.7 Pos i t ion ing of electrodes Placement of electrodes was done with the aid of a d issect ing microscope. The st imulat ing electrodes were posit ioned in the stratum radiatum or in the CA3 region for st imulating Schaffer c o l l a t e r a l s . In some cases, st imulat ing electrodes were placed in the alveus or the stratum oriens for the st imulat ion of CA1 pyramids axons or commissural afferents respect ive ly . The terminal region e x c i t a b i l i t y was examined with st imulat ion electrodes placed in the Schaffer-CAl dendrites synaptic regions to evoke discharges in CA3 pyramids. These st imulat ion and recording electrode posit ions are shown in f i g . 6A and 6B. The recording micropipettes, i . e . , s ingle b a r r e l , double-barrel ion s e l -ective electrode or seven-barrel iontophoretic e lectrode, were positioned in the stratum o r i e n s , stratum pyramidale or stratum radiatum in the CAlb sub-f i e l d (Masukawa, Bernado and Pr ince , 1982). This arrangement permitted the recording from basal dendr i tes, CA1 c e l l bodies and apical dendr i tes. Once posi t ioned, the depth of the t i p of the micropipette from the s l i c e surface was adjusted to maximise the observed responses evoked by st imulation of Stimulating Recording Fig. 6 . Positioning of stimulating and recording electrodes. Stimulating: 1. Commissural afferents. 2. Schaffer collaterals. 3 . Sch/CAlb synaptic regions. 4. CAlb axons. Recording: 5 . Antidromic single spikes in CA3. 6 . PS with central barrel; ejecting. L-glutamate with side barrels. 7. CAlb PS. 8. EPSP in dendritic regions. 9. Monitoring K+ or Ca in cell body layer. A. B. - 60 -CAlb axons, commissural and/or Schaffer c o l l a t e r a l s . During Schaffer c o l l a -te ra l boutons e x c i t a b i l i t y t e s t i n g , the s ing le -bar re l recording micropipette was positioned in the CA3 s u b f i e l d , for the recording of antidromic s ingle c e l l discharges. 12.8 Laboratory e l e c t r i c a l instruments The main e l e c t r i c a l apparatus/instruments used are b r i e f l y summarised below. Square wave pulses were del ivered through an i s o l a t i o n unit type DS2 regulated by the d ig i t imer programmer D4030. A l t e r n a t i v e l y , the Grass S88 stimulator was used to drive ei ther the stimulus i s o l a t i o n unit S1115 or the photoelectr ic constant current u n i t , which was used in terminal region ex-c i t a b i l i t y t e s t i n g . Recording electrode s ignals were amplif ied on the Neurolog systems or the Dam-5A d i f f e r e n t i a l p r e - a m p l i f i e r . The amplif ied signals could be viewed on any of the three types of osc i l loscopes namely; the Data 6000 programmable acqu is i t ion u n i t , the Tektronix dual beam storage type 565 or type 5113. Ion se lect ive s ignals were led into the Vibronmeter which, in tu rn , fed signals to the Grass polygraph to be amplif ied and plotted out . Typ ica l l y , permanent records of a l l observed s ignals could be plotted on the HP 7404A recorder and the HP 7470A p l o t t e r . This last p lo t te r was driven and con-t r o l l e d by the Data 6000 programmable acqu is i t ion u n i t . Signals could also be plotted out on the Grass polygraph. In add i t ion , displayed signals on the osc i l loscope type 5113 could be photographed d i r e c t l y . In some cases complete experiments, (especia l ly those examining indiv idual pulses during tetanic st imulat ions) were taped on Sony PR-150 magnetic tape using the HP-3968A instrument recorder. Then selected segments of these tapings could be charted out on the HP-7404A p lo t te r for analysis or played back on one of - 61 -the osc i l loscopes . Las t l y , a s i x channel Neurophore BH-2 (Medical Systems) was used for iontophoretic appl icat ions of selected ion ic species. Among other features, t h i s Neurophore gave control led current ejections of s e l e c t -ed ion ic species as well as induction of set backing and/or balancing cur -rents . The a v a i l a b i l i t y of these e l e c t r i c a l instruments made i t possible to combine two or more units and independently stimulate and/or record from more than one hippocampal s i t e . 13. EXPERIMENT SCHEMES 13.1 Induction of evoked responses . Depolarizing square wave pulses (2-15 V; 0 .02 -0 .8 msec) del ivered at 0.2 Hz test frequency were used to stimulate CAlb c e l l axons or commissural (Com) or Schaffer c o l l a t e r a l s (Sch). Components of the somatic and dendr i t ic recorded potent ia ls were ex-amined in standard medium and in Mg+ +-medium. This l a t t e r procedure per-mitted the v i s u a l i z a t i o n of the presynaptic vol ley as well as antidromic components or non-Ca + + dependent evoked potent ia ls . Furthermore, depolar iz ing square wave pulses of 4-15 uA amplitudes and 0 . 2 - 0 . 8 msec durations (test frequency), 0.2 Hz) were applied in the Sch-CAlb apical dendr i t i c synaptic regions to evoke antidromic s ingle c e l l discharges in CA3 pyramids. These a l l - o r -none discharges could be evoked in ++ ++ standard medium and in Mg or Mn media. Subsequent use of these antidromic s ingle c e l l discharges was dependent on the demonstration of (a) constant onset latency, which was the time from onset of st imulat ion a r t i -fac t to peak s ingle discharge negat iv i ty (b) constant shape and amplitude in - 62 -standard medium, comparable to spontaneous discharges in these CA3 pyramids and (c) d i s t i n c t thresholds of a c t i v a t i o n . The selected sets of e l e c t r i c a l parameters in each experiment were those that e l i c i t e d PS amplitudes of 1 .0 -1 .5 mV or dendr i t ic EPSP amplitudes of approximately 1.0 mV. Once these i n i t i a l population responses were ob-ta ined, p a r t i c u l a r l y those of orthodromic s t imulat ion , the i r amplitudes were constantly monitored (at f i xed pulse amplitude and durat ion; 0.2 Hz) for a minimum 30 minutes. These were the baseline responses. Subsequent exper i -mentation was conducted only i f these baseline responses remained unchanged for at least 30 minutes. Then changes to evoked potent ia ls caused by e l e c -t r i c a l condit ioning and/or pharmacological manipulations were compared to baseline responses and where appl icable these changes were expressed as f ract ions of the baseline responses respect ive ly . 13.2 Tetanic frequencies Studies on potentiat ion and depression of evoked responses were con-ducted by using one or more of the fo l lowing frequencies: 20 Hz, 200 or 600 pulses; 100 Hz, 100 or 500 pulses and 400 Hz, 200 pulses. These tetanic t ra ins were evoked using the same st imulation strengths and durations as was used to obtain baseline (control) responses respect i ve ly . Tetanic st imulations were del ivered to any of the fo l lowing inputs (a) commissural (b) Schaffer c o l l a t e r a l s (c) Sch-CAlb synaptic regions and (d) CAlb c e l l axons. To f a c i l i t a t e analysis indiv idual pulses in the tetanic t ra ins were taped and charted out at the end of the experiment. 13.3 4-AP dose-response curves In a given s l i c e , dose response curves were constructed by the method of stepwise cumulative addit ion of the drug. After a control period the i n i -- 63 -t i a l concentration of 4-AP used was 25 uM. The amount of 4-AP was then doubled af ter each 5 minute interva l to a maximum of 400 pM. On completion of the dose-response curve, 4-AP was washed out from the s l i c e s by cont inu-ous perfusion with standard medium. A l l responses to 4-AP and the subse-quent recovery were expressed as a percentage of the control responses ob-tained in standard medium pr io r to the determination of the dose response curves. 13.4 Ef fects of 4-AP A concentration of 100 uM 4-AP was selected from the dose-response curve above and used for subsequent s tudies . In one ser ies of experiments, each s l i c e was perfused with 4-AP fo r 5 minutes only and then the drug was washed out with standard medium. A second 5-minute 4-AP perfusion was started after an in terva l of 40-60 minutes. Responses in 4-AP and during washing out were monitored. S imi lar experiments were conducted (d i f ferent s l i c e s ) with the fol lowing modi f icat ions . After obtaining control responses, st imulat ion was stopped fo r 15 minutes and then resumed again. This was done to check for the e f -f e c t s , i f any, of temporarily stopping s t imulat ion . Spontaneous a c t i v i t y was also monitored. After a minimum of 30 minutes st imulat ion was stopped again in the same s l i c e , but t h i s time 4-AP was perfused for 5 minutes. ++ Effects of 4-AP in calc ium-free medium was examined as fo l lows : Mg -med-ium was prefused to block synaptic transmission for 15 minutes. 100 pM 4-AP (previously added to a second M g + + medium) was perfused for 5 minutes during synaptic blockade i . e . , 5 min. af ter synaptic blockade was estab-l i s h e d ) . 13.5 Verapamil studies Dose-response curves to 0.083-2.64 pM verapamil were obtained using the s ingle appl icat ion method ( i . e . , with washing between doses). After - 64 -exposure to the i n i t i a l 0.083 yM for 3-4 min, the verapamil dose was doubled. This process was repeated u n t i l a maximum 2.64 yM verapamil was appl ied . From the completed dose-response curves, an appropriate verapamil concentration (0.33 yM) was selected and used in subsequent experiments. The ef fects of a s ingle concentration (0.33 yM) verapamil were tested as fo l lows . After establ ish ing baseline responses to Sch s t imulat ion , 0.33 yM verapamil in standard medium was perfused for 3 minutes and then washed out with just standard medium perfusions. A s i m i l a r appl icat ion was repeated some 40-60 minutes after the f i r s t app l i ca t ion . In both cases, evoked res -ponses during verapamil were monitored. 13.6 Iontophoretic L-glutamate Using eject ion currents of 50-200 nA, L-glutamate was l o c a l l y applied for 1 minute to the CAlb pyramidal layer . In these experiments the Sch were continuously stimulated at 0.2 Hz. In another set of experiments, modified standard medium containing 0.33 yM verapamil preceded the ejection of L - g l u -tamate by 1 minute This modified standard medium with verapamil was l e f t on for a to ta l 3 minutes. L a s t l y , te tanic st imulations (100 Hz, 1 sec) to Sch were evoked (a) while L-glu was ejected alone or (b) when L-Glu was ejected concurrently with the verapamil medium. 13.7 Effects of B a + + At pre-determined i n t e r v a l s , B a + + medium was perfused for 3 , 10 or 15 minutes. Each selected perfusion time constituted a complete experiment in a given s l i c e . The B a + + medium perfusion was ins t i tu ted while the Sch were being stimulated at 0.2 Hz. ++ + 13.8 Monitoring ex t race l lu la r Ca r and K changes Changes in soma ex t race l lu la r C a + + or e x t r a c e l l u l a r K + during te tan -ic st imulations (20 Hz or 100 Hz) were recorded using the ion -sens i t i ve - 65 -electrodes (ISE). These ISE were positioned in the pyramidal layer as previously described (see section 12 .7 ) . 13.9 Sch boutons e x c i t a b i l i t y tes t ing Control thresholds in standard medium fo r the i n i t i a t i o n of antidromic CA3 s ing le c e l l discharges were f i r s t determined in standard medium. The threshold was defined as the st imulat ion strength used to evoke 4-5 a n t i -dromic discharges out of 10 possible attempts. The thresholds were then determined during as well as after 4-AP or B a + + perfusions. 13.10 Homo- and heterosynaptic p l a s t i c i t y To invest igate homo- and heterosynaptic p l a s t i c i t y the commissural and the Schaffer c o l l a t e r a l s were stimulated as fo l lows . I n i t i a l l y baseline responses and the i r appropriate st imulat ion parameters were determined for each input independently. Thereafter, 10-15 depolar iz ing pulses at 0.2 Hz were given to the Sch while the Com was not st imulated. Af ter these pulses, st imulation was stopped and then another 10-15 depolar iz ing pulses at 0.2 Hz were given to the commissural input. This alternate manner of st imulat ing these two inputs was repeated during the whole experiment. However, at appropriate i n t e r v a l s , selected tetanic t ra ins were evoked in the Sch or the Com. In some experiments tetanic st imulations were evoked while 0.33 uM verapamil was perfused for 3 minutes. In these experiments, tetanic st imu-lat ions were evoked during the last minute of verapamil perfusions. 14. RESULTS 14.1 Evoked potent ia ls The evoked potent ia ls recorded in CAlb c e l l layer had a biphasic p o s i -t i v e wave (PW) that could be interrupted with a negative going peak which - 66 -was the population spike (PS). The presence of the PS and i t s increasing magnitude, i . e . , more neurons recrui ted was dependent on the stimulus strength used. Stimulus strengths between 2-15 volts (0 .02-0.8 msec; 0.2 Hz) to Sch or Com e l i c i t e d PS amplitudes of 1 .0 -1 .5 mV. Figure 7 shows how the PS amplitude and i t s latency was measured. The observed latencies from onset of a r t i f a c t to peak negat iv i ty of the orthodromic.PS were 6-10 msec. The longer latencies were associated with st imulat ing electrodes positioned farthest from the recording electrodes in CAlb. S i m i l a r l y , antidromic a c t i -vation of CAlb axons in the alveus, used lower stimulus strengths but e l i c -i ted comparable PS amplitudes (1 .0 -1 .5 mV) that had shorter latencies (1-3 msec). Orthodromic potent ia ls recorded at the CAlb dendrites in stratum oriens or stratum radiatum induced a slow, negative going potent ial which was the population EPSP. Depending on the st imulat ion strength, a pos i t i ve going peak was sometimes superimposed on th is dendr i t i c EPSP wave, which was the soma generated PS. The re la t ionsh ip of st imulation strength and ampl i -tude of PW, PS and EPSP obtained in a t yp ica l experiment are given in f igure 8 . In M g + + or M n + + media, orthodromic potent ia ls were abolished but not the antidromic evoked po ten t ia l s . During M g + + or M n + + perfusions, the presynaptic v o l l e y , espec ia l l y in dendr i t ic recordings, was c l e a r l y d i s c e r -n i b l e . L a s t l y , v isual inspection of the continuous osc i l loscope signals revealed l i t t l e or no spontaneous a c t i v i t y in standard medium. In M g + + or M n + + media, however, the spontaneous a c t i v i t y increased and sometimes led to burst ing a c t i v i t y . 14.2 4-AP dose-response curves Figure 9 shows the dose-response curves obtained from the cumulative addit ion of 4-AP. Maximal enhancements in evoked PS were obtained with con - 67 -F i g . 7. PS amplitude and latency measurement. V e r t i c a l l i n e corresponds to PS amplitude measure-ment. Horizontal l i n e represents PS onset latency. Diagonal arrow shows onset of stimulus a r t i f a c t . Negat iv i ty i s downwards. Abbreviat ions: A, st imu-lus a r t i f a c t ; PS, population sp ike, 2PW, second component of pos i t i ve wave. - 68 -dendritic EPSP soma PS weak moderate strong •i I. F i g . 8 . Stimulus strength and evoked p o t e n t i a l s . Terms on side re fe r to r e l a t i v e stimulus strength used to evoke the presented p o t e n t i a l s . Note the presence of soma generated PS in the dendr i t i c EPSP (*). Sca le : 1.0 mV by 10 msec for EPSP; 1.5 mV by 10 msec fo r PS. Negat iv i ty i s downwards. - 69 -4-AMINOPYRIDINE ( p M ) F i g . 9. Dose ^response curves to the cumulative  addit ion of 4-AP showing increases in the ampl i - tudes of evoked population sp ikes . Responses are expressed as percentages of the responses obtained in standard medium. Each value represents mean ± S.E .M. (n = 5) . - 70 -centrations between 100-200 uM 4 -AP. Cumulative drug concentrations above 200 uM often caused reductions in the evoked responses during perfusions. In add i t ion , a second spike was often associated with cumulative doses above 100 yM 4 -AP. The last two records in f igure 10 show these spikes which rapid ly diminished with washing. Further analysis of the ef fects of 4-AP were conducted using 100 yM concentrations. 14.3 Verapamil From the verapamil studies ( for methods see section 13.5) i t was found that verapamil concentrations greater than 0.66uM tended to induce some dep-ression (down to 85-95% of control PS) of the evoked PS. Yet verapamil con-centrations less than 0.66|»M did not cause a suppression of these poten-t i a l s . Hence 0.33uM verapamil was selected and used in the remaining exper-iments. During 0.33uM verapamil applied for 3-4 min, there was l i t t l e or no increase in the evoked PS. After verapamil perfusions, the evoked PS was s l i g h t l y enhanced as i l l u s t r a t e d in f igure 11. 14.4 LLP In the experiments described here, LLP induction t r i a l s were considered successful or not after one tetanic s t imulat ion . As stated previously , antidromic tetanic st imulations at a l l tested frequencies, f a i l e d to e l i c i t LLP. Only orthodromic tetani evoked in standard medium induced LLP. This LLP was seen as an increase in PS, PW and EPSP amplitudes with reduced l a t -encies. Figure 12 depicts these potentiated p o t e n t i a l s . The potentiated responses (PS increases of 200-500% or EPSP increases of 150-200% r e la t i ve to controls) showed l i t t l e or no decay even after 60 min. Interest ing ly , the best LLP ( in terms of magnitude) was e l i c i t e d by 400 Hz then 100 Hz and l a s t l y 20 Hz tetanic st imulations in that order. In fac t the higher tetanic - 71 -CONTROL 4-AMINOPYRIDINE •HN- -^j^- VjN. +j 20 ms 25 50 100 200 400JJM F i g . 10. Representative enhancements in evoked  population spikes of CAlb pyramidal c e l l s to cumu- l a t i v e addit ion of 4-AP during the determination of  the dose response curves/ St imulat ion was in the stratum radiatum. The las t t race c l e a r l y shows a second spike (arrow). Negat iv i ty i s downwards; each trace i s an average of eight consecutive sweeps. - 72 -frequencies were associated with an immediate post - tetanic expression of LLP (100 Hz, LLP in 7/9 s l i c e s ; 400 Hz, LLP in 6/6 s l i c e s ) . Yet after 20Hz, LLP expression was gradual and tended to peak at 15-20 min. af ter tetanus and here the success rate was low (20Hz, LLP in 5/9 s l i c e s ) . 14.5 Homo- and heterosynaptic p l a s t i c i t y In prel iminary experiments i t was found that 20Hz for 30 sec could pro-duce a post - te tan ic depression (PS, 20-80% Of cont ro ls ; n = 14) that lasted for 10-110 min. In some cases, th i s depressive phase was followed by a return to pre-tetanus PS or LLP. Yet tetanus of 100-150 Hz for 1-5 sec con-s i s t e n t l y caused the development of LLP (PS, 100-720% of cont ro ls ; in 20/22 s l i c e s ) without a depressive phase. LLP produced by 100-150Hz could be interrupted temporarily by a subsequent 20Hz tetanus ( f ive s l i c e s ) fo r 10-20 min. Thereafter, LLP could be seen again. This phenomena (potentiat ion and depression). was further investigated on two separate inputs, Com and Sch afferents to the same output CAlb neurons, monitored simultaneously. In add i t ion , the involvement of C a + + in potentiat ion and depression was tested by using 0.33uM verapamil, which s e l e c t i v e l y blocked C a + + entry into the postsynaptic neurons (see section 11.3) . High frequency tetanus (400Hz, 200 pulses) to Com or Sch e l i c i t e d a rapid development of LLP of the CAlb PS that was l imited to the tetanized input. This LLP after 400Hz was preceded by a post - te tan ic potentiat ion (PTP, discussed in section 10.1) that quickly decayed down in 2-3 min, r e -vealing the underlying LLP. The tetanus given in the presence of verapamil (0.33uM) also induced an i n p u t - s p e c i f i c LLP. Table 1 gives a summary of the resul ts on LLP fo l lowing 400Hz. The PTP preceding LLP e l i c i t e d by 400Hz during verapamil perfusions was much greater than PTP obtained with - 73 -150-1 o a: Z o o 100-50 I I control YA i n verapamil fBk post-verapamil X U F i g . 11. Verapamil and evoked p o t e n t i a l s . Bars represent mean amplitude of population spike Small v e r t i c a l l ines are +S.E.M. for n=7 s l i c e s . The post-verapamil recordings were taken 15 and 30 min after 0.33 micromolar verapamil was applied for 3 min. -74 -control Fig. 12. Illustration of hippocampal long lasting  potentiation. Note that even after 60 min there is no decrement in potentiated population spike. Negativity is downwards. Each record is an average of four sweeps. - 75 -400Hz alone. Furthermore, during the LLP of Sch PS, i f Com was tetanized with 400Hz to induce LLP, there was no detectable homo- or heterosynaptic depression (c f . with 20Hz tetanus) . Thereafter, the second LLP in Com co -existed with the LLP previously i n i t i a t e d in the Sch input. The resu l ts obtained with low frequency tetanus (20Hz, 200 pulses) are compiled in table 2. I t was found that 20Hz to Com or Sch i n i t i a l l y induced a post - te tan ic depression in both inputs (homo- and heterosynaptic depres-sion), that was followed by return to pre-tetanus PS in the non-tetanized input (heterosynaptic) and sometimes in the tetanized (homosynaptic) input or LLP in the tetanized input only . Though not ca re fu l l y characterized in the present s tud ies , the 'PTP' associated with 20Hz seemed to be an exten-sion of the frequency potent iat ion during the tetanic t r a i n s . This 'PTP' after 20Hz (cf . B l i s s and L^rno, 1973) quickly decayed down to within 1-1.5 min to reveal a marked depression of evoked responses. When 20Hz tetanus was evoked while verapamil (0.33 uM) was perfused, t h i s verapamil was found to counteract the homo- and heterosynaptic depression (see table 2) . A r e -markable f ind ing was the observation that an establ ished LLP could tempor-a r i l y be masked by a subsequent 20Hz to the same input (homosynaptic) or to a non-potentiated (heterosynaptic) input. The above resu l t s prompted the need to examine the c h a r a c t e r i s t i c s of indiv idual pulses associated with the tetanic t ra ins used in these studies. The f indings from such analyses are presented in the fol lowing sec t ion . 14.6 Character is t ics of tetanic frequencies Figure 13 depicts the indiv idual PS evoked in a representative s l i c e , during antidromic tetanizat ion of CAlb axons. These PS associated with each pulse in the tetanus could be evoked in standard s o l u t i o n , M g + + or Mn + + - 76 -Sch-1nduced population spike Comm-induced population spike (X of control) (X of control) 30 min post-tetanus 30 min post-tetanus 400 Hz tet Sch 243 * 56 (n = 7) 103 * 4 (n = 7) 400 Hz tet Sch 494 * 95 (n = 5) 108 * 4 (n = 5) with verapamil 'Al l values are mean ± SEM. Table 1. Effects of verapamil on long l a s t i n g p o t e n t i a t i o n . Note that only Schaffer (Sch) was tetanised and t h i s input developed LLP. Verapamil d id not block the i n i t i a t i o n of LLP but was found to enhance LLP development (bottom row). Sch-induced population spike (X of control) Ccmm-lnduced population spike (% of control) 5 min 10 min 20 min 5 min 10 min 20 min (post-tetanus) (post-tetanus) 20 Hz (Sch) 8 56 * 6 77 * 3 79 * 3 47 * 6 64 * 5 70 * 3 20 Hz tet (Sch) 8 93 * 7 118 * 16 139 * 21 81 * 10 89 * 7 101 * 5 with verapamil (p < 0.01) (p < 0.05) (p < 0.02) (p < 0.02) (p < 0.02) (p < 0.01) 20 Hz tet (Sch) 10 53 * 10 80 * 7 94 * 7 59 * 18 68 * 12 88 * 13 during 400 Hz Induced LLP 20 Hz tet (Sch) 8 98 * 4 101 * 4 102 * 3 103 * 5 101 * 4 114 * 7 with .verapamil..' (p < 0.01) (p < 0.05) (p < 0.05) (p < 0.05) durinq LLP •All values are mean * SEM. p values are obtained using unpaired t-test between verapamil-treated and untreated responses. Table 2. Ef fects of verapamil on homosynaptic and heterosynaptic  depression. In f i r s t row, note the time-course of the depression of evoked PS in the tetanised (Sch) and non-tetanised (Com) inputs after 20Hz tetanus. This depression was counteracted i f the 20Hz tetanus was evoked during verapamil perfusions (second and fourth rows). The 20Hz-induced depression masked developed LLP ( th i rd row; pre-20Hz data of LLP not given). See text for discussions. - 78 -media. The amplitudes and latencies of indiv idual PS evoked during 20 Hz ( 200-600 pulses) or 100 Hz (100-500) pulses) were e s s e n t i a l l y unaltered. In a few experiments the amplitudes of some indiv idual PS during tetanic s t imulat ions , p a r t i c u l a r l y with 100 Hz, were i n s i g n i f i c a n t l y depressed. This ef fect was apparent in the l a t t e r phases of a long antidromic tetanus and only in standard medium. A s i g n i f i c a n t f ind ing was that none of the antidromic tetani in more than twenty separate attempts was associated with frequency potent ia t ion . Above a l l , antidromic tetanic st imulat ion at a l l ++ ++ tested frequencies, evoked during standard, Mg or Mn perfusions, f a i l e d to e l i c i t LLP. Orthodromic 400 Hz tetanus to Sch evoked 2-4 rapid ly decl in ing synchron-ously discharged PS only ( f i g . 14A). The remaining pulses in the 400 Hz t r a i n were not associated with a c lear PS or PW. Without exception, the f i r s t pulse in the 400 Hz tetanic st imulation gave the maximum PS amplitude, i . e . , no frequency potent ia t ion . In contrast , 100 Hz t ra ins ( f i g . 14B) t y p i c a l l y gave a synchronous PS associated with the i n i t i a l 1-50 pulses of the t r a i n . Thereafter, the PS were usually absent. Sometimes, PS to the 2-4 pulses in the 100 Hz t r a i n were s l i g h t l y bigger than that evoked by the f i r s t pulse. Even after the PS were no longer evoked in the l a t t e r pulses of the 100 Hz t r a i n , a gradually decaying PW could s t i l l be observed (cf . with 20 Hz). Rarely did a l l the pulses in the 100 Hz t ra in evoke a PS. 20 Hz tetanic st imulations t y p i c a l l y induced greatly f a c i l i t a t e d PS (frequency po tent ia t ion ) . in a manner shown in f igure 14C. In the 20 Hz t r a i n s , pulses after 150-250 tended to evoke less frequency potent ia t ion . During frequency potent ia t ion , the PS was greatly enhanced (increases of 500-900% r e l a t i v e to the f i r s t pulse in the t r a i n ) , with associated two or more secondary - 79 -100 20Hz to CA1 axons c 50 u 10 20 30 40 50 60 70 80 no. of pulse 90 100 100Hzto CA1 axons 100 I 50 o 10 20 30 40 50 60 no. of pulse 70 80 90 100 F i g . 13. Records of i n d i v i d u a l pulses during a n t i -aromic low and high frequency te tan ic s t imu la t ions . 10 In each case, only the f i r s t 0 pulses of each t r a i n are shown. Data was obtained from a s ingle represen-t a t i v e s l i c e . See text for complete desc r ip t ions . - 80 -spikes. The PS during 20 Hz eventually declined below the amplitude of the PS evoke by the f i r s t pulse within the 300-350 pulse range. Thereafter, the PS was abolished. Though not shown in f igure 14C the PS and PW components during the 20 Hz tetanus seemed to decay at d i f fe rent rates (c f . with 100 Hz above). I n i t i a l l y , both the PS and PW were greatly f a c i l i t a t e d . Then even though the PS could s t i l l be seen with the late pulses pr ior to being blocked there was no discernable second PW component associated with these sp ikes . Las t l y , in some orthodromic tetanic experiments, the c l a r i t y of the pre-++ synaptic vo l ley was enhanced by blocking synaptic potent ia ls with Mg or ++ Mn media. Under these condi t ions , i t was found that the presynaptic vol ley (PV) followed both the 20 Hz or 100 Hz t e t a n i . However, during the l a t t e r phases of the 100 Hz tetanic s t imulat ion , the PV began to taper down. Figure 15 shows the resu l ts on PV during 20 Hz and 100 Hz t ra ins ob-tained from two d i f fe rent s l i c e s . 14.7 Ex t race l lu la r C a + + and K + Among other th ings, the preceding resu l ts (pa r t i cu la r l y those on tetanic t ra ins and potentiation/depression with verapamil) indicated that the syn-chronous discharge of PS consistent ly followed the low and not the high frequency tetanus. Yet LLP was better e l i c i t e d with the high frequency t e t -anic st imulations i . e . , there was l i t t l e or no synchronous discharge of the postsynaptic CAlb neurons during tetanus. It was the depression which was associated with low tetanic st imulations (or the continuous discharges of CAlb neurons during the tetanus) . It also became c lear that interference with Ca entry to postsynaptic CAlb neurons during 20Hz tetanus counter-acted the depression associated with t h i s tetanus. These observations - 81 -400 Hz i _ ; _ 10 20 30 40 50 60 70 80 90 100 no. of pulse 100 Hz 10 20 30 40 50 60 70 80 90 100 no. of pulse 20 Hz 10 20 30 40 50 60 70 80 90 100 no. of pulse F i g . 14. Records of ind iv idual pulses during ortho- dromic Tow and high frequency te tan ic s t imulat ions . In each case, only the f i r s t 100 pulses of each t r a i n are shown. Data was obtained from a s ing le represen-t a t i v e s l i c e . See text for complete descr ip t ions . Shaded parts indicate presence of secondary spikes. - 82 -presynaptic volley •  20Hz _ 150 o ~ 100 o M 5 0 10 20 30 40 50 60 70 80 90 100 no. of pulse presynaptic vol!ey:100Hz 100 o o 50 u ~HJ Ln_n 10 20 30 40 50 60 70 80 90 100 no. of pulse F i g . 15. Records of ind iv idua l presynaptic vol leys  during low ano high frequency te tan ic s t imulat ion of  bchaffer c o l l a t e r a l s . In each case, only the f i r s t 100 pulses of each t r a i n are shown. Data was obtained from a s ing le representat ive s l i c e . See text for complete d e s c r i p t i o n . - 83 -formed the basis of a formulated hypothesis that stated that low frequency tetanic stimulations caused an in f lux of Ca into CAlb neurons which med-iated the development of a generalised postsynaptic depression. In order to substantiate th i s working hypothesis , studies were conducted that monitored changes in e x t r a c e l l u l a r C a + + during low and high frequency tetanic st imu-l a t i o n s . The resul ts obtained from these experiments are presented here. Using ca l ibrated Ca se lect ive e lectrodes, i t was determined that 20Hz given for 20-40 sec produced a reduction in the ex t race l lu la r C a + + (from 2 to 0 .7 -1 .2 mM; n = 5: CAlb pyramidal layer recordings) . In con-t r a s t , 100Hz given for 5-10 sec did not produce a s i g n i f i c a n t change in C a + + leve ls (n = 5 ) . The PS was depressed af ter the 20Hz tetanus but not after 100Hz. Yet the 100Hz e l i c i t e d LLP. E x t r a c e l l u l a r K + . l e v e l s increase during tetanus (Benninger, Kadis and Pr ince , 1979). It was therefore l i k e l y that the observed depression f o l l o w -ing 20Hz (and even the LLP i n i t i a t i o n for that matter!) could have been caused by elevated e x t r a c e l l u l a r K + during tetanus. This p o s s i b i l i t y was examined by f i r s t determining the ex t race l lu la r K + levels with K + s e l e c -t i ve e lectrodes. Then in separate experiments, hippocampal s l i c e s were ex-posed to modified medium with substituted K ( i . e . , K medium) in con-centrations comparable to those present during 20Hz tetanus. 20Hz tetanus for (20-40sec) produced an increase in ex t race l lu la r K of 10-15mM (recorded in the CAlb c e l l body; n = 6) . Consequently, the K + medium pre-pared contained 15mM K + . when t h i s K + medium was perfused for 10 min i t caused a sustained increase in evoked PS (PS, 300-400% times controls ; n = 4) during the app l i ca t ion . Spontaneous a c t i v i t y increased and secondary spikes were present. Yet af ter K + medium perfusions, on average, the evoked PS returned to the control l e v e l s . These resul ts are presented in f igure 16. - 84 -14.8 B a + + and evoked potent ia ls Figure 17 i l l u s t r a t e s the sequence of changes in evoked potent ia ls ++ ++ associated with Ba perfusions. Ba appl icat ions caused an i n i t i a l increase in the amplitudes of these po tent ia l s . The onset latency for the PS and PW were greatly enhanced. The delayed and widened PW was inundated with a multitude of s ingle neuron spikes (see f i g . 17). With continued Ba perfusions, the PS and PW were abolished. Where the presynaptic vol leys were discernable these showed increases in amplitudes but the i r on-set latencies seemed unaltered. These e f fec ts to the a r t i f a c t and pre-syn-++ ++ aptic vol ley were reversed to pre-Ba levels after Ba perfusions. Upon returning to standard medium, there was an immediate recovery of evoked potent ia ls and these potent ia ls were s t i l l great ly potent iated. Gradually these potent ia ls decayed resu l t ing in one of the fo l lowing out -comes. After 60 minutes, which was the selected p o s t - B a + + monitoring per -iod , evoked potent ia ls (a) remained augmented or (b) were depressed. The average changes, for d i f ferent B a + + appl icat ion times are summarised in f i g . 18 and 19. Furthermore, Sch terminal region e x c i t a b i l i t y was tested in f i v e f ibe rs (d i f ferent s l i c e s ) before, during and after Ba perfusions. Interest ingly i t was found that the Sch antidromic thresholds were greatly ++ ++ increased only during the Ba perfusions. Yet the post-Ba Sch a n t i -dromic threshold were e s s e n t i a l l y the same (n = 5) as the p r e - B a + + l e v e l s . 14.9 Ef fects of 4-AP Single 5 minute perfusions with 100 uM 4-AP caused larger enhancements (400-800%; n = 4) in evoked PS than those obtained with cumulative additions -85 -400n 15 30 60min F i g . 16. Effects of ra ised potassium concentrations  on evoked population sp ike . Bars represent mean amplitude of evoked population sp ike . V e r t i c a l l i n e s are +S.E.M. (n = 4 ) . K + medium was perfused fo r 10 min. - 86 -15min control post-Ba 10 f 15 min | ^ F i g . 17. Ef fects of barium on evoked population sp ike . Numbers on side indicate elapsed time during barium per -f u s i o n . Ce l l disharges are shown by arrows. See text for d i s c u s s i o n . soo Ba+*perf usion (min) 400-g 300* Z ' O u 200-100-• 3 010 E 3 « I T 15 30 60 > > X X < < x < < < x X '.X / y > > >< < < < < < x X X * X X > 15 30 60 MIN POST-BARIUM 15 30 < < < > 60 F i g . 18. Changes In evoked population spike fol lowing 3,  10 an<1 15 i n . exposures to barium medium. Ver t i ca l l ines represent S . l . M . \n-b). F i g . 19. L inear regression p lots of decay times of evokert  population spike fo l lowing barium app l i ca t ions . ' ' - 88 -up to 400 uM 4-AP '(300-400% ; n = 5 ) . Furthermore, maximal enhancements with 100 yM 4-AP were obtained i f the drug was perfused for 5 minute during s t imulat ion . However, absence of synaptic transmission ( i . e . , Mg + + b lock-ade or temporary cessation of st imulat ion ) when the drug was applied did not prevent subsequent enhancement of the evoked PS (see table 3 and f igure 20) . Presumably the s i tes of action for 4-AP are located on the inner parts of the membranes (Mashal l , 1981). The resu l ts in table 3 seem to indicate that 4-AP movement across the membrane may not be e n t i r e l y dependent on v o l -tage -sens i t i ve channels. A l t e r n a t i v e l y , i t may be that s u f f i c i e n t 4-AP was present ( i . e . , not completey washed) when synaptic transmission was r e - i n -s t i tu ted which then f a c i l i t a t e d movement across the membranes. It is also ++ possible that the incubation for 5 minutes with Mg was inadequate to completely remove e x t r a c e l l u l a r C a + + and, therefore, block a l l synaptic t ransmission. In addi t ion , repet i t i ve bursting a c t i v i t y at 1-2 minutes in terva ls were observed during perfusion with 4-AP. I n i t i a t i o n of t h i s bursting behavior i s shown in f igure 21. Bursting a c t i v i t y was not observed i f 4-AP was added ++ during synaptic transmission blockade with Mg medium. Effects of 4-AP including the bursting a c t i v i t y when present were revers ib le by washing for 30-60 minutes. However, in some s l i c e s , depressions of evoked responses occurred fol lowing reversal of 4-AP ef fects with washing. Repeated a p p l i c a -t ions did not a l t e r the recovery t imes. As has been shown in e a r l i e r sect ions , e f fec ts of 4-AP were revers ib le by washing for 30-60 minutes. In contrast , when the stratum radiatum or the CA3 region afferents to CAlb were tetanized (100 Hz, 5 sec) during 4-AP per-fus ions , the subsequent enhancements in evoked PS were not reversed to con - 89 -Post 4-AP time (min) Recovery time to contro l 15 30 l e v e l (min) St imulat ion 4 6.75 ± 0.75 1.65 ± 0.15 45- ± 12.72 No s t imulat ion 3 5 .8 ± 1.06 0 .8 ± 0.95 40 / * 6.26 In M g ^ 4 2.89 ± 1.18 1.98 :± 0.83 45.75 ± 10.46 Table 3. Drug induced enhancements associated with 4-AP perfusions during st imulat i o n , no stimu lat ion and magnesium-meciated synaptic blockade. Evoked responses reported as a r a t i o (a) of population spike in s tand-ard medium. Ear ly time (b) chosen for making comparisons since rever -sal of synaptic transmission blockade with magnesium medium occurred within 10 min (Maret ic , Chirwa and Sastry , unpublished observat ions) . - 90 -i 200 c o o o 111 a » z • 2 100 < a o a a. < yy ZmM Mg** 0 L \ \ \ 0 20 40 60 80 100 120 TIME (min) F i g . 20. Plot of 4-aminopyridine perfused during synaptic  transmission blockade with raised magnesium medium. - 91 -t r o l l e v e l s . Figure 22 i l l u s t r a t e s the methods used and resu l t s obtained from one s l i c e . Large enhancements (400-500% ; n = 4) in evoked PS were obtained immediately af ter the 4-AP/100 Hz, 5 sec treatment and decayed to 150-200% l i m i t s within 30-40 minutes. The i n i t i a l enhancements probably were due to 4-AP ef fects and decayed as the drug was washed out. The second phase presumably represented LLP induced by tetanus that could now be seen af ter washing out of 4-AP. These potentiated responses could be followed for two or more hours (see f igure 22) . 14.10 Iontophoretic glutamate L-glutamate ejected with 200nA currents for 1 min on CAlb c e l l bodies i n i t i a l l y f a c i l i t a t e d then rapid ly diminished the PS evoked by Sch st imula -t ion (test pulse,0.2Hz) . Spontaneous a c t i v i t y was greatly increased by L-glutamate. After L-glutamate iontophoresis, the depressed PS recovered slowly (PS, 11-90% of control at 5 min post L-glutamate; 6/8 s l i c e s ) Sur-p r i s i n g l y , i f the Sch was tetanized with 100Hz (100 pulses) during the last seconds of L-glutamate e ject ions , the post-drug depression was much more pronounced (PS, 1-12% of control at 5 min post -drug; 4/4 s l i c e s ) and t h i s depression was s t i l l present even after 30 min. In some experiments, verapamil perfusions preceded the L-glutamate e jec -t i o n s . If t h i s treatment was i n s t i t u t e d , verapamil counteracted the post -L -glutamate depression (PS, 84-130% of controls at 5 min post -L-glutamate; 4/5 s l i c e s ) . Even more s t r i k i n g was the f inding that verapamil counteracted the depression fo l lowing L-glutamate /100Hz treatments and actual ly 'unmasked' the tetanus induced LLP to Sch (PS, 130-325% of controls at 5 min postdrugs; 5/5 s l i c e s ) . - 92 -Y control in i-AP F i g . 21 . 4-aminopyridine induced burst ing a c t i v i t y in  CAlb s u b f i e l d . Ve r t i ca l l i n e is spikes per second. Arrow indicates time when 4-AP was s t a r t e d . The post -4-AP records were taken at the 10 min i n t e r v a l . Figures were re-touched to enhance c l a r i t y . post -4 -AP 1 min - . '93 -£ 400 o u o 111 a w z o < _ l 3 a O & 300 200 > 3 in tn X o o 4-AP v. V B 100 W 40 80 120 160 200 240 TIME (min) F i g . 22. Maintained enhancements of evoked population  spike fo l lowing 4-raminopyridine and 100Hz treatment. The two rates of decay are indicated by the l e t t e r s A and B (see text for f u l l d i scuss ion ) . Results shown were obtained from one s l i c e . 0.1mV 5 ms F i g . 23. I l l u s t r a t i o n of an antidromic s ing le spike  ( • ) evoked in f i e l d CA3. St imulat icn was at Sch/CAlb synaptic region. - 94 -14.11 E x c i t a b i l i t y of Schaffer c o l l a t e r a l terminals Figure 23 i s a record of the CA3 single spike evoked by the antidromic st imulat ion of Sch. The threshold for th i s antidromic act ivat ion was deter -++ ++ mined during Ba , and 4-AP. Ba caused more than a 3 - f o l d increase in threshold during perfusions. Yet t h i s increase in Sch e x c i t a b i l i t y th res -hold was reversed to p r e - B a + + leve ls once standard medium was r e - i n s t i t u t -ed. On the other hand, 4-AP perfusions s l i g h t l y decreased (93-97% of con-t r o l threshold , n = 4) the Sch e x c i t a b i l i t y threshold during appl ications but t h i s was reversed to pre-4-AP levels within 10-15 min after 4-AP perfus-ions . 15. DISCUSSION 15.1 LLP A l l the evoked potent ia ls reported in t h i s thes is had the c l a r i t y and charac te r i s t i cs of s imi la r potent ia ls reported in the l i t e r a t u r e (Swanson, Teyler and Thompson, 1983). The e l e c t r i c a l l y evoked LLP was seen as a r e -duction in PS latency and/or a net increase in the amplitude of PS, Pw or EPSP. Many overlapping currents , both inward and outward to CAlb pyramids, interneurons and possibly g l i a c e l l s , contributed to the global shape of the evoked potent ia ls (see section 6 ) . However, the s p e c i f i c contr ibut ion of each ion ic current i s indeterminate and therefore a detai led account of the exact ionic conductance changes associated with LLP cannot be given. It i s conclusively c lear that antidromic tetanic st imulations f a i l e d to e l i c i t LLP. Only orthodromic t ra ins evoked in standard medium e l i c i t e d LLP. It became c lear during the analysis of indiv idual pulses of the varied tetanic st imulations that LLP was best e l i c i t e d by high frequency t ra ins - 95 -that caused l i t t l e or no discharges of the postsynaptic CAlb neurons. Yet the presynaptic vo l ley under these conditions could fo l low the high frequen-cy tetanus. Since C a + + inf lux i s a prerequis i te for LLP development, c l e a r l y the C a + + inf lux must have been largest in the presynaptic t e r -minals as these were continuously activated by a l l orthodromic tetanus exa-mined. It was possible that asynchronous release of neurotransmitters, though i n s u f f i c i e n t to discharge the postsynaptic neurons, s t i l l depolarised the local dendr i tes. This could have led to the in f lux of C a + + into the dendr i t ic regions which might have mediated LLP development. A few observa-t ions negate t h i s p o s s i b i l i t y . F i r s t l y , twin-pulse studies show a marked reduction in the second PS i f the interva l between the two pulses is 2.5 msec, i . e . , 400Hz (Chirwa, unpublished; Turner, Richardson and M i l l e r , 1983). This is due to the depletion of terminal neurotransmitters caused by the f i r s t pulse and incomplete neurotransmitter recovery ( i . e . , rep len ish -ment) a f ter th i s pulse (cf . Elmqvist and Quastel , 1965). Secondly, i t is known that eight pulses given at 400Hz can e l i c i t LLP (Swanson, Teyler and Thompson, 1983 review). Obviously, the last 4-6 pulses in th i s t r a i n would cause l i t t l e or no release of neurotransmitters since they are probably evoked when the terminals (a) have been emptied and (b) the interva l i s small for complete replenishment of neurotransmitter substances. If i t i s argued that s u f f i c i e n t C a + + in f lux to postsynaptic dendrites s t i l l occur under these condit ions (400Hz, 8 pulses) then i t remains to be explained why strong s ingle shocks to Sch do not e l i c i t LLP. L a s t l y , verapamil in doses ++ that s e l e c t i v e l y interfered with the Ca in f lux to postsynaptic neurons (see l a t e r sections) augmented PTP and LLP development. These resu l ts are consistent with a presynaptic locus for LLP development. - 96 -15.2 Tetanic st imulations The major differences between antidromic and orthodromic tetanus were that (a) synchronous discharges accompanied a l l pulses in the antidromic tetanus, whereas (b) few, i f any synchronous PS followed the high frequency orthodromic tetanus and (c) only the low frequency orthodromic tetanus gave well developed frequency potentiat ion and secondary PS. Many hypothetical schemes can be given that can account for these d i f fe rences . However, when the known i n t r i n s i c c i r c u i t r y of the CAlb region are taken into considera-t ion (Knowles and Schwartzkroin, 1981) then the fo l lowing ser ies of events seem to occur. The intense depolarizations on CAlb dendrites caused by repet i t i ve or -thodromic st imulat ion made neurons to f i r e repeatedly. These CAlb neurons in turn fed s ignals to inh ib i to ry interneurons (v ia the recurrent inh ib i to ry loop) intensely depolar iz ing them. Hence the interneurons then generated huge IPSPs in CAlb pyramids (Andersen and L/mo, 1967). Conceivably these huge IPSPs could have reversed and became depolar iz ing influences during the t r a i n and 'potent iated' on-going CAlb dendr i t ic depolar izat ions . A l t e r n a -t i v e l y , the intense depolar izat ions on interneurons during 20Hz may have caused depolar izat ion blockade of interneurons, thereby e f f e c t i v e l y abo l i sh -ing IPSPs, i . e . , i nh ib i to ry interneurons inact ivated (Andersen and Lfftno, 1967). In any case, the net e f fec t was a greatly enhanced CAlb neuron e x c i -t a b i l i t y resu l t ing in the recruitment of inact ive CAlb neurons. In fact the increased e x t r a c e l l u l a r K + during tetanus possibly reduced CAlb neuron e x c i t a b i l i t y and thereby f a c i l i t a t e d CAlb discharges (Alger and Teyler , 1978). These processes probably accounted for the frequency potentiat ion and secondary spiking seen with 20Hz. Blockade of these processes set in - 97 -when (a) inact i vat ion or depolar izat ion blockade in CAlb dendrites/soma com-ponents occured or (b) neurotransmitters were depleted or (c) action poten-t i a l s were blocked in Sch boutons. It seemed l i k e l y that high frequency orthodromic tetanus rapidly caused depolar izat ion blockade and/or terminal neurotransmitter depletion and therefore not a l l of the above events, i . e . , synchronous discharges, frequency potentiat ion or secondary spikes, were seen. From the above descr ipt ions , i t can also be seen why antidromic tetanus did not cause frequency f a c i l i t a t i o n or secondary spikes. These events were mainly generated and/or perpetrated by synaptic in te rac t ions . Las t l y , the reduction in PS sometimes seen with antidromic tetanus in standard medium could be explained as fo l lows . While the CAlb neurons were being ant idromical ly act ivated, c o l l a t e r a l s of these axons also fed ortho- dromic s ignals to inh ib i to ry interneurons, which then caused IPSPs that subsequently shunted some CAlb neuron discharges. Following the antidromic tetanus, i t was l i k e l y that more inh ib i to ry interneurons were recruited since the CAlb axon c o l l a t e r a l s must have developed PTP immediately af ter the tetanus. This then would explain the t ransient depression to Sch evoked potent ia ls af ter antidromic tetanus (Chirwa, Murali Mohan and Sastry , un-publ . ) A l t e r n a t i v e l y , since the s l i g h t reduction in PS sometimes seen dur-ing antidromic tetanus was not present in M g + + or M n + + media, i t could ++ be that the reductions were caused by Ca in f lux into CAlb neurons, an e f fect that was extended to the post-tetanus period. 15.3 Homo- and heterosynaptic depression It i s evident that homo- and heterosynaptic depression i s a general -ised phenomena occuring in the postsynaptic CAlb neurons. I n i t i a t i o n of t h i s depression i s dependent on the CAlb neuronal accumulation of C a + + , - 98 -which is greater with low frequency tetanus. In support of t h i s conclusion were the fo l lowing r e s u l t s . Using C a + + se lect ive electrodes i t was found that the largest drop in e x t r a c e l l u l a r Ca in the pyramidal layer oc -curred with low frequency tetanus. Iontophoresed C a + + on CAlb c e l l bodies i s known to induce homo- and heterosynaptic depression (Sastry, Chirwa, Goh and Maret ic , 1983). Iontophoresed glutamate on CAlb c e l l bodies i n i t i a t e d a post-drug depression. This depression was more pronounced af ter glutamate/lOOHz treatments. The simplest and p laus ib le explanation of the resu l ts was that glutamate depolarized CAlb c e l l bodies and le t in C a + + . Tetanus caused even more C a + + to enter the dendri tes. That indeed C a + + mediated the above changes was confirmed by the discovery that verapamil could counteract the glutamate-induced depressions. In fac t i t was p red ic t -ed and subsequently confirmed that reduction of the glutamate/lOOHz post-synaptic depression by verapamil led to the unmasking of a possibly pre-synaptic mediated LLP. Both LLP and depressions co-existed together. In low tetanus, depressions were more pronounced and invar iab ly masked the ex-pression of LLP. At higher frequencies, LLP development was favoured which masked the less favoured depressions. These conclusions are consistent with the f indings that homo- and heterosynaptic i n i t i a t i o n could revers ib ly i n -terrupt establ ished LLP. Such an ef fect was not seen with high frequency tetanus to a potentiated input or a separate input to the same output neur-ons. 15.4 Bari urn Turner, Baimbridge and M i l l e r (1982) caused a long term increase in syn-aptic e f f i cacy by the t ransient exposures to elevated ex t race l lu la r C a + + . In s i m i l a r experiments, Sastry, Chirwa, Goh and Maretic (1983) reported that - 99 -a sustained potentiat ion followed a 10 min exposure of hippocampal s l i c e s to Ca + + /K + combinations of 4 and 5 mM and not 4 and 3.1 mM respect i ve ly . It seemed that a voltage-dependent C a + + channel was involved in the deve l -opment of the potent iat ion during these exposures to C a + + . A major out -come of the above studies included the demonstration of a technique, i . e . , exposure to elevated C a + + , that seemed to be e l i c i t i n g a potentiat ion s im-i l a r to that evoked with e l e c t r i c a l s t imulat ions . In the present s tudies , the method of exposing hippocampal s l i c e s to B a + + medium was used to test B a + + capab i l i t y to induce LLP. Other f a c -tors that governed the se lect ion of these technique included the fo l lowing . From the i n i t i a l experiments, i t was c lear that B a + + caused a massive but asynchronous release of neurotransmitters which led to the observed delay in the latency of the evoked PS and i t s PW. Further evidence for the asynchro-nous neurotransmitter release became apparent when i t was noted that super-imposed along the ent i re length of the delayed PW, were numerous s ingle c e l l discharges. Besides, i t i s known that Ba causes an increase in the t e r -minal action potential ref ractory period (Sastry, 1979). Since the duration of a s i m i l a r Ba induced terminal action potent ia l refractory period in the hippocampus was not known, i t seemed inappropriate to evoke tetanic st imulations in B a + + medium and thereby test fo r post - tetanic LLP. The p o s s i b i l i t y existed that an increase in terminal action potent ia l refractory period could cause the f a i l u r e of some axonal action potent ia ls generated in Sch to invade the Sch synaptic boutons (which are a possible loc i for LLP; Sastry, 1982) in a manner necessary to induce LLP. On average, a l l exposures to B a + + medium for 3 , 10 or 15 min. led to the p o s t - B a + + potentiat ion of the evoked PS. A huge asynchronous release - 100 -++ in neurotransmitter was evident in the i n i t i a l phases of Ba perfusions. Continuation of B a + + appl icat ions led to the blockade of synaptic r e s -ponses. The mechanisms mediating t h i s blockade were not c l e a r , since B a + + i s known to support neurotransmitter re lease. It can be speculated that B a + + caused the depletion of releasable neurotransmitter pools. If th i s is cor rect , then i t must be argued that unl ike Ca , Ba possibly h i n -ders the replacement of depleted releasable neurotransmitter pools. Second-l y , a combination of C a + + / B a + + , unlike C a + + alone, that must have existed in the i n i t i a l p o s t - B a + + periods greatly enhanced the to ta l neuro-ne transmitter released. After a l l , Ba prolongs the terminal action potent ia l duration (Sastry, 1979) and t h i s could account for the better e f -f i cacy in neurotransmitter release caused by C a + + / B a + + combinations. Given that neurotransmitter release was augmented, th i s could therefore account for the observed p o s t - B a + + potent iat ion . An a l ternat ive explana-t ion such as an increase in postsynaptic receptors is unattainable since B a + + exposures presumably do not increase glutamate receptors (Baudry and Lynch, 1979). Since there were no changes to amplitudes of the st imulat ion a r t i f a c t or the presynaptic vol ley after B a + + prefusions, i t was un l ike ly that there were a l terat ions to the st imulat ion electrodes that could account for the potent ia t ion . What seemed l i k e l y was that the actions of a B a + + increase in neurotransmitter release were further 'ampl i f ied ' by the post -synaptic e f fects of t h i s i on . B a + + is known to augment the Ca + + -med ia t -ed depolar izat ions and also blocked the outward K + currents (Hotson and Pr ince, 1981). These actions could conceivably f a c i l i t a t e the recruitment of CAlb neurons during synaptic t ransmission. However, the potentiat ion seen with B a + + d i f fered from the e l e c t r i c a l l y induced LLP since the former - 101 -quickly decayed in t ime. There was a re lat ionship between the level of potent iat ion and decay times to the B a + + exposure t imes. It seemed that long exposures to B a + + led to increased accumulation of B a + + in s l i c e s , which subsequently took longer to passively wash out after B a + + appl icat ions . Moreover, LLP is known to be associated with a decrease in the e x c i t a b i l i t y of Sch terminals fo l lowing an LLP-inducing tetanus to t h i s input (Sastry , Murali Mohan and Goh, 1985). Yet B a + + did not a l t e r the p o s t - B a + + threshold for the antidromic act ivat ion of Sch. The increase in Sch threshold seen during B a + + perfusions further con -firmed the presynaptic actions of th i s ion . However, under the conditions employed with B a + + in the present study, i t i s concluded that B a + + did not e l i c i t LLP. Perhaps the most ideal exposure times or methods for B a + + exposures that could e l i c i t LLP were not attained in the present s tud ies . 15.5 4-Aminopyridine The bursting a c t i v i t y induced by 4-AP perfusions may have originated from i n t r i n s i c s i tes on the CAlb pyramidal c e l l s . It i s conceivable that substances such as 4-AP enhance C a + + in f lux into the soma-dendritic s i tes and promote bursting a c t i v i t y (Wong and Pr ince , 1978; Simmons and Dun, 1984). However, since t h i s bursting a c t i v i t y was not detected during 4-AP/Mg + + medium perfusions i t seemed l i k e l y that they were caused by syn-aptic in te ract ions . In any case, C a + + binding s i tes presumably increase along the stimulated afferents and the i r synaptic regions with 4-AP t r e a t -ment (Kuhnt, Mihaly and Joo, 1983). Hence these regions could be the source of ectopic action potent ials in these af ferents , leading to bursting a c t i -v i t y in CAlb neurons. Besides, i t was observed that 4-AP caused a s l ight decrease in Sch e x c i t a b i l i t y threshold during appl icat ion an e f fec t that could have enhanced spontaneous transmitter re lease. - 102 -Single 100 yM 4-AP perfusions caused larger PS enhancements than cumula-t i ve doses of 400 yM 4-AP. It was possible that the long exposure times needed to reach a cumulative dose of 400 yM 4-AP made i t possible for secon-dary interact ions to occur that diminished synaptic responsiveness. Yet again i t i s known that 4-AP f a c i l i t a t e d neurotransmitter release at lower concentrations and exerted a c u r a r e - l i k e e f fect at higher concentrations (Van Der Sprong and Voskuyl , 1982; Simmons and Dun, 1984). In addition s i n -gle 100 yM 4-AP perfusions e l i c i t e d maximum enhancements greater than those i n i t i a t e d in 4-AP/100Hz treatments. Tetanic st imulations are known to p a r t -l y reverse the 4-AP mediated blockade of fas t K + channels and t h i s e f fect could account for the differences in enhancements reported above (Yeh, Oxford, Klu and Naharashi, 1976). More importantly, the 4-AP mediated en-hancements were revers ib le with washing for 30-60 min. In contrast , the 4-AP/100Hz treatment led to enhancements that exhibited two rates of decay. The f i r s t enhancement decayed within 40 min to reveal the second potentiated phase, which was maintained throughout the duration of the experiments. It i s proposed here that the second potentiated phase was the LLP (caused by 100Hz in the 4-AP/100Hz combination) that could now be seen after 4-AP had washed out. 4-AP has mostly dose-dependent presynaptic and some postsynaptic actions (Ikemoto, Klee and Daunicht, 1982). In the 4-AP doses used in the present ++ studies , i t was expected that enhanced Ca in f lux into presynaptic t e r -minals as a resu l t of blockade of K + conductance by 4-AP could i n i t i a t e LLP. However i t was found that 100 yM 4-AP added to medium containing 0 to 2 mM C a + + (test frequency, 0.2Hz) did not induce LLP. A possible post -synaptic locat ion for LLP i n i t i a t i o n could not explain the absence of 4-AP - 103 -mediated LLP since t h i s drug also had revers ib le ef fects on CAlb neurons (Buckle and Haas, 1982; Van Harreveld, 1984). It was possible that the 4-AP mediated increase in C a + + in f lux though s u f f i c i e n t to f a c i l i t a t e t rans -mit ter re lease, may not have attained c r i t i c a l intraterminal C a + + levels that were maintained for a period necessary to e l i c i t LLP. In fact 4-AP may have been enhancing transmitter release in both active and mostly inact ive Sch boutons. 4-AP f a c i l i t a t e s afferent t ransmission, as evidenced by i n -creased presynaptic vol leys (Kocsis, Malenka and Waxman, 1980; Haas, Wieser and Yasarg i l , 1983) and could resu l t in the recruitment of inact ive synaptic boutons. The net resul ts would be an apparent increase in the neurotrans-mitter re leased. In contrast , e l e c t r i c a l l y induced LLP might involve (a) an improved in f lux of C a + + into terminals and (b) an actual increase in terminal neurotransmitter replacement and/or increase in releasable pools (Voronin, 1983 review). The resu l t s reported by Sastry (1982), though not att r ibuted to such an e f f e c t , are suggestive of a possible increase in t e r -minal boutons volume during LLP and hence the associated decrease in t e r -minal e x c i t a b i l i t y . This is mere speculat ion. 15.6 Miscellaneous Raised e x t r a c e l l u l a r K + in concetrations that mimicked increases in K + during tetanus did not induce s i g n i f i c a n t changes. These resu l ts seem to suggest that increased ex t race l lu la r potassium i s not involved in the i n i t i a t i o n of LLP. A l t e r n a t i v e l y , the raised K + which must have depolar-ized presynaptic neurons, may have induced potent iat ion and depression that then ' cance l led ' each other . Su f f i c ien t experiments were not done to exa-mine these p o s s i b i l i t i e s . - 104 -16. CONCLUSION The resu l t s from the studies presented in t h i s thes is i l l u s t r a t e that continuous synchronous discharge of CAlb neurons during tetanus, in the presence of e x t r a c e l l u l a r C a + + i . e . , in standard medium, correlated with the development of post - te tan ic homo- and heterosynaptic depressions. L-glutamate appl icat ions to CAlb neurons, which i s known to cause C a + + entry into neurons, led to depressions. Verapamil in doses that se lec t i ve l y i n -terfered with postsynaptic entry of Ca counteracted the late depression associated with Glu or low frequency induced depressions. While the exact mechanisms of these depressions are uncertain , the fo l lowing statements can be made. Ce l l death cannot account for the depressions since i t i s known that subsequent development of LLP to the same input (and other inputs) can be induced with high frequency tetanus (Dunwiddie and Lynch, 1978; Chirwa, Goh, Maretic and Sastry , 1983). In any case, complete recovery of evoked potent ia ls af ter the heterosynaptic depression usual ly occured. Depolar iza-t ion blockade and/or increased p o s t - a c t i v i t y hyperpolarizations contributed towards the ear ly phase of the depressions and th is could not be reversed by verapamil. It seemed therefore that CAlb repe t i t i ve c e l l discharges led to ++ the entry of Ca into these neurons, which then effected a generalised postsynaptic depressions. The dependency of LLP i n i t i a t i o n on e x t r a c e l l u l a r C a + + i s well docu-mented. Yet verapamil did not in ter fere with the development of LLP. It could be that the subsynaptic C a + + current responsible for LLP development was insens i t i ve to verapamil. A l t e r n a t i v e l y , and t h i s i s the favoured i n -te rp re ta t ion , verapamil did not antagonise neurotransmitter re lease. This - 105 -was suggestive of the fact that verapamil did not in ter fe re with C a + + i n -f lux into presynaptic regions. A pertinent observation was that the pre -synaptic vol ley followed low and high frequency tetanic st imulat ions . The high frequencies must have p r e f e r e n t i a l l y caused C a + + i n f lux into cont inu -ously activated synaptic boutons. Yet high frequencies continuously e l i c i t e d LLP. C lear ly t h i s i s strong evidence for a presynaptic mediated LLP. Barium and 4-aminopyridine induced enhancements of evoked potent ia ls d i f fe red from the e l e c t r i c a l l y induced LLP, i . e . , drug induced enhancements were reversed with washing. F i r s t l y , these resu l t s are suggestive of the fac t that LLP is not mediated by blockade and/or a l te ra t ions to K + f l u x e s . This seemed l i k e l y since elevated K + medium did not induce LLP. S t i l l , i t i s not c lear why the enhanced C a + + in f luxes that was secondary to the actions of these drugs did not e l i c i t LLP. It i s tentat i ve ly sug-gested here, that at the level of the presynaptic terminals , while these drugs enhanced neurotransmitter re leased, more importantly they f a c i l i t a t e d the recruitment of subthreshold Sch boutons ( i . e . , through K + blockade and at t h i s level the afferent sections are not myelinated). In add i t ion , the depletion of neurotransmitter pools that may have accompanied B a + + a p p l i -cations was not read i l y replenished as occurs with C a + + . It i s proposed here that the changes in LLP possibly involve (a) presynaptic a l terat ions of the active boutons that f a c i l i t a t e s improved invasion of terminal action potent ia ls and (b) a more e f f i cac ious neurotransmitter mob i l i za t ion , which then t rans lates into an increase in quantal content. 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