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Studies on the effects of saccharin on synaptic transmission in the hippocampus Morishita, Wade Katsuji 1992

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STUDIES O N T H E E F F E C T S O F SACCHARIN O N S Y N A F n C TRANSMISSION IN THE HIPPOCAMPUS By WADE KATSUJI MORISHITA B. Sc., (Pharmacology and Therapeutics), The University of Bri t ish Columbia, 1989 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR T H E D E G R E E OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology and Therapeutics, Faculty of Medicine. The University of Bri t ish Columbia) We accept this thesis^s conforming to the requiredi standard in THE UNIVERSITY OF BRITISH COLUMBIA Apr i l 1992 © Wade Katsuji Morishita, 1992 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that pemiission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pharmacology and Therapeutics The University of British Co lumbia Vancouver, Canada Date May 6 , 1992 DE.6 (2/88) ABSTRACT Saccharin has been shown to block long-term potentiation (LTP) of the hippocampal C A l neuronal excitatory postsynaptic potentials (EPSPs) (Chirwa, 1988; Morishita et. al.. 1992). The mechanisms involved in this action were, however, unknown. The present electrophysiological investigation on guinea-pig hippocampal slices was undertaken to determine whether saccharin interfered with LTP by modulating the excitatory and the inhibitory synaptic transmission or the excitability of the CAlb, neurons. Application of 10 m M saccharin for 10 minutes did not alter the field or intracellular EPSPs in C A l b neurons elicited by low frequency stimulation of the stratum radiatum but prevented LTP of the EPSPs following a brief tetanic stimulation of the afferents. A post-tetanic application of saccharin did not prevent LTP from developing, indicating that the induction and not the maintenance of LTP was blocked by the drug. This agent also inhibited LTP induced by pairing sustained postsynaptic depolarization with low frequency activation of the stratum radiatum. Saccharin, at the concentrations that blocked LTP, did not alter the membrane potential or input resistance of the neurons. Since the induction of LTP appears to require the activation of N-methyl-D-aspartate (NMDA) receptors (CoUingridge et. al.. 1983) and is modulated by activation of the A and B subtypes of y-aminobutyric acid (GABA) receptors (Wigstrom and Gustafsson, 1988; Davies et. al.. 1991). a variety of intracellular experiments were conducted to determine whether the blockade of LTP by saccharin was the result of the drug acting on these receptor systems. The depolarization of CA lb neurons produced by a tetanic stimulation in normal medium or by brief applications of NMDA in either a Mg2+-free medium or a Mg2+-free medium containing 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a drug that antagonizes non-NMDA glutamate receptors, was not significantly altered in the presence of saccharin. Moreover, the slope and the height of the intracellular EPSP evoked in a Mg2+-free medium containing CNQX as well as in a normal medium containing 2-amino-5-phosphonovalerate (APV), a drug that antagonizes NMDA receptors, were also not significantly altered by the drug. These results suggested that saccharin blocked the induction of LTP by mechanisms that did not involve a blockade of the NMDA and the non-NMDA glutamate receptors. "Input-output" (I-O) curves constructed from the EPSPs and the inhibitory postsynaptic potentials (IPSPs) revealed that saccharin selectively increased the height of the IPSP. Pharmacological separation of the IPSP into its GABA receptor-mediated fast, and GABAg receptor-mediated slow components revealed that saccharin significantly increased the duration and height of the fast IPSP but decreased the height of the slow IPSP. In neurons injected with QX-314 (to block the postsynaptic GABAB receptor-mediated IPSPs), paired-pulse depression of the fast IPSP evoked in a CNQX and APV containing medium was not significantly altered in the presence of saccharin, suggesting that the drug did not interfere with the presynaptic GABAB receptors. Saccharin prevented LTP of the field and intracellular EPSP when the fast IPSP was blocked by picrotoxinin, suggesting that the alteration of the fast IPSP by saccharin was not responsible for the ability of the drug to block LTP. Taken togther, the results from the present study suggest that saccharin blocks the induction of hippocampal LTP at a step beyond the activation of the NMDA and non-NMDA glutamate receptors. Actions of this agent on the GABAA and GABAB receptor-mediated responses also appear not to be responsible for its LTP-blocking action. It is possible that saccharin might have interfered with LTP-inducing growth-related substances (Chirwa and Sastiy, 1986; Morishita et. al.. 1992; Sastiy et. al.. 19$8a; Sastiy et. al.. 1988b; Xle et. al.. 1991) or with intracellular facilitators of LTP. Date: r\<^ ô Researc TABLE OF CONTENTS Chapter Title Page A. ABSTRACT i i B. TABLE OF CONTENTS V C. LIST OF FIGURES x i D. LIST OF TABLES xi i i E. LIST OF PUBLICATIONS xiv F. ACKNOWLEDGEMENTS X V G. DEDICATION xvi L INTRODUCTION 1 2. THE HIPPOCAMPAL FORMATION 3 2.1. Development of the hippocampal formation 3 2.2. Basic Topography of the hippocampus 4 2.2.1. The dentate gyrus 4 2.2.2. The hippocampus proper 6 2.2.3. The subicular complex 6 3. STRATIFICATION, SUBFIETDS AND ASSOCIATED CELL TYPES OF T H E HIPPOCAMPUS 6 3.1. The dentate gyms 8 3.2. The hippocampus proper 8 4. INTRINSIC AFFERENTS OF THE HIPPOCAMPUS 9 4.1. Intrinsic afférents within the same subfleld 9 4.2. Intrinsic afférents from different fields 10 4.3. Commissural afférents 12 5. PROJECTION PROFILE OF INTRINSIC HIPPOCAMPAL AFFERENTS 12 5.1. Distribution of perforant path fibers 13 5.2. Distribution of fibers from dentate gyrus 13 5.3. Distribution of fibers from CA3 14 6. EXTRINSIC AFFERENTS TO THE HIPPOCAMPAL FORMATION 15 6.1. Entorhinal projections 15 6.2. Septal projections 15 6.3. Isocortical projections 16 7. EXTRINSIC EFFERENTS FROM THE HIPPOCAMPAL FORMATION 16 7.1. Fornix-fimbria system 16 7.1.1. Pre- and post-commissural fornices 17 7.2. Entorhinal projections 17 7.3. Isocortical projections 17 8 • ELECTROPHYSIOLOGY OF HIPPOCAMPAL NEURONS 17 8.1. Bursting activity of hippocampal neurons 19 8.2. Hippocampal intemeurons 19 8.2.1. Basket cells 19 8.2.2. Oriens/alveus (O/A) intemeurons 21 8.2.3. Lacunosum-moleculare (L-M) intemeurons 21 9. IONIC CURRENTS IN HIPPOCAMPAL NEURONS 23 9.1. Na+-currents 24 9.2. K+-currents 24 9.2.1. K+-currents activated through depolarization 25 9.2.2. K+-currents activated through h5^erpolarization 27 9.3. Voltage-dependent Ca2+-currents 27 9.4. Ca2+-activated K+-currents 29 9.5. Voltage-dependent Cl"-current 31 9.6. Miscellaneous currents 31 9.7. Passive "leak" currents 31 10. FIELD POTENTIALS IN THE HIPPOCAMPUS 32 11. EXCITATORY POSTSYNAPTIC POTENTIALS (EPSPs) 33 11.1 NMDA receptors and synaptic transmission 34 11.1.1. Allosteric modulation by glycine 36 11.1.2. Modulation by zinc 37 11.1.3. NMDA antagonists 37 11.1.4. Glycine antagonists 38 11.2. Non-NMDA receptors and synaptic transmission 38 11.2.1. The non-NMDA component of the EPSP 38 11.2.2. The non-NMDA ACPD receptor 40 11.2.3. The non-NMDA L-AP4 receptor 41 11.2.4. Non-NMDA receptor antagonists 41 12. INHIBITORY POSTSYNAPTIC POTENTIALS (IPSPs) 43 12.1. Recurrent and feed-forward inhibition 43 12.2. Properties of the GABA^ receptor-mediated fast IPSP 44 12.3. Properties of the GABAg receptor-mediated slow IPSP 4g 12.3.1. Presynaptic GABAg receptors 49 12.4. Properties of the GABA^ receptor-mediated depolarizing IPSP 50 13. LONG-TERM POTENTIATION IN THE HIPPOCAMPUS 52 13.1. Properties of LTP 52 13.1.1. Population responses 52 13.1.2. Single neurons 53 13.2. Projections in the hippocampus supporting LTP 54 13.3. Homo- and heteros)maptic LTP 54 13.4. Cooperativity 55 13.5. Associativity 55 13.6. Requirement for calcium 56 13.6.1. Source of calcium entry 57 13.7. Induction and Maintenance of LTP 57 13.7.1. Induction of LTP 57 13.7.1.1 Modulation by GABA 58 13.7.2. Maintenance of LTP 59 13.7.2.1. Posts5niaptic mechanisms 59 13.7.2.2. Presynaptic mechanisms 62 13.7.2.3. Pre- and postsynaptic mechanisms 65 14. SACCHARIN AS A PHARMACOLOGICAL TOOL OF INVESTIGATION 67 14.1. History 67 14.2. Chemistry and physical properties 68 14.3. Pharmacodisposition 68 14.4. Tumor promotor 69 14.5. Interaction with growth factors 70 14.6. Involvement in LTP 71 15. MATERIALS AND METHODS 73 15.1. Animal source and care 73 15.2. Slice preparation 73 15.3. Slice chamber 75 15.4. Perfusion media 77 15.5. Recording and stimulating equipment 79 15.5.1. Recording electrodes 79 15.5.2. Amplifiers 79 15.5.3. Recording systems 81 15.5.4. Stimulation and isolation units 81 15.5.5. Stimulating electrodes 82 15.5.6. Positioning of electrodes 82 15.5.7. Arrangement of the recording set up 82 15.6. Extracellular recording 83 15.7. Intracellular recording 83 15.8. Induction of LTP 84 15.8.1. Tetanic stimulation 84 15.8.2. Pairing 84 15.9. Measurements and statistics 85 16. EXPERIMENTAL PROTOCOLS 86 16.1. Saccharin and LTP of the field EPSP 87 16.2. Saccharin and LTP of the intracellular EPSP 88 16.3. Saccharin and maintenance of LTP 88 16.4. Saccharin and pairing 88 16.5. Specific intracellular studies on saccharin 89 16.5.1. Saccharin and NMDA receptor-mediated responses 89 16.5.2. Saccharin and input-output (I-O) relationships of the EPSP and IPSP 90 16.5.3. Saccharin and IPSPs 91 16.5.4. Saccharin and QX-314 injected neurons 92 16.5.5. Saccharin and paired-pulse depression of the fast IPSP 92 16.5.6. Saccharin and paired-pulse responses 93 16.5.7. Saccharin and LTP in picrotoxinin containing media 94 17. RESULTS 95 17.1. Saccharin and LTP of the field EPSP 95 17.2. Saccharin and LTP of the intracellular EPSP 95 17.3. Saccharin and maintenance of LTP 99 17.4. Saccharin and pairing 99 17.5. Saccharin and NMDA receptor-mediated responses 99 17.5.1. Saccharin and tetanus-induced depolarizations 99 17.5.2. SacchEirin and depolarizations produced by applied NMDA 102 17.5.3. Saccharin and NMDA and non-NMDA components of the EPSP 104 17.6. Saccharin and input-output relationships of the EPSP and the IPSP 106 17.7. Saccharin and postsynaptic potentials recorded in a Mg2+-free medium 106 17.8. Saccharin and IPSPs 108 17.9. Sacharin and fast IPSPs recorded in neurons injected with QX-314 111 17.10. Saccharin and its effects on paired-pulse depression of the fast IPSP 111 17.11. Saccharin and paired-pulse responses of postsynaptic potentials 115 17.12. Saccharin and LTP in picrotoxinin containing media 118 18. DISCUSSION 120 18.1. Effects of saccharin on excitatory synaptic transmission 120 18.2. Effects of saccharin on inhibitory synaptic transmission 121 18.3. Possible mechanisms of action of saccharin 126 19. CONCLUSIONS 128 20. REFERENCES 129 LIST OF FIGURES Figure Page 2.1. Orientation of the hippocampal formation 5 2.2. Organization of the hippocampal formation 7 15.1. Schematic representation of the slice chamber used to study in vitro electrophysiological potentials from guinea-pig hippopcampal slices 76 17.1. Saccharin blocks LTP of the field EPSP slope 96 17.2. LTP of the intracellular EPSP slope is not blocked by saccharin 97 17.3. Maintenance of LTP is not blocked by saccharin 98 17.4. Saccharin reversibly blocks pairing-induced LTP of the intracellular EPSP slope 100 17.5. Depolarizations induced by tetanic stimulations are not blocked by saccharin 101 17.6. Depolarizations induced by applied NMDA are not significantly altered in the presence of saccharin 103 17.7. The intracellular EPSP slope recorded in the presence of excitatory amino acid antagonists is not significantly altered by saccharin 105 17.8. The effects of saccharin on the input-output (I-O) relationship of the EPSPs and IPSPs recorded from CAl^j neurons 107 17.9. Saccharin potentiates the IPSP during activation of the stratum radiatum 109 17.10. Saccharin anatagonizes the late component of the IPSP/C 110 17.11. The slow I P S P / C ( I PSP/CB ) is decreased by saccharin 112 17.12. QX-314 blocks the slow IPSP 113 Figure Page 17.13. Saccharin increases the height and duration of the fast IPSP in neurons injected with QX-314 114 17.14. Paired-pulse depression of the fast IPSP (IPSP^) is not blocked by saccharin 116 17.15. Effects of saccharin on paired-pulse facilitation of the EPSP and paired-pulse depression of the fast IPSP in neurons injected with QX-314 117 17.16. LTP in picrotoxinin-treated slices is not blocked by saccharin 119 LIST OF TABLES Table Page 17.1. Effects of saccharin on some properties of the IPSP/Cs 110 17.2. Effects of saccharin on some properties of the I P S P / C B 112 17.3. Effects of saccharin on some properties of the IPSP^ 114 LIST OF PUBLICATIONS 1. Morishita, W., Z. Xie, S. S. Chirwa, P. B. Y. May, B. R. Sastry. The blockade of hippocampal long-term potentiation by saccharin. Neuroscience 4 7 (1992) 21-31. 2. Morishita, W., B. R, Sastry, Chelation of posts5niaptic Ca2+ facilitates long-term potentiation of hippocampal IPSPs. NeuroReport2 (1991) 533-536, 3. Sastry, B. R., H . Maretic, W. Morishita, Z, Xie, Modulation of the induction of long-term potentiation in the hippocampus. In: Advances in Experimental Medicine and Biology. 2 6 8 (1991) 377-386. 4. Xie, Z., W. Morishita, T, Kam, H. Maretic, B, R. Sastry. Studies on substances that induce long-term potentiation in guinea-pig hippocampal slices. Neuroscience 4 3 (1991) 11-20. 5. Morishita, W., B. R. Sastry. Actions of ginseng and gotu kola on C A l neurones in guinea-pig hippocampal slices. Can. Fed. Biol. Soc. Abstr. 3 2 (1989) 55. 6. Kam, T., H. Maretic, W. Morishita, B. R. Sastry, Z. Xie. Induction of long-term potentiation in guinea-pig hippocampal slices by substances released from rabbit neocortex. Can. Fed. Biol. Soc. Abstr. 3 2 (1989) 55. 7. Sastry, B. R., H. Maretic, W. Morishita, T. Kam, Z. Xie. The involvement of glial cells and endogenous peptides in long-term potentiation. International Workshop on Plasticity of Synaptic Transmission. Kanagawa, Japsm, 1989. 8. Morishita, W. The involvement of glial cells in the induction of long-term potentiation in the hippocampus. The University of U. B. C. Health Sciences Student Research Forum Abstract. 1989. 9. Morishita, W., S, S, Chinva, P, B, Y. May, Z, Xie, B. R. Sastry. Saccharin blocks the induction of long-term potentiation (LTP) in the hippocampus. U. B. C. Health Sciences Student Research Forum Abstract. 1990. 10. Morishita, W., S. S. Chirwa, P. B. Y. May, B, R, Sastry. Saccharin blocks the induction of long-term potentiation (LTP) in the hippocampus. Soc. Neurosci. Abstr. 1 6 (1990) 981. 11. Xie, Z., W. Morishita, T. Kam, H. Maretic, B. R. Sastry, Studies on substances that induce long-term potentiation (LTP), Soc. Neurosci Abstr. 1 6 (1990) 981. 12. Morishita, W., B. R. Sastry. Chelation of postsynaptic Ca2+ facilitates long-term potentiation of inhibitory postsynaptic potentials in hippocampal C A l neurons. U. B. C. Health Sciences Stxuient Research Forum Abstract. 1991. ACKNOWLEDGEMENTS I would like to thank my supervisor Dr. Bhagavatula R. Sastry for his emotional support and academic guidence throughtout this study. 1 am also greatful to my collègue Dr. Zheng Xie for his participation in some of the experiments conducted in this study. 1 wish to thank all the members of the Department of Pharmacology and Therapeutics, in particular, Drs. David Godin and Dick Wall for their encouragement, and academic guidence during my undergraduate years in this department. DEDICATION 1. INTRODUCTION In the m£immalian central nervous system (CNS), repetitive activation of certain types of excitatory synapses results in a long-term potentiation [LTP) of synaptic transmission. Though LTP has been demonstrated in a number of areas in the CNS, the hippocampus has received the most attention since it is an area where learning and memory are thought to occur. Consequently, this use-dependent form of synaptic plasticity has attracted the interest of neurobiologists because LTP subserves as a viable mechanism for learning and memory. LTP was first described by Lomo in 1966, however, subsequent and more detailed accounts were reported by Bliss and Lomo as well as Bliss and Gardner-Medwin in 1973. LTP is characterized by an increase in synaptic efficacy lasting from several minutes to hours In vitro (Schwartzkroin and Wester, 1975) and hours to days in vivo (Bliss and Lomo, 1973). The most common method for initiating LTP relies on a tetanic stimulation of an afferent pathway. This results i n a long-lasting increase in postS5niaptic response to a test stimulus given to the same pathway after the tetanus and is characterized by, 1) an enhancement in the amplitude of the population spike, 2) decreased latency of the population spike, 3) an increase in the size and rate of rise of the population and intracellular excitatory postsynaptic potential (EPSP), 4) an increase in the population spike amplitude or the probability of spike discharge for a constant EPSP (E-S) potentiation. LTP can be differentiated into two stages, induction and maintenance. Recent evidence has suggested that the induction of LTP is triggered by an accumulation of posts5maptic Ca2+ through channels coupled to N-methyl-D-aspartate (NMDA) receptors (Regehr and Tank, 1990) (for reviews see, Collingridge and Bliss, 1987; CoUingridge and Singer, 1990; Gustaffson and Wigstrom, 1988; Nicoll et. al.. 1989; Watkins and CoUingridge, 1989). Ca2+ entry into the posts5maptic site initiates other biochemical processes, not yet fully understood, leading to the maintenance of LTP, Proteins are released into the extracellular fluid following brief tetanic stimulations of in vivo preparations of the mammalian hippocampus (Charriault-Marlangue et. al.. 1988; Chirwa and Sastry, 1986; Duffy et. al . . 1981) and neocortex (Sastry et. al.. 1988a). Evidence has been provided that some of these proteins possess growth factor-like properties (Sastry et. al . . 1988a) and are capable of producing LTP in hippocampal slice preparations (Sastry et. al.. 1988a; Sastry et. al.. 1990; Xie et. al.. 1991). The release but not the LTP-inducing effects of these proteins also depends upon the activation of NMDA receptors (Sastry et. al.. 1990; Xie et. al.. 1991). One interpretation of these findings is that during activation of postS5maptic NMDA receptors certain event(s), as yet unknown, occur resulting in the release of proteins into the extracellular fluid that interact with either pre- or postsynaptic elements or both, resulting in LTP. A n understanding of where and how these proteins interact with their primary site(s) of action may introduce new insights regarding the mechanisms involved in the induction of LTP. Saccharin interferes with the binding of nerve-growth factor (NGF) to its receptors (Ishii, 1982). It has been shown to reversibly block LTP produced by application of exogenous NGF and endogenous tetanized neocortical samples (TNS) as well as LTP produced through high frequency activation of the stratum radiatum (Sastry et. al.. 1988b; Sastry et. al.. 1990). Whether or not this antagonism occurs by an interference with the actions of NGF or other growth-related substances that are released during the train or with receptors directly involved in synaptic transmission is unclear. Preliminary investigations have suggested that saccharin does not block the NMDA receptors, however, this conclusion relied on the observation that the depolarization of C A l neurons produced by 25 to 50 |LIM iV-methyl-D,L-aspartate (NMDLA) in the absence and presence of saccharin remained unchanged (Chirwa, 1988). Since this experiment was not performed in a Mg^^-free medium which would reduce the voltage-dependent block of the NMDA receptor channel by Mg2+ (Mayer et. al.. 1984; Nowak et. al.. 1984) it is unknown whether saccharin interferes with the NMDA receptors. Saccharin's effects were also not examined on the y-aminobutyric acid (GABA) receptors which have been suggested to play an important modulatory role i n the induction of LTP (Davies et. al.. 1991; Mott and Lewis, 1991; Wigstrom and Gustaffson, 1988). In order to clarify whether saccharin exerts its action by affecting glutamatergic and GABAergic neurotransmission or at steps beyond these sites, experiments were conducted to evaluate the actions of saccharin on both NMDA and non-NMDA receptor-mediated and GABA^ and GABA^ receptor-mediated synaptic transmission. 2. THE HIPPOCAMPAL FORMATION In review of the literature, much work has been done by different scientists to characterize the architecture of the hippocampal formation. Consequently, the terminology describing the morphology of the hippocampus and its associated structures varies and, as a result, one structure may be characterized by several different names. Thus, the following review wi l l contain terminology most frequently used by such authors as: Cajal, Doinikow, Lorente de No and Blackstad. 2.1. Development of the Hippocampal Formation During ontogenic development, the hippocampal formation arises from two cortical structures: the allocortex and the periallocortex (Chronister and White, 1975). The allocortex is separated into the paleocortex and archicortex. The paleocortex is comprised of the olfactory bulb, accessory olfactory bulb, anterior olfactory nucleus, the olfactory tubercle, the periamygdala region, the septum, the diagonal region, and the periform region (Schwerdtfeger, 1984). The archicortex or hippocampus is comprised of the dentate gyrus (fascia dentata), the hippocampus proper (Ammon's horn, C o m u Ammonus or CA), and the subicular complex (Chronister and White, 1975; Schwerdtfeger, 1984; Teyler and DiSenna, 1985). The periallocortex consists of the entorhinal region, the peripalaeocortical region, the claustral region, the presubicular region, the retrospinal region, and the periarchicortical cingulate region (Schwerdtfeger, 1984). 2.2. Basic Topography of the Hippocampus The hippocampus is an elongated symmetrical structure which lies below the cortical surface and hugs the floor of the descending horn of each lateral ventricle (Figure 2.1A and 2. IB). It is oriented such that the "long" or septo-temporal axis runs from the septal nuclei rostrally to the temporal cortex ventrocaudally. The transverse axis of the hippocampus lies perpendicular to the septo-temporal axis (Amaral and Witter, 1989) (Figure 2.2A). The major fields of the hippocampus can be easily viewed from coronal (transverse) slices cut midway along the septo-temporal axis. Examination of a transverse slice reveals two interdigitating " U " shaped archicortical fields (Teyler and DiSenna, 1984) (Figure 2.2B). 2.2.1. The dentate gyrus. The primary cell type of the dentate gyrus is the granule cell (Golgi, 1886). The ovoid shaped somata of granule cells typically range from 15 to 25 \im in diameter (Figure 2.2C). The somata of granule cell bodies form one "U " of the hippocampus (Figure 2.2B). The axons of granule cells termed, mossy fibers (Blackstad et. al.. 1970; Cajal, 1893; Lorente de No. 1934), are polarized towards the center of the "U" to form an upper (suprapyramidal) and lower (infrapyramidal) blade (Teyler and DiSenna, 1985) . Between the blades is a region called the hilus. The hilus contains several layers of polymorphic cells in addition to basket, horizontal, mossy and Figure 2.1. Orientation of the hippocampal formation. A is a lateral view of the guinea-pig brain with part of the neocortex removed to illustrate the position of the hippocampus relative to other bra in formations. B shows a hippocampus fully excised from the brain. Adapted from Andersen et. al. (1971). sep, septal pole; tem, temporal pole. the modified pyramids of CA4 (Amaral, 1978; Cajal, 1893; Lorente de No, 1934). However, because of differences seen from species to species it is difficult to determine whether the CA4 region belongs to the dentate gyrus or the hippocampus proper (Blackstad, 1956; Lorente de No, 1934). Consequently, it is associated with both regions and subserves as a zone of transition between the two hippocampal fields. 2.2.2. The hippocampus proper. The hippocampus proper comprises the other "U" of the hippocampus (Figure 2.2B). It is composed of 3 blades: the lower blade (regio superior) containing the C A l subfield, the transition blade (regio inferior) containing the CA2 and CA3 subfields and the end blade (hilus) containing the CA4 subfield. The lower blade extends towards the subiculum while the transition blade extends toward the dentate gyrus with the end blade terminating abruptly in the hilus of the dentate gyrus. The principle cell type occupying this subfield is the pyramidal cell (Golgi, 1886). These cells are pear-shaped and range from 25 to 40 |j.m in diameter with the largest cells located in field CA3 (Figure 2.2B and 2.2C). Pyramidal cells possess both basal and apical dendrites which traverse radially i n the different strata of the hippocampus (Figure 2.2C). 2.2.3. The subicular complex. The subicular complex is comprised of the prosubiculum and the subiculum. They lie between the C A l subfield of the hippocampus proper and the presubicular region of the periallocortex. Though dispersed, these two regions are occupied primarily by pyramidal cells exhibiting similiar patterns of stratification to the hippocampus proper. 3. STRATIFICATION. SUBFIELDS AND ASSOCIATED CELL TYPES OF T H E HIPPOCAMPUS Based on the observations of Kolliker (1896) and Cajal (1911), the fields of the dentate gyrus and hippocampus proper are divided into different laminae containing distinct cell types and their fiberous connections. Figure 2.2. Organization of the hippocampal formation. A illustrates a cross-section of the hippocampus resulting from a perpendicular cut along its septo-temporal axis. B is a three dimensional representation of the major subfields and intrinsic afferent pathways In a transverse hippocampal slice. C illustrates a pyramidal neuron and a granule cell along with the different strata in which their processes radially distribute, sep, septal pole; tem, temporal pole; alv, cilveus; DG, dentate gyrus; HF, hippocampal fissure; sub, subiculum; fim. fimbria; pp. perforant path; mf. mossy fiber; Sch, Schaffer collaterals; comm/assc, commissural/assoclatlonal fibers. B Adapted from Maclver and Roth (1988). 3.1. The Dentate Gyrus The dentate gyrus is divided into three strata or laminae (layers); they are, stratum granulosum, stratum moleculare and stratum polymorphe. The stratum granulosum is composed of granule cell bodies. The cell's processes are oriented such that their axons (termed mossy fibers) project through the pol5miorphe layer and their dendrites extend into and fill the moleculare layer (Figure 2.2C). The stratum polymorphe which lies in the hilus possesses a wide variety of cells most notably, basket cells and modified pyramidal cells. The statum moleculare lies adjacent to the stratum granulosum and extents towards the hippocampal fissure. Cells in this layer have previously been divided into those which exist in superficial and deep zones (Cajal, 1893; Sala, 1892). 3.2. The Hippocampus Proper The hippocampus proper is commonly differentiated into three subfields: C A l , CA2 and CA3 (Lorente de No, 1934). The CA2 subfield, which lacks innervation by mossy fibers, is small and serves as a zone of transition between the C A l and CA3 subfields (Chronister and White, 1975; Lorente de No, 1934). The C A l and CA3 subfields can be further subdivided into C A l a, C A l b , C A l c , CA3a, CA3b and CA3c (Lorente de No, 1934) (Figure 2.2B). There are 6 laminae extending throughout subfields C A l and CA3 becoming diffuse and less apparent in subfield CA4. The strata that traverse these subfields are: alveus, stratum oriens, stratum pyramidale, stratum lucidum, stratum radiatum, stratum lacunosum and stratum moleculare (Cajal, 1911; Kolliker, 1896; Lorente de No, 1934) (Figure 2.2C). Lorente de No noted, however, that in certain mammals the stratum lacunosum cannot be distinguished from the stratum radiatum and should, therefore, be considered as part of the stratum radiatum. Similar observations were observed between stratums lucidum and pyramidale, lucidum being indistinguishable from pyramidale. In the C A l subfields the lamina located immediate to the ventricle is called the alveus which contains primary efferent axons of pyramidal cells, though some afferent processes are present. Below this lies the stratum oriens which consists the basal dendrites of pyramidal cells as well as invading axons from the alveus. Deeper to this lies the stratum pjnramidale containing the soma of pjnramidal cells. Beneath this is a dense lamina called the stratum radiatum where the apical dendrites of the pyramidal cells lie. Below this and bordering the hippocampal fissure is the stratum lacunosum-moleculare which contains the distal dendrites of the pyramidal cells as well as intemeurons which appear to possess axonal and dendritic processes that traverse the hippocampal fissure into the dentate gjnnas. In the CA3 subfleld, the laminae are arranged in a similiar order to the C A l subfield, however the mossy fibers of granule cells produce a distinct band of transversely oriented axons just below the stratum pyramidale. This region constitutes the stratum lucidum. Thus, the most important feature of the laminae is that they represent the various afferent connections to the hippocampus proper and the dentate gyrus which terminate on restricted portions on the dendrites of p5n:amidal and granule cells. 4. INTRINSIC AFFERENTS OF T H E HIPPOCAMPUS In the hippocampus proper there are two principle sources of intrinsic afferent input to the pyramidal cells they are, 1) afferent input from cells located within the same subfield, 2) afferent input to cells from different fields. Together, these systems enable the hippocampus to integrate and process a vast array of neural input from extrinsic sources. 4.1. Intrinsic Afférents Within the Same Subfield Both inhibitory and excitatory afferent input has been shown to occur between the pyramids of C o m u Ammonus. In the CA3 and, similarity, C A l subfields antidromic activation studies have revealed that afferent inhibition between pyramidal cells is indirect and arises through activation of interneurons (Kandel et. al.. 1961) identified as basket cells (Andersen et. al.. 1964a), Based on histological and physiological findings, basket cells exert their inhibition near the soma of the p3nramidal cells (Andersen et. al.. 1964b) by recurrent (feed-back) inhibition (Spencer and Kandel, 1961) and activity of one interneuron may inhibit many pyramidal cells (Andersen et. al.. 1969). Recurrent inhibition has also been demonstrated in the granule cells of the dentate gyrus (Andersen et. al.. 1966a). Based on antidromic activation studies, excitatory connections between the pyramids of CA3 have been demonstrated to exist (Andersen et. al.. 1969; Lebovitz et. al.. 1971). The presence of such connections has been established using various histological techniques. Important features of these connections are that they occur extensively in the CA3 subfields (Gottlieb and Cowan, 1973; Laurberg and Sorensen, 1981) from highly collateralized axons which project along the septo-temporal axis (Laurberg and Sorensen, 1981; Lorente de No, 1934; Swanson et. al.. 1981) and terminate almost exclusively in strata oriens and radiatum but not in lacunosum-moleculare (Hjorth-Simonsen, 1973). These longitudinal associational projections are thought to participate in integrating information from different levels along the long-axis of the hippocampus (Lorente de No, 1934). 4.2. Intrinsic Afférents From Different Fields The different fields of the hippocampus are interconnected by afferent projections that are, in general, unidirectional starting in the dentate gyrus continuing through CA3 and ending in C A l (Figure 2.2B). In the dentate g3^s , perforant path fibers originating from the entorhinal cortex (Cajal, 1911; Blackstad, 1958; Hjorth-Simonsen and Jeune, 1972; Lorente de No, 1934) distribute within the molecular layer where they synapse with the dendrites of granule cells (Fifkova, 1975; HJorth-Simonsen, 1972). The granule cells send out mossy fibers which collateralize in the pol3miorph layer in the hilus of the dentate gyrus before projecting to the CA3 subfield. The mossy fibers form two highly laminated separate bundles. Mossy fibers which arise from the infrapyramidal blade travel in stratum oriens and terminate within subfields CA3b and CA3c, and those arising from the suprapyxamidal blade travel in stratum lucidum, course through CA3 and end in CA2 (Blackstad et. al.. 1970; Chronister and White, 1975; Lorente de No, 1934). Throughout the length of their travel, the mossy fibers make numerous excitatory en passant S5niapses with the dendrites of pyramidal cells i n CA3 (Andersen et. al.. 1966). The mossy fibers also synapse with basket cells in this field and with neurons in the hilus of the dentate gyrus. Mossy fibers, which never leave the ipsilateral hippocampus, constitute the major intrinsic afferent pathway to CA3. CA3 cells also receive projections from the perforant path. These fibers synapse with the distal apical dendrites of CA3 pyramidal cells (Hjorth-Simonsen and Jeune, 1972). The giant pyramidal cells of field CA3 possess associational projections which run along the transverse and septo-temporal axis. The_most prominant projections arising from CA3 are the Schaffer collaterals (Schaffer, 1892), This fiber system courses through the C A l field and synapses with pyramidal cells en passant on their basal dendrites in stratum oriens and proximal 3/4 of the apical dendrites in stratum radiatum (Gottlieb and Cowan, 1973; Lorente de No, 1934). Ipsilateral projections from CA3 to CA4 to the dentate gjnnas, opposite to the main direction of flow, are also present (Gottlieb and Cowan, 1973; Swanson et. al.. 1981). 4,3. Commissural Afférents The two hippocampi are interconnected by a vast network of fibers which cross the midline in the ventral and dorsal hippocampal commissures (psalleria). The majority of commissural fibers course through the ventral commissure. While all regions of the hippocampus receive commissural input from their respective fields, in some fields the origin of these inputs is not entirely homotropic. Studies by Gottlieb and Cowan (1973) revelled that, commissural projections from CA3 to C A l and CA3 to dentate gyrus exist and, therefore contribute heterotropic inputs to these regions. Such a pattern, however, would attribute an associational rather than a commissural nature to these connections. The significance of this arrangement is not clear at present. 5. PROJECTION PROFILE OF INTRINSIC HIPPOCAMPAL AFFERENTS In general, the overall organization of afferent projections that course each hippocampal field assumes a transverse or trisynaptic circuit (Andersen et. al.. 1971; Teyler and DiScenna, 1984). That is, the main direction of flow within the hippocampus starts as input to the dentate granule cells from the entorhinal cortex via the perforant path. The granule cells then project mossy fibers to the p5a-amidal cells of CA3. The CA3 pyramidal cells in turn send Schaffer collaterals to the pjnramidal cells of C A l . Physiologically, this arrangement appears to be functional throughout the transverse axis (Andersen et. al.. 1971). Consequentiy, the hippocampus has been described as being organized in a lamellar fashion (Andersen et. al.. 1971) with each lamina functioning independent from one another. However, subsequent studies (Hjorth-Simonsen, 1973; Laurberg, 1979; Swanson et. al.. 1978) suggest that the organization of the intrinsic circuitry of the hippocampus extends as much in the septo-temporal axis as it does in the transverse axis. Recent anatomical and physiological studies (Amaral and Witter, 1989; Buzsaki et. al.. 1990; Ishizuka et. al.. 1990) using the "extended" hippocampal preparation (in this procedure the septal to temporal curvature of the hippocampus is corrected by flattening or extending it resulting in a more accurate representation of fibers being distributed in the septo-temporal plane when transverse slices are cut) in combination with the descrete anteriorgrade tracer, Phaseolus vulgarus leucoagglutinin (PHA-L) have shown that the 3-dimensional organization of the intrinsic hippocampal circuitry (with the exception of the mossy fibers) is not restricted to a lamellar pattern but a more diffuse one which favours a septo-temporal distiribution. 5.1. Distribution of Perforant Path Fibers The major extrinsic input to the dentate gyrus arises from the entorhinal cortex. Perforant path fibers from this region enter the dentate gyrus through the perforant path where they distribute in the subiculum, stratum lacunosum-moleculare of the hippocampus proper and the molecular layer of the dentate gyrus (HJorth-Simonsen and Jeune, 1972; Witter et. al.. 1988). Recent studies have shown either through autoradiographic or PHA-L tracing techniques that small injections of the anteriorgrade tracers result i n widespread termingil labelling in the molecular layer along the septotemporal axis (Amaral and Witter, 1989; Ruth et. al.. 1988; Steward, 1976; Wyss, 1981). The projection profile of these fibers originates from highly collateralized axons of entorhinal cortical cells rather than from axons that project to restricted and different areas in the dentate gyrus. Thus, perforant path input to the dentate gyrus assumes a highly dispersed pattern which projects septo-temporally to innervate distant levels of the dentate gyrus. 5.2. Distribution of Fibers from the Dentate Gyrus The dentate gyrus provides both mossy fiber projections which synapse within CA3 and associational projections which s)mapse within the dentate gyrus. The trajectory and general distribution of mossy fibers have been extensively studied (Amaral and Witter, 1989; Blackstad et. al.. 1970; Gaarskjaer, 1986; Swanson et. al.. 1978). With the exception of a bend toward the long axis near the CA3 - C A l boarder, the mossy fibers are organized in a lamellar fashion. This pattern, however, is not observed when the trajectories of associational fibers are traced. The associational projections originate i n the pol5niiorphic region of the dentate hilus (Laurberg and Sorensen, 1981) and are confined within the limits of the dentate gyrus. These fibers travel parallel to the long axis and are arranged so they do not provide feedback to the granule cells (Amaral and Witter, 1989). Therefore, projections within the dentate gyrus are coordinated septotemporally as well as transversely. 5.3. Distribution of Fibers from CA3 CA3 cells possess highly collateralized axons which project in both transverse and septo-temporal axes (Ishizuka et. al.. 1990). The Schaffer collaterals constitute the major projection to C A l . Contrary to current belief, the Schaffer collaterals do not l ink CA3 to C A l at the same hippocampal level. In fact, CA3 cells located near the dentate gyrus project their collaterals more heavily in the septal direction while those close to CA2 have collaterals that project more frequent to the temporal surface (Amaral and Witter, 1989). Furthermore, in transverse slices, CA3 cells injected with horse radish peroxidase, to reveal their distribution of axon collaterals, send few axons to C A l (Ishizuka et. al.. 1990). Together, these patterns imply that the trajectories of these collaterals do not run parallel to the transverse axis but more in the septo-temporal plane. In addition to the Schaffer collaterals. CA3 cells also possess other associational projections which run parallel to the long axis of the hippocampus. These longitudinal associational projections have already been discussed. Thus, the hippocampus is not organized to function in discrete lamellar units, but rather as a whole unit capable of processing information along both axes. 6. EXTRINSIC AFFERENTS TO THE HIPPOCAMPAL FORMATION The hippocampal formation receives afferent innervation from both parahippocampal and, to a lesser extent, isocortical regions. The major parahippocampal projections arise from the entorhinal cortex and the medial septal nucleus. 6.1. Entorhinal Projections The entorhinal cortex projects to the dentate gyrus via the perforant path fibers. While the origin of these projections has been found to arise from neurons located mainly in lamina II of the entorhinal cortex (Witter and Groenewegen, 1984), some cells in deeper layers have been reported to project to the dentate gyrus (Kohler et. al.. 1984). As in the dentate gyrus, subfleld CA3 also receives extrinsic input from the entorhinal cortex (Swanson and Cowan, 1977). This pathway originates i n the superficial laminae and is considered to be sparcer than the perforant path projection. The entorhinal cortex also supplies efferents to C A l (Nafstad, 1967; Steward, 1976). These projections originate in lamina III in the rostral and lateral portions of the entorhinal cortex (Witter and Groenewegen, 1984; Witter et. al.. 1988), 6.2. Septal Projections The dentate gyrus is also innervated by flbers from the lateral and medial septal nucleus and nucleus of the diagonal band. Consisting of primarily cholinergic efferents, these septo-hippocampal pathways are responsible for initiating rhythmic theta activity in the hippocampus (Petsche et. al.. 1962). Some neurons projecting from the medial septal nucleus and diagonal band to the dentate gyrus are glutamic acid decarboxylase (GAD) positive (Kohler et, al.. 1984; Panula et. al.. 1984) suggesting the presence of GABAergic input to the dentate gyrus. The medial septal nucleus also supplies input to CA3, Though these fibers project to all layers of CA3, they are most concentrated in stratum oriens (Nyakas et. al.. 1987). Experiments combining lesion studies with choline acetyl transferase (ChAT) labelling suggest these pathways are cholinergic (Frotscher et. al.. 1986; Houser et. al.. 1983) though the presence of septal input from GAD positive neurons implys a GABAergic input also exists (Kohler et. al.. 1984). The existence of a pathway to C A l from the medial septal nucleus is controversal. While some investigators have reported the presence of a septal projection to C A l (Monmaur and Thompson, 1983; Sakanaka et. al.. 1980) others have not (Powell, 1963; Rose et. al.. 1976; Swanson and Cowan, 1979). 6.3. Isocortical Projections Isocortical projections to the hippocampus are of particular importance because they serve to l ink the hippocampus with different regions of the cortex and illustrate that hippocampal function is influenced from cortical sensory input. Projections from the isocortex to the hippocampus terminate primarily in C A l , occasionally in CA3, and not in the dentate gyrus. They originate i n parietal, temporal, and perirhinal cortices (Schwertfeger, 1979; Schwertfeger, 1984). 7. EXTRINSIC EFFERENTS F R O M T H E HIPPOCAMPAL FORMATION The hippocampal formation not only receives input from parahippocampal and isocortical regions, but also projects to these areas. These projections are of significant importance because they subserve as a anatomical substrate for interplay between the hippocampus and cortex. 7.1. Fornix-Fimbria Svstem The most well characterized extrinsic pathway from the hippocampus is the forntx-fimbria system (O'Keefe and Nadel, 1978). Efferent fibers from the hippocampus and adjacent allocortical areas gather in the fimbria where they meet at the rostral part of the hippocampus to become the columns of the fornix. The main portion of the fibers course through the septo-fimbrial nucleus and split to form the post-commissural and pre-commissural fomices. 7.1.1. Pre- and post-commissural fornices. The pre-commissural fornix contaiins fibers from fields C A l and CA3 that distribute in the lateral septal nucleus, the diagonal band of Broca, the bed nucleus of the anterior commissure, the lateral preoptic region, and the lateral hypothalmus (Swanson and Cowan, 1977). The post commissural fornix sends projections originating from the presubiculum, parasubiculum and subiculum to the thalmus, mammillary bodies and rostral brain stem (Chronister and DeFrance, 1979). 7.2. Entorhinal Protections The entorhinal cortex (especially lamina IV), subiculum and to a lesser extent pre- and parasubiculum also receive afferent input from the hippocampal formation. While fibers from C A l constitute the major projections to the entorhinal cortex (Swanson and Cowan, 1977; Swanson et. al.. 1981), those from ipsilateral and contralateral CA3 provide the major projection to the subiculum (Swanson and Cowan, 1977), 7.3. Isocortical Projections Efferent projections from C A l serve to l ink the hippocampal formation with the isocortex. Connections have been established between C A l and various parts of the frontal (Swanson, 1981) and temporal cortices (Schwerdfeger, 1979), the retrosplenial and perihinal cortices (Swanson and Cowan, 1979). 8. ELECTROPHYSIOLOGY OF HIPPOCAMPAL NEURONS A number of in vitro slice studies have been performed to investigate the electrophysiological properties of the various cell t3rpes in the hippocampus (Brown et. al.. 1981; Brown and Johnston, 1983; Durand et. al.. 1983; Johnston, 1981; Lambert and Jones, 1990; Masukawa et. al.. 1982; Randall et, aL, 1990; Schwartzkroin, 1975, Schwartkroin, 1977; Turner and Schwartzkroin, 1983). As expected, the recordings not only reflect differences in the electrophysiological properties of neurons from different subfields, but also tJie quality of the intracellular recording. Consequently, the electrophysiological parameters of these neurons are associated with upper and lower limits. Typical resting membrane potentials (RMPs) for pjnramidal cells range from -50 to -70 mV and for granule cells between -60 and -85 mV. Action potentials evoked at RMP by injecting current into the cell range from 50 to 110 mV for p3n:amidal cells and 70 to 140 mV for granule cells. The input resistance calculated from the slope of the linear portion of current-voltage plots varies from 25 to 40 MQ for p5nramidal cells and 40 to 55 MQ. for granule cells. The membrane time constant measured as the time required for peak voltage defections to settle to l-(l/e) in response to short current pulses are on average 25 msec for CA3 cells, 15 msec for C A l cells and 11 msec for granule cells (Brown et. al.. 1981; Durand et. al.. 1983; Schwartzkrion and Mueller, 1987). Based on the single dendrite (equivalent cylinder) model (Rail, 1969; Rail, 1974; Rail, 1977), other electrotonic parameters not available by direct intracellular measurements have been calculated. Estimated values for the ratio of dendritic to somatic conductance and electrotonic length of hippocampal neurons are 1.0 to 1.5 and 0.9 to 1.2, respectively (Brown et. al.. 1981; Durand et. al.. 1983; Schwartzkroin and Mueller, 1987). Recent studies based on the multipolar cylinder model have re-calculated the electrotonic length and found it to be only 1/3 that of previous estimates (Glen, 1988). Indeed, studies utilizing the "on slice" whole-cell patch clamp technique (Blanton et. al.. 1989; Edwards et. al.. 1989; Randal et. al.. 1990; Zafir et. al.. 1990) have reported input resistances of p5n:amidal neurons to be 200 to 500 MQ (the discrepencies between values obtained with patch electrodes and conventional microelectrodes may lie in the seal which develops between the electrode-membrane juncture ) (Storm, 1990). These results imply that hippocampal neurons are even more electrically compact than was previously thought. 8.1. Bursting Activity of Hippocampal Neurons Hippocampal pyramidal cells are endowed with the ability to fire action potentials and burst discharges either spontaneously or during current evoked depolarizations. Variations in burst discharge have been demonstrated in the different subfields of regio superior (Masukawa et. al.. 1982; Wong et. al . . 1979). C A l pyramidal cells fire both accomodating trains and bursts of action potentials where as CA3 pyramidal cells burst i n response to artificial depolarizing current injections (Masukawa et. al.. 1982; Wong et. al.. 1979). In addition, CA3 neurons readily support spontaneous burst discharges which develop from activation of a T-type calcium current (Traub, 1982; Wong and Prince, 1978). The physiological significance for bursting activity in the CA3 region may be to amplify excitatory signals from incoming afferent pathways to other regions in the hippocampus or, alternatively, to act as a pacemaker for interictal discharge in the hippocampus (Schwartzkroin et. al.. 1990). Granule cells, on the other hand, rarely exhibit spontaneous activity because of the cell's high negative resting membrane potential and high threshold for spike generation. 8.2. HIPPOCAMPAL INTERNEURONS The hippocampus is endowed with a variety of intemeurons which have been characterized anatomically (Cajal, 1911; Lorente de No, 1934). Their morphological attributes include: large somata (35 to 50 jim on average) identified as either p5n-amidal, horizontal, fusiform, inverted fusiform, or multipolar. They possess aspinous dendrites and locally arborizing axons (Riback and Andersen, 1980). The most well characterized intemeurons, are those located in the stratum p5n:amidale, near the oriens/alveus border and near the stratum radiatum/lacununosum-molecular border. 8.2.1. Basket cells. The most studied hippocampal intemeuron is the basket cell which is so called for the shape of its axonal plexus which resembles a "basket" around its target cell somata (Cajal, 1911; Lorente de No, 1934). Basket cells are present in both strata p5n:amidale and granulosum. Their somata average 45 |im and project dendrites which are aspinous, show periodic swellings (appear beaded) and receive multiple synaptic contacts. Early studies by Kandel et. al. (1961) and Andersen et. al. (1964b) revealed that basket cells exert a powerful feedback (recurrent) inhibition on pyramidal neurons. Since these intemeurons are immunoreactive for y-aminobutyric acid (GABA) (Gamrani et. al.. 1986) and GAD (Ribak et. al.. 1978), basket cell inhibition is presumed to involve the inhibitory neurotransmitter GABA, Electrophysiologically, basket cells differ from pyramidal cells and granule cells in that they have very short membrane time constants (approximately 3 msec), fire very brief action potentials (approximately 0,8 msec) that are associated with large after-h3^erpolarizing potentials (5 to 10 mV), produce non-accomodating spike discharges in response to tonic depolarization and exhibit a high degree of spontaneous S5niaptically driven EPSPs. In addition to the antidromic activation studies Kandel et. al. (1961), experiments by Knowles and Schwartzkroin (1981) involving simultaneous intracellular recordings between basket cell and pyramidal cell pairs have confirmed that basket cells can exert feed-back inhibition on pyramidal cells. They demonstrated that a pyramidal cell can directly excite a basket cell which, in turn, produces a h5^erpolarization in the p5n:amidal cell sufficient to raise the rheobasic threshold for spike generation. Basket cells have also been shown to be activated by stimulation of Schaffer coUateral/commissural afférents (Alger and NicoU, 1982a; Ashwood et. al.. 1984; Busaki and Eidelberg. 1982) supporting the view that these intemeurons are involved in both feed-forward and feed-back inhibition. 8.2.2. Oriens/alveus fO/A) interneurons. Another type of interneuron sharing similiar ultrastructural properties with basket cells is found in C A l between stratum oriens and the alveus (Lacaille et. al.. 1987). The oriens/alveus (O/A) interneuron is multipolar having a soma that measures 20 to 30 |j,m in diameter. O/A interneurons, like basket cells, possess aspinous "beaded" dendrites. The majority of the dendrites are arranged parallel to the alveus, however, some turn and project into strata oriens, pyramidale, radiatum and lacunosum-moleculare where they engage in forming numerous S3^aptic contacts. The axons of O/A interneurons branch and distribute in strata oriens and P3n:amidal. O/A interneurons display both GABA-like (Gamrani et. al.. 1986) and somatostatin-like immunoreactivity (Kohler and Chan-Palay, 1982; Morrison et. al.. 1982; Sloviter and Nilaver, 1987) and are, therefore, considered inhibitory interneurons. In addition to ultrastructural properties, O/A interneurons share similiar electrophysiological properties with basket cells. The membrane time constant is short (approximately 6 msec), action potentials are brief (approximately 1 msec) and associated with large after-hyperpolarizations and non-accomodating spike discharges develop in response to tonic depolarizations. In addition, O/A interneurons are continually barraged with excitatory sjmaptic activity (Lacaille et. al.. 1987; Lacaille et. al.. 1989). They can be activated either by stimulation of the Schaffer coUateral/commissural afférents or by depolarizing pyramidal cells that are synaptically paired with them. Both forms of activation result i n pyramidal cell inhibition. Thus, like the basket cells, O/A interneurons are capable of producing both feed-forward and feed-back inhibition. 8.2.3. Lacunosum-moleculare (L-M) interneurons. The lacunosum-moleculare (L-M) interneurons are located at the border between strata lacunosum-moleculare and radiatum (Kawaguchi and Hama, 1987; Lacaille and Schwartzkroin, 1988). L-M intemeurons morphologically resemble the stellate cells of stratum lacunosum and are characterized by fusiform or multipolar shaped somata. The main dendritic portion of a typical L-M interneuron runs parallel to and branches out in stratum lacunosum-moleculare projecting into stratum pyramidal and stratum oriens of the hippocampus proper and stratum moleculare of the denate gyms. The main axon usually branches immediately and adopts a similar projection profile to that taken by its dendrites (Kunkel et. al.. 1988; Lacaille and Schwartzkroin, 1988). L-M intemeurons exhibit GABA-like immunoreactivity and are therefore considered as GABAergic inhibitory intemeurons. L-M intemeurons share similar electrophysiological properties with basket and O/A intemeurons in that they exhibit spike after-hyperpolarizations and show little spike accomodation to depolarizing current injection. However, unlike basket and O/A intemeurons, L-M intemeurons do not support spontaneous action potentials nor do they exhibit a high degree of spontaneous synaptic activity. Their action potential duration (approximately 2 msec) and membrane time constant (approximately 9 msec) are relatively long when compared to the other intemeurons. L -M intemeurons receive both excitatory and inhibitory input. While evidence has implicated the Schaffer coUateral/commisural afférents to be responsible for generating S5niaptic excitation in L-M intemeurons, the source of inhibition is less clear. Because L-M intemeurons, S5niaptically paired with pyramidal cells, are neither excited nor inhibited by the pyramidal cells, they are thought to mediate feed-forward inhibition (Lacaille and Schwartzkroin, 1987). To what extent O/M and L-M intemeurons contribute to inhibitory synaptic transmission is not yet fully understood. However, preliminary investigations performed by Samulack and Lacaille (1991) and Williams and Lacaille (1991) have demonstrated that during activation of the Schaffer coUateral/commissural pathways, orthodromically evoked slow IPSPs in hippocampal p5nramidal neurons appear to be mediated through activation of these interneurons. 9. IONIC CURRENTS IN HIPPOCAMPAL NEURONS Hippocampal neurons support a variety of different ionic conductances that are mediated either through opening of voltage-gated channels, of ion-gated channels or of non-voltage-gated "leak" channels. Studying the physiological properties of ionic conductances is complicated by a variety of factors. For example, their activation and inactivation may depend not only on the temperature (QIQ) and the potential of the cell membrane or the ionic state of the intraceUular milieu but also on the presence of neurotransmitters and second messengers. Furthermore, resolution of some currents may not be uniform since channels may be preferentially localized on restricted portions of the soma or dendrite. Indeed, to circumvent these problems experimenters have supplemented their recording solutions with second messengers; their perfusion media with certain neurotransmitters. Others have successfully recorded from the dendrites of pyramidal cells and the soma dissociated from its dendrites. Further complicating the study of ionic currents is the lack of space clamp that arises when using a point somatic current source. Fortunately, this problem can be reduced using patch-clamp techniques. But even with this technique care must be taken so as not to upset the intracellular enviroment by dialysing the cell with the recording solution. Despite these problems, recordings of different ionic currents from hippocampal neurons have been obtained. The most common currents associated with hippocampal neurons are those mediated by Na+, K+, Ca+ or CI' ions. The nomenclature for the different currents described in the following sections characterizes those found in hippocampal neurons (for reviews see. Brown et. al.. 1990; Llinas, 1988; Ruby, 1988; Schwartzkroin and Mueller, 1987; Storm, 1990). 9.1. Na+-Currents Two types of voltage-dependent Na+-currents are present in hippocampal neurons. They are the fast activating Na+-current (lNa(fast)^ slow inactivating Na^-current I(Na(slow)) (Brown et. al.. 1990). The fast activating Na"*"-current underlies the classic action potential and has been observed i n both the soma and dendrites of hippocampal neurons (Hugenard et. al.. 1989; Sah et. al.. 1988). iNa(fast) a Hodgkin-Huxley type current with an activation threshold of -60 mV at and a time to peak of 0.9 msec. Inactivation is complete throughout the whole activation range. iNa(fast) blocked by extracellular application of tetrodotoxin (TTX) or intracellular injection of QX-314. The slow inactivating Na+-current may be confined to the soma of hippocampal neurons (Benardo et. al.. 1982; French et. al.. 1990). Triggered and sustained by depolarizing pre-potentials, the activation threshold for %a(slow) is -70 mV (French and Gage, 1985; French et. al.. 1990; Lanthom et, aL, 1984). Like iNa(fast)' iNa(Slow) is blocked by TTX or QX-314. This current may participate in hippocampal pacemacker activity (Brown et. al.. 1990) or repetitive firing of action potentials in response to prolonged depolarizations produced from intense synaptic activity (French et. al.. 1990). 9.2. K+-Currents Six voltage-dependent K+-currents have been identified in hippocampal neurons. These include the delayed rectifier (Ij^ ), the transient A-current (%(A) the slowly-inactivating delay current (1K(D))' the non-inactivating M-current (^K(M))' the inward rectifying K+-current (IK{IR)) and the inward rectifying Q-current (Ig) (Storm, 1990). These currents can be divided into two groups, those that are activated by events which depolarize the cell from its rest potential and those that are activated by events which hyperpolarize the cell from its rest potential. 9.2.1. K+-currents activated through depolarization (IR. IK(A)- IK(D)^K(M)1-IK is the classic delayed rectifier previously described by Hodgkin and Huxley (1952). This high threshold slowly inactivating K+-current is activated by depolarizing potentials beyond -40 mV {Neumann et. al., 1988; Segal and Barker, 1984) and has a time to peak ranging from 150 to 200 msec at 0 mV. IK is blocked by tetraethylammonium [TEA) in m M concentrations and insensitive to 4-aminopyridine {4-AP) and Cs"*" when externally applied (Segal and Barker, 1984). Both the soma and dendrites of hippocampal neurons exhibit % currents (Masukawa and Hansen, 1987). Though relatively slow, the delayed rectifier may participate in repolarization of the action potential (Ruby, 1988; Storm, 1987a). The fast transient A-current is rapidly activated (5 to 10 msec) by depolarizing steps from a holding potential negative to -60 mV (Gustaffson et al.. 1982; Zbicz and Weight, 1985). Its inactivation is also rapid (time constant, 10 to 20 msec) and is complete throughout its activation range (Sah et. al.. 1988). ^(A) is characterized by being blocked by externally applied 4-AP in low m M concentrations (Numann et. al.. 1987; Segal and Barker, 1984; Storm, 1988a) and internally applied TEA and Cs+ (Ruby, 1988). It is also blocked by |iM concentrations of dendrotoxin (DTX) (Halliwell et. al.. 1986), suppressed by elevated concentrations of internal Ca2+ (Chen and Wong, 1991) and enhanced by }j.M concentrations of GABA and baclofen (Saint et. al.. 1990). The A-current is fast enough to contribute to Na+ spike repolarization (Storm, 1987) and has been implicated in regulating neuronal firing rate (Segal et. al.. 1984). This current has been detected primarily in the soma of pyramidal neurons. The slowly Inactivating delay current is largely inactivated at resting potentials (-60 to -65 mV) but is activated by depolarizations from holding potentials more negative to -70 mV with a time to peak reaching 20 ms (Storm, 1988a). Inactivation starts around -120 mV and is complete at -60 mV and consists of several time constants altogether lasting 3 to 5 seconds (Storm, 1988a). 1K(£)) is blocked by |J,M concentrations of 4-AP (Storm, 1988) and by n M concentrations of DTX and Toxin 1 (Storm, 1990). The current appears to be insensitive to m M concentrations of TEA and Cs+. Fast activation of the current suggests that it may participate in Na+ spike repolarization (Storm, 1987b). 1K(D) is capable of producing long delays in firing patterns induced by "just-threshold" depolarizations. Its slow recovery from inactivation enables recruitment of repetitive depolarizing inputs and, therefore, may participate i n dendritic processing (Hounsgaard and Midtgaard, 1988; Hounsgaard and Midtgaard 1989). The M-current is a subthreshold voltage-gated K+-current (Brown and Adams, 1980; Brown and Griffith, 1983a; Brown, 1988). Activation of 1K(M) is slow and is produced by depolarizations beyond -60 mV. Deactivation is also slow (decay time, 50 msec); the current does not inactivate (Adams et. al . . 1982; Brown and Adams, 1980; Brown and Griffith, 1983a). IK(M) is dubbed the M-current because it is inhibited by muscarinic receptor agonists (Brown and Adams, 1980; Halliwell and Adams, 1982). The current is also modulated by other neurotransmitters. For example, it is reduced by 5-hydroxytryptamine (5-HT) (Colino and Halliwell, 1987), trans-1-aminocyclopentyl-l,3-dicarboxylate (ACPD) (Charpak et. al.. 1990) and is increased by somatostatin (Moore et. al.. 1988; Watson and Pittman, 1988), an effect which may be mediated through activation of arachidonic acid metabolites (Schweitzer et. al.. 1990). The M-current has also been shown to be blocked by mM concentrations of externally applied Ba"*" and TEA but not by 4-AP or Cs+ (Halliwell and Adams, 1982; Storm, 1989). In hippocampal neurons, the M-current contributes to stabilizing the membrane potential, produces spike frequency adaptation and mediates muscarinic and peptidergic excitation (Brown et. al.. 1990). 9.2.2. K+-currents activated through hyperpolarization (IK(IR). IQ). The fast inward rectifying K+-current (IK(IR)) is partially activated at -60 mV and inactivates at membrane potentials more negative to -100 mV (Owen, 1987). The current, which rapidly activates (<10 msec) upon membrane hyperpolarization, is blocked by Cs+ and Ba+ (Brown et. al.. 1990). IK(IR) contributes to stabilizing the resting potential in hippocampal neurons. The inward queer or Q-current (IQ) is a mixed cationic current being carried by both K+ and Na+ ions (Adams and Halliwell, 1982). Ig is activated by hyperpolarizations beyond -80 mV. Activation of IQ is relatively slow and increases with increasing hyperpolarization (Halliwell and Adams, 1982). The current is reduced by tetrahydroaminoacradine (THA) (Brown et. al.. 1988) and completely blocked by 1 m M Cs+ (Halliwell and Adams, 1982). Activation of Ig prevents hippocampal neurons from h5rperpolarizing insults and may contribute to spike after-hyperpolarization when spikes are initiated at more negative potentials to the resting potential of the neuron (Storm, 1989). 9.3. Voltage-Dependent Ca2+-Currents (T. L. N.) Three types of voltage-dependent Ca2+-currents have been demonstrated in hippocampal neurons (Brown and Griffith, 1983; Fischer et. al.. 1990; Gahwiler and Brown, 1987; Halliwell, 1983; Johnston et. al.. 1980; Mogul and Fox, 1991; Ozawa et. al.. 1989; Takahashi et. al.. 1989). They are analogous to those previously described in chick sensory neurons (Fox et. al.. 1987; Nowycky et. al.. 1985) which include the T-, L- and N-types of Ca2+-currents. The T-tj^e or transient inward Ca2+-current is a low threshold fast inactivating current, activated at -60 mV from holding potentials > -100 mV (Halliwell. 1983; Ozawa et. al.. 1989; Takahashi et. al.. 1989; Takahashi et. al.. 1991). Inactivation begins at -60 mV and is complete at -40 mV (decaying with a time constant of 16 msec at -40 mV), In addition to Ca2+ ions, T-type channels equally conduct Ba2+ ions. Depending on the charge carrier, the single channel conductance ranges from 7 to 8 pS (Fischer et. al.. 1990, Mogul and Fox, 1991). The T-type current is relatively resistant to dihydrop5nidine Ca2+ antagonists (Tsien et. al.. 1988) and is blocked by |iM concentrations of Ni2+ and Cd2+ (Halliwell, 1983; Mogul and Fox, 1991; Yaari et. al.. 1987). T-type Ca2+-currents have been implicated in participating i n the initial phase of burst potentials (Brown and Griffith, 1983; Traub, 1982), the genesis of Ca2+-dependent low threshold spikes (Coulter et. al.. 1989) and in pace-maker depolarizations (Llinas and Yarom, 1981). The high threshold slowly inactivating "long-lasttng" or L-type inward Ca2+-current is activated at potentials between -20 mV and -10 mV and inactivated at potentials between -60 mV and -40 mV (Ozawa et. al.. 1989; Takahashi e t al.. 1989). Compared to the other types of voltage-dependent Ca2+-currents, the L-type current inactivates very slow during maintained depolarizations decaying with a time constant > 500 msec to reach 0 mV (Ozawa et. al.. 1989). L-1ype channels conduct Ba2+ greater than Ca2+ (Fox et. al.. 1987; Tsien et. al.. 1988). With Ca2+ as the charge carrier, the single channel conductance is 25 pS (Fischer et. al.. 1990; Mogul and Fox, 1991). The current is blocked by Cd2+, Ni2+ and |J.M concentrations of co-conotoxin (Mogul and Fox, 1991), is sensitive to low |iM concentrations of the dihydropyridine Ca2+ antagonists, nimodipine and nifedipine and is increased by the dihydropyridine agonist. Bay K-8644 (Docherty and Brown, 1986; Gahwiler and Brown, 1987a; Tsien et al.. 1988). Somatic L-type currents have been recorded from freshly dissociated adult neurons (Kay and Wong, 1987) and both the soma and its processes have been shown to bind fluorescent derivatives of co-conotoxin (Jones et. al., 1989), suggesting that throughout the length of the neuron, the relative distribution of these channels is not confined to any one region. However, since co-conotoxin binds to both T- and N-type Ca2+ channels (Jones et. al.. 1989), a localization of L-type channels on specific portions of the soma or its processes cannot be ruled out. The high threshold moderately inactivating inward Ca2+-current is dubbed the N-type current because it exhibits activities that are neither characterized by the L- or T-type currents. For example, unlike the L-type current it requires stronger depolarizations (cell membrane depolarized more positive to -10 mV) for activation (Madison et. al.. 1987; Ozawa et. al.. 1989; Takahashi et. al.. 1989) and unlike the T-type current the N-type current inactivates at more negative potentials (approximately -70 mV), decaying from prolonged depolarizations with a time constant of 30 msec at 0 mV (Ozawa e t al.. 1989). The N-t3rpe channel conducts Ba^+ greater than Ca2+ ions (Tsien et al.. 1988) and possesses a single channel conductance of 13 pS (Fischer et. al.. 1990; Mogul and Fox, 1991). The current is blocked by Cd^+, Ni2+ and co-conotoxin, and is relatively insensitive to dihydropyridine Ca2+ antagonists. The N-type current is also modulated by neurotransmitters as it is reduced by both adenosine (Madison et. al.. 1987a) and muscarine (Gahwiler and Brown, 1987b). 9.4. Ca2+-Activated K+-Currents (In^. l^p) Two Ca2+-activated K+-currents have been identified in hippocampal neurons. I^a is a large time- and voltage-dependent K+-current which activates rapidly (1 to 2 msec) in response to a depolarizing current pulse or during an action potential (Brown and Griffith, 1983b; Lancaster and Adams, 1986; Lancaster and Nicoll, 1987; Storm, 1987). Activation of l^a requires high concentrations of internal Ca^+ (threshold at > 1 |iM) (Franciolini, 1988). Depending on the magnitude of depolarization, deactivation is voltage-dependent taking 50 to 150 msec when the cell is repolarized from depolarized holding potentials (Brown and Griffith, 1983). ÏQ^ is blocked by n M concentrations of charybdotoxin and externally applied TEA at concentrations (1 to 10 mM) below that needed to block % (Storm, 1987). I^a is also sensitive to blockade if exposed to Ca2+-free medium as well as the Ca2+ channel blockers, Mn2+, Cd2+ and Co2+ or intracellular injections of the fast Ca?+ chelator BAPTA but not EGTA (chelates Ca2+ too slow for it to effect Ica) (Lancaster and Nicoll, 1987; Storm, 1987c). The current has been implicated in Na+ spike repolarization, and contributes to the generation of the medium after-hyperpolarization (mAHP) that accompanies spike bursts (Lancaster and Nicoll, 1987; Storm, 1987c; Williamson and Alger, 1990). In contrast to l^a» ^AHP is a smaller voltage-dependent current which is activated by lower (30 to 60 nM) concentrations of Ca2+. is resistant to concentrations of TEA which block l^a (Lancaster and Adams, 1986). Like I^^, l ^ H P is blocked by Ca^^-free media as well as Mn2+, Cd2+, and Co2+. The current is also blocked by intracellular injections of both BAPTA and EGTA (Lancaster and Nicoll, 1987). I ^ p is also modulated by neurotransmitters, being increased by adenosine (Haas and Greene, 1984); and reduced by noradrenaline (via receptors) (Haas and Konnerth, 1983), histamine (via H2 receptors) (Haas and Konnerth, 1983), 5-HT (Andrade and Nicoll. 1987), ACPD (via quisqualate metabotropic receptors) (Charpak et. al.. 1990) and blocked by muscarinic agonists at concentrations 10 times lower than that needed to block Ijyi (Dutar and Nicoll, 1987). Modulation of I ^HP by neurotransmitters may involve phosphatidylinositol turnover (Charpak et. al.. 1990) and activation of protein kinase C (Baraban et. al.. 1985; Malenka et. al.. 1986) as well as activation of a cyclic AMP-dependent protein kinase (Nicoll, 1988). The current, which underlies the slow Ca2+-dependent AHP, accompanies single spikes and spike bursts and increases in amplitude with increasing number of spikes (Lancaster and Adams, 1986). Unlike Ica. IAHP ^O^S not contribute to repolarization of single action potentials (Lancaster and NicoU, 1987). 9.5. Voltage-Dependent Cl '-Current ilçi^y)) Present in hippocampal neurons is a voltage-dependent chloride-current (lci(V)) which activates by h5rperpolarizing steps between -20 and -100 mV (Madison et. al.. 1986). The current is blocked by Cd+ ions and phorbol dibutjnrate (Madison et. al.. 1986). lc\{y) is thought to contribute to the resting dendritic conductance since the current is detected intrasomatically only when K+ channels are blocked with Cs+, TEA and carbachol (Madison et. al.. 1986). 9.6. Miscellaneous Currents Hippocampal pyramidal cells possess a Ca2+-activated Cl"-current (Ici(ca)^  and a Na+-activated K+-current. Ici(Ca) is a voltage insensitive Ca^"^-activated Cl"-current which activates at intracellular Ca2+ concentrations approaching 0.5 |iM (Owen et. al.. 1988). This current probably contributes to the Ca2+-dependent tall currents which develop following chloride loading or suppresion of Ca2+-activated K+-currents in pyramidal neurons (BroAvn and Griffith, 1983). The Na+-activated current is thought to arise from electrogenic extrusion of Na+ following intense synaptic activity (Gustaffson and Wigstrom, 1983). The resulting prolonged after-h3^erpolarization is thought to be generated by a Na+-dependent K+-current (Gustaffson and Wigstrom, 1983). The significance of this current to hippocampal function has yet to be resolved. 9.7. Passive "Leak" Currents In hippocampal neurons, leak currents are present at rest potential when the contribution of currents by voltage-gated and ion-gated channels are minimal. One current is generated by a voltage-insensitive K+ conductance which can be reduced by muscarinic agonists (Madison et. al.. 1987b) through activation of a pertussis toxin résistent GTP binding protein (Brown et. al.. 1988). Another current is mediated by a Cl--dependent conductance (Franciolini and Nonner, 1987). Contributions of both CI' and K+ leak currents stabilize the membrane potential between -60 mV and -70 mV. 10. FIELD POTENTIALS IN THE HIPPOCAMPUS Because both somata and dendrites of pyramidal and granule cells are aligned in a uniform parallel fashion the activation of one or many afferent en passant fibers results in a near s)mchronous activation of a population of cells (Schwartzkroin and Mueller, 1987; Teyler and DiSenna, 1986). Therefore, unlike other areas in the cortex, the hippocampus is capable of generating relatively large field potentials. Synaptic excitation is reflected in field potential recordings by an excitatory postsynaptic potential (EPSP). The population (or field) EPSP is characterized by a large negative wave when recording at the level of the dendrites (active sink) and reverses in polarity when recording at the cell somata (source). When the sjmaptic excitation exceeds the threshold in individual cells for spike generation, the field EPSP is interrupted by a population spike. This is represented in field potential recordings as a sharp negative wave when recorded near the cell body layer and as a sharp positive wave when recording near the dendrites. The amplitude and width of the population spike reflects the number of S5mchronously discharging neurons (Andersen et. al.. 1971b). For a fixed stimulation strength, the relative amplitudes of the field EPSP and the population spike vary depending on the location of the active source with respect to the recording electrode. These discrepencies reflect the length and passive cable properties of the dendrites (Andersen, 1975). Because the afferent input into these regions is primarily excitatory, attempts to measure inhibitory field potentials have been less successful. However, recent experiments using pharmacological agents that antagonize synaptic excitation, have shown that it is possible to record monosynaptic GABA-mediated inhibitory field potentials from hippocampal pyramidal cells fBorroni et. al.. 1991). 11. EXCITATORY POSTSYNAPTIC POTENTIALS (EPSPs) Excitatory postsynaptic potentials have been demonstrated in the three principle cell types of the hippocampus. The granule cell EPSPs arise from activation of perfor£int path fibers which sjniapse on the middle and distal third of their dendritic tree (Blackstad, 1958; Hjorth-Simonsen and Jeune, 1972; McNaughton and Barnes, 1977). Synapses formed by commissural, associational and septal afférents on the proximal portions of the granule cell dendrites also support the generation of EPSPs (Steward et. al.. 1977; Fantie and Goddard, 1982). The CA3 pyramidal cell EPSPs arise from activation of the mossy fibers which synapse on the proximal dendrites of CA3 neurons (Andersen and Lomo, 1966c; Yamamoto, 1972). Commissural, entorhinal and septal excitatory inputs also S3niapse on specific portions of the CA3 dendritic tree and, therefore, when activated also produce EPSPs. The EPSPs of C A l neurons develop from activation of proximal and distal synapses by the Schaffer collaterals, commissural, and septal afférents (Andersen, 1960; Andersen, 1975; Stanley et. al.. 1979). Stimulation of the distal portions of the pyramidal and granule cell dendrites produces EPSPs with slower rise times than those generated at more proximal locations. Langmoen and Andersen (1981), Andersen et. al. (1980a) and Abraham (1982) have investigated a number of factors which may have contributed to the descrepency between EPSP rise times in response to proximal and distal stimulations. They attribute these observations to the electrotonic decay which occurs as the EPSP travels from the distal dendrites to the soma as well as to the different characteristics of the inputs synapsing with these dendritic portions. Considerable evidence supports L-glutamate as being the endogenous neurotransmitter which underlies the EPSP in both pyramidal and granule cells (Fagg, 1985; Fonnum, 1984; Hablitz and Langmoen, 1982; Watkins and Evans, 1981). Synaptic release of glutamate activates two classes of glutamate receptors co-localized on the subs5maptic membrane (Bekkers and Stevens, 1989). They are the NMDA and the non-NMDA receptors. The resulting increase in conductance through NMDA channels mediates the slow EPSP where as the rise in conductance through non-NMDA channels generates the fast EPSP. 11.1. NMDA Receptors and Synaptic Transmission The NMDA receptor is an ionotropic receptor selectively activated by N-methyl-D-aspartate (NMDA), a structural analogue of glutamate (Watkins and Evans, 1981). The ion channel associated Avith the NMDA receptor is permeable to both Na+ and Ca^^ ions and possesses a single channel conductance between 40 and 50 pS with an open time duration of 5 to 10 msec (Ascher and Nowak, 1988; Jahr and Stevens. 1987). The current-voltage (I-V) relationship from neurons exposed to NMDA over a holding potential range of -100 to +30 mV exhibit both positive and negative slope behaviours (Mayer et al.. 1984). This observation is due to the voltage-dependent non-competitive block of the channel by Mg2+ at relatively negative potentials (Mayer et. al.. 1984; Mayer and Westbrook. 1987; Nowak et al.. 1984). Consequently, at resting potentials, the channel exhibits very low conductance to Na"*" and Ca^^ which progressively decreases as the neuron is depolarized and the Mg2+ block is removed. The maximum inward current produce by NMDA occurs around -30 mV and decreases upon further depolarization reversing in direction at approximately 0 mV. 1-V curves constructed from whole-cell recordings of excitatory posts3niaptic currents (EPSCs) from hippocampal slices also exhibit a similar voltage-dependence when the non-NMDA receptors are pharmacologically blocked (CoUingridge et. al.. 1988; Hestrin et. al.. 1990), These observations Ulustrate that S5aiaptic activation of the NMDA receptors mediate at least one component of the EPSP. The NMDA component of the EPSC has rise times between 8 to 20 msec and exponential decay rates lasting from 60 to 150 msec (CoUingridge et. al . . 1988; Forsythe and Westbrook, 1988; Hestrin et. al.. 1990). The slow rise times may indicate that network related factors, Uke polysynaptic pathways, are activated (Mayer and Westbrook, 1987). Recent experiments have demonstrated that spontaneously released quanta from a single central excitatory sjmapse are capable of producing fast followed by slow NMDA receptor-mediated currents (D'Angelo et. al.. 1990). The slow current generated a "hump" that peaked 20 to 25 msec after the start of the fast component. The time for the slow current to peak in these experiments closely resembles the time for the NMDA EPSC to peak. One plausible explanation for such slow rise times is that once the channels mediating the fast component are closed, glutamate dissociates and then rebinds to its receptor in an asynchronous fashion (D'Angelo et. al.. 1990), However, experiments conducted by Lester et, al, (1990) have demonstrated that because of the slow unbinding of glutamate from the NMDA receptor, the NMDA channel activity outlasts glutamate application. Therefore, the slow onset and long duration of the NMDA EPSC may be accounted for by its prolonged activation by glutamate. Because the NMDA EPSC is voltage-dependent and long in duration, it increases with increasing depolarizations and is capable of regenerating itself, much like the Na+-dependent action potential. The receptor may even be tonically activated by ambient levels of endogenous glutamate which would act to increase the excitability of the neuron and, hence, its susceptibility to excitatory synaptic input (Sah et. al.. 1989). Such unique properties are valuble for the expression of activity-dependent processes including, temporal integration, rh3rthmic firing and synaptic plasticity. 11.1.1. Allosteric modulation by glycine. Glycine plays an important role in inhibitory neurotransmission. Glycinergic inhibition has been well characterized in the brainstem and the spinal cord, where its actions are selectively blocked by strychnine. However, experiments conducted by Johnson and Ascher (1987) demonstrated that submicromolar concentrations of glycine were sufficient to significantly potentiate NMDA induced currents recorded from cultures of central neurons. Single channel analysis from membrane patches in the outside-out configuration, revealed that glycine increases the opening frequency but not the open time or the conductance of the channels when exposed to either NMDA or L-glutamate (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988; Maver et. al.. 1989; Reynolds et al.. 1987). Glycine, therefore, may exert is actions by regulating the transition states that are intermediate between binding of NMDA receptor agonists and ion channel gating. Indeed, in some preparations, channel openings are not detected if glycine is absent from the incubation medium (Kleckner and Dingledine, 1988; D'Angelo et al.. 1990), which suggests that glycine may be essential for NMDA receptor activation. Glycine also reduces desensitization of the NMDA current by speeding up the rate constant of recovery from desensitization (Mayer et. al.. 1989). In the presence of glycine, the slow rate of desensitization of the NMDA receptor complex may contribute to the slow decay constants observed for NMDA currents. In support of this view, Forsythe et. al. (1988) have demonstrated that the rate of decay for monosynaptic EPSPs recorded from pairs of hippocampal neurons is significantly increased if glycine is added to the perfusion media. 11.1.2. Modulation by zinc. In hippocampal neurons, zinc induces a noncompetative voltage-independent inhibition of NMDA responses (Westbrook and Mayer, 1987). At the same concentration required to antagonize NMDA responses, responses to kainate or quisqualate are unaffected by Zn2+ (Peters et. al.. 1987). Experiments conducted by Westbrook and Mayer (1987) showed that the fast EPSP and the somatic action potential were not altered by Zn2+ but the slow NMDA EPSP was indicating that the antagonism of the slow EPSP by Zn2+ was selective and did not reflect a failure of synaptic transmission. So far the role of endogenous zinc in modulating NMDA receptor activity remains unclear. However, evidence for its role as an endogenous modulator of synaptic transmission is supported by it being presence in the nerve terminals of mossy fibers (Haug, 1967) where it can be co-released with glutamate (Aniksztejn et. al.. 1987). However, because few NMDA receptors exist at mossy fiber-CA3 pyramidal cell synapses (in stratum lucidum), it is possible that Zn2+ may exert its effects by diffusion to nearby commissural-CA3 P5n:amidal cell synapses where NMDA receptors have been demonstrated to participate in synaptic transmission (Westbrook and Mayer, 1987). 11.1.3. NMDA antagonists. A large number of compounds that possess activity as NMDA antagonists now exist (for reviews see, CoUingridge and Lester, 1989; Watkins and Olverman, 1987; Watkins et. al.. 1990). The role of NMDA receptors in neurotransmission has been elucidated primarily on the bases of the use of 2-£imino-5-phosphonovalerate (APV). APV is a potent competitive antagonist of NMDA receptor-mediated responses (Davies et. al . . 1981; Evans et. al.. 1982). Because of its high degree of specificity and rapid dissociation rate, APV can quickly block NMDA responses and, in turn, be rapidly removed upon washout (a valuble asset for demonstrating the degree of contribution of NMDA receptors to synaptic transmission). Consequently, it is the NMDA antagonist of choice unless penetration into the brain is desired. In in vivo experiments, where accessibility of some drugs to the brain can be limited by the blood brain barrier, psychomimetic compounds comprising the Sigma opioids and dissociative anaesthetics which act as non-competitive IMMDA antagonists are used. Although mechanisms by which some of these agents exert their antagonism is not well understood, compounds like PCP, ketamine and MK-801 have been reported to block NMDA receptor-mediated responses by entering the open channel in a highly voltage- and use-dependent manner (Hicks and Guedes, 1981; MacDonald et. al.. 1987). 11.1.4. Glycine antagonists. Recently, compounds including, 7-chloro-kyneurenic acid and HA-966 (Donald et. al.. 1988; Kemp et. al.. 1988), have been developed which act to specifically antagonize the allosteric glycine receptor on the NMDA receptor-channel complex. These compounds have been primarily used to demonstrate that glycine fully occupies the allosteric site in physiological preparations of brain slices (Fletcher and Lodge, 1988; Kemp et aL, 1988) (but see, Minota et. al.. 1989). 11.2. Non-NMDA Receptors and Synaptic Transmission Four classes of non-NMDA receptors have so far been identified and are named after the agonists (all structural analogues of glutamate) which selectively activate them. The kainate and a-amino-3-hydroxy-5-methyl-4-isoazolepropionate (AMPA) receptors are associated with channels that possess a mixed cationic conductance; synaptic activation of these receptors underlies the fast EPSP. The ACPD receptor is a metabotropic receptor linked to inostitol triphosphate (IP) turnover and the L-AP4 receptor is a recently discovered non-NMDA receptor thought to be located presynaptically (for review see, Collingridge and Lester, 1989; Watkins e t al.. 1990). 11.2.1. The non-NMDA component of the EPSP. Although NMDA receptors play a critical role in synaptic function, their voltage-dependent block by magnesium limits their participation in mediating the EPSP resulting from a unitary synaptic activation. Instead, the non-NMDA kainate and AMPA ionotropic receptors are responsible for the voltage-independent portion of the EPSP which underlies the fast synaptic response in many hippocampal pathways. The ion channels linked to the kainate and AMPA receptors are equipermeant to Na+ and Cs+ ions (Ascher and Nowak, 1988; Jahr and Stevens, 1987; Vyklicky et. al.. 1988) and are therefore believed to be relatively non-selective between Na+ and K+ (or Cs+). Unlike NMDA channels, the kainate and AMPA channels exhibit poor permeability to Ca^"^ and are not blocked by Mg2+ (Jahr and Stevens, 1987; Mayer and Westbrook, 1987). Consequently the I-V relationships from neurons exposed to either kainate or AMPA exhibit relatively linear negative slope behaviours when examined over membrane potentials between -90 mV and +30 mV (Mayer and Westbrook, 1984). The reversal potential for both kainate and quisqualate induced currents occurs around 0 mV (Mayer and Westbrook, 1984), Such properties have also been demonstrated for the non-NMDA EPSC (Hestrin et. al.. 1990). Kainate and AMPA channels exhibit complex interactions which may be explained if both agonists act at the same receptor-channel complex. Indeed, kainate-induce depolarizations often require concentrations of kainate that are more comparable to kainate's affinity for AMPA receptors (Mayer and Westbrook, 1987). Channels activated by either kainate or quisqualate (an agonist at AMPA receptors) produce conductances between 5 to 15 pS, lasting 0.5 to 2 msec, though kainate preferentially generates conductances of 5 pS and quisqualate of 10 pS and 15 pS (Ascher and Nowak, 1984; Jahr and Stevens, 1987). These events have also been observed in the presence of L-glutamate (Jahr and Stevens, 1987) In addition to the 10 pS and 15 pS conductance, quisqualate also activates ion channels which exhibit a 35 pS conductance and mean open times between 3 to 8 msec (Tang et. al.. 1989). This high conductance channel has several properties which suggest it may underlie the the fast EPSP. Kainate has also been reported to activate a high conductance channel (30 to 50 pS) (Cull-Candy and Usowicz. 1987; Jahr and Stevens, 1987). however, this may reflect a nonspecific action of kainate at the NMDA receptor channel complex. The non-NMDA component of the EPSC exhibits rise times of 1 to 3 msec and an exponential decay rate 7 to 8 msec (Hestrin et. al.. 1990). Because of their similiar time courses, the time constant of decay of the non-NMDA component of the EPSC may actually reflect the mean open-time of AMPA channels. Since the concentration of glutamate in the synapse remains high enough to persistently activate the NMDA receptor-channel complex, desensitization at AMPA receptors may account for the fast decay time of the non-NMDA EPSC. Indeed, desensitization of the AMPA receptor is rapid to exogenous application of glutamate (Kishkin et. al.. 1986), a property which appears to be determine by the kinetics of the receptor channel complex (Tang et. al.. 1991). Alterations in the desensitization process would, therefore, subserve as a mechanism by which fast excitatory synaptic transmission could be modified. 11.2.2. The non-NMDA ACPD receptor. Quisqualate activates another non-NMDA receptor, however, rather than being associated with an ionotropic receptor, it is linked to a metabotropic one. Since trans-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD) is the most potent agonist for this receptor, it is termed the ACPD receptor. In hippocampal neurons, activation of the ACPD receptor by L-glutamate, ibotenate and quisqualate but not NMDA, AMPA, or kainate produces phosphoinositide (PI) turn-over (Baudry et. al.. 1986; Nicoletti et. al.. 1986; Palmer et. al.. 1988; Schoepp and Johnson. 1988) which involves a pertussis-toxin sensitive G-protein (Masu et. al.. 1991; Nicoletti et. al.. 1988). The quisqualate induced PI formation is blocked by L-AP3 (Irving et. al.. 1990; Schoepp et. al.. 1990), L-AP4 (Nicoletti et. al.. 1986; Schoepp and Johnson, 1988) and NMDA receptor activation (Palmer et. al.. 1988). Such negative regulation by NMDA receptor activity may provide a potential mechanism for regulation of synaptic plasticity as well as other activity-dependent processes. 11.2.3. The non-NMDA L-AP4 receptor. Contrary to its blocking action at ACPD receptors, L-AP4 activates a fourth class of non-NMDA receptor. The L-AP4 receptor produces physiological responses consistent with a presynaptic locus. Hence, activation of the receptor is associated with a decrease in the EPSP due to a reduction in the frequency but not the size of mini EPSPs (Cotman et. al.. 1986; Harris and Cotman, 1985). The presynatic inhibiton of excitatory synaptic responses by L-AP4 receptors is also mimicked by L-glutamate and they are, therefore, thought to promote autoinhibition at glutamatergic synapses (Forsythe and Clements, 1988). Thus, the L-AP4 receptors may regulate neurotransmission when conditions of hyperexcitability exist. 11.2.4. Non-NMDA receptor antagonists. In the past, only the broad spectrum antagonists y-D-glutamylglycine (DGG) and kyneurenic acid were available to study non-NMDA receptor-mediated neurotransmission at glutamatergic S5niapses. However, being broad spectrum, these antagonists not only blocked non-NMDA receptors but NMDA receptors as well. TTius, their use in delineating the non-NMDA from the NMDA components of synaptic transmission has produced little success. Recently, Honore et. al. (1988) reported that the quinoxaline derivatives 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) selectively blocked quisqualate and kainate induced responses at low ()j.M) concentrations. Schild analysis revealed that antagonism of both kaiinate and quisqualate responses was competative (Blake et. al.. 1989; Drejer and Honore, 1988). At higher concentrations, both CNQX and DNQX have been reported to block NMDA responses by acting at the strychnine-insensitive glycine receptor (Birch et. al . . 1988) . In the C A l region of the hippocampus, the use of CNQX and DNQX to block non-NMDA component of synaptic transmission, has demonstrated that the non-NMDA receptors but not the NMDA receptors predominantly mediate EPSPs during low frequency stimulation under "normal" conditions (conditions where Mg2+ is in the perfusate) (Andreasen et. al.. 1988; Blake et. al.. 1988; Collingridge et. al.. 1988; Neuman et. al . . 1988). The finding that excitatory synaptic transmission can be completely blocked by CNQX when elicited at a low intensity stimulation suggests that synaptic activation of the NMDA receptor need not be required to mediate the EPSP (Collingridge and Lester, 1989) . The quinoxaline derivatives have also been used to demonstrate the NMDA component of sjmaptic transmission. Since Mg2+ is a potent cintagonist of channels coupled to the NMDA receptor, under conditions where Mg2+ i n the perfusate is omitted, NMDA EPSPs can be evoked by low frequency stimulation when non-NMDA EPSPs are blocked (Andreasen et. al.. 1988; Collingridge et aL, 1988; Hestrin et al.. 1990; Lambert and Jones, 1990). The quinoxaline derivatives have proven to be valuble pharmacological tools in studying excitatory neurotransmission. However, because they exert poor selectivity towards antagonizing either kainate or AMPA receptor-mediated responses it is difficult to determine the relative contribution of each receptor to the non-NMDA EPSP. Undoubtedly, antagonists specific for the kainate and the AMPA ionotropic receptors will be developed in the near future and questions pertaining to the relative contributions of either receptor to the fast EPSP wil l be answered. 12. INHIBITORY POSTSYNAPTIC POTENTIALS flPSPs) In the hippocampus, y-aminobutyric acid (GABA) is the major inhibitory neurotransmitter (Curtis et. ai.. 1970; Curtis et. al.. 1971). Synaptic release of GABA activates at least three types of GABA receptors. They include two subtypes of GABA^ receptor and a subtype of GABAg receptor. While the former receptors are associated with channels that conduct chloride ions, the latter receptor is coupled to an ionophore that conducts potassium ions. Orthodromic stimulation of the stratum radiatum elicits a triphasic synaptic potential characterized by an EPSP-fast IPSP-slow IPSP sequence. In some preparations a depolarizing IPSP is observed between the fast and slow IPSPs. Based on pharmacological, biochemical and electrophysiological techniques, the fast and depolarizing IPSPs have been shown to be mediated by actvation of GABA^ receptors while the slow IPSP by activation of GABAg receptors. Though IPSPs are displayed in both granule and pyramidal cells, much of the work done to characterize the GABA mediated synaptic potentials has been performed on the latter. Therefore, unless otherwise stated, the following chapter will discuss the properties of different IPSPs obtained from pjn-amidal neurons. 12.1. Recurrent and Feed-Forward Inhibition Hippocampal IPSPs can be evoked by either orthodromic or antidromic stimulation. Antidromic stimulation of alvear fibers produces prominant IPSPs in the pyramidsil cells through activation of a recurrent inhibitory circuit (Andersen et. al.. 1964a; KEindel et. al.. 1961). In contrast, orthodromic stimulation of the stratum radiatum elicits IPSPs through activation of a feed-forward inhibitory system (Alger and NicoU, 1982a). The former inhibitory system has been described to be primarily mediated through activation of basket cells (Andersen et. al., 1963; Andersen et. al.. 1964b; Knowles and Schwartzkroin, 1981) which form a dense axo-somatic plexus around the somata of p5n:amidal cells (Lorente de Nô, 1934), The latter inhibitory system activates Interneurons which synapse with the somata and dendrites of pyramidal neurons (Alger and Nicoll, 1982a; Fujita, 1979), The interneurons supporting this type of inhibition may be those residing in the strata along the oriens/alveus boarder or in stratum lacunosum-moleculare (see chapter 8.2). While antidromic stimulations of the alveus primarily elicit fast IPSPs (Alger and Nicoll, 1982a; Andersen et. al.. 1964a; Dingledine and Langmoen, 1980), orthodromic stimulations of the stratum radiatum induce both fast and slow IPSPs (Alger and Nicoll, 1982a; Fujita, 1979). This pattern suggests that fast IPSPs are evoked near the pyramidal cell soma and dendrites, where as, slow IPSPs are generated at the dendrites. In support of this view, autoradiographic studies have demonstrated that the distribution of GABA^ binding sites in C A l to CA3 are moderate where as GABAg binding sites in the same subfields are primairily observed in strata oriens and radiatum but not i n stratum p5n'amidale (Bowerv et. al.. 1987). 12.2. Properties of the GABA^ Receptor-Mediated Fast IPSP The fast (or early) IPSP can be evoked by orthodromic and antidromic stimulations of the stratum radiatum and alveus. respectively. The properties of the antidromic IPSP are similar to the orthodromic IPSP. The antidromic IPSP has a threshold of activation lower than the antidromic spike (Andersen et. al.. 1964b), has a latency to peak of 15 to 25 msec and last between 50 to 150 msec (Dingledine and Langmoen, 1980). It is characterized by a reversal potential of -65 to -75 mV (Alger and Nicoll, 1982b; Dingledine and Langmoen, 1980), which is associated with a transmembrane chloride flux since substitution of part of the extracellular CI" concentration with the impermeant anion isethionate produces depolarizing shifts in its reversal potential (Knowles et. al.. 1984) and injecting the neuron with CI' ions reverses the IPSP from a hyperpolarizing potential to a depolarizing one (Alger and Nicoll. 1981). The fast IPSP is blocked by the GABA^ receptor antagonists bicuculline (Curtis e t al.. 1971) and picrotoxin (Olsen, 1982). Some investigators have also noted the existence of a slow IPSP following the antidromic fast IPSP when high stimulation strengths are used (Alger, 1984; Proctor et. al.. 1986). However, this is thought to reflect a nonspecific activation of orthodromic fibers i n stratum oriens since the slow IPSP is not observed when a lesion is made along the width of the stratum oriens between the recording and stimulating electrodes. (Dingledine and Langmoen, 1980; Alger and Nicoll, 1982a; Newberry and Nicoll, 1984). The orthodromic fast IPSP displays similar properties to those observed for the antidromic IPSP but, because it occurs in sequence with the EPSP and the slow IPSP accurate measurements are more difficult to obtain. Nevertheless, the orthodromic IPSP has been reported to have a latency of onset of 20 ms and a chloride dependent reversal potential of -75 mV (Knowles et. al.. 1984). Like the antidromic IPSP, the orthodromic fast IPSP is blocked by bicuculline and picrotoxin (Knowles e t al,. 1984; Nicoll and Alger, 1981; Newberry and Nicoll, 1985) and is therefore a GABA^ receptor-mediated potential. Recently, Davies et. al. (1990) demonstrated that the orthodromic fast IPSP could be pharmacologically separated from the EPSP and the slow IPSP. In their experiments, the fast IPSP exhibited a latency to peak of 17 msec, a duration of 225 msec and a reversal potential of -75 mV. Results from patch clamp studies performed on hippocampal pyramidal cells have demonstrated that the GABA mediated CI" channel exhibits multiple conductance states in the presence of the agonist with the principle conductance being between 16 to 20 pS (Edwards et. al.. 1989). Recently, Ropert et. al. (1990) reported that the conductance of a unitary hippocampal IPSC ranges from 237 to 321 pS. Therefore, based on their results, a single quantum of GABA would be sufficient to activate 13 to 20 channels, far lower than prevous predictions (Collingridge et. al.. 1984). In the same experiment, the time constant of decay for the IPSC was 21 to 28 msec. Since this approaches the observed mean open-time (30 msec) of the GABA mediated chloride chginnel it appears that for a unitary IPSC, GABA molecules bind only once to the receptor. Differences exist between unitary IPSCs and antidromically evoked IPSPs. For example, while unitary IPSCs last 21 to 28 msec, IPSPs elicited antidromically last 50 to 150 msec (Dingledine and Langmoen, 1980). Furthermore, while unitary IPSCs peak around 3 msec antidromic IPSPs peak between 20 msec to 30 msec. Therefore, it is unlikely that the antidromic IPSP arises from an "all or none" release of GABA but rather from a sequential release of quanta. Indeed, the h)^erpolarization in a pyramidal cell exhibits a faint ripple that is synchronized with the repetitive firing of a S5niaptically paired basket cell (Knowles and Schwartzkroin, 1981; Miles and Wong, 1984). 12.3. Properties of the GABAg Receptor-Mediated Slow IPSP The orthodromically evoked slow IPSP (also termed late IPSP or late hyperpolarizing potential) has a latency to peak of 200 to 300 msec and is associated with an increase in membrane conductance (Alger, 1984; Hablitz and Thalmann, 1987; Knowles et. al.. 1984). The reversal potential for the slow IPSP is more difficult to obtain than for the fast IPSP since K+-dependent inward rectification (see chapter 9.2.2) usually counteracts membrane polarities beyond -90 mV. Hablitz and Thalmann (1987) reported that a clear reversal potential could be attained for the slow IPSP if the inward rectification was blocked by externally applied Cs"^ ions. However, their results should be taken with caution since others have reported that the slow IPSP itself is blocked by similar concentrations of external Cs"*" (Alger, 1984; Gahwiler and Brown, 1985). Nevertheless, the reversal potential of the slow IPSP obtained either by extrapolation or directly from neurons that exhibit little inward rectification ranges from -80 to -95 mV (Alger, 1984; Davies et. al.. 1990; Hablitz and Thalmann, 1987; Knowles et. al.. 1984; Newberry and Nicoll, 1985; Nicoll and Alger, 1981). The observation that the reversal potential of the slow IPSP shifts to values predicted by the Nernst equation when the extracellular potassium concentration is changed supports the view that it is a K+-dependent potential (Alger, 1984; Hablitz and Thalmann, 1987; Knowles et. al.. 1984) . Similar slow hyperpolarizing potentials are observed in p5nramidal neurons after a brief train of action potentials which are dependent upon intracellular Ca2+ concentrations (Alger, 1984, Knowles et. al.. 1984). However, the slow IPSP is not a Ca2+-dependent potential since injection with the Ca2+ chelator EGTA fails to alter it (Knowles et. al.. 1984). Furthermore, the slow IPSP can be elicited without an EPSP or action potentials (Knowles et al.. 1984). GABA^ receptors appear not to participate in generating the slow IPSP since neither bicuculline nor picrotoxin block the S5niaptically evoked potential or baclofen-induced hjrperpolarization (Newberry and Nicoll, 1984; Newberry and Nicoll, 1985). The slow IPSP shares many physiological and pharmacological similarities to the slow K"^-dependent hj^erpolarization produced by the GABAg receptor agonist baclofen (Inoue et al.. 1985; Newberry and Nicoll, 1985) . This led to the hypothesis that the slow IPSP was mediated by activation of GABAg receptors and was later confirmed by the development of specific centrally acting G A B A Q antagonists. Phaclofen (Kerr et. al.. 1987) was one such antagonist which blocked the slow IPSP (Dutar and Nicoll, 1988) and provided definitive pharmacological evidence for a GABAg receptor mediated IPSP in the hippocampus. Other more potent antagonists have since been synthesized; these include saclofen (Kerr et. al.. 1989), 2-hydroxy-saclofen (Kerr et. al.. 1988b; Lambert et. al.. 1989) and CGP 35348 (Olpe et al.. 1990). There is groAving evidence which suggesting that the GABAg receptor is linked to a second messenger system. The observation that Pertussis toxin blocks the slow IPSP and baclofen-induced slow hyperpolarization (Dutar and Nicoll, 1988) and that the non-hydrolysable form of guanosine 5'-triphosphate (GTPyS) maximally activates a baclofen-induced K+-dependent hyperpolarization (Andrade et. al.. 1986) suggests the involvement of guanine nucleotide binding proteins (G-proteins) in mediating GABAg responses. In support of these observations, single channel saclofen-sensitive potassium currents recorded from hippocampal neurons have been reported to appear after a delay of 30 sec when exposed to either GABA or baclofen. However, when the cells were pre-equilibriated with Pertussis toxin or when the membrane patches were excised, these events did not occur (Gage, 1992; Prekumar et. al.. 1990). In addition, since application of the phorbol ester, phorbol-12,13-dibutyrate also blocks the hj^erpolarizing response to baclofen, protein kinase C (PKC) may also regulate GABAg receptor mediated responses. Whether activation of G-proteins and PKC converge to a common biochemical or whether they operate separately to modulate the receptor remains to be determined. The functional role of the slow IPSP is at present uncertain, however, it has been implicated in a variety of physiological and pathophysiological states. For example, because the potential produces a decrease in membrane conductance, it may subserve to shunt excitatory signals associated with dendritically localize synapses (Hablitz and Thalmann, 1987). Alternatively, the slow IPSP may be important in regulating seizure activity since the h5^erpolarization occurs when epliliptiform burst potentials cire maximal (Stelzer et. al.. 1987). The observation that blockade of the slow IPSP by zinc i n the immature rat hippocampus induces spontaneous giant depolarizing potentials (GDPs) which underlie seizure activity in immature animals supports this view (Xie and Smart, 1991), Since the slow IPSP appears to regulate the activation of the NMDA receptor it may also play an important role i n regulating synaptic plasticity (Morrisett et, al,. 1991) or other events which require the participation of NMDA receptors. 12.3.1, Presynaptic GABAg receptors. There is growing evidence that GABAg receptors function in reducing s)niaptic potentials through a mechanism independent of its postsynaptic effects. This type of inhibition is thought to involve activation of presynaptically located GABAg receptors (Bowery et. al.. 1981). Initial experiments suggested that only EPSPs were blocked by the pres)niaptic actions of baclofen (Ault and Nadler, 1982; Lanthorn and Cotman, 1981; Olpe et. al.. 1982), but later experiments demonstrated IPSPs were affected as well (Misgeld et. al.. 1989; Peet and McLennan, 1986). Recent experiments have further supported the existence of presynaptic GABAg receptors (Davies et. al.. 1990; Dutar and Nicoll, 1988; Thompson and Gahwiler, 1989). While activation of these receptors involves a reduction of neurotransmitter release (Anderson and Mitchell, 1985; Bowery e t al. 1980; Potashner, 1979), the mechanism(s) that mediate this effect is at present unknown. However, if these receptors operate i n an analogous fashion to the postsjmaptic GABAg receptors then an increase in K"*" conductance leading to a hyperpolarization in or near the pres5niaptic terminal could act to reduce the excitability of the terminal and, hence, reduce the release of neurotransmitter provided that the action potentials invading the presynaptic terminal become subthreshold for transmitter release, (Gahwiler and Brown, 1985). Indirect evidence for a presynaptic GABAg receptor-mediated K+ conductance is supported by the observation that the presynaptic actions of baclofen are blocked by barium (Misgeld et. al.. 1989), an ion which has been demonstrated to block the postsynaptic GABAg receptor-mediated K"*" conductance (Gahwiler and Brown, 1985; Newberry and Nicoll, 1985). However, Lambert et. aL (1991) did not observe an antagonism of the baclofen-induced pres3niaptic GABAg receptor-mediated responses to barium. Therefore, the existence of a pres5niaptic GABAg receptor-mediated K+-current remains to be determined. Several indirect lines of evidence suggest that pre- and postsynaptic GABAg receptors are not identical. For example, desensitization occurs more rapidly at the postsynaptic but not the presynaptic GABAg receptors (Thompson and Gahwiler, 1989). Furthermore, presynaptic GABAg responses are more resistant than postS)maptic GABAg responses to GABAg antagonists (Dutar and Nicoll, 1988; Davies et. aL. 1990; Davies et. al.. 1991; Gage, 1992). The observation that the postsynaptic effects mediated by GABAg receptors are blocked by Pertussis toxin while the presynaptic effects are not suggests these receptor systems are linked to different second messenger systems (Dutar and Nicoll, 1988). 12.4. Properties of the GABA^ Receptor-Mediated Depolarizing IPSP Following orthodromic stimulation of the stratum radiatum, a depolarzing potential can sometimes be observed between the fast IPSP-slow IPSP (Perreault and Avoli, 1988). Application of either pentobarbitone (Alger and Nicoll, 1982a) or 4-AP (Avoli and Perreault, 1987) potentiates the depolarizing potential. This pharmacologically induced potentiation is characterized by a large and long-lasting depolarization (LLD) that often masks the slow IPSP. The observation that the orthodromically evoked depolarizing potential persists when TTX is iontophoresed onto the soma but not when iontophoresed onto the dendrites of pyramidal neurons suggests a dendritic site of origin (Alger and Nicoll, 1982a). This is further supported by current-source density (CSD) analysis of field potentials which reveal that during maximal activation of the potential the active sink occurs in the middle portion of the stratum radiatum (Perreault and Avoli, 1989). The depolarizing IPSP is mediated by activation of GABA^ receptors since it is mimicked by dendritic applications of GABA (Alger and Nicoll, 1982b; Andersen et. al.. 1980b; Thalmann et. al.. 1981) and is abolished by either bicuculline or picrotoxin (Alger and Nicoll, 1982a). It appears that this potential is generated by pres)maptic release of GABA since spontaneous GABA receptor-mediated depolarizations have been observed (Alger and Nicoll, 1980) and depolarizing responses to iontophoretically applied GABA are not significantly altered when 4-AP is applied (Perreault and Avoli, 1991). Though not conclusive, the depolarizing response to GABA is thought to be mediated by activation of extrasynaptic GABA receptors (Alger and Nicoll, 1981; Alger and Nicoll, 1982b). The reversal potential of the depolarizing IPSP is between -55 and -65 mV. It is associated with an increase in membrane conductance during its peak activation. The observation that decreasing the extracellular 01" concentration (Perreault and Avoli, 1987) increases the amplitude of the depolarization suggest that the depolarizing potential is mediated by an outward directed CI" conductance. In support of this, application of the CI" pump blocker furosemide abolishes the spontaneous and evoked depolarizing potentials in pyramidal cells while having little effect on the increased conductance (Perreault and Avoli, 1987), If different CI" concentration gradients exist in the soma and the dendrites of pyramidal cells then this would explain the opposing effects of GABA when applied to the dendrites and soma of the pyramidal cells. The physiological significance for a GABA mediated depolarizing potential i n pyramidal cells is unclear. These may produce the GDPs observed when the slow IPSP is blocked by Zn2+ (Xie and Smart, 1991). Recently, Michelson and Wong (1991) reported that GABA^ mediated depolarizing potentials are responsible for the recruitment and synchronus activation of GABAergic interneurons which generate high amplitude IPSPs in neighboring granule and pyramidal cells. 13.0. LONG-TERM POTENTIATION IN THE HIPPOCAMPUS Long-term potentiation (LTP) of S3maptic transmission was first demonstrated by Lomo in 1966 and later analyzed by Bliss and Lomo; Bliss and Gardner-Medwin in 1973. LTP has since been a topic of great interest to many neurobiologists because it displays use-dependent properties that resemble certain physiological processes such as learning and memory. No where else in the mammalian central nervous system has LTP been as intensely studied as in the hippocampus, the presumed locus for information storage. Consequently, much progress has been made on characterizing the nature of hippocampal LTP. However, despite these advancements, the mechanism(s) underlying LTP have yet to be elucidated. The following chapter describes some of the major discoveries made in the LTP field which have contributed to our present understanding of the phenomenon. 13.1. Properties of LTP 13,1.1. Population responses. Tlie experiments conducted by Bliss and collègues (1973) examined the long-lasting enhancement of synaptic transmission at the perforant path to dentate synapse resulting from repetitive stimulations of the perforant path. These in vivo experiments were the first detailed accounts of what is now known as long-term potentiation (LTP). They demonstrated that conditioning trains 10 to 20 Hz lasting 10 to 15 seconds or 100 Hz lasting 3 to 4 seconds potentiated the population spike and population EPSP 50 to 100 percent of the control pre-tetanic responses. While the latency of the population spike decreased by 25 percent. The duration of the potentiations lasted 3 to 12 hours in their anaethestized preparations while in their unanaesthetized preparations lasted up to 16 weeks. In some cases, following the conditioning tetanus, the amplitude of the population spike exhibited potentiations that could not be explained by an increase in the field EPSP. This phenomenon was later examined by Andersen et. al. (1980) who demonstrated that even when the amplitude of the field EPSP was kept constant, potentiations of the population spike were still observed. This property of LTP was termed, EPSP-spike (E-S) potentiation. Long-term potentiation also induces changes in the slope of the field EPSP measured in the dendrites. Following a high frequency tetanic stimulation of the hippocampal afférents, the slope of the field EPSP is increased (Alger and Teyler, 1976). This has been described as a more efficient method for measuring the degree of synaptic potentiation (see chapter 15.9). Hence, based on these results the common features of LTP are a long-lasting increase in the amplitudes of the population spike and population EPSP, a prolonged decrease in population spike latency, the production of E-S potentiation and an increase in the slope of the field EPSP. 13.1.2. Single neurons. In C A l neurons, LTP induced by tetanic stimulations of the Schaffer coUateral/commissural fibers is characterized by an increase in the height and slope of the intraceUular EPSP (Andersen et. al.. 1977; Schwartzkroin, 1975) with no detectable changes in membrane potential, input resistance or excitability (Andersen et. al., 1977). Following LTP, synaptic activation induces an increased probability for neurons to discharge action potentials (Andersen, 1980c; Schwartzkroin, 1975), a property which supports the E-S potentiation observed in field potential recordings. Voltage-clamp experiments performed on pyramidal cells associated with the mossy fiber-CA3 synapse and the Schaffer coUateral/commisural-CAl synapse also exhibit some of these properties and further demonstrated that LTP of the EPSP is accompanied by an increase in synaptic conductance that is not associated with a shift in the EPSP reversal potential (Barrionuevo et. al.. 1986). 13.2. Projections in the Hippocampus Supporting LTP The initial finding of Bliss and co-workers demonstrated the medial perforant pathway was capable of supporting LTP. Since this observation, LTP has been described in other afferent pathways in the hippocampus including the lateral perforant pathway (McNaughton et. al.. 1978). mossy fiber projection to CA3 (Alger and Teyler. 1976). Schaffer collateral projection to C A l (Schwartzkroin, 1975) commissural projection to C A l (Buzaki. 1980) as well as septal projections to C A l and dentate gyrus (McNaughton and Miller, 1984; Racine et. al.. 1983). While the LTP displayed at these synapses is mediated by excitatory S5nnaptic transmission, reports suggest that LTP also occurs at feed-forward inhibitory synapses in the dentate gyrus (Kairiss et. al.. 1987) and C A l (Buzaki and Eidelberg, 1982). 13.3. Homo- and Heterosvnaptic LTP During LTP only those inputs that have previously been tetanized display LTP (Andersen et. al.. 1977; Bliss et. al.. 1973; Lynch et. al.. 1977; McNaughton and Barnes, 1977; Schwartzkroin and Wester, 1975). This input specificity describes the "homos5niaptic" nature of LTP. Homos5niaptic LTP has been observed in C A l (Andersen et. al.. 1979; Lynch et. al.. 1977; Schwartzkroin and Wester, 1975) and dentate gyrus (Bliss et. al.. 1973; McNaugton and Barnes, 1977). A n exception to this generalzed observation occurs in CA3 where tetanization of mossy fiber input not only produces homosynaptic LTP at the mossy fiber-CA3 pyramidal cell synapse but also LTP of the non-tetanized input converging on the same population of cells (Yamamoto and Chujo, 1978; Misgeld et. al.. 1979). That LTP can be induced in an untetanized non-overlapping pathway characterizes the "heteros5niaptic" nature of LTP. While the input specificity of homos3niaptic LTP presumably arises from activation of postsynaptic NMDA receptors associated with the tetanized pathway, heterosynaptic LTP (at least for mossy flber-CA3 pyramidal cell synapses) appears to involve either an enhancment of synaptic interactions or electrotonic coupling among individual CA3 neurons (Higashima and Yamamoto, 1985). 13.4. Cooperativitv Only when a sufficient number of afferent fibers are co-activated can LTP be induced. This threshold intensity for LTP describes the "cooperative" nature of LTP (McNaughton et. al.. 1978). Cooperativity has been demonstrated in the major fields of the hippocampus which include the dentate gyrus (McNaughton et. al.. 1978), CA3 (Yamamoto and Sawada, 1981) and C A l (Lee, 1983). The threshold for LTP induction has been reported to require stimulation intensities that elicit a population spike implying a necessity for sufficient postsynaptic depolarization (McNaughton et. al.. 1978). However, LTP has been reported to occur at stimulation intensities that are subthreshold for producing a population spike (Barrionuevo and Brovm, 1983). Furthermore, the observation that intracellular injection of QX-222 (to block Na+-dependent action potentials) does not block tetanus-induced LTP suggests that the depolarization rather than Na+-dependent action potentials are important for LTP induction (Kelso et. al.. 1986). 13.5. Associativitv When separate weak and strong synaptic inputs converge on the same population of cells, tetanic stimulation of both the weak and strong inputs not only induces LTP in the strong input but also in the weak input, which, when tetanized alone does not display LTP (Barrionuevo and Brown, 1983; Levy and Steward, 1979; McNaughton et. al.. 1978). This property describes the "associative" nature of LTP. Associative LTP can occur when the separate inputs are located at different positions along the dendritic tree, but occurs maximally when they synapse on the same region of the dendrite (Moore and Levy, 1986; White et. al.. 1986). A n important aspect of associative LTP is that it is not observed when stimulation of the inputs is separated by at interstimulus intervals greater than 200 msec (Levy and Steward, 1983). This temporal restraint appears to coincide with the duration of the NMDA EPSP (see chapter 11.1). The associative property of LTP induction also emphasizes the importance of interactions between pre- and postsynaptic elements, where strong postsynaptic depolarizations paired with low frequency afferent activation induces LTP once a critical number of pairings is performed (Kelso et. al.. 1986; Sastry et. al.. 1986; Wigstrom et. al.. 1986). The observation that LTP is not induced if afferent activation is paired when the cell is hyperpolarized (Mallnow and Miller, 1986) demonstrates the voltage-dependence of associativity and further supports a role for NMDA receptors i n associative LTP. 13.6. Requirement for Calcium The observations that hippocampal slices exposed to a Ca2+-free perfusate were incapable of supporting S3niaptic transmission and, not suprisingly, LTP suggest an essentlEil role for Ca2+ in LTP (Dundwiddle et. al.. 1978; Wigstrom et. al.. 1979). Even when the Ca^+ concentrations were reduced so synaptic transmission was not blocked, the probability of LTP induction was greatly reduced (Dundwiddle and L5nich, 1979). The finding by Turner et. al. (1982) that hippocampal slices briefly exposed to elevated Ca2+ (4 mM) exhibit LTP in the C A l region demonstrated the importance of Ca2+ i n the induction of LTP. Consistent with this finding, intracellular injections of the Ca2+ chelators EGTA (Lynch et. al.. 1983) and BAPTA (Morishita and Sastry, 1991) block the induction of LTP in C A l neurons. A similar Ca2+-dependent form of LTP has been reported to occur at the mossy fiber-CA3 pyramidal cell synapse CWilliams and Johnston. 1989; but see Zalutsky and Nicoll, 1990). Whether or not calcium-induced LTP or tetanus induced LTP are equivalent is uncertain. However, the observation that calcium-induced LTP could not be further potentiated by tetanic stimulations of the afferent input suggested that both forms of LTP share similar inductive mechanisms (Rejmiann et. al.. 1986). 13.6.1. Source of calcium entry. During the induction of LTP, the major conduit for calcium is the ionophore coupled to the NMDA receptor. But what about S3mapses that do not display NMDA receptor-dependent LTP? Do they require an posts)maptic increase intracellular Ca2+? The apparent answer to this question is yes! In fact, NMDA receptor independent LTP has been described at the mossy fiber-CA3 p5n-amidal cell S5niapse (Kauer and Nicoll, 1986; Williams and Johnston, 1989) and more recently at the Schaffer coUateral/commissural-CAl pyramidal cell S3niapse (Grover and Teyler, 1990; Regher and Tank, 1990). The latter being blocked by antagonists of voltage-dependent Ca2+ channels and the former by intracellular injections of BAPTA or Quin-2. However, whether or not mossy fiber-CA3 pyramidal cell synapses are entirely dependent upon a rise in postsynaptic Ca2+ concentration remains controversial since recent experiments conducted by Zalutsky and Nicoll (1990) demonstrate that LTP at mossy fiber-CA3 pyramidal cell S5niapses is a presynaptic phenomenon independent of elevations in postsynapic Ca2+ concentrations. 13.7. Induction and Maintenence of LTP LTP can be separated into two temporally distinct events, 1) those events that occur during and immediately following a high frequency train which induce a brief décrémentai increase in synaptic efficacy and 2) those events that transpire following the tetanus producing a stable non-decremental increase in sjmaptic efficacy. These events constitute the induction and maintenance phases of LTP, respectively. 13.7.1. Induction of LTP. The induction of LTP begins with a tetanic stimulation delivered to the the afferent input. Immediately, the effected S5niapses display a post-tetanic potentiation (FTP) characterized by a brief (time course, 3 to 5 min) increase in S5niaptic efficacy (McNaughton et. al.. 1978). At this point, depending on the stimulating parameters constituting the train, one of three events may occur, 1) the FTP may fail to develop into LTP, 2) The PTP may develop into a short-term potentiation (STP) (time course, 10 to 15 min), 3) the PTP may convert to LTP, At the Schaffer coUateral/commissural-CAl pyramidal cell sjmapse, one factor which determines the expression of these events is the degree of NMDA receptor activation and, hence, the magnitude of calcium entry into the neuron (Malenka, 1991), 13.7.1.1. Modulation of the induction of LTP bv GABA receptor-mediated neurotransmission. It is widely accepted that NMDA receptors play a paramount role in the induction of LTP (CoUingridge et, al.. 1983; CoUingridge and BUss, 1987; Wigstrom and Gustaffson, 1988). Because of its voltage-dependence, however, the degree of NMDA receptor activation depends on factors that regulate membrane depolarization. Of particular interest is the influence of inhibitory synaptic transmission on this process. For example, application of GABA to the perfusing media blocks the induction of LTP (Scharfman and Sarvey, 1985). This effect has been attibuted to the inability of the dendritic region of the neuron to depolarize, as a result of the h5^erpolarization and shunting effect produced by activation of GABA^ receptors. The finding by Wigstrom and Gustaffson (1983) that picrotoxin greatly facUitates the induction of LTP further emphasizes the importance of inhibition in regulating the induction of LTP. Recently, presynaptic GABAg receptors have been demonstrated to play an important modulatory role in the induction of LTP in both dentate gyrus (Mott and Lewis, 1991) and C A l (Davies et. al.. 1991). This is supported by previous observations that application of baclofen to the perfusate faciUtates the induction of LTP (Mott et. al.. 1990) and that the specific GABAg receptor antagonists 2-hydro3^-saclofen and CGP 35348, at concentrations that antagonize paired-pulse depression of the fast IPSP, also block the induction of LTP (Davies et. al.. 1991; Mott and Lewis, 1991). Presynaptic GABAg receptor-mediated autoinhibition is one mechanism which can explain why IPSPs are suppressed during high frequency trains used to induce LTP (Lynch and Larson, 1986; Pacelli and Kelso, 1989). Clearly, the overall effect of suppressing inhibitory influences on the postsynaptic cell is a greater probability of the membrane to depolarize following repetative stimulation of the afférents, a manoeuvre that favors LTP induction. 13.7.2. Maintenance of LTP. The maintenance (or expression) of LTP is characterized by a non-decremental increase in synaptic efficacy lasting hours in vitro and days in vivo. Recently, much controversy has surrounded the issue on whether maintenance of LTP is supported by pre- and/or postsynaptic mechanisms. The following section discusses evidence supporting either mechanism and the controversies accompan5àng some of these findings. 13.7.2.1. Postsynaptic mechanisms. How might an enhanced S3niaptic response be expressed during LTP? One possible explanation is an increase in receptor number. This hypothesis was proposed by Lynch and Baudry (1984) when they discovered that the number but not affinity of glutamate binding sites increased in hippocampal slices that were tetanically stimulated (Lynch et. al., 1982). They proposed that a tetanically-induced increase in postsynaptic Ca2+ activated a protease (calpain) which in turn degraded a cytoskeletal protein (fodrin) resulting in unmasking a subpopulation of dendritically localized glutamate receptors. However, the strength of this hypothesis was questioned when two independent groups separately reported no increase in glutamate binding in potentiated hippocampal tissue (Goh et a i , 1986; Sastry and Goh, 1984; Lynch et. al.. 1985). Furthermore, Sastry (1985) demonstrated that Leupeptin, a calpain antagonist, did not block LTP. Another finding inconsistent with Lynch and Baudiy 's hypothesis is the distribution of calpains 1 and 11 appear not to be present in axo-dendritic synapses (Hamakubo et. al.. 1986). The involvement of specific phosphorylating enz5mies (kinases) i n the maintaining the efficacy of excitatory synaptic transmission has also been proposed following the induction of LTP. The most studied kinases are the Ca2+ and phospholipid dependent protein kinase C (PKC) and the Ca2+-calmodulin dependent protein kinase 11 (CaMKll). Evidence for participation of PKC in hippocampal LTP stems from the observations that phosphorylation of protein F l by membrane bound PKC increases Avith increasing potentiation of the perforant path (Lovinger et. al.. 1986; Routtenberg et. al.. 1985). Consistent with this finding is the observation that other substrate proteins for PKC including B50 and GAP 43 are phosphorylated during LTP. Furthermore, translocation of cytosolic PKC to the membrane bound form has been reported to be enhanced in potentiated tissue (Akers et. al.. 1986) and phorbol esters, activators of PKC (Malenka e t aL. 1986; Gustaffson et al.. 1988) as well as injection of PKC (Hu et al.. 1987) induce an LTP-like potentiation. In addition to PKC, activation of CaMKII kinase also appears to be activated following the induction of LTP. CaMKII kinase has been detected postsynaptically particularly in the dendritic spine (Kennedy et al.. 1983). Hippocampal slices perfused with calmodulin antagonists have been reported not to support LTP (Dundwiddle e t aL, 1982; Mody et. al.. 1984; Turner et. al.. 1982). However, because of their lack of selectivity for a posts5niaptic locus of action, a presynaptic site cannot be ruled out. The observation that specific calmodulin antagonists when injected into the postsynaptic cell blocks LTP offers more direct evidence for participation of a postS5aiaptic CaMKII kinase in LTP (Malinow et. al.. 1989; Malenka et. al.. 1989). Furthermore, pseudosubstrate proteins that inhibit posts5niaptic CaMKII kinase activity have also been reported to block LTP (Malenka et. al.. 1989). The involvement of PKC and CaMKl l activity in the maintenance of LTP was reported by Malinow et. al. (1988). They demonstrated that sphingosine, an inhibitor of diacylglycerol and calmodulin (both activators of PKC and CaMKl l kinase, respectively), blocked the induction but not the expression of LTP. In addition to this, the non-specific protein kinase inhibitor H-7 blocked the induction of LTP but reversibly antagonised the maintenace of LTP, Taken together, these findings suggested that once induced, LTP is sustained by persistent protein kinase activity independent of the presence of substrate activators. The observation that CaMKl l kinase can be activated by auto-phosphorylation (Saitoh and Schwartz, 1985) and reports suggesting that PKM, the active form of PKC, may be derived from a Ca2+-dependent proteolysis of the parent enzyme (Inoue et. al.. 1977; Pontremoli et. al.. 1986) supports this idea. Whether the site of persistent change was pre- or postsynaptic could not be discerned in their study since both sphingosine and H-7 were added to the perfusion medium. Subsequent experiments conducted by the same group suggested that both postsynaptic PKC and CaMKl l kinase were required for the induction of LTP where as activation of presynaptic protein kinase was responsible for its expression (Malinow et. al.. 1989). These conclusions were based on the observation that intracellular injection of H-7 blocked the induction of LTP but had no effect on LTP that had previously been estabished. Although such findings imply a presynaptic involvment for PKC and CaMKl l kinase in the expression of LTP of excitatory synaptic transmission, participation of postsynaptic protein kinases appears to modulate long-term changes in inhibitory synaptic transmission. For example, Xie and Sastry (1991) demonstrated that LTP of the slow IPSP occured if posts)maptic protein kinase activity was inhibited by intracellular injection of K-252b, This posts5niatlc modification also appeared to be Ca^^-dependent since the potentiation was produced only when the posts5aiaptic cell was injected with EGTA or BAPTA (Morishita and Sastiy, 1991). Kauer et. al. (1988) demonstrated that LTP induced by either tetanic stimulations or pairing produced an increase in the non-NMDA but not the NMDA component of the EPSP in C A l neurons. Similar results were also reported by MuUer et. al. (1988) (but see Bashir et. al.. 1989). Davies et. al. (1989) reported that the sensitivity of C A l neurons to iontophoretically applied quisqualate and AMPA slowly increased following the induction of LTP supporting the view that LTP is associated with a persistent postsjmaptic modification. 13.7.2.2. Presynaptic mechanisms. Following the induction of LTP a decrease in presynaptic terminal excitability has been reported in both dentate gyrus (Sastiy, 1982) and C A l (Goh and Sasty, 1985a; Sastry et. al.. 1986). One possible explanation that may account for this observation is if a hyperpolarization occurs in the presynaptic terminal during LTP. Consequently, the driving force for extracellular Ca2+ would increase and, provided that somatically generated action potentials invading the terminal are not suppressed, the presynaptic Ca2+ concentration would increase resulting in a greater facilitation of neurotransmitter release. Such a presynaptic mechanism would be consistent with the finding of Baimbridge and Miller (1981) where the uptake and retention of labelled calcium in C A l was reported to be increased following the induction of LTP. Though such experiments did not clarify whether the accumulation occured pre- or postsynaptically, subsequent experiments by Agoston and Kuhnt (1986) reported increased Ca^"^ uptake in synaptosomes (derived from pinched off nerve terminals) isolated from potentiated slices suggesting a presynaptic accumulation of the divalent cation. In CA3, additional evidence for a presjmaptic mechanism comes from observations that LTP of the mossy fiber-CA3 pyramidal cell S5niapse occurs even when EGTA is present i n the postsynaptic cell (Bradler and Barrionuevo, 1988, but see Williams and Johnston, 1989). Furthermore, dialysis of the postsynaptic cell with a BAPTA-containing patch electrode appears not to prevent LTP at this sjmapse and further eliminates any argument for Ca?^-dependent postsynaptic involvement in the maintenance of mossy fiber LTP (Zalutsky and Nicoll, 1990). Recently, Regehr and Tank (1992) combined microfluorimetry with local perfusion techniques to assess the degree of calcium accumulation in the mossy fiber terminals following the induction of LTP at mossy fiber-CA3 p5n-amidal cell synapses. Suprisingly, they reported that increased Ca2+ levels were not sustained during the maintenance of LTP. These results, however, do not rule-out the involvement of pres5niaptic Ca2+ i n LTP since the authors noted that, with the methods employed, Ca2+ accumulation produced by an increase in pres5maptic Ca2+ could go undetected. Further evidence for a presynaptic involvement in the expression of LTP arises from observations that tetanus-induced as well as Ca2+-induced LTP results in a sustained increase in the release of glutamate and aspartate in the dentate gyrus (Bliss et. al.. 1986, but see Aniksztejn et. al.. 1989). A similar long-lasting increase for aspartate has been observed in CA3 (Feasely et. al.. 1985) and C A l (Lynch et. al.. 1989). In support of this view. Chirwa and Sastry (1988) reported that following the induction of LTP the frequency of miniature EPSPs that followed the EPSP (presumably due to an asynchronous release of transmitter) was increased in hippocampal slices incubated in Ba2+. These observations, however, do not confer any assurance that the increased release of neurotransmitter arises from the presynaptic terminals of the effected synapse. For example, other sources including the postsynaptic cell and surrounding glia may release neurotransmitter thereby confounding an accurate assessment of presynaptic neurotransmitter release. In order to accurately determine whether maintenance involves an increase in neurotransmitter release, a quantal analysis could be conducted on the synapse in question. It should be noted, however, that this technique when applied in its conventional form also has its weaknesses. Quantal analysis has been performed on the neurons of CA3 (Voronin, 1983; Yamamoto et. al.. 1987) and C A l (Hess et. al.. 1987; Friedlander et. al. 1990; Sayer et. al.. 1989; Voronin et. al.. 1990). However, results from these studies are limited to the detectability of presumed descrete quantal events. Because conventional microelectrode techniques were employed in these studies some quantal events may have gone undetected due to the poor signal to noise ratio of this recording technique. Recent advancements in microelectrode recording techniques, particularly the "on-slice" whole cell voltage clamp recording technique (Blanton et. al.. 1989) have enabled investigators to assess whether expression of LTP resides pre- or postsynaptically. The advantage the whole cell microelectrode technique has over the conventional microelectrode technique is that it offers a higher signal to noise ratio, thus, permitting better resolution of quantal events. Moreover, the recordings of currents would minimize the problems associated with non-linear summations. Malinow and Tsien (1990) were the first to incorporate the whole cell technique to examine LTP at Schaffer collateral/commissural-CAl pyramidal cell synapses. They demonstrated that associative LTP was primarily mediated by a presynaptic mechanism. Of particular interest, was the observation that intracellular dialysis of the postsjniaptic cell prevented the induction but not the maintenance of LTP supporting previous hypothesis of a possible involvement of retrograde factors in the initiation of LTP (Chiwa and Sastiy, 1988; Goh and Sastry, 1985; Goh and Sastiy, 1985; Malinow et. al . . 1989; Sastry et. al.. 1988; Sastry et. al.. 1986; Williams et. al.. 1989). Subsequent studies incorporating the whole cell voltage clamp technique demonstrated that LTP in C A l neurons induced by either tetanic stimulations of the Schaffer coUateral/commissural fibers (Bekkers and Stevens, 1990) or by depolarization of CA3 neurons synaptically paired with the C A l neurons (Malinow, 1991) are also due to a presynaptic enhancement of neurotransmitter release. 13.7.2.3. Pre- and postsynaptic mechanisms. Hebb (1949) proposed that interactions between pre- and postsynaptic elements were responsible for changes in synaptic efficacy. Indirect evidence for such interactions in hippocampal LTP is supported by the associative property of LTP. This is best characterized by the observation that LTP can be induced when low frequency activation of afferent input is paired with a sustciined postsynaptic depolarization. The fact that no LTP occurs when the two events occur independently suggests a postS3niaptic contribution to the associative Induction of LTP. Furthermore, the observation that associative LTP is primarily presynaptic in origin and is blocked when the intracellular contents of the postsynaptic cell are dialysed prior to but not after LTP induction, supports the h)rpothesis that LTP is triggered by some diffusable factor released from the postsynaptic cell which then acts on the presynaptic cell to produce an increased release of neurotransmitter. Possible candidates for such a retrogade messenger include trophic factors, arachidonic acid and nitric oxide. Before a substance can be considered a retrograde messenger, it should possess at least three important properties, 1) it should be present in the postsynaptic cell, 2) it should be a highly diffusable substance capable of rapidly crossing the cell membrane and 3) it should enhance low frequency sjmaptic transmission. Based on these properties, the validity for some of the proposed retrograde messengers becomes questionable. Trophic factors have been suggested as possible retrograde messengers. The observation that neurite inducing substances in samples collected during tetanic stimulations of the guinea-pig hippocampus (Chirwa and Sastiy, 1986) or rabbit neocortex (Sastry et. al.. 1988a; Sastry et. al.. 1988b) are capable of inducing LTP during low frequency S5niaptlc transmission supports this idea. Furthermore, if the neocortical samples are separated into various molecular weight fractions only some of these fractions induce LTP, the potentiations of which are smaller than the ones produced by the unfractionated samples (Xie et. al.. 1991). These findings support a synergystic effect between the trophic factors in regulating the magnitude of LTP. However, whether these factors are released from the postS5maptic cell or from the pres3niaptic cell of from other sourses remains to be determined. Arachidonic acid has also been suggested as a retrograde messenger. For example, arachidonic acid can be can be generated postsjmaptically, is highly diffusable and is elevated in the extracellular fluid during LTP (L5aich e t al.. 1989). However, application of it onto hippocampal slices fails to potentiate low frequency synaptic transmission unless the application is accompanied by a weak tetanus (L5nich et. al.. 1989). This observation does not rule out arachidonic acid as a retrograde messenger but implies additional factors may be required for it to exert a potentiating effect on low frequency sjniaptic transmission. Recently, nitric oxide (NO) has been purported to be a retrograde messenger for the expression of LTP in area C A l of the rat hippocampus (Haley et. al.. 1992; Schuman and Madison. 1991). However, the observation that NO synthase is not present in C A l p5n:amidal neurons (Bredt et. al.. 1992; Vincent and Kimura, 1992) contradicts an involvement of NO in Schaffer collateral/commissural-CAl pyramidal cell LTP. Thus, while many studies support the occurance of a retrograde interaction between pre- and postsynaptic elements following the induction of LTP, identification of the substances mediating such an interaction is confounded by the strict requirements they must possess. Recently, Ballyk and Goh (1991) adveinced the hypothesis by Goh and Sastry (1985) that potassium ions (thought to be involved in promoting cooperativiy among pres5niaptic nerve terminals) may act as possible retrograde messengers. This is a particularly attractive h5^othesis since K+ can be released from the postsynaptic cell, is a diffusable substance, is capable of potentiating low frequency synaptic transmission (May et. al., 1987) and is briefly elevated following tetanic stimulation of afferent fibers (McCarren and Alger, 1985). However, since K+ may also be released from the presynaptic terminals, contributions of postsynaptically derived K+ may act to further enhance cooperativity between presynaptic flbers. Future studies investigating the involvement of K+ in LTP may substantiate the monovalent cation as a retrograde messenger. 14. SACCHARIN AS A PHARMACOLOGICAL TOOL OF INVESTIGATION 14.1. History Since its discovery by Remsen and Fahlberg in 1879, saccharin has been used as a non-caloric sweetner in nearly all countries of the world. Its use alone or in combination with cyclamate was so popular in the food industry that by 1967, it was estimated that nearly 75% of the United States population consumed food products containing the artiflcial sweetner (Arnold et. al.. 1983; Rubini, 1969). The first evidence revealing the potential carcinogenicity of saccharin was put forth by Allen et. al. in 1957. In their single animal study, bladders of mice implanted with a pellet containing saccharin were found to have a significant incidence of tumors than those that were implanted with pellets not containing the drug. However, since only one species of animal was used in the study and the evidence for saccharin as a potential carcinogen in man was lacking, the public heath authorities of many countries felt that this finding was not enough to warrent a ban on consumption of saccharin. It was not unti l 1970 that B i yan et. al. confirmed the initial finding of Allen and co-workers. Their results were supplemented with histological evidence that so lacked in the 1957 study. In the next 7 years evidence was put forth by a variety of investigators varifying the carcinogenic effects of saccharin in other mammalian species. Though by this time, conclusive evidence for its carcinogenicity in humans was still lacking, results from the animal studies prompted the Canadian government to ban the use of saccharin in the food, drug and cosmetic industries in March 1977. Because of the controversy surrounding saccharin as a potential agent for carcinogenicity in humans, other counties have yet to follow Canada's suit. 14.2. Chemistry and Physical Properties Saccharin is a 2,3-dihydro-3-oxobenzisosulfonazole which is commercially synthesized from 0-sulfanoylbenzoic acid. It is considerably stable under normal physiological conditions. Even when temperatures exceed 100 ^C for 1 hour no detectable decomposition is observed. The melting point of saccharin occurs at 228 to 229 ^c. Saccharin itself is insoluble (1 g dissolves in 290 mL of H2O), however, its sodium salt is not (1 g dissolves i n 1.2 mL of H2O). Because the pKa of saccharin is approximately 1.8, almost all of the drug is ionized at neutral pH. The potential for saccharin as a artificial sweetner was based on the finding it exhibited a sweetness 200 to 300 times that of sugar. 14.3. Pharmacodisposition Saccharin is not metabolized in humans and is excreted essentially unchanged in the urine (Lethco and Wallace, 1975). It has been detected in highly perfused organs including fetal tissue and urinary bladder (Ball et. al.. 1977; Matthews et. al.. 1973; Pitkin et. al.. 1971); whether it crosses the blood brain barrier remains to be determined. The observations that both fetal and adult urinary bladders concentrate high levels of radioactive saccharin being detectable even after the radioactivity in other tissues subsides (Ball et. al.. 1977) along with previous findings that saccharin significantly increases the incidence of urinary bladder cEirclnomas further perpetuated the causal relationship between the drug and bladder carcinogenesis. 14.4. Tumor Promoter Since its discovery as a potential bladder carcinogen, numerous studies have been conducted to determine the mechanism(s) by which saccharin induced tumors are expressed (for review see Arnold et. al.. 1983; Ellwein and Cohen, 1990). Consequently, a number of initiation/promotion bioassays have been conducted in an attempt to understand the carcinogenic nature of saccharin. That saccharin acted as a tumor promoter was first demonstrated by Hicks et. al. (1973). In their experiments, the weak carcinogen N-methyl-N-nitrosourea (MNU) when combined with saccharin produced a greater incidence of bladder tumors than when MNU was used alone. The results of this study were highly controversial since it could not be repeated by others i n succeeding studies (Green et. al.. 1980; Soudah et. al.. 1981). However, other weak carcinogens such as N-[4-(5-nitro-2 furyl)-2-thia2olyl] formamide (FANFT) and N-butyl-iV-(4-hydroxybutyl) nitrosamine (BBN) have since been demonstrated to initiate the carcinogenic process when co-administered with saccharin (Cohen et. al.. 1983; Cohen, 1985; Cohen et. al . . 1987). Unlike other chemicals, the actions of saccharin as a tumor promoter cannot be explained by the classical paradigm for chemical carcinogens which involves metabolic activation to reactive electrophiles that interact with DNA to induce genetic alterations (Ellwein and Cohen, 1990) since the drug does not interact with DNA (Lutz and Schlatter, 1977) nor is it metabolized to a reactive electrophile (Sweatman and Renwick, 1979). Furthermore, at a p H approximating the pH of bladder urine, saccharin is a strong nucleophile (EUwein and Cohen, 1990). It therefore appears that the carcinogenic actions of saccharin cannot be attributed to a genotoxic mechanism. A number of chemical carcinogens including saccharin have been shown to inhibit the activity of guanylate cyclase (Vesely and Levey, 1978), an enzyme that converts guanosine triphosphate (GTP) to guanosine 3', 5'-cyclic monophosphate (cGMP). This is particularly interesting since changes in the intracellular levels of cGMP have been implicated in normal and abnormal cell growth (Kram and Tompkins, 1973; Kimura and Murad, 1975; Vesely et. al.. 1976). The actions of saccharin are, however, not confined to one enzyme system, infact other systems not implicated in tumerogenesis are also altered by saccharin. For example, changes in intracellular adenosine 3', 5'-cyclic monophosphate (cAMP) concentration in taste cells are thought to be involved sweet taste transduction (Avenet et. al.. 1988; Tonosaki and Fumakoshi. 1988). The observation that saccharin alters adenylate cyclase activity in rat musle and liver membrane preparations suggests that the taste transduction process may be affected in a similiar manner (Striem et. al.. 1990). In other studies, the preventiation of dental caries produced by streptococcus mutans has been explained through saccharin inhibiting the activities of specific glycolytic enzymes responsible for cell growth and fermentative acid production (Brown and Best, 1986; Linke and Kohn. 1984). Saccharin has also been found to stimulate protein kinase C activity in liver cells (Kleine et. al.. 1986). 14.5. Interaction With Growth Factors Certain growth factors are potent stimulators of cell differentiation and neurite out-growth. This and the fact that tumor promoters inhibit neurite out-growth in an irreversible manner prompted Lee (1981) and later Ishii (1982) to conduct experiments examining the effects of saccharin on the binding and production of neurite out-growth of different growth factors. Lee demonstrated that binding of murine epidermal growth factor (EGF) to the membranes of various cell culture lines including HeLa (human carcinoma), HTC (rat hepatoma), K22 (rat liver) and 3T3-L1 (mouse fibroblasts) was inhibited by saccharin in a dose-dependent manner. Subsequent experiments by Ishii demonstrated that saccharin inhibited the binding of nerve growth factor (NOP) to membranes of chick dorsal root ganglia (DRG) cells and NGF-induced neurite out-growth in the DRG cells (an effect that was not observed if the neurite out-growth had already been established). Recently, saccharin (10 mM) has been reported to inhibit neurite out-growth in PC-12 cells exposed to samples collected from tetanically stimulated rabbit neocortices (Chirwa, 1988; Sastry et. al.. 1988a). The observation that these samples induced LTP in guinea-pig hippocampal slices raised the prospect for a possible involvement of endogenous growth factors in the development of LTP (Chirwa, 1988; Sastry e t aL, 1988a; Sastry et. aL. 1988b; Xie et. al.. 1991) 14.6. Involvement in Long-Term Potentiation Based on the premise that saccharin was capable of inhibiting neurite out-groAvth induced by the neurite-inducing neocortical samples, Sastry et. al. (1988a) examined the effects of saccharin on LTP. It was found that 10 m M saccharin reversibly blocked hippocampal LTP induced by either a high frequency tetanus or by application of the samples collected during tetanic stimulations of the rabbit neocortex. This effect was attributed to a specific action of the drug rather than a non-specific effect since saccharin did not, 1) alter the population spike or field EPSP, 2) alter the membrane potential, input resistance and the ability of C A l neurons to induce action potentials, 3) produce significant effects on paired-pulse facilitation of the population spike, 4) alter the frequency of Ba.^+ induced miniature EPSPs, and 5) block the depolarizations induced by exogenously applied NMD LA (Chirwa, 1988). Furthermore, the observation that saccharin blocked LTP induced by applied NGF further supported the involvement of growth factors in LTP. 15. MATERIALS AND METHODS The following pages are organized in sections describing in order, animal care, the hippocampal slice preparation, perfusion chamber, the perfusion media, the recording and stimulating equipment, intra- and extracellular recording methods, LTP induction, statistics and, finally, a description of the experimental protocols used in this study. 15.1. Animal Source and Care Male Hartley guinea-pigs weighing 175-200 gms were obtained from the Animal Care Centre of the University of Brit ish Columbia. The guinea-pigs arrived at the Department of Pharmacology and Therapeutics at the beginning of each week and were transferred to the animal care room where they were kept in a communal wire "housing" cage (dimensions: 59 x 54 x 23 cm). Animals arriving the following week were housed in a separate cage from those remaining from the previous week. The living environment for the animals was carefully controlled. The temperature of the animal care room was kept between 20 to 22 ^C and the lights in the room were on between 6 am to 6 pm. The guinea-pigs had free access to Guinea-pig Chow contained in a compartment located on the side of the cage and water dispensed through a small drinking tube connected to an inverted 250 ml bottle that was also kept on the side of the cage. Throughout the week, food and water supplies were maintained and on the last working day of each week, enough food and water were supplemented to maintain the animals' diet through the weekend. The litter trays of the housing cages were cleaned and relined once every two days. 15.2. Slice Preparation Care was taken to reduce any stress or discomfort the guinea-pig might encounter during the pre-operative preparations. For this reason, the guinea-pig was transfered from the housing cage to a temporary holding cage and quickly brought up to the lab. In the lab, the animal was gently placed in a dessicator jar on top of a pack of ice (to lower the body's metabolic demand), A mixture of halothane (2%) and carbogen (95% O2 5% CO2 mixture) was then introduced into the jar. The anaesthetic was delivered to the animal unt i l surgical anaesthesia was achieved. This was established if the animal appeared unconscious, respiration was predominantly thoracic in origin and the animal did not exhibit the corneal and the hindleg Avithdrawal reflex when challenged with the approriate stimuli. The guinea-pig was then placed on a clean flat surface so that it could be easily positioned for the subsequent operation. The scalp was exposed by making a medial incision at the top of the animal's head and pulling the skin laterally. A pair of bone nippers was used to create a small hole at the base of the skul l . This enabled a pair of scissors to be inserted under the skul l case and a subsequent cut along the sagital suture line to be performed. A pair of ronguers were then used to peel away the bone and the dura so that a small spatula could be inserted behind the cerebellum and underneath the brain. The brain was then gently pryed out of the skul l case and was immediately placed on dissecting paper (ventral side-down) and washed with cold (4 ^C) physiological control (or normal) medium saturated with carbogen. The hippocampus was exposed by separating the two cerebral hemispheres laterally. Cold o:jQrgenated medium was poured over the hippocampus and using a fine spatula, one or both sides of the hippocampus were dissected free. The freed hippocampus was gently placed on dissecting paper that was mounted on the cutting platform of a Mcllwain tissue chopper. The platform was oriented such that the septo-temporal axis of the hippocampus was perpendicular to the blade of the tissue chopper. Transverse slices were cut to a thickness of 400 |j,m and were transfered to a petri dish (containing the cold oxygenated normal medium), where they were separated with a fine spatula. Slices obtained from the mid-portion of the hippocampus were placed on a partially submerged nylon net that was attached to a plexiglass ring. In order to prevent movement of the slices, another nylon net mounted on a slightly larger plexiglass ring was placed over the smaller one. The two rings were then carefully pressed together. The tight fit between the outer side of the smaller ring and the inner side of the larger ring enabled the nylon nets to be lowered just enough to sandwich the slices without damaging them. The secured slices were then transfered from the petri dish to the slice chamber where they were submerged and superfused with oxygenated normal medium at a rate of 1 to 1.5 ml per minute. Carbogen mixture was introduced over the top of the slice chamber to maintain a oxygen-saturated environment. The whole procedure from the start of surgery to placement of the slices into the slice chamber took no more than 5 minutes. The slices were allowed to equilibriate with the superfusate for at least 1 hour. At the end of the equilibration period, only those slices in which the stratum p5n:amidal could be clearly discerned from the rest of the surrounding tissue (the cell body layer appeared as a well defined line with sharp borders) were selected for recording. Slices selected in this manner were capable of supporting physiological responses for at least 8 hours. Even though slice viability exceeded the duration of the experiments performed, only one slice was used per animal per experiment to minimize any variability that may arise during prolonged superfusion times. 15.3. Slice Chamber A detailed description of the slice chamber has previously been published (Panaboina and Sastiy, 1984). Briefly, the chamber consisted of a raised rectangular stage constructed from plexiglass as illustrated in Figure 15.1, A circular chamber was milled into the top surface of the plexiglass stage and a straight narrow trough was bored into the stage entering the chcimber at the same depth. Glued to the top of chamber near the edge of the trough was a Figure 15.1. Schematic representation of the slice chamber used to study in vitro electrophysiological potentials from guinea-pig hippocampal slices. small plexiglass box. A small hole was drilled into the box and angled so that a suction "waste" line could be inseri;ed into the trough and adjusted to control the level of the bathing medium. Another hole was drilled at an angle into the top of the slice chamber unt i l it entered the base of the circular chamber. The main inlet line was inserted through this hole. The inlet line fed through an aluminum heating block that was attached to the underside of the slice chamber. The temperature of the heating block was continually monitored by a temperature sensing device placed in the heating block. Because the temperature sensing device fed-back to a heater control panel, the superfusate in the chamber could be monitored and maintained at a constant temperature, A gold "female" grounding pin was inserted into the bottom of the circular chamber though a hole drilled just below the inlet line. The main inlet line was connected to a manifold which was fed, in turn, by eight lines each connected to a separate superfusion barrel (60 ml syringe tube). The superfusion barrels were mounted on an adjustable a luminum bracket that was positioned above the slice chamber. The superfusate was gravity fed from the barrels to the slice chamber £ind the flow rate of the superfusate could be increased or decreased by adjusting the height of the aluminum bracket. 15.4. Perfusion Media The physiological normal media was prepared daily and contained (in mM): NaCl 120, K C l 3.1, NaHCOa 26. NaH2P04 1.8. MgCl2 2, CaCl2 2, and dextrose 10. The pH of the medium was 7.4 when bubbled with carbogen. In some experiments, saccharin (sodium salt; Sigma), 6-cyano-7-nitroquinoxallne-2,3-dione (CNQX; Tocris Neuramin), 2-amino-5-phosphonovalerate (APV; Sigma), D(-)2-amino-5-phosphonovalerate [(-)APV; Sigma], N-methyl-D-aspartate (NMDA; Sigma), tetrodotoxin (TTX; Sigma) and picrotoxinin (Sigma) were added to the medium. With the exception of saccharin, concentrated stock solutions of all the drugs were made and diluted with the normal medium to the desired concentrations. When saccharin (10 mM) was made, the concentration of NaCl i n the normal medim was decreased to 110 m M to protect against a potential rise in total osmolarity. In addition to the normal physiological medium, two other media were used in this study. Slices requiring prolonged exposure to picrotoxinin were superfused with a modified medium containing 20 |iM picrotoxinin. In this medium, the concentrations of Mg2+ and Ca^+ were increased to 4 m M each and NaH2P04 was omitted. Omission of the phosphate buffer did not alter the pH of the ox/genated media. The Mg2+ and Ca2+ concentrations were elevated to reduce the likelihood of inadvertantly inducing LTP (Wigstrom and Gustaffson, 1983) or epileptiform activity in C A l (Hablitz, 1984) arising from excessive spontaneous sjmaptic excitation due to picrotoxinin-induced CA3 cell bursting. In addition to this, a surgical cut was made between the CA3 and the C A l subfields to prevent the latter from occuring, . In Mg2+-free medium, Mg2+ was omitted from the normal medium. Because traces of Mg2+ ions are detectable in the chemicals constituting the normal medium, conceivably, there still exists the potential for Mg2+ contamination. However, the contribution of Mg2+ due to contamination would be very low and, therefore, the medium would be relatively Mg2+-free. Because the adjustable bracket could hold up to eight superfusion barrels, up to eight different media could be superfused in one experiment. However, in a typical experiment, only 3 to 4 barrels were used. The main superfusion barrel, which contained the normal medium, was kept full by a line which syphoned the medium from a reservoir located above the barrels. Media in all the barrels as well as the reservoir were aereated with carbogen through lines that were connected to a common plexiglass tube that was, in turn, connected to a regulator attached to a carbogen tank. To minimize "dead space" in the superfusing apparatus, the superfusing lines were opened to remove any trapped air bubbles. The process was accelerated by momentarily attaching a suction line to the mouth of the inlet line. Except for the line which contained the normal medium, all other lines were sequentially closed with butterfly clips. Set up in this particular manner, the slice chamber offered rapid exchange of different media with minimal flow disturbance. 15.5. Recording and Stimulating Equipment 15.5.1. Recording Electrodes. Extracellular recording electrodes were made from standard Kwik-FilTM borosilicate glass cappilaries (inner wall diameter, 0.68 mm; outer wall diameter, 1.2 mm). The microelectrodes were pulled on a Narshige PE-2 vertical puUer and filled with 4 M NaCl. Tip resistances of these electrodes ranged from 1 to 3 MQ. Intracellular recording electrodes were fabricated from Kwik-Fil™ micropipettes (inner wall diameter, 0.58 mm; outer wall diameter, 1.0 mm). The microelectrodes were pulled on a programmable Flaming/Brown (model P-87) micropipette puller. The microelectrodes were filled with either, 4 M C H 3 C O O K (tip resistances, 70 to 85 MQ) or a solution containing 4 M C H 3 C O O K and 100 m M QX-314 (Astra Pharmaceuticals) (tip resistance, 85 to 120 MQ). 15.5.2. Amplifiers. Extracellular field potentials were amplified with a DAM-5A differential pre-amplifier (World Precision Instruments). Dur ing operation, the low frequency (10 Hz) and high frequency (10 KHz) filters were set at 0.1 Hz and 1 KHz, respectively. Intracellular potentials were recorded with an Axoclamp 2A microelectrode clamp (Axon Instruments) where use of the bridge current-clamp (CC), discontinuous current-clamp (DCC) and discontinuous single electrode voltage-clamp (dSEVC) modes was employed. The headstage located in the microelectrode probe contained a precision resistor (R=100 MQ) that set the headstage current gain (H) to 0.1. At this particular value, the bridge balance range was 0 to 1000 MQ; the maximum DC current command was ±10 nA; the maximum gain in dSEVC was 10 nA/mV and the capacitance neutralization range was -1 to 4 pF, In the CC mode, the microelectrode resistance was determine by the null-bridge method. Briefly, this procedure involved eliminating the voltage response developed by a 0,5 nA, 200 msec square pulse delivered to the microelectrode by adjusting the bridge balance of the axoclamp. The value of the bridge balance at which no voltage response was observed, was an estimate of the microelectrode resistance. This procedure was done, first in the recording medium and while i n the impaled neuron (the difference between these two values gave an approximation of the cell's membrane resistance). This was also performed after "puUing-out" of the cell to determine if the electrode resistance strayed away from its pre-experiment value. While the majority of intacellular responses were recorded in the CC mode, some were monitored in the DCC and dSEVC modes. In these particular cases, in addition to monitoring the voltage and current responses of the cell, the switching frequency of the sample and hold circuit and voltage drop across the microelectrode were monitored on a separate oscilloscope. The duty cycle used in both DCC and dSEVC modes was 30% current passing and 70% voltage recording for each cycle. In both DCC and dSEVC modes, the capacitance neutralization was set Just below the level which caused the sample and hold circuit to "ring". At this setting the voltage arising from brief current pulses recorded by the microelectrode decayed rapidly (usually between 10 to 50 p-s) before the beginning of the next current passing cycle. When recording in dSEVC mode, both phase and gain controls were adjusted to obtain optimal voltage-clamping of the cell. More often than not, these procedures (used to improve the clamping performance of the microelectrode) often introduced high frequency noise into the voltage and current recordings. This could be reduced by anti-aliasing. However, increasing the amount of filtering by this method lowered the djoiamic response of the microelectrode and, hence, its clamping performance. Thus, the phase, gain and anti-alias were adjusted to reach a compromise between the noise and the d5niamic response of the microelectrode. While in the impaled neuron, by observing the period between voltage decay of the microelectrode at the end of each cycle and the voltage rise of the microelectrode at the beginning of each cycle, the sampling frequency could be adjusted between 2 to 3 KHz. This was sufficient enough for the membrane capacitance to smooth the voltage and current response of the cell to direct current and pulsed current. The signals from the Axoclamp were amplified ten times and filtered between 0.1 KHz and 1 KHz before they were fed into the recording systems. 15.5.3. Recording Systems. Intracellular potentials were monitored with a Tektronix 5113 dual beam storage oscilloscope and a Data Precision DATA 6000 wave form analyzer. Signals fed into the DATA 6000 were digitized and plotted on a Hewlett Packard 7470A graphics plotter or stored on a Hewlett Packard 3968A magnetic tape recorder. Extracellular field potentials were monitored and plotted in a similar manner except the signals were fed into the DATA 6000 only. 15.5.4. Stimulation and Isolation Units. Current was generated by a Grass S88 stimulator coupled to two photoelectric constant current units (model PS1U6). One of the units supplied current to the Axoclamp so that square wave current pulses or, alternatively, DC current could be injected through the microelectrode. The other unit supplied current to the stimulating electrode. In some cases, as an alternative current source, a programmable square wave generator was used that attached to a Digitimer isolation unit (model DS2) which, in turn, was connected to the Axoclamp. 15.5.5. Stimulating Electrodes. Bipolar concentric electrodes (SNEX-100) were obtained from David Kopf Instruments (DKl). These stimulating electrodes had shaft lengths and contact lengths of 50 mm and 0.75 mm, respectively. The contact diameter of the electrodes (0.1 mm) was small enough, so that the damage sustained to the surrounding tissue was minimal. The electrodes typically had resistances of 1 MQ and were replaced if their resistance exceeded 7 MQ. 15.5.6. Positioning of Electrodes. Extracellular recording electrodes were positioned using an Optikon micropositioner which was capable of moving the microelectrode in the X, Y, and Z coordinate planes. Intracellular electrodes were advanced with a D K l hydraulic microdrive unit (model 607 WCP) attached to the micropositioner. The microdrive was capable of lowering the microelectrode in 1 |im steps at a rate of 200 steps/second. Stimulating electrodes were mounted on DK l electrode carriers. Like the Optikon micropositioner, the electrode carriers were capable of positioning the stimulating electrodes in all three coordinate planes. A maximum of two electrode carriers could be mounted on one carrier stand. 15.5.7. Arrangement of the Recording Set-up. The recording set-up consisted of one carrier stand equipped with two electrode carriers, the micropositioner and the slice chamber all mounted on a thick aluminum base plate. The set-up was covered by an aluminum wire-mesh screen to shield the recording apparatus from electrical noise that arose from external electrical sources (eg. lights). The whole recording set-up sat on a vibration-free, air isolated, table. Positioning the recording and stimulating electrodes to their desired locations on the slice was facilitated by a Zeiss gross dissecting microscope that was mounted on an adjustable floor stand. 15.6. Extracellular Recording Population spikes and excitatory postsynaptic potentials (EPSPs) were recorded in the CAl]^ region of the guinea-pig hippocampus. A l l extracellular potentials were elicited orthodromically with square-wave pulses (10-150 foA, 0.2 msec, 0.1-0.2 Hz) delivered through a stimulating electrode placed in the stratum radiatum. The stimulation strengths were adjusted so that the height of population spikes measured 1 to 1.5 mV or the amplitude of the field EPSPs were between 0.5 to 1.0 mV. Only those responses that exhibited a 1,5 fold or greater increase in amplitude to a twin "test" pulse (interstimulus interval, 40 msec) were used for the experiments. After these conditions were met, the somatic or the dendritic responses were monitored for stability. The responses had to be stable for at least 10 minutes (they constituted the control for the subsequent experiment) before the experiment commenced, 15.7. Intracellular Recording Intracellular recordings were obtained from CAl^j neurons of the hippocampus. Unless otherwise stated, the synaptic potentials were elicited using the same stimulation paradigm employed to elicit the extracellular field potentials. Before the intracellular S3niaptic potentials could be recorded certain criteria had to be met, 1) the resting membrane potential (RMP) of the neurons had to be more negative thcin -55 mV, 2) the input resistance (R|nOf the impaled neurons was more than 25 MQ, and 3) the action potentials produced by a depolarizing rectangular current pulse (0.1 to 0.3 nA, 200 msec) were greater than 65 mV. The stimulation strength deliver to the electrode was adjusted to elicit EPSPs between 5 to 10 mV and inhibitory postsynaptic potentials (IPSPs) of 2 to 5 mV. EPSPs and IPSPs had to be stable for at least 10 minutes before they could be subjected to any experimental manipulations. These responses served as the control for the experiment. Paired-pulse facilitation (interstimulus interval, 40 msec) of the EPSP was used as a test to determine the viability of the neuron and its associated excitatory S5niapses. The neuron was considered for use in the experiment if the second pulse generated an EPSP that was at least 1.5 times greater than the one generated by the first pulse. Paired-pulse depression of the IPSP (interstimulus interval, 100-400 msec) was used to assess the viability of the inhibitory synapses. Cells were considered acceptable for use in the experiment if the second pulse produced an IPSP that was smaller than the one generated by the first. 15.8. Induction of LTP 15.8.1. Tetanic Stimulation. Two types of tetanic stimulations were delivered to the stratum radiatum. The first type of stimulation was one train at 400 Hz consisting of 200 pulses. The second type of stimulation comprised two trains of 100 Hz consisting of 100 pulses each separated by 20 seconds. Though, each were capable in inducing LTP, the latter stimulation was employed in some of the intracellular experiments conducted because LTP of the EPSP slope could be induced more reliably than if the former stimulation was used. 15.8.2. Pairing. Experiments conducted by Sastry et. al. (1986) demonstrated that LTP could be induced in individual hippocampal pyramidal neurons if the stratum radiatum was stimulated in conjunction with sufficient postsynaptic depolarization. The number of pairings determined whether the intracellular EPSP exhibited short-term potentiation (STP) or long-term potentiation (LTP). In the present study, LTP of the intracellular EPSP slope was induced in slices superfused with 20 \iM picrotoxinin by performing 25 consecutive pairings. The pairings involved injecting DC current (3 nA to 4 nA) through the recording electrode to depolarize the neuron near the reversal potential of the EPSP while at the same time activating the stratum radiatum at 0.2 Hz. 15.9. Measurements and Statistics The population spike amplitude was measured as follows, a tangent line was drawn from the tip of the first EPSP peak to the second EPSP peak then a vertical line was draAvn from the mid-point of the tangent to the tip of the population spike. The length of the vertical line represented the amplitude of the population spike when measured and expressed in millivolts. The init ial slopes of the extracellular and intracellular EPSPs were computed on-line by the DATA 6000 and, unless otherwise stated, expressed in mV/msec. Because the stimulator was set for a delay (40 msec to 100 msec) prior to each stimulation of the stratum radiatum, on-line recordings of intracellular responses consisted of a baseline followed by a stimulus artifact and then the synaptic response. To assess the heights of either the EPSPs or the IPSPs, a parallel line was drawn to extend the baseline the length of the control synaptic response. The length of a perpendicular line drawn from the extended baseline to the peak of the positive or negative going wave when expressed in mV represented the height of the EPSP and IPSP, respectively. For each experiment, the time measured from the stimulus artifact to the peak of the control sjniaptic response (latency to peak) acted as a reference point for subsequent measurements. Data from some of the experiments, were displayed as coordinate graphs and histogram plots. In the coordinate graphs, the slopes of the field and intracellular EPSPs were expressed as a percent of the averaged control responses. In the histogram plots, the control responses were taken as 100% and all other corresponding responses were expressed as a percent of the controls and then averaged. Two statistical tests were employed to analyse the data in this study. The Student's t-test was used when comparisons were made between two related samples. The hypothesis for each experimental paradigm determined if the direction of the t-test was one-tailed or two-tailed. When comparisons were made between multiple means which only involved one criteria for classification (ie. mean of the field EPSP) a one-way ANOVA was employed. To determine whether the differences that occured between pairs of means were statistically significant, the data was subjected to Duncans' multiple comparisons test. In both statistical tests, the level of significance (p) was arbitrarily chosen at 0.05. Coordinate graphs and histogram plots were constructed such that each point or bar represented the mean ± S E M of the responses. 16. EXPERIMENTAL PROTOCOLS Previous experiments demonstrated that 10 m M saccharin when applied for 5 or 10 minutes blocked LTP of the population spike in the CAl^j region of the guinea-pig hippocampus without significantly affecting the resting membrane potential, the input resistance, the ability of the neuron to generate action potentials and the amplitude of the EPSP (Chirwa, 1988). Saccharin also appears not to affect transmitter release since the frequency of Ba2+-induced miniature EPSPs and paired-pulse facilitation of the population spike are not significantly altered (Chirwa, 1988). Despite these findings, the mechanism(s) by which the drug produces its LTP blocking effect remains to be elucidated. In the present study, specific experiments were conducted to ascertain if saccharin (10 mM, applied for 10 minutes) effects excitatory and inhibitory synaptic transmission and whether such effects could account for it blocking LTP in guinea-pig hippocampal CAljr, neurons. 16.1. Saccharin and LTP of the Field EPSP Previous experiments demonstrated that saccharin blocked LTP of the population spike (Chirwa, 1988). However, population spike measurements are plagued by a number of problems, all of which, affect its accuracy in quantitating LTP-induced changes in S5maptic transmission. For example, one can never observe the population spike in isolation since it is always superimposed upon the field EPSP. The fact that the population spike undergoes changes in latency also affects its appearance relative to the field EPSP. Furthermore, the measurements are complicated because the field EPSP is also susceptible to change. Therefore, it is difficult to find a stable baseline from which the population spike can be measured (Alger and Teyler, 1976). Recording dendritic EPSPs may eliminate some of these problems, however, contamination by an interrupting population spike may also distort EPSP measurements. Furthermore, after the development of LTP, the later component of the EPSP may be persistently enhanced from contributions of potentiated polysynaptic pathways (Miles and Wong, 1987; Malinow et. al . . 1989). To avoid these problems, the initial slope of the EPSP is measured. Thus, an increase in the slope, as opposed to an increase in the height, of the EPSP was taken to reflect a potentiation of relatively pure monosynaptic transmission. Since the present study was interested in examining the effects of saccharin on synaptic transmission, the slopes of the fleld EPSPs were recorded. The fleld EPSPs were evoked in the CAl^j area of the guinea-pig hippocampus by stimulating the stratum radiatum at 0.2 Hz. After obtaining control population EPSPs, saccharin (10 mM) was applied to the slices for 10 minutes. During the last minute of saccharin application, the stratum radiatum was tetanized (400 Hz, 200 pulses) upon which the slices were resuperfused with the normal media. The post-tetanic responses were then monitored for 60 minutes. 16.2. Saccharin and LTP of the Intracellular EPSP In order to establish that saccharin blocked LTP of the field EPSP by-acting on the individual CAl^j neurons, experiments identical to those used to demonstrate the effects of saccharin on the extracellular EPSP slope were also conducted on the intracellular EPSP slope. In order to minimize the standard error, whenever possible, the experiments were conducted on a slightly larger sample size than those used in the extracellular experiments. 16.3. Saccharin and Maintenance of LTP In these experiments, hippocampal slices were exposed to a post-tetanic application of saccharin to test whether its LTP blocking effect was specific to the induction of LTP. Population spikes were recorded from the CAl^^ region of the hippocampus and elicited by a 0.2 Hz stimulation of the stratum radiatum. After obtaining stable control responses for 30 minutes, a tetanic stimulation (400 Hz, 200 pulses) was delivered to the afférents. The post-tetanic responses were followed for 10 minutes before applying saccharin. Saccharin was superfused at a concentration of 10 m M for 10 minutes. After termination of drug application the population spike was monitored for an additional 30 minutes. 16.4. Saccharin and Pairing Experiments were done to examine whether saccharin interfered with LTP induced by pairing prolonged posts5maptic depolarizations with concommitant low frequency activation of the stratum radiatum. In these experiments, the slices were equilibriated with the picrotoxinin containing medium. Intracellular EPSPs were recorded from CAly^ neurons in response to 0.2 Hz stimulation of the stratum radiatum. Membrane potentials of the neurons were clamped at -80 mV so if any potentiation of the EPSP occured after pairing, it would not result in the generation of action potentials (contamination by action potentials prevents an accurate assessment of the slope of the potentiated EPSP). Before saccharin was applied, control EPSP slopes were recorded for 10 minutes. Saccharin (10 mM) was then superfused onto the slices for 10 minutes. During the 8th minute of drug superfusion, pairing was performed. At the end of the pairing, the superfusing media containing saccharin was exchanged for the normal superfusing media. Two additional pairings were executed after a 20 minute and 50 minute recovery from the drug. 16.5. Specific Intracellular Studies on Saccharin 16.5.1. Saccharin and NMDA receptor-mediated responses. Since the induction of LTP by a high frequency tetanus is dependent upon an influx of calcium through ionophores coupled to the NMDA receptor (CoUingridge et. al.. 1983; CoUingridge and Bliss, 1988; Regher and Tank, 1990), experiments were conducted to determine if saccharin altered responses mediated by activation of NMDA receptors. To examine this possibility, three different experiments were performed. During a tetanic stimulation of the C A l neuron, a depolarizing plateau develops. The late component of this depolarizing plateau has been shown to be reduced by the NMDA receptor antagonist, APV and has been interpreted as being necessary for the induction of LTP in these neurons (CoUingridge et. al.. 1988; Haung et. al.. 1986). Whether saccharin interfered with the late NMDA dependent component of the depolarizing plateau was tested. The depolarization produced by a tetanic stimulation (400 Hz, 200 pulses) of the stratum radiatum was monitored before and during the last minute of a 10 minute appUcation of 10 m M saccharin. For comparisons, identical tetanic stimulations were given to the same CAlj^ neuron while superfused with media containing either 10 ^ M CNQX or 25 |iM D(-)APV. In impaled CAl-^ neurons exposed to a Mg2+-free medium, depolarizations induced by bath applied NMDA (10 |xM; 30 sec) were monitored in the presence and absence of a 10 minute application of saccharin. The input resistance of the cells were also monitored by injecting hyperpolarizing rectangular current pulses (-1 nA; 200 msec) at a frequency of 0.1 Hz. In separate experiments, to reduce the possibility that the NMDA induced depolarizations were not contaminated by depolarizations produced by a non-specific action of NMDA on non-NMDA receptors, the non-NMDA receptor antagonist, CNQX (10 ^M) was added to the Mg2+-free media. In this experiment, 0.1 |J.M TTX was also present in the media Experiments were also conducted to examine the effects of saccharin on NMDA and non-NMDA components of the intracellular EPSP slope. In these experiments, EPSPs were evoked by stimulating the stratum radiatum at 0.2 Hz. The EPSPs were evoked in either a normal medium, a normal medium containing 40 |J.M APV or in a Mg2+-free medium containing 10 |iM CNQX. Stable control responses were obtained for 10 minutes before the slices were exposed to saccharin. Records were acquired at the 10th minute of a 10 minute application of 10 m M saccharin after which the slices were resuperfused with their respective control media. Additional responses were recorded after a 20 minute recovery from saccharin. 16.5.2. Saccharin and input-output (I-O) relationships of the EPSP and the IPSP. Experiments were conducted to investigate the "input-output" (I-O) relationship of a CAl^^ pyramidal cell EPSP and IPSP in the presence and absence of saccharin. Control EPSPs were evoked in a picrotoxinin containing medium by stimulating the stratum radiatum at 0.1 Hz. The stimulation strength was adjusted from 20 to 80 |JA and the corresponding height of the evoked EPSP was measured at fixed values set within this range. A n identical stimulating protocol was performed during the last minute of a 10 minute application of 10 m M saccharin. l-O curves were constructed by plotting EPSP height against stimulation intensity for the control responses and responses to saccharin. In separate experiments, the I-O relationship of a CAl]^ pyramidal cell IPSP was determined using the experimental protocol described for the EPSP. I-O plots were made for the IPSP evoked in the normal (control) superfusing media and the superfusing media containing saccharin. The stimulation intensities for this particular experiment were set between 20 to 40 \xA. 16.5.3. Saccharin and IPSPs. Because the inhibltoiy control exerted by the local intemeurons is quite significant to determining the overall excitability of the pyramidal cell, the effects of saccharin on GABA-mediated inhibitory synaptic transmission was also examined in the CAl]-, neurons. For these particular experiments it was necessary to eliminate all excitatory neurotransmission so that only the IPSPs and not the EPSPs were elicited during activation of the stratum radiatum. Therefore, the slices were superfused with medium containing 20 |iM CNQX and 40 fxM APV, In order to record IPSPs in this medium, the stimulating electrode was placed in the stratum radiatum approximately 0.5 mm from the cell under investigation, IPSPs recorded in this manner have been interpreted as resulting from activation of inhibitory pathways through stimulation of local intemeurons (CoUingridge et. al.. 1988; Davies et. al.. 1990). The resulting blphasic IPSP, characterized by a fast and a slow component, has previously been shown to arise from activation of GABA^ and GABAg receptors, respectively (Newberry and Nicoll, 1985). The effects of saccharin on the fast and slow components of the IPSP were examined. In these experiments the stratum radiatum was stimulated at 0.1 Hz and control IPSPs were monitored for stability for at least 10 minutes. Saccharin (10 mM) was then applied for 10 minutes. At the end of the application, the slices were resuperfused Avith the CNQX and APV containing medium. Permanent records were taken before, during the last minute of, and following a 15 minute recoveiy from saccharin application. Separate experiments using the same protocol were conducted except 20 |iM picrotoxinin was added to the modified superfusing media in order to investigate the effects of saccharin on the slow IPSP. 16.5.4. Saccharin and QX-314 injected neurons. The quartemary lidocaine derivative, QX-314 has has been reported to block fast Na"^-dependent action potentials (Conners and Prince, 1982). Recently, QX-314 has been reported to block the slow IPSP when injected into hippocampal neurons (Nathan et. al.. 1990). This effect appears to be confined to the postsynaptic cell since the molecule is positively charged, under normal physiological conditions. Thus, to test whether saccharin could alter the fast IPSP in the absence of the slow IPSP, CAlj^ neurons were injected with QX-314. In these experiments, the stratum radiatum was stimulated at 0.1 Hz to elicite an EPSP-fast IPSP-slow IPSP sequence. QX-314 was injected into the neuron through the microelectrode AVith depolarizing current (+1 nA, DC current). Stimulation of the stratum radiatum was stopped during the injection procedure to prevent pairing. After obtaining stable control records, saccharin (10 mM) was applied for 10 minutes. Traces of the postsynaptic potentials were taken before and during the 9th minute of saccharin application for subsequent analysis. 16.5.5. Saccharin and paired-pulse depression of the fast IPSP. Paire d-Pulse depression of the fast IPSP has been extensively studied in the hippocampus and is thought to involve a decrease in release of GABA due to activation of GABAg autoreceptors on the pres5niaptic terminals of inhibitory interneurons (see chapter 12.3.1). Presumably, GABA released by the first pulse activates presynaptic GABAg receptors which, in turn, reduces transmitter release evoked by the second pulse. This appears as a decrease in amplitude of the fast IPSP arising from the second pulse when compared to the one evoked by the first. Recently, the induction of LTP in the C A l region of the hippocampus has been shoAvn to be blocked by a selective GABAg antagonist (Davies et. al.. 1991) the effect, of which, has been attributed to a reduction in GABAg mediated autoinhibition of GABA release. In view of the above, the possibility that saccharin blocked the induction of LTP through an action at the pres5niaptic GABAg receptors was examined. Paired-pulse depression of the fast IPSP was performed to assess the effects of saccharin on the presynaptic activity mediated by activation of the GABAg receptors. In these experiments, slices were superfused with medium containing 20 |iM CNQX and 40 |iM APV. IPSPs recorded from C A l ^ neurons injected with QX-314 were elicited by paired-pulse stimulations (interstimulus interval, 300 msec) of the stratum radiatum approximately every 30 seconds (0.033 Hz). IPSP pairs were recorded in the control superfusing media, during the last minute of a 10 minute application of 10 m M saccharin, 16.5.6. Saccharin and paired-pulse responses. During paired-pulse facilitation of the EPSP, the EPSP evoked by the second pulse is superimposed upon the fast IPSP arising from the first pulse. The facilitation of the second EPSP with respect to the first may not solely depend upon an augmentation of transmitter release. Instead, the hyperpolarization produced by the IPSP may also contribute to the increased height of the EPSP since it is moving the membrane potential to a more negative potential and, therefore, further away from the equilibrium potential of the EPSP. This has the effect of increasing the ionic driving force for the EPSP and is reflected as an increase in EPSP amplitude. Alternatively, the hyperpolarization can also temporarily increase the firing threshold of the neuron. If saccharin can potentiate either the amplitude or the duration of the fast IPSP then it may block tetanus induced LTP by preventing the neuron from depolarizing to the voltage which the NMDA receptor is activated. In addition, since the fast IPSP has also been reported to shunt the EPSP in pyramidal neurons (Dingledine and Langmoen, 1980), summation of EPSPs during tetanic stimulation of the affererent may not be sufficient to depolarize the neuron to the voltage range nessesaiy for activation of the NMDA receptor. To investigate this possibility, experiments were conducted to examine the effects of the fast IPSP on paired-pulse facilitation of the EPSP, first, in normal (control) medium and then in medium containing saccharin. For these experiments, the EPSP-fast IPSP-slow IPSP sequence was recorded from C A l ^ neurons in response to stimulating the stratum radiatum at 0.033 Hz. Paired-pulse facilitation of the EPSP was elicited by delivering two identical shocks (interstimulus interval, 40 msec) to the stratum radiatum. Records were obtained in the control superfusing media and during the 9th minute of a 10 minute application of 10 m M saccharin. In these experiments, neurons were injected with QX-314 to block the slow IPSP. 16.5.7. Saccharin and LTP in picrotoxinin containing media. In order to determine if saccharin blocked tetanus induced LTP by augmenting the fast IPSP, the following experiments were performed. Hippocampal slices were expose to the picrotoxinin containing media. Simultaneous extracellular and intracellular EPSPs were recorded from the CAl^ region of the guinea-pig hippocampus. The EPSPs were evoked by stimulating the stratum radiatum at 0.1 Hz. After obtaining control EPSPs, the slices were superfused with 10 m M saccharin for 10 minutes. During the last minute of application, a tetanic stimulation (two trains: 100 Hz, 100 pulses each separated by 20 sec) was delivered to the stratum radiatum. The slices were then resuperfused with the picrotoxinin media and the post-tetanic responses were monitored for an additional 30 minutes. In control experiments the same protocol was used except the slices were not superfused with saccharin. 17. RESULTS 17.1. Saccharin and LTP of the Fleld EPSP When slices were superfused with 10 m M saccharin for 10 minutes a tetanus (400 Hz, 200 pulses) delivered to the stratum radiatum during the last minute of drug application produced a post-tetanic potentiation (PTP) of the field EPSP slope that quickly decayed within 10 minutes. Thereafter, LTP of the EPSP slope was not observed in the 6 slices tested (Figure 17.1b). In separate control experiments, slices not exposed to saccharin exhibited a PTP of the EPSP slope following an identical tetanus which was approximately two times greater than that observed from slices exposed to the drug (Figure 17,1a). In all of the control experiments (n=6), the PTP decayed to form a stable LTP of the population EPSP which was on average twice the pre-tetanic control responses (Figure 17,1a). These results demonstrate that saccharin blocks LTP of the field EPSP an effect that parallels the action of the drug on LTP of the population spike as previously described by Chirwa (1988). 17.2. Saccharin and LTP of the Intracellular EPSP Following a 10 minute application of 10 m M saccharin a tetanic stimulation (400 Hz, 200 pulses) of the stratum radiatum produced a PTP of the intracellular EPSP slope that rapidly decayed to levels approaching the pre-tetanic control values which persisted unti l the experiment was terminated (Figure 17.2b). This was observed for all 9 slices tested. However, in control experiments where the slices were not superfused with sacchcirin, the same tetanic stimulation induced a PTP that was, on average, two times greater than that observed in saccharin treated slices. Following the PTP, LTP was observed in all 9 slices tested (Figure 17.2a). It, therefore, appears that saccharin blocks LTP of the population spike and field EPSP by exerting its effects on the individual CAl-^ neurons. 3 5 0 T CL O 3 0 0 - -m o i 250-. ^ 2 0 0 -3 ^ 05 ° - CO 1 5 0 -p o uX U J 1 0 0 8 0 O a 1 mV 20 ms i h à- T. T. 1 i 1 i i i i i i ^ I I O y 0 2 0 3 0 4 0 5 0 6 0 T i m e ( m i n ) 7 0 a 8 0 9 0 Figure 17.1. Saccharin blocks LTP of the field EPSP slope. The field EPSP slope was recorded prior to and after a tetanic stimulation (arrow) of the stratum radiatum. This procedure was executed in slices exposed to 10 m M saccharin for 10 minutes (closed circles) and i n normal medium without saccharin (open circles). Note that LTP does not develop in the presence of saccharin but does i n the normal medium. Superimposed traces at the top of the graph illustrate field EPSPs averaged fix)m four consecutive responses i n control a and saccharin b treated slices taken at times denoted by x and y. Solid bar on the abscissa represents the duration of saccharin application. Calibration bar represents 1 mV and 20 msec. Data are represented as mean ± S E M . Asterisks tadlcate significant differences from the control veilues taken prior to the tetanus as calculated by a one-way ANOVA with Duncan's multiple comparison test (p<0.05, n=6 sUces). 0) û. _o _ CO o û- c 00 o CL CJ LxJ o ° 13 ° u en o o 2 5 0 T 2 0 0 - -1 5 0 - -O T O y y b 4 T* T * o A^* y O X* T: 5 m V 20 ms l o o i o î Q o ^ • • i é ? ^ i ? 9*9 g .* T , O 0 O ' • i 1 • • i T y 0 10 2 0 3 0 4 0 5 0 6 0 T i m e ( m i n ) -H h-7 0 8 0 9 0 Figure 17.2. LTP of the intracellular EPSP slope is blocked bv saccharin. The slope of the intracellular EPSP was recorded before and after a tetanic stimulation (arrow) of the stratum radiatum i n the presence (closed circles) and absence (open circles) of 10 m M saccharin applied for 10 minutes (solid bar on the abscissa). Note that LTP is present only tn those slices not exposed to saccharin. A t the top of the graph, superimposed traces represent EPSPs averaged from four consecutive réponses In control a and saccharin b treated slices recorded at times denoted by x and y. Calibration bar represents 5 mV and 20 msec. Data are represented as mean ± S E M . Asterisks Indicate significant differences from the control responses taken prior to the tetanus as calulated by a one-way ANOVA with Duncan's multiple comparison test (p<0.05, n=9 slices). 3 . 5 0 0 T ^ 3 . 0 0 0 i E 2 . 5 0 0 ^ 2 . 0 0 0 -c o 'o 1 . 5 0 0 - -3 ^ 1 . 0 0 0 + 0 . 5 0 0 0 O o 2 0 y o o ^ o o =(t T O o y 0 1 4 0 6 0 T i m e ( m i n ) O 8 0 0.5 mV 20 ms 1 0 0 Figure 17.3. Maintenance of LTP is not blocked bv saccharin. A post-tetanic application of saccharin (solid bar on the abscissa) failed to prevent LTP of the population spike from developing. Traces located at the top of the graph are averaged from 8 consecutive responses taken from a typical experiment at times denoted by x, y and z. Calibration bar represents 0.5 mV and 20 msec. Data are presented as meein ± S E M . Asterisks Indicate significant difierences from the control responses as calculated by a one-way ANOVA with Duncan's multiple comparison test (p<0.05. n=6 slices). 17.3. Saccharin and Maintenance of LTP In all 6 slices tested PTP of the population spike was observed following a single tetanic stimulation (400 Hz, 200 pulses) of the stratum radiatum. A subsequent application of 10 m M saccharin for 10 minutes failed to prevent LTP of the population spike from developing. Significant potentiation was still present after a 30 minute recovery from the drug (Figure 17,3). Though significant differences were observed between control responses and post-tetanic responses, differences between the responses obtained during the post-tetanic application of saccharin and the responses recorded after its withdrawal were not significant. These results demonstrate that saccharin selectively interferes with the induction and not the maintenance of LTP. 17.4. Sacchcirin and Pairing When postsjmaptic depolarization was paired with activation of the stratum radiatum during the last minute of a 10 minute application of 10 m M saccharin, LTP of the intracellular EPSP slope was not observed (Figure 17.4). If the pairing was performed after a 20 minute recovery from the drug, LTP occured (Figure 17.4). A subsequent pairing done 30 minutes later, however, produced no further lasting increases in the EPSP slope (Figure 17.4). Similar results were observed in two other CAlj-, neurons each obtained from different slices. 17.5. Saccharin and NMDA Receptor-Mediated Responses 17.5.1. Saccharin and tetanus-induced depolarizations. In normal medium, a tetanic stimulation of the stratum radiatum (400 Hz, 200 pulses), produced a postsynaptic response which consisted of a series of action potentials superimposed upon a depolarizing plateau as shown in Figure 17.5A. The depolarization persisted unti l the end of the train upon which a slow hyperpolarization ensued. In the presence of saccharin, an identical tetanus produced a depolarization that was longer in duration than if) E > c Q-O (71 iX (y") CL y+z x + z 5 m V 50 ms 3 i 2 -2 A A 3 • A • y 0 10 2 0 3 0 —I 1 1 1 1^ 1 5 0 5 0 7 0 8 0 9 0 1 0 0 4 0 T i m e ( m i n ) Figure 17.4. Saccharin reversibly blocks pairing-induced LTP of the intracellular EPSP slope. The slope of the intracellular EPSP was measured and plotted as a function of time (triangles). During the 8th minute of application of 10 m M saccharin (solid line on the abscissa), pairing was done (arrow). Two additional pairings were carried out in normal medium after a 20 minute and 50 minute recovery from the drug. Traces at the top of the graph are averages of four consecutive responses taken at times denoted by x, y and z. Some traces are superimposed for comparisons of the EPSP slopes recorded at their respective times. The membrane potential of the CA l^ , neuron was clamped at -80 mV. A B C Control Saccharin (10 mM) - A P V (25 nM) '^f/^ ' -^—'^L/^'^t CNQX ( 10 jiM) E 30 min Recovery A+B 5mvL_ 400 Hz, 0.2 s 200 pulses Figure 17.5. Denolarlzations induced bv tetanic stimulations are not blocked bv saccharin. A shows the depolarization of a C A l neuron recorded tn normal (control) medium arising from a tetanic stimulation of the stratum radiatum (solid bar below the trace). A n Identical tetanus was given to the same neuron superfused with media containing B, 10 m M saccharin; C, 25 pM D(-)APV; D. 10 nM CNQX and E, normal medlvmi after a 30 minute recovery from D. A+B are superimposed traces of the depolarizations recorded in the control medium and tn medium containing saccharin. Note that action potentials Induced by the tetanus are cut-off i n the traces. the control response and the post-tetanic hyperpolarization did not occur (Figure 17.5B and 17.5A+B). For comparisons, an identical tetanic stimulation was administered to slices superfused with media containing either 25 |J,M D(-)APV or 10 |iM CNQX (Figure 17.5C and 17.5D, respectively). Compared with the control response, the depolarization and post-tetanic h5^erpolarization produced in APV containing medium were slightly reduced and those in CNQX containing medium were completely abolished. Following a 30 minute resuperfusion with the normal medium, the depolarization and post-tetanic hyperpolarization were restored (Figure 17.5E). 17.5.2. Saccharin and depolarizations produced by applied NMDA. Because depolarizing responses in C A l neurons decrease to repeated applications of exogenously applied NMDA (a phenomenon thought to reflect desensitization), care was taken to allow sufficient time between repeated NMDA applications to prevent desensitization from occuring. In a Mg2+-free medium, consistent non-decrementing depolarizations were obtained if the 30 second applications of 10 |iM NMDA were separated by at least 5 minutes. When slices were superfused with 10 m M saccharin for 10 minutes, no change in membrane potential or input resistance was observed (Figure 17.6A). A subsequent application of NMDA during the last minute of saccharin application produced a depolarization that did not differ significantly from the depolarization produced by NMDA prior to application of the drug (peak depolarization to NMDA in mV, control: 16 ± 4; during the last min of saccharin application: 15 ± 4; n=6 slices). The ratio of input resistances (R^n) measured just prior to and at the peak of the NMDA depolarization was also not significantly altered by saccharin (ratio of Rj^ to applied NMDA. control: 0.6 ±0.1; during the last min of saccharin application: 0.6 ± 0.2; n=6 slices). Similar results were also obtained for slices exposed to a Mg2+-free medium containing 10 |iM CNQX and 0.1 |iM TTX (peak depolarization to NMDA in mV. A Mg2+-free Sacdiafta(IOinM) -APV(25(iM) J* NMOA(IOtiM) 60 mV 3min B Mg2+-frae and CNQX (10 MM) Saccharin (10 mM) -APV(25MM) NM0A(10|iM) 30 mV 3min Figure 17.6. Depolarizations Induced bv applied NMDA are not significantly altered to the presence of saccharin. A shows depolarizations of a CA l^ , neuron recorded to a Mg^^-free medium to response to bath applications of 10 |JM NMDA superfused for 3 0 seconds (soUd bar below the trace). NMDA was applied before, during and after a 10 mtoute application of 1 0 m M saccharin. The neuron was later exposed to 25 \M D(-)APV for 5 mtoutes to block the NMDA toduced depolarization. B. the depolarizations of a CAl^ neuron toduced by applied NMDA were performed to a Mg2'''-free medium contatotog 10 nM CNQX and 0.1 \iM tetrodotoxto. Traces to A and B were recorded from two different cells. control: 19 + 2; during the last min of saccharin application: 18 ± 2; ratio of Rin to applied NMDA, control: 0.7 ± 0.1; during the last min of saccharin application: 0.7 ± 0.1; Figure 17.6B, n=6 slices). 17.5.3. Saccharin and NMDA and non-NMDA components of the intracellular EPSP. Previous experiments demonstrated that saccharin did not alter the height of the intracellular EPSP in slices superfused with a normal media (Chirwa, 1988). The same was observed for the slope of the intracellular EPSP (slope of the EPSP in V/sec, control: 1.94 ± 0.50; during the last min of saccharin application: 1.88 ± 0.51; Figure 17.7A and 17.7D, n=6 slices). Additional experiments were conducted to examine if saccharin altered the slope of the non-NMDA and NMDA components of the intracellular EPSP. To isolate the non-NMDA component, slices were superfused with a normal medium containing 40 |iM APV. In these experiments, a 10 min application of 10 m M saccharin did not significantly alter the non-NMDA EPSP slope (slope of the EPSP in V/sec, control: 1.71 ± 0.25; during the last min of saccharin application: 1.58 ± 0.22 ; Figure 17.7B and 17.7D, n=6 slices). After a 20 min recoveiy from saccharin, a subsequent application of 10 |iM CNQX blocked the EPSP, confirming it was dependent upon S5niaptic activation of the non-NMDA receptors (last trace in Figure 17.7B), The NMDA component of the EPSP was obtained from slices superfused with a Mg2+-free medium containing 10 ^iM CNQX. During a 10 minute application of 10 m M saccharin, the slope of the NMDA mediated EPSP was not significantly altered (slope of the NMDA EPSP i n V/sec, control: 0.52 ± 0.05; during the last min of saccharin application: 0.53 ±0.05; Figure 17.7C and 17.7D, n=6 slices). After a 20 minute recovery from the drug, addition of 40 |iM APV to the superfusing media completely abolished the EPSP and, therefore, demonstrated the EPSP resulted from synaptic activation of the NMDA receptors (last trace. Figure 17.7C). Saccharin (10 mM) ±APV and CNQX NoftnaJ medium B Normal medium and±APV(40nM) r C Mg2+-free and CNQX (10 |iM) Control 10 min 20 min Recovery 10 mV 100 ms 3.000 T Q. O 2.000 •• ^ 1.000 Q. U 0.000 Normal medium Normal medium and ±APV Mg free and CNQX c 2 2 O o o u o 11 c o o a U CO Figure 17.7. The intracellular EPSP slope recorded in the presence of excitatory amino acid antagonists is not significantly altered by saccharin. In A, the EPSPs were recorded i n a normal meditun. The first trace In A was taken prior to a 10 minute application of 10 m M saccharin. The following trace shows the EPSP recorded at the 10th minute of saccharin application. The last trace Illustrates the EPSP recorded in the normal medium after a 20 minute recovery from the drug. EPSPs were also recorded tn normal medium and 40 |iM APV, B and in Mg2+-free medium containing 10 pM CNQX. C. The last trace In B and C Illustrate the block of synaptic responses when both APV and CNQX were present In their respective media. D, shows a histogram of the EPSP slopes recorded in the different media In the absence (blank histogram) and presence (cross-hatched histogram) of saccharin. Traces in A to C are averaged from 4 consecutive responses. Data are represented as mean ± S E M . Asterisks indicate significant differences from the control as calculated by a Student's t-test (p<0.05, n=6 slices each) Taken together, these results demonstrate that saccharin does not interfere with responses mediated through sjmaptic activation of the NMDA and the non-NMDA receptors. 17.6. Saccharin and Input-Output fl-O) Relationships of the EPSP and the IPSP In normal medium containing 20 |iM picrotoxinin, activation of the stratum radiatum with increasing stimululus intensities (20 to 80 |aA) evoked intracellular EPSPs recorded from a CAl^^ neuron that correspondingly-increased in amplitude as illustated in Figure 17.8A. A plot of the maximal EPSP height against stimulation strength produced a non-linear I-O plot which plateaued as the higher stimulation strengths were approached (Figure 17.8B). When the same stimulating protocol was performed during the last minute of a 10 m M application of saccharin, the I-O relationship of the EPSP remained primarily unchanged (Figure. 17.8B). In contrast, stimulation of the stratum radiatum (20 to 40 (iA) i n media which did not contain picrotoxinin resulted i n the maximal height of the IPSP being increased during the application of saccharin (Figure 17.8C). The resulting non-linear I-O plot constructed for the intracellular IPSPs was proportionately shifted upwards in the presence of saccharin as illustrated in Figure 17.8D. These results indicate that saccharin preferentially affects IPSPs and further demonstrates its lack of effect on EPSPs. 17.7. Saccharin and Postsynaptic Potentials Recorded in Mg^+-free Medium To further show that saccharin selectively affects the IPSP, the following experiments were performed. In a Mg2+-free medium containing 10 |j,M CNQX, stimulation of the stratum radiatum produced intracellularly recorded postsynaptic potentials consisting of an NMDA EPSP followed by an IPSP (Figure 17.9A). Application of 10 m M saccharin decreased the duration of the NMDA EPSP and increased the amplitude of the IPSP (Figure 17.9B). After a Control Saccharin B > J, Q. t/) Q. 20 15 10 Control Saccharin 10 mV 40 ms 20 40 60 Stimulation ( ^ ) 80 100 > to Û-10 4-10 mV zooms 10 20 30 40 Stimulotion (/iA) 50 Figure 17.8. The effects of saccharin on the input-output (I-O) relationship of EPSPs and IPSPs recorded from C A l ^ neurons. A illustrates a series of EPSPs recorded from a CA l^ , neuron each being evoked by stimulating the stratum radiatum with increasing stimulation strengths. Traces i n A depict EPSPs elicited i n picrotoxtntn containing medium before and during the last minute of a 10 minute application of 10 m M saccharin. B illustrates the corresponding I-O plot for the EPSPs recorded in the picrotoxinin containing medium (open circles) and medium containing saccharin (closed circles). C shows a series of IPSPs evoked i n normal medium and during the last minute of the saccharin application using a similar stimulation protocol as that described In A. D Illustrates the corresponding I-O graph for the IPSPs evoked i n the normal medium (open circles) and saccharin contcdning medium (closed circles). Traces tn A and C represent responses evoked by their corresponding stimulation intensities (in pA) located to the left of the stimulation artifacts (arrow). I-O graphs are constructed by plotting the height of the responses against their corresponding stimulation Intensities. 10 minute recoveiy in the CNQX containing medium, 40 ^iM APV was added. The NMDA EPSP was completely abolished revealing an underlying IPSP (Figure 17.9C). A subsequent application of saccharin enhanced the height and the duration of the IPSP (Figure 17.9D). When all four traces were superimposed upon one another, it became apparent that the distortion in the shape of the NMDA EPSP by saccharin was, in fact, produced by the increase in duration of the fast IPSP (Figure 17.9E). 17.8. Saccharin and IPSPs In order to examine the effects of saccharin on the IPSPs, excitatory synaptic transmission was blocked by superfusing the slices Avith normal media containing 40 |j.M APV and 20 |iM CNQX. Simulation of the stratum radiatum by an electrode positioned in close proximity to the recording electrode evoked monosynaptic IPSPs characterized by a fast IPSP (IPSP^) -slow IPSP (IPSPg) sequence as seen in Figure 17.1 OA. During the last minute of a 10 minute application of 10 m M saccharin, the peak height of the fast IPSP and similarity the fast inhibitory postsynaptic current (IPSC^) was not significantly altered from the control response as shown in Figure 17.10A, 17.10B and Table 17.1. At the same time, however, the time for the fast IPSP/C (IPSP/C^) to peak (latency to peak) after stimulation of the stratum radiatum was significantly increased (Table 17.1). In contrast, the height of the slow IPSP/C (IPSP/Cg) which followed the fast IPSP/C was reduced in the presence of saccharin (Figure 17.1 OA and 17.1 OB). Because the decay of the fast IPSP was increased in saccharin, it was difficult to assess the drug's effects on the slow IPSP/C since the decay of the fast IPSP/C interfered with the onset of the slow IPSP/C. Therefore, in separate experiments, 20 \iM picrotoxinin was added to the superfusate to block the fast IPSP. In these experiments, the latency to peak of the slow IPSP/C was not significantly altered from the control value in the presence of saccharin (Figure 17.11A and Mg2+-free + CNQX (10 jiM) 10 mV 200 ms Figure 17.9. Saccharin potentiates the IPSP during activation of the stratum radiatum. In a Mg2''"-free medium containing 10 nM CNQX. stimulation of the stratum radiatum produced an EPSP followed by an IPSP In the CAl i^ neuron, A. B shows the response In the presence of 10 m M saccharin applied for 10 minutes. After a 10 minute recovery in the CNQX containing Mg2"'"-free medium, 40 pM APV was added which blocked the NMDA-dependent EPSP and revealed an underlying IPSP, C. In D, saccharin was applied again. Note that compared to C, the ampUtude and duration of the IPSP in D were both enhanced. These changes are clearly illustrated when the traces are superimposed In E. Each trace represents the average of four consecutive responses recorded from the same cell. 4mV 0.2 nA 400 ms B Figure 17 .10 . Saccharin antagonizes the late component of the IPSP/C. A shows IPSPs recorded from a CAl^^ neuron i n normal (control) medium, during the last minute of a 10 minute application of saccharin and following a 10 minute recovery In the normal medium. B shows similar effects of saccharin on the IPSC. Stimulus artifacts in B are blanked for clarity. The normal medium and saccharin containing medium contained 4 0 yM APV and 2 0 |JM CNQX to block excitatory synaptic transmission. Traces are the average of four consecutive responses. Traces In A and B were recorded from two different cells. T A B L E 17.1. Effects of saccharin on some properties of the IPSP/Cs. IPSP Height Latencv to Peak fms) Control Saccharin Control Saccharin n I P S P / C A 100 96 ± 2 16 ± 1 26 ± 4* 6 I P S P / C g 100 57 ± 5* 245 ± 8 6 I P S P / C A and I P S P / C g were evoked In media containing 40 pM APV and 20 pM CNQX to block synaptic excitation. Control responses were taken as 100%. Responses to saccharin were expressed as a percent of the controls and then averaged. Data are presented as mean + S E M . Asterisks Indicate signtflcant differences from the control responses as calculated by Student's t-test (two-tailed: p<0.05). 17.1 IB, Table 17.2). In addition, the time for the slow IPSP/C to reach half its maximal response (duration at 1/2 max) was also not significantly altered by the drug (Figure 17.1 l A and 17.1 IB, Table 2). However, the peak height of the slow IPSP/C was significantly reduced during the application of saccharin (Figure 17.1 l A and 17.1 IB, Table 2). 17.9. Saccharin and Fast IPSPs Recorded in Neurons Injected With QX-314 In order to determine whether saccharin could alter the decay phase of the fast IPSP, C A l ^ neurons were injected with QX-314 to block the slow IPSP. Neurons injected with QX-314 produced a fast IPSP upon stimulation of the stratum radiatum which was not followed by a slow IPSP as illustrated in Figure 17.12. In these cells, compared to the control IPSPs, application of 10 m M saccharin for 10 minutes caused a significant increase in the maximal height of the fast IPSP as well as the time for the fast IPSP to reach half its maximal height (duration at 1/2 max) (Figure 17.13, Table 17.3). 17.10. Saccharin and its Effects on Paired-Pulse Depression of the Fast IPSP Because saccharin was capable of antagonizing the postS3niaptic G A B A Q responses, experiments were conducted to determine if the drug was capable of blocking responses mediated by activation of the pres3maptic GABAg receptors. In these experiments, excitatory S3niaptic transmission was blocked by superfusing the slices with a normal medium containing 40 |iM APV and 20 |J.M CNQX. Since previous results showed that the peak amplitude of the slow IPSP/C occured between 250 to 300 msec (see Tables 1 and 2), the interstimulus Interval was adjusted so that the stimulating electrode delivered two identical pulses to the stratum radiatum each separated by 300 msec. In both current clamp and voltage clamp recordings, during the last minute of a 10 minute application of 10 m M saccharin, the height of the fast IPSP resulting from the second pulse (P2) when expressed as a percent of the response arising from the first pulse (PI), was not significantiy altered from the controls B 400 ms 4mV 0.2 nA Figure 17.11. The slow IPSP/C flPSP/CB) is decreased bv saccharin. A illustrates a slow IPSP recorded from a CA l^ , neuron In normal (control) medium, during the last minute of a 10 minute application of 10 m M saccharin and following a 10 minute recovery from the drug. B shows the effects of saccharin on the slow IPSC. St imulus artifacts In B were blanked for clarity. The slow IPSP/C were evoked in normal media containing 40 nM APV, 20 ^lM CNQX and 20 |iM picrotoxinin. Traces represent the average of four consecutive responses. Traces In A and B were recorded from two different cells. TABLE 17.2. Effects of saccharin on some properties of the IPSP/Cg. IP§P Hglght Latencv to Peak fms) Durat ion at 1II max fms) Control Saccharin Control Saccharin Control Saccharin n 100 53 ± 5* 247 ± 10 249 ± 12 503 ± 28 503 ±25 7 IPSP/Cg was Isolated using a media containing 20 \M. picrotoxinin. 40 pM APV and 20 |iM CNQX. The height of the control responses were normalized to 100% and responses to saccharin were expressed as a percent of the controls and then averaged. Data are presented as mean ± S E M . Asterisks Indicate signtflcant differences from the control values as calculated by Student's t-test (two-tailed; p<0.05). -62 i -70 4 10 mV 400 ms Figure 17.12. QX-314 blocks the slow IPSP. The top trace Illustrates a typical postsynaptic potential recorded from a CAl^-^ neuron to response to stimulation of the stratum radiatum. Note the slow IPSP Is reduced after 10 mtoutes and blocked after 20 mtoutes of tojecting QX-314 toto the neuron. The ntmabers to the left of the traces represent the potentials (to mV) at which the neuron was clamped. Traces are averaged from four consecutive responses. 10 mV 400 ms Figure 17.13. Saccharin increases the height and duration of the fast IPSP i n neurons injected with QX-314. In A, prior to a 10 minute application of 10 m M saccharin, a C A l ^ neuron injected with QX-314 produced a synaptic potential consisting of an EPSP followed by a fast IPSP when the stratum radiatum was stimulated. The trace In B shows the postsynaptic potential during the last minute of saccharin application. Note that In B, the peak height and duration of the IPSP are Increased when exposed to saccharin. This Is more apparent when the two responses are superimposed In A+B. Traces are averaged from four consecutive responses. T A B L E 17.3. Effects of saccharin on some properties of the IPSP^. IPSP Heiçht Duration at 111 max fmsl Control Saccharin Control Saccharin n 100 127 ± 7* 202 ± 14 235 ±12* 6 IPSP;^ was recorded from cells injected with QX-314 to block postsynaptic GABAQ charmels. The height of the control IPSPs were normalized to 100% and the corresponding responses to saccharin expressed as a percentage of the controls emd then averaged. Data are presented as mean ± S E M . Asterisks indicate significant differences from the control response as calculated by Student's t-test (two-tailed: p<0.05). measured prior to the drug application. In fact, rather than being decreased, saccharin increased the paired-pulse depression of the fast IPSP (P2 as a percent of PI , control: 63 + 3; during the last min of saccharin application: 54 ± 4; Figure 17.14A, 17.14B and 17.14C, n=6 slices). These results demonstrate that saccharin does not block paired-pulse depression of the fast IPSP and it is, therefore, unlikely that it antagonizes the presynaptic GABAg receptors. 17.11. Saccharin and Paired-Pulse Responses of Postsynaptic Potentials Since saccharin could potentiate the amplitude and duration of the fast IPSP, experiments were conducted to determine if such chainges could contribute to a reduction of paired-pulse faciliation of the EPSP. For these experiments, an interstimulus interval of 40 msec was chosen to separate the first pulse from the second in the stimulus pair. At this interstimulus interval, paired-pulse stimulation of the stratum radiatum produced a large facilitation of the second EPSP compared to the first in slices exposed to a normal medium (Figure 17.15A). However, in the presence of 10 m M saccharin applied for 10 minutes, this facilitation was not significantly decreased (P2 as a percent of P I , control: 687 ± 135; during the last min of saccharin application: 592 ± 145; Figure 17.15A and 17.15B, n=6 slices). In contrast, at the same interstimulus interval paired-pulse depression of the fast IPSP was significantly increased during the drug application (P2 as a percent of P I , control: 30 ± 6; during the last min of saccharin application: 14 ± 2; Figure 17.15A and 17.15B, n=6 slices). It, therefore, appears that by increasing the amplitude and duration of the fast IPSP, saccharin does not significantly alter paired-pulse facilitation of the EPSP but significantly decreases paired-pulse depression of the fast IPSP when 40 msec separate the first and second pulses of a paired-pulse. Control Saccharin (10 mM) 5mV B 0.1 nA 200 ms o à? o CO O CN CL IOOt 7 5 -5 0 - -2 5 -c o o o o o o CD Figure 17.14. Paired-pulse depression of the fast IPSP (IPSPji,^ ) Is not blocked bv saccharin. A shows current-clamp responses to a palred-pvilse stimulation (Interstlmulus Interval. 300 msec) of the stratum radlatima. These responses were evoked In normal (control) medlvim and during the last minute of a 10 minute application of 10 m M saccharin. B shows voltage-clamp responses In the control medium and saccharin containing medium. C Is a histogram representing the the peak amplitude evoked by the second pulse (P2) as a percent of the peak amplitude evoked by the first pulse (PI) In the control medium (blank histogram) and In saccharin containing medium (cross-hatched histogram). Asterisks Indicate significant differences from the control response as calculated by a one-tailed Student's t-test (p<0.05. n=6 slices). The normal medium and saccharin containing medlima contained 40 [iM APV and 20 nM CNQX. Traces In A and B are recorded from two different cells injected with QX-314. A 200 ms Figure 17.15. Efifects of saccharin on paired-pulse facilitation of the EPSP and palred-pulse depression of the fast IPSP i n neurons injected with QX-314. A illustrates both the EPSP and the fast IPSP arising from two Identical pulses (Interstimulus Interval 40 msec) delivered to the stratum radiatum. The responses were recorded In normal (control) medium and during the last minute of a 10 minute application of 10 m M saccharin. B shows a histogram representing the peak amplitude of the response evoked during the second pulse (P2) as a percent of the peak response arising from the first pulse (PI). Histograms were constructed for the EPSP and the fast IPSP evoked In normal medium (blank histograms) and i n medium containing saccharin (cross-hatched histograms). Data are represented as mean ± S E M . Asterisks Indicated significant differences from the control values as calculated by a two-tailed Student's t-test (p<0.05. n=6 slices). A l l neurons were Injected with QX-314 prior to taking the control resonses. 17.12. Saccharin and LTP in Picrotoxinin Containing Media Finally, to examine the possibility that saccharin blocked the induction of LTP through its potentiating effects of the fast IPSP, experiments were conducted to determine if LTP of both the extracellular and intracellular EPSP slopes could be tetanically induced in the presence of 10 m M saccharin when the fast IPSP was blocked by picrotoxinm. In these experiments, after the slices had been equilibriated with picrotoxinin containing medium, LTP of both the extracellular and intracellular EPSP slopes could not be induced after a tetanic stimulation (two trains of 100 Hz, 100 pulses each separated by 20 sec) was delivered to the stratum radiatum during the last minute of a 10 minute application of 10 m M saccharin (Figure 17.16b). However, in picrotoxinin treated slices not exposed to saccharin, robust LTP occurred for both the extracellular and intracellular EPSP slopes (Figure 17.16a). When comparing the PTP of both the extracellular and intracellular EPSP slope in control slices and slices exposed to saccharin, the PTP occuring immediately after the tetanic stimulation was decreased in the slices treated with saccharin. It, therefore, appeared that saccharin could still block LTP in the CAljh> region even if the fast IPSP was blocked by picrotoxinin. a M c o O o m O (U Q. _o CL CL 450 1 350-250-150-Extracellular ^ i 9 • 50 J 300 1 250-200-150-100O 0 m t r a c e M u l a r 2 » 8 10 o b -0 O O O T* 1 i 1 i O .a 1 mV 10 mV 20 ms 1 O 20 30 Time (min) 40 50 60 Figure 17.16. LTP i n plcrotoxliiln-treated slices is not blocked bv saccharin. Simultaneous extracellular and Intracellular EPSPs were recorded in the CAl]^ subfleld. In slices exposed to 10 m M saccharin for 10 minutes a tetanic stimulation (arrow) delivered to the stratum radlatiam failed to produce LTP of both the extracellular as well as the Intracellular EPSP slopes (closed circles). However, In slices not exposed to saccharin, LTP of both the extracellular and intracellular EPSP slopes occured following the tetanic stimulation (open circle). Data are represented as mean ± S E M . Asterisks Indicate significant differences from the control responses as calculated by a one-way ANOVA with Dimcan's multiple comparislons test {P<0.05, n=8 sUces). Superimposed traces at the top of the graphs are averages of four consecutive responses taken at times x and y in picrotoxinin containing media a and In picrotoxinin containing media with saccharin b. Calibration bar represents I mV, 20 msec for the extracellular EPSPs and 10 mV, 20 msec for the Intracellular EPSPs. 18. DISCUSSION The results presented in this study demonstrate that the induction of LTP in the CAl]-) region of the guinea-pig hippocampus is blocked by saccharin. This is supported by the obseryation that saccharin significantly decreased the duration of synaptic enhancement that followed a tetanic stimulation i n recordings of both the extracellular and the intracellular EPSP slope. The fact that LTP persisted after a post-tetanic application of saccharin suggested that the drug selectiyely interferred with some process inyolyed in the induction of LTP. Pairing sufficient postsynaptic depolarization with concommitent low frequency actiyation of the afférents has also been shown to produce LTP (Kelso et. al.. 1986; Sastry et. al.. 1986; Wigstrom et. al.. 1986). The associative nature displayed in the induction of this LTP has been attributed to the relief of the yoltage-dependent block of NMDA channels by extracellular Mg2+ and their simultaneous actiyation by stimulation of the afferent input (Gustaffson and Wigstrom, 1988; Collingridge and Singer, 1990). This results in a rise i n postsynaptic calcium, postulated to be a nessesary requirement for the induction of LTP (Lynch et. al.. 1983; Malenka et. al.. 1988; Malenka, 1991). The possibility that saccharin could interfere with pairing-induced LTP was tested and it was found that the drug reversibly interfered with the development of LTP in the CAlj-, neuron. Whether this effect was due to an action of the drug on the NMDA receptors was examined. Because the induction of LTP is thought to be a threshold phenomenon often requiring high frequency activation of a critical number of afferent fibers (McNaughton et. al.. 1978), the resultant summation of the postsynaptic depolarization also becomes an important factor in determining whether or not LTP is expressed in the neuron. Indeed, when the late NMDA-dependent component of the tetanus-induced depolarizing plateau is blocked by APV, subsequent development of LTP in these neurons is not observed (CoUingridge and Lester, 1988; Huang et. al.. 1988). Whether saccharin blocked the induction of LTP by interfering with the late NMDA component of the depolarizing plateau that arises from high frequency activation of the stratum radiatum was examined and it was found that saccharin, rather than depressing the depolarization, actually prolonged it. During a tetanic stimulation of the afférents, IPSPs become suppressed resulting in a prolonged decay phase of the non-NMDA EPSP (Larson and Lynch, 1986). The depolarization produced by repetitive activation of the non-NMDA receptors has been demonstrated to prime the membrane potential to the voltage range where the NMDA receptors are activated (Pacelli and Kelso, 1989). The possibility that saccharin could interfere with the activation of the NMDA receptors in this manner was unlikely since the early component of the depolarizing plateau was not blocked. The finding that the depolarizations and associated changes in input resistance produced by exogenously applied NMDA in a Mg2+-free medium or a Mg2+-free medium containing CNQX were not significantly altered in the presence of saccharin and that the slopes of the non-NMDA EPSP or NMDA EPSP were also not significantly altered by the drug further supported the notion that saccharin did not antagonize the NMDA as well as the non-NMDA receptors. 18.2. Effects of Saccharin on Inhibitory Svnaptic Transmission The first evidence which suggested that saccharin preferentially affected inhibitory synaptic transmission was from intracellular recordings where IPSPs were prominant in the synaptic responses. For example, with respect to the I-O plot for the EPSP, the I-O plot for the IPSP was proportionately shifted upward in the presence of saccharin. The idea that saccharin exerted a selective action on the IPSP was further supported when it was demonstrated that saccharin decreased the duration of the NMDA EPSP by enhancing the duration of the underlying IPSP. It appeared that this effect was, in fact, due to the IPSP and not to a depression of NMDA receptor-mediated synaptic transmission per se because only the duration and not the slope of the NMDA EPSP was reduced in the presence of saccharin. It was possible that saccharin decreased the duration of the NMDA EPSP, however, in recordings where the contribution of the IPSP was minimal (neurons with RMPs approaching -70 mV or in picrotoxinin-containing media), the duration of the NMDA EPSP was not reduced in the presence of the drug. Because recent investigations have provided evidence suggesting that S)niaptic activation of GABA^ receptors (Wigstrom and Gustaffson, 1983 Wigstrom and Gustaffson, 1986) and presynaptic GABAg receptors play an important role in modulating the induction of LTP in the hippocampus (Davies et. al., 1991; Mott and Lewis, 1991; Olpe and Karlsson, 1990), it was necessary to study the actions of saccharin on inhibitory neurotransmission and to determine if these effects had any influence on blocking the induction of LTP. The enhanced duration of the fast IPSP induced by saccharin is similar to the effects of pentobarbitone on the fast IPSP (Nicoll et. al.. 1975). However, unlike pentobarbitone which produces a depolarizing IPSP (Alger and Nicoll, 1982), no such potential was observed when saccharin was present. Diazepam produces a similiar effect to pentobarbitone on hippocampal neurons, but the depolarizing IPSP is only observed when the membrane is polarized to the reversal potential of the fast IPSP. However, no depolarizing IPSP was observed in the presence of saccharin when the membrane potential was clamped at the fast IPSP's reversal potential. Therefore, it was unlikely that saccharin altered the IPSP in the same manner as the barbiturate or benzodiazepene. Alternatively, saccharin may have interfered with GABA uptake. Indeed nipecotic acid, an inhibitor of GABA uptake, also enhances the IPSP (Alger and Nicoll, 1982). Saccharin caused some suppression of the GABAg receptor-mediated slow IPSP. It was unlikely that this effect was due to a shift in extracellular potassium levels since such an effect would alter the resting membrane potential; saccharin did not exhibit this effect. Recently, Davies et. al. (1991) and Mott and Lewis (1991) demonstrated that pharmacological blockade of the presjmaptic GABAg receptors rather than the postsynaptic GABAg receptors inhibits tetanus-induced LTP in hippocampal neurons through a mechanism involving autoinhibition of GABA release. Based on these reports, it was conceivable that saccharin blocked the induction of LTP by antagonizing the presynaptic GABAg receptors. However, saccharin did not reduce the paired-pulse depression of the fast IPSP, an effect that is thought to reflect blockade of the presynaptic GABAg receptors (Davies et. al.. 1990; Thompson and Gahwiler, 1989). Thus, an antagonism of presynaptic GABAg autoreceptors appeared not to account for the ability of saccharin to block the induction of LTP. These results taken together with results previously presented by Chirwa (1988) demonstrate the lack of effects of saccharin on paired-pulse depression and paired-pulse facilitation both, of which, are thought to be mediated at least partly by presynaptic mechanisms. Alternatively, postsynaptic modifications may also contribute to changes in the ratio of responses to paired-pulse facilitation or to paired-pulse depression. Because paired-pulse depression of the fast IPSP was not significantly altered in the presence of saccharin, it appeared that the enhanced duration and, though not always observed, the Increased height of the fast IPSP, were due to the drug's effects on the postsynaptic GABA receptors rather than on the presjmaptic GABA receptors. Whether or not the saccharin-induced changes in the fast IPSP could alter paired-pulse facilitation or paired-pulse depression was, therefore, examined. Results from paired-pulse facilitation experiments demonstrated that in the presence of saccharin a slight decrease in paired-pulse facilitation of the EPSP occured. Though this effect was insignificant when statistically compared to its control, if the amplitude of the potentiated fast IPSP produced by the first pulse i n the presence of saccharin was compensated to match the amplitude of the IPSP evoked by the first pulse before application of the drug, no change in paired-pulse facilitation of the EPSP was observed. A similar effect was also exhibited for the saccharin-induced increase in paired-pulse depression of the fast IPSP. One possible explanation which may account for an increase in paired-pulse depression of the IPSP is the enhanced fast IPSP arising from the first pulse may have exerted conductance changes in the CAl^ neurons which were capable of adjusting the membrane potential close to the equilibrium potential of the fast IPSP (Ejpsp) evoked by the second pulse (Dingledine and Langmoen, 1980). Such a postsynaptic modification is supported by several indirect lines of evidence. At an interstimulus interval of 40 msec, the fast IPSP evoked by the second pulse occured on top of the hyperpolarization produced by the fast IPSP arising from the first pulse in the stimulus pair. Paired-pulse depression was greatly reduced in this particular experiment when compared to the experiments where an interstimulus interval of 300 msec was employed because, in the latter case, the fast IPSP evoked by the first pulse had already decayed to a greater extent. The change in conductance associated with the IPSP has also been demonstrated to decrease the amplitude of the dendritic EPSP by a shunting effect (Dingledine and Langmoen, 1980). In the present studies maximal shunting of the EPSP occured only when the peak IPSP amplitude was attained (less than 20 msec). Because saccharin increased the latency to peak and duration of the fast IPSP to a period approaching the interstimulus interval used in the paired-pulse facilitation experiments it would be expected that the shunting effect be maximal in the presence of the drug rather than in its absence. This effect could explain why paired-pulse facilitation of the EPSP was decreased when saccharin was present. Therefore, it could be argued that saccharin blocked the induction of LTP by increasing the shunting effect of the fast IPSP on the EPSPs elicited during the high frequency tetanus. The net effect would result in a decreased depolarization due to an inefficient summation of EPSPs during the train, a manouver disfavouring the the activation of the NMDA receptor-gated channels. Alternatively, by adjusting the membrane potential close to the Ejpsp, the potentiated fast IPSP may regulate the activity of the NMDA receptor-operated channels by moving the membrane potential further away from the voltage nessesary to remove the Mg2+ block of the ionophore. Indeed, the fact that the enhanced duration of the fast IPSP distorted the duration of the NMDA EPSP does provide some merit to this postulation. Furthermore, the latency to peak of the fast IPSP increased in the presence of saccharin to a time which fell within the range of reported rise times of the NMDA EPSP (CoUingridge et. al.. 1988; Hestrin et. al.. 1990). However, several indirect observations suggests that at least during high frequency trains, the detrimental contribution of the fast IPSP to the overall depolarization was minimal. For example, during the 400 Hz, 0.5 sec train, the resulting depolarizations produced from CAl^ neurons were not decreased the presence of saccharin but were actually increased. In addition, neither the extracellular EPSP slope nor the intracellular EPSP slope exhibited LTP in the presence of sacchgirin when the fast IPSP was blocked by picrotoxinin. The possibility that saccharin overcame the antagonism by picrotoxinin was unlikely since there was no evidence which suggested a reemergence of the fast IPSP when saccharin was present. Furthermore, IPSPs tend to "run-down" when evoked at frequencies greater than 0.1 Hz (Ben-Ari et. al.. 1978), an effect, presumably due to the pres5niaptic mechanism discussed earlier. Taken together, the results of the present study demonstrate a general lack of effects of saccharin on both excitatory and inhibitory synaptic transmission. Moreover, based on our experiments, the blockade of the induction of LTP appears not to depend upon an antagonism of the NMDA receptors, GABA^ receptors or the GABAg receptors, all of which have been implicated in the induction of LTP. The mechanisms through which saccharin blocks the induction of LTP remains unresolved. However, it is important to note that the post-tetanic potentiation (PTP) exhibited from slices treated with saccharin was reduced following a tetanic stimulation of the stratum radiatum or foUoAving the pairing of strong postsynaptic depolarization with low frequency activation of the stratum radiatum. Because saccharin did not interfere with the depolarization produced by either LTP-inducing method, it was possible that the drug interfered with some diffusible substance released during the depolarization that was responsible for producing the non-decremental enhancement of synaptic transmission. Alternatively, saccharin could have prevented the release of the substance or interfered with its locus of action, however, such a retrograde message has yet to be identified. 18.3. Possible Mechanisms of Action of Saccharin Previous experiments conducted in this laboratory have demonstrated that 10 m M saccharin applied for 10 minutes inhibits LTP induced by nerve growth factor (NGF) (Chirwa, 1988; Sastiy et. al.. 1988). Based on these results and results from other studies originating from this laboratory, it is possible that saccharin blocked the induction of LTP by interfering with the actions of NGF of other growth-related substances. Growth factors are believed to be released from the postsjmaptic cell and taken up by the presynaptic terminals where they produce sprouting of boutons and cause biochemical changes in the presynaptic terminals (Hendry et. al.. 1974; Springer and Loy, 1985). The ability of saccharin to interfere with the actions of a specific growth factor was first demonstrated when tetanic stimulations of a weak input that only induced a short-term potentiation of the field EPSP induced a long-term potentiation when nerve growth factor (NGF) was present. The effect of NGF was. however, antagonized when saccharin was present (Sastrvet. al.. 1988). Other studies conducted in this laboratory supported the involvement of trophic factors in the induction of LTP. That saccharin was capable of antagonising their LTP-inducing actions in a reversible manner suggested a specific interaction existed between them. In support of this view, subsequent studies demonstrated that the blockade of LTP by saccharin and the action of the growth-related substances to induce LTP were both independent of NMDA receptor activation (Morishita et. al.. 1992; Xie et. al.. 1991). Taken together, these findings suggest that the growth-related substances and saccharin may share a common site of action. Whether or not this site is located presynaptically or postsynaptically remains to be determined. Thus far, the actions of saccharin have been explained through its known antagonizing effects on NGF and on endogenus growth-related peptides. Because saccharin inhibits the activities of specific phosphoiylating enz5aiies, if it also interferes with the activities certain kinases implicated in the induction of LTP then, conceivablely it could block the induction of LTP without antagonizing the NMDA or the GABAg receptors. In summary, the present studies have demonstrated that saccharin blocks the induction of LTP in the C A l region of the guinea-pig hippocampus through a mechanism that does not involve an interference Avith the NMDA and the non-NMDA glutamatergic receptors as well as the GABA^ and the GABAg receptors. 19. CONCLUSIONS Based on the experiments conducted in this study the major findings are summarized as follows: 1. Saccharin blocked the induction of LTP by either, (a) a tetanic stimulation or, (b) pairing a strong postsynaptic depolarization with concommitant activation of the afférents. 2. Saccharin did not block the maintenance of LTP. 3. In agreement with previous findings, saccharin did not significantly alter the electrical properties of the C A l ^ neurons nor did it significantly change responses mediated by activation of the NMDA and the non-NMDA glutamate receptors. 4. 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