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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 T H E HIPPOCAMPUS  By W A D E K A T S U J I MORISHITA B . Sc., (Pharmacology and Therapeutics), The University of British Columbia, 1989 A THESIS S U B M I T T E D IN PARTIAL F U L F I L L M E N T O F THE REQUIREMENTS FOR T H E D E G R E E OF MASTER OF SCIENCE in T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Pharmacology and Therapeutics, Faculty of Medicine. The University of British Columbia)  We accept this t h e s i s ^ s conforming to the requiredi standard  T H E UNIVERSITY O F BRITISH C O L U M B I A A p r i l 1992 © Wade Katsuji Morishita, 1992  In  presenting  degree freely  this  thesis  at the University  in of  available for reference  copying  of  department publication  this or  thesis by  his  for or  partial  fulfilment  British C o l u m b i a , and study. scholarly her  of  I agree  I further  purposes  the  agree  representatives.  It  Pharmacology and  The University of British C o l u m b i a Vancouver, Canada  (2/88)  that  an  May 6 , 1992  Therapeutics  advanced  shall make it  pemiission for extensive by  understood  the head that  of this thesis for financial gain shall not be allowed without  Department of  DE.6  is  for  that the Library  may be granted  permission.  Date  requirements  of  my  copying  or  my written  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 i n this action were, however, unknown. The present electrophysiological investigation on guineapig 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 i n 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-methylD-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 C A l b neurons produced by a tetanic stimulation i n normal medium or by brief applications of N M D A i n 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 i n the presence of saccharin. Moreover, the slope and the height of the intracellular E P S P evoked i n a Mg2+-free medium containing C N Q X as well as i n a normal medium containing 2-amino-5-phosphonovalerate (APV), a drug that antagonizes N M D A 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 N M D A 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. its  GABA  receptor-mediated  Pharmacological separation of the IPSP into 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 G A B A B receptor-mediated IPSPs), paired-pulse depression of the fast IPSP evoked i n a C N Q X and A P V containing medium was not significantly altered i n the presence of saccharin, suggesting  that the drug did not interfere  with the presynaptic G A B A B  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 G A B A B receptor-mediated  responses  responsible for its LTP-blocking action.  It is possible that saccharin  have interfered with LTP-inducing growth-related  also appear not to  be  might  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  T A B L E O F CONTENTS Chapter  Title  Page  A.  ABSTRACT  ii  B.  T A B L E O F CONTENTS LIST O F F I G U R E S  V  xi  LIST OF T A B L E S  xiii  LIST O F PUBLICATIONS ACKNOWLEDGEMENTS DEDICATION INTRODUCTION  xiv  T H E HIPPOCAMPAL FORMATION  3 3 4  C. D. E. F. G. L 2.  2.1. 2.2.  3.  4.  5.  Development of the hippocampal formation Basic Topography of the hippocampus  2.2.1. 2.2.2. 2.2.3. STRATIFICATION,  The dentate gyrus The hippocampus proper The subicular complex S U B F I E T D S A N D ASSOCIATED C E L L  XV  xvi 1  4 6 6  TYPES OF T H E HIPPOCAMPUS 3.1. The dentate gyms 3.2. The hippocampus proper  6 8 8  INTRINSIC A F F E R E N T S O F T H E HIPPOCAMPUS  9  4.1. 4.2.  9 10 12  Intrinsic afférents within the same subfleld Intrinsic afférents from different fields  4.3. Commissural afférents PROJECTION PROFILE O F INTRINSIC HIPPOCAMPAL AFFERENTS  12  5.1.  Distribution of perforant path fibers  5.2.  Distribution of fibers from dentate gyrus  13 13  5.3.  Distribution of fibers from C A 3  14  6.  7.  8•  9.  EXTRINSIC A F F E R E N T S TO T H E HIPPOCAMPAL FORMATION 6.1. Entorhinal projections 6.2. Septal projections 6.3. Isocortical projections EXTRINSIC E F F E R E N T S F R O M T H E HIPPOCAMPAL FORMATION 7.1. Fornix-fimbria system 7.1.1. Pre- and post-commissural fornices 7.2. Entorhinal projections 7.3. Isocortical projections ELECTROPHYSIOLOGY O F HIPPOCAMPAL N E U R O N S 8.1. Bursting activity of hippocampal neurons 8.2. Hippocampal intemeurons 8.2.1. Basket cells  11.  16 16 17 17 17 17 19 19 19  8.2.2.  Oriens/alveus (O/A) intemeurons  21  8.2.3.  Lacunosum-moleculare (L-M) intemeurons  21  IONIC C U R R E N T S IN HIPPOCAMPAL N E U R O N S  23  9.1. 9.2.  Na+-currents K+-currents  24 24  9.2.1. 9.2.2.  25  9.3.  10.  15 15 15 16  K+-currents activated through depolarization K+-currents activated through h5^erpolarization  Voltage-dependent Ca2+-currents  27 27  9.4. Ca2+-activated K+-currents 9.5. Voltage-dependent Cl"-current 9.6. Miscellaneous currents 9.7. Passive "leak" currents FIELD POTENTIALS IN T H E HIPPOCAMPUS  29 31 31 31 32  EXCITATORY POSTSYNAPTIC POTENTIALS (EPSPs) 11.1 N M D A receptors and synaptic transmission  33 34  11.2.  11.1.1. Allosteric modulation by glycine 11.1.2. Modulation by zinc 11.1.3. N M D A antagonists 11.1.4. Glycine antagonists Non-NMDA receptors and synaptic transmission 11.2.1. The non-NMDA component of the E P S P 11.2.2. The non-NMDA A C P D receptor 11.2.3.  12.  The non-NMDA L-AP4 receptor  11.2.4. Non-NMDA receptor antagonists INHIBITORY POSTSYNAPTIC POTENTIALS (IPSPs) 12.1. Recurrent and feed-forward inhibition 12.2. Properties of the G A B A ^ receptor-mediated fast IPSP 12.3. Properties of the G A B A g receptor-mediated slow IPSP 12.3.1. 12.4.  Presynaptic G A B A g receptors  41 41 43 43 44 4g 49  Properties of the G A B A ^ receptor-mediated depolarizing IPSP  13.  36 37 37 38 38 38 40  L O N G - T E R M POTENTIATION IN T H E HIPPOCAMPUS 13.1. Properties of LTP 13.1.1. Population responses 13.1.2. Single neurons 13.2. Projections i n the hippocampus supporting LTP 13.3. Homo- and heteros)maptic LTP 13.4. Cooperativity 13.5. Associativity 13.6. Requirement for calcium 13.6.1. Source of calcium entry 13.7. Induction and Maintenance of LTP 13.7.1. Induction of LTP 13.7.1.1 Modulation by G A B A 13.7.2. Maintenance of LTP  50 52 52 52 53 54 54 55 55 56 57 57 57 58 59  13.7.2.1. 13.7.2.2. 13.7.2.3.  14.  15.  Posts5niaptic mechanisms Presynaptic mechanisms Pre- and postsynaptic mechanisms SACCHARIN AS A PHARMACOLOGICAL TOOL OF INVESTIGATION  67  14.1. 14.2. 14.3. 14.4. 14.5. 14.6.  67 68 68 69 70 71  History Chemistry and physical properties Pharmacodisposition Tumor promotor Interaction with growth factors Involvement i n LTP  59 62 65  MATERIALS A N D M E T H O D S  73  15.1. 15.2. 15.3. 15.4. 15.5.  A n i m a l source and care Slice preparation Slice chamber Perfusion media Recording and stimulating equipment 15.5.1. Recording electrodes 15.5.2. Amplifiers  73 73 75 77 79 79 79  15.5.3. 15.5.4. 15.5.5.  81 81 82  15.6. 15.7. 15.8.  15.9.  Recording systems Stimulation and isolation units Stimulating electrodes  15.5.6. Positioning of electrodes 15.5.7. Arrangement of the recording set up Extracellular recording Intracellular recording Induction of LTP  82 82 83 83 84  15.8.1.  Tetanic stimulation  84  15.8.2.  Pairing  84  Measurements and statistics  85  16.  E X P E R I M E N T A L PROTOCOLS 16.1. Saccharin and LTP of the field E P S P 16.2. Saccharin and LTP of the intracellular E P S P 16.3. Saccharin and maintenance of LTP 16.4. Saccharin and pairing 16.5. Specific intracellular studies on saccharin 16.5.1. 16.5.2. 16.5.3. 16.5.4. 16.5.5. 16.5.6. 16.5.7.  17.  Saccharin and N M D A receptor-mediated responses Saccharin and input-output (I-O) relationships of the E P S P and IPSP Saccharin and IPSPs Saccharin and QX-314 injected neurons Saccharin and paired-pulse depression of the fast IPSP Saccharin and paired-pulse responses Saccharin and LTP i n picrotoxinin containing media  86 87 88 88 88 89 89 90 91 92 92 93 94  RESULTS 17.1. Saccharin and LTP of the field E P S P  95 95  17.2. 17.3. 17.4.  Saccharin and LTP of the intracellular E P S P Saccharin and maintenance of LTP Saccharin and pairing  95 99 99  17.5.  Saccharin and N M D A receptor-mediated responses 17.5.1. Saccharin and tetanus-induced depolarizations 17.5.2. SacchEirin and depolarizations produced by applied N M D A 17.5.3. Saccharin and N M D A and non-NMDA components of the E P S P  99 99 102 104  17.6. 17.7. 17.8. 17.9.  18.  106 108  Sacharin and fast IPSPs recorded i n neurons injected with QX-314  111  17.10.  Saccharin and its effects on paired-pulse depression  17.11.  of the fast IPSP Saccharin and paired-pulse responses of postsynaptic potentials  17.12. Saccharin and LTP i n picrotoxinin containing media DISCUSSION 18.1. Effects of saccharin on excitatory synaptic transmission 18.2. 18.3.  19. 20.  Saccharin and input-output relationships of the E P S P and the IPSP Saccharin and postsynaptic potentials recorded i n a Mg2+-free medium Saccharin and IPSPs  106  111 115 118 120 120  Effects of saccharin on inhibitory synaptic transmission  121  Possible mechanisms of action of saccharin  126  CONCLUSIONS REFERENCES  128 129  LIST O F F I G U R E S Figure  2.1. 2.2. 15.1.  17.1. 17.2. 17.3. 17.4. 17.5. 17.6. 17.7.  17.8. 17.9.  Page  Orientation of the hippocampal formation Organization of the hippocampal formation Schematic representation of the slice chamber used to study in vitro electrophysiological potentials from guinea-pig hippopcampal slices Saccharin blocks LTP of the field E P S P slope  5 7  76 96  LTP of the intracellular E P S P slope is not blocked by saccharin Maintenance of LTP is not blocked by saccharin  97 98  Saccharin reversibly blocks pairing-induced LTP of the intracellular E P S P slope Depolarizations induced by tetanic stimulations are not blocked by saccharin Depolarizations induced by applied N M D A are not significantly altered i n the presence of saccharin  103  The intracellular E P S P slope recorded i n the presence of excitatory amino acid antagonists is not significantly altered by saccharin  105  The effects of saccharin on the input-output (I-O) relationship of the EPSPs and IPSPs recorded from CAl^j neurons  107  100 101  Saccharin potentiates the IPSP during activation of the stratum radiatum  109  17.10. 17.11.  Saccharin anatagonizes the late component of the IPSP/C The slow I P S P / C ( I P S P / C B ) is decreased by saccharin  110  17.12.  QX-314 blocks the slow IPSP  113  112  Figure  17.13. 17.14. 17.15.  17.16.  Page  Saccharin increases the height and duration of the fast IPSP in neurons injected with QX-314 Paired-pulse depression of the fast IPSP (IPSP^) is not blocked  114  by saccharin  116  Effects of saccharin on paired-pulse facilitation of the E P S P and paired-pulse depression of the fast IPSP i n neurons injected with QX-314  117  LTP i n picrotoxinin-treated slices is not blocked by saccharin  119  LIST O F T A B L E S Table  17.1. 17.2. 17.3.  Page  Effects of saccharin on some properties of the IPSP/Cs Effects of saccharin on some properties of the I P S P / C B Effects of saccharin on some properties of the IPSP^  110 112 114  LIST O F 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 longterm 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 i n the hippocampus. In: Advances i n 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 i n 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 i n 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 longterm potentiation i n 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 i n long-term potentiation. International Workshop on Plasticity of Synaptic Transmission. Kanagawa, Japsm, 1989. 8. Morishita, W. The involvement of glial cells i n the induction of long-term potentiation i n 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) i n 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) i n 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 i n 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 i n some of the experiments conducted i n this study. 1 wish to thank all the members of the Department of Pharmacology and Therapeutics, i n particular, Drs. David Godin and Dick Wall for their encouragement, and academic guidence during my undergraduate years i n this department.  DEDICATION  1. INTRODUCTION In the m£immalian central nervous system (CNS), repetitive activation of certain types of excitatory synapses results i n a long-term potentiation [LTP) of synaptic transmission. Though LTP has been demonstrated i n a number of areas i n 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 i n 1966, however, subsequent and more detailed accounts were reported by Bliss and Lomo as well as Bliss and Gardner-Medwin i n 1973.  LTP is characterized by a n increase i n synaptic  efficacy lasting from several minutes to hours In vitro (Schwartzkroin and Wester, 1975) and hours to days i n 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 i n postS5niaptic response to a test stimulus given to the same pathway after the tetanus and is characterized by, 1) an enhancement i n the amplitude of the population spike, 2) decreased latency of the population spike, 3) an increase i n the size and rate of rise of the population and intracellular excitatory postsynaptic potential (EPSP),  4)  an  increase i n the population spike amplitude or the probability of spike discharge for a constant E P S P (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-methylD-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  i n 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 i n hippocampal slice preparations (Sastry et. al.. 1988a; Sastry et. al.. 1990; Xie et. al.. 1991). The release b u t not the LTP-inducing effects of these proteins also depends upon the activation of N M D A receptors (Sastry et. al.. 1990; Xie et. al.. 1991). One interpretation of these findings is that during activation of postS5maptic N M D A receptors certain event(s), as yet unknown, occur resulting i n the release of proteins into the extracellular fluid that interact with either pre- or postsynaptic elements or both, resulting i n 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 i n 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 N G F 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 N G F or other growthrelated substances that are released during the train or with receptors directly involved i n synaptic transmission is unclear. Preliminary investigations have suggested that saccharin does not block the N M D A 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) i n the absence and  presence of saccharin remained unchanged (Chirwa, 1988).  Since this  experiment was not performed i n a Mg^^-free medium which would reduce the voltage-dependent block of the N M D A receptor channel by Mg2+ (Mayer et. al.. 1984; Nowak et. al.. 1984) it is unknown whether saccharin interferes with the N M D A receptors.  Saccharin's effects were also not examined on the y-  aminobutyric acid (GABA) receptors which have been suggested to play a n 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 N M D A and non-NMDA receptormediated and G A B A ^ and G A B A ^ receptor-mediated synaptic transmission. 2. T H E HIPPOCAMPAL FORMATION In review of the literature, m u c h 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 will  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 a n 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. is the granule cell (Golgi, 1886).  The primary cell type of the dentate gyrus The ovoid shaped somata of granule cells  typically range from 15 to 25 \im i n 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 i n 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 b r a i n 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 C A 3 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 i n 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 i n diameter with the largest cells located i n field C A 3 (Figure 2.2B and 2.2C).  Pyramidal cells possess both  basal and apical dendrites w h i c h 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. S U B F I E L D S A N D A S S O C I A T E D C E L L T Y P E S O F 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 i n which their processes radially distribute, sep, septal pole; tem, temporal pole; alv, cilveus; D G , 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 i n 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 i n this layer have previously been  divided into those which exist i n 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 C A 3 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 C A 3 becoming diffuse and less apparent i n 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 w h i c h 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 i n a similiar order to the C A l subfield, however the mossy fibers of granule cells produce a distinct band of transversely oriented axons j u s t 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 A F F E R E N T S O F 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 i n 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 i n 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 i n strata oriens and radiatum but not i n lacunosum-moleculare (Hjorth-Simonsen,  1973).  These longitudinal associational projections are thought to participate i n 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, i n general, unidirectional starting i n the dentate gyrus continuing through CA3 and ending i n C A l (Figure 2.2B).  In the dentate  g 3 ^ 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 i n the pol3miorph layer i n the hilus of the dentate gyrus before projecting to the C A 3 subfield. The mossy fibers form two highly laminated separate bundles.  Mossy fibers which arise from the  infrapyramidal blade travel i n stratum oriens and terminate within subfields CA3b and CA3c, and those arising from the suprapyxamidal blade travel i n stratum lucidum, course through C A 3 and end i n 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 C A 3 (Andersen et. al.. 1966).  The  mossy fibers also synapse with basket cells i n this field and with neurons i n 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 C A 3 pyramidal cells (Hjorth-Simonsen and Jeune, 1972). The giant pyramidal cells of field C A 3 possess associational projections which r u n along the transverse and septo-temporal axis. The_most prominant projections arising from C A 3 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 i n stratum oriens and proximal 3/4 of the apical dendrites i n stratum radiatum (Gottlieb and Cowan, 1973; Lorente de No, 1934).  Ipsilateral projections from C A 3 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 w h i c h cross the midline i n the ventral and dorsal hippocampal (psalleria).  commissures  The majority of commissural fibers course through the ventral  commissure. While all regions of the hippocampus receive commissural input from their respective fields, i n 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 C A 3 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. P R O J E C T I O N PROFILE O F INTRINSIC HIPPOCAMPAL A F F E R E N T S 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 C A 3 pyramidal cells i n turn send Schaffer  collaterals to the pjnramidal cells of C A l .  arrangement  appears  to  be  functional throughout  Physiologically, this the  transverse  axis  (Andersen et. al.. 1971). Consequentiy, the hippocampus has been described as being organized i n a lamellar fashion (Andersen et. al.. 1971) with each lamina functioning independent from one another. studies (Hjorth-Simonsen, suggest  However, subsequent  1973; Laurberg, 1979; Swanson et. al.. 1978)  that the organization of the intrinsic circuitry of the hippocampus  extends as m u c h i n the septo-temporal axis as it does i n the transverse axis. Recent  anatomical and physiological studies (Amaral and Witter,  1989;  B u z s a k i 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 i n a more accurate representation of fibers being distributed i n the septo-temporal plane when transverse slices are cut) i n combination with the descrete anteriorgrade tracer, Phaseolus vulgarus leucoagglutinin (PHA-L) have shown that the 3dimensional 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 i n 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 i n the molecular layer along the septotemporal axis (Amaral and Witter, 1989; R u t h 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 i n 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 C A 3 - C A l boarder, the mossy fibers are organized i n a lamellar fashion. This pattern, however, is not observed when the trajectories of associational fibers are traced. The the pol5niiorphic region of the  associational projections originate i n  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 C A 3 CA3 cells possess highly collateralized axons which project i n 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 link C A 3 to C A l at the same hippocampal level. In fact, C A 3 cells located near the dentate gyrus project their collaterals more heavily i n the septal direction while those close to CA2 have collaterals that project more frequent to the temporal surface (Amaral and Witter, 1989). Furthermore, i n transverse slices, C A 3 cells injected with horse  radish  peroxidase, to reveal their distribution of axon collaterals, send few axons to CAl  (Ishizuka et. al.. 1990).  Together, these patterns imply that the  trajectories of these collaterals do not r u n parallel to the transverse axis but more i n the septo-temporal plane. In addition to the Schaffer collaterals. C A 3 cells also possess other associational projections which r u n parallel to the long axis of the hippocampus.  These longitudinal associational projections have  already been discussed. Thus, the hippocampus is not organized to function i n discrete lamellar units, but rather as a whole unit capable of processing information along both axes.  6. EXTRINSIC A F F E R E N T S TO T H E HIPPOCAMPAL FORMATION The  hippocampal formation receives  afferent  innervation from  parahippocampal and, to a lesser extent, isocortical regions.  The  both 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 i n lamina II of the entorhinal cortex (Witter and Groenewegen, 1984), some cells i n deeper layers have been reported to project to the dentate gyrus (Kohler et. al.. 1984). As i n 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 i n lamina III i n 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 i n 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 C A 3 , Though these fibers project to all layers of CA3, they are most concentrated i n stratum oriens (Nyakas et. al.. 1987).  Experiments combining lesion studies w i t h  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 G A D 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 link 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 i n C A l , occasionally i n CA3, and not i n the dentate gyrus. They originate i n parietal, temporal, and perirhinal cortices (Schwertfeger, 1979; Schwertfeger, 1984). 7. EXTRINSIC E F F E R E N T S 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 i n 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 C A 3 that distribute i n 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 s u b i c u l u m to the thalmus, mammillary bodies and rostral b r a i n stem (Chronister and DeFrance, 1979). 7.2. Entorhinal Protections The entorhinal cortex (especially lamina IV), s u b i c u l u m 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 link the hippocampal formation with the isocortex. various  parts  of  Connections have been established between C A l and the  frontal  (Swanson,  1981)  and  temporal  cortices  (Schwerdfeger, 1979), the retrosplenial and perihinal cortices (Swanson and Cowan, 1979). 8. ELECTROPHYSIOLOGY O F HIPPOCAMPAL N E U R O N S A number of i n vitro slice studies have been performed to investigate the electrophysiological properties of the various cell t3rpes i n the hippocampus (Brown et. al.. 1981; Brown and Johnston, 1983; D u r a n d 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, b u t  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 m V and for granule cells between -60 and -85 mV. Action potentials evoked at R M P by injecting current into the cell range from 50 to 110 m V for p3n:amidal cells and 70 to 140 m V for granule cells. The input resistance calculated from the slope of the linear portion of current-voltage plots varies from 25 to 40 M Q 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) i n 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. ratio  of  dendritic  to  somatic  conductance  Estimated values for the  and  electrotonic  length  of  hippocampal neurons are 1.0 to 1.5 and 0.9 to 1.2, respectively (Brown et. al.. 1981; D u r a n d 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 i n 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 i n burst discharge have been demonstrated i n 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 C A 3 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 w h i c h develop from activation of a T-type calcium current (Traub, 1982; Wong and Prince, 1978).  The physiological significance for bursting activity i n the C A 3  region may be to amplify excitatory signals from incoming afferent pathways to other regions i n the hippocampus or, alternatively, to act as a pacemaker for interictal discharge i n 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). morphological attributes include:  Their  large somata (35 to 50 jim on average)  identified as either p5n-amidal, horizontal, fusiform, inverted fusiform, multipolar.  or  They possess aspinous dendrites and locally arborizing axons  (Riback and Andersen, 1980). The most well characterized intemeurons, are those located i n 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 w h i c h  resembles a "basket" around its target cell somata (Cajal, 1911; Lorente de No, 1934).  Basket cells are present i n 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 G A D (Ribak et. al.. 1978), basket cell inhibition is presumed to involve the inhibitory neurotransmitter GABA, Electrophysiologically, basket cells differ granule  cells i n that they  have  very  from pyramidal cells and  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  i n 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 i n 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; B u s a k i and Eidelberg. 1982) supporting the view that these intemeurons are involved i n both feedforward 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 i n 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 i n 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 i n forming numerous S3^aptic contacts.  The axons of O/A interneurons branch and distribute i n  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 i n 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. forms of activation result i n pyramidal cell inhibition.  Both  Thus, like the basket  cells, O/A interneurons are capable of producing both feed-forward and feedback inhibition. 8.2.3. moleculare  Lacunosum-moleculare (L-M) interneurons. (L-M)  interneurons are located  The lacunosum-  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 i n stratum lacunosummoleculare projecting into stratum pyramidal and stratum oriens of the hippocampus proper and stratum moleculare of the denate gyms.  The m a i n  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 basket  and  O/A  share similar electrophysiological  intemeurons  in  that  they  exhibit  properties  with  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 i n 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 coUateral/commissural  pathways,  that during activation of the orthodromically  evoked  slow  Schaffer IPSPs  in  hippocampal p5nramidal neurons appear to be mediated through activation of these interneurons. 9. IONIC C U R R E N T S IN HIPPOCAMPAL N E U R O N S Hippocampal neurons support a variety of different ionic conductances that are mediated either through opening of voltage-gated channels, of iongated 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  that  clamp  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 hippocampal neurons have been obtained.  ionic currents from  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 i n the following sections characterizes those found i n 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 i n hippocampal  neurons.  They are the fast activating Na+-current  slow  (lNa(fast)^  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 m V 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 m V (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 i n hippocampal pacemacker activity (Brown et. al.. 1990) or repetitive firing of action potentials i n 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 i n 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 b y 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)1IK 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 m V {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)  i n m M concentrations  and  insensitive to 4-aminopyridine {4-AP) and Cs"*" when externally applied (Segal and Barker, 1984).  B o t h the soma and dendrites of hippocampal neurons  exhibit % currents (Masukawa and Hansen, 1987). Though relatively slow, the delayed rectifier may participate i n 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 m V (Gustaffson e t 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 i n low m M concentrations (Numann et. al.. 1987; Segal and Barker, 1984; Storm, 1988a) and internally applied T E A 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 G A B A and baclofen (Saint et. al.. 1990). The A-current is fast enough to contribute to Na+ spike repolarization (Storm, 1987) and has been implicated i n regulating neuronal firing rate (Segal et. al.. 1984).  This current has been detected primarily i n 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 m V with a time to peak reaching 20 ms (Storm, 1988a).  Inactivation starts around -120 m V 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 D T X and Toxin 1 (Storm, 1990). The current appears to be insensitive to m M concentrations of T E A and Cs+.  Fast activation of the  current suggests that it may participate i n Na+ spike repolarization (Storm, 1987b). 1K(D) is capable of producing long delays i n 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). the M-current because it is inhibited by muscarinic  IK(M) is dubbed  receptor agonists (Brown  and Adams, 1980; Halliwell and Adams, 1982). The current is also modulated by  other  neurotransmitters.  hydroxytryptamine  (5-HT)  For (Colino  aminocyclopentyl-l,3-dicarboxylate  example, and  it  is  Halliwell,  reduced  by  5-  1987),  trans-1-  (ACPD) (Charpak et. al.. 1990) and is  increased by somatostatin (Moore et. al.. 1988; Watson and Pittman, 1988), a n 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 m M concentrations of externally applied Ba"*" and T E A 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 i n 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). hyperpolarizations beyond -80 mV.  Ig is activated by  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; J o h n s t o n et. al.. 1980; Mogul and Fox, 1991; Ozawa et. al.. 1989; Takahashi et. al.. 1989). They are analogous to those previously described i n 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 m V  (Halliwell. 1983; Ozawa et. al.. 1989; Takahashi et. al.. 1989; Takahashi et. al.. 1991). Inactivation begins at -60 m V and is complete at -40 m V (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 p S (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). Ttype Ca2+-currents have been implicated i n 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 i n 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 m V and -10 m V and inactivated at potentials between -60 m V and -40 m V (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 m V (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 e t 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 m V (Ozawa e t al.. 1989). The N-t3rpe channel conducts Ba^+ greater than Ca2+ ions (Tsien e t 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 i n hippocampal  neurons. I^a is a large time- and voltage-dependent K+-current which activates rapidly (1 to 2 msec) i n response to a depolarizing current pulse or during a n 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  nM  concentrations of charybdotoxin and externally applied T E A 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 E G T A (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 T E A 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 E G T A (Lancaster and Nicoll, 1987).  I ^ p is also modulated by neurotransmitters,  being increased by adenosine (Haas and Greene, noradrenaline (via  1984); and reduced by  receptors) (Haas and Konnerth, 1983), histamine (via H2  receptors) (Haas and Konnerth, 1983), 5-HT (Andrade and Nicoll. 1987), A C P D (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).  may  involve  Modulation of I ^ H P by neurotransmitters  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 i n 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 i n hippocampal neurons is a voltage-dependent chloride-current (lci(V)) which activates by h5rperpolarizing steps between -20 and -100 m V (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+, T E A and carbachol (Madison et. al.. 1986). 9.6. Miscellaneous Currents Hippocampal  pyramidal  cells  possess  (Ici(ca)^ and a Na+-activated K+-current. activated  Cl"-current which activates  a Ca2+-activated Cl"-current  Ici(Ca) is a voltage insensitive Ca^"^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 i n pyramidal neurons (BroAvn a n d 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 minimal.  channels are  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 m V and -70 mV. 10. FIELD POTENTIALS IN T H E HIPPOCAMPUS Because both somata and dendrites of pyramidal and granule cells are aligned i n a uniform parallel fashion the activation of one or many afferent en passant fibers results i n a near s)mchronous activation of a population of cells (Schwartzkroin and Mueller, 1987; Teyler and DiSenna, 1986).  Therefore,  unlike other areas i n the cortex, the hippocampus is capable of generating relatively large field potentials. Synaptic excitation is reflected i n 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 i n polarity when recording at the cell somata (source).  When the sjmaptic excitation exceeds the threshold i n  individual cells for spike generation, the field E P S P is interrupted by a population spike. This is represented i n 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 E P S P 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 i n 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 C A 3 pyramidal cell EPSPs arise from activation of the mossy fibers which synapse on the proximal dendrites of C A 3 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 E P S P rise times i n response  to  proximal and distal stimulations. They attribute these observations to the electrotonic decay which occurs as the E P S P 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 E P S P i n 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 N M D A and the non-NMDA receptors.  The resulting  increase i n conductance through N M D A channels mediates the slow E P S P where as the rise i n conductance through non-NMDA channels generates the fast EPSP. 11.1. N M D A Receptors and Synaptic Transmission The N M D A receptor is a n ionotropic receptor selectively activated by Nmethyl-D-aspartate (NMDA), a structural analogue of glutamate (Watkins and Evans,  1981).  permeable  The ion channel associated Avith the N M D A receptor  to both Na+ and Ca^^ ions and possesses  is  a single channel  conductance between 40 and 50 pS with an open time duration of 5 to 10 msec (Ascher and Nowak, 1988; J a h r and Stevens. 1987). The current-voltage  (I-V)  relationship from neurons exposed to N M D A over a holding potential range of 100 to +30 m V exhibit both positive and negative slope behaviours (Mayer e t 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 e t 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 N M D A occurs around 30 m V and decreases upon further depolarization reversing i n 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 N M D A receptors mediate at least one component of the EPSP. The N M D A component of the E P S C 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 N M D A 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 i n these experiments closely resembles the time for the N M D A E P S C 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 i n a n 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 N M D A receptor, the N M D A channel activity outlasts glutamate application. Therefore, the slow onset and long duration of the N M D A E P S C may be accounted for by its prolonged activation by glutamate. Because the N M D A E P S C is voltage-dependent and long i n 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. role i n inhibitory neurotransmission.  Glycine plays  an  important  Glycinergic inhibition has been well  characterized i n 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 N M D A induced currents recorded from cultures of central neurons. membrane  Single channel analysis from  patches i n 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 N M D A or L-glutamate (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988; Maver et. al.. 1989; Reynolds e t al.. 1987). Glycine, therefore, may exert is actions by regulating the transition states that are intermediate between binding of N M D A receptor agonists and ion channel gating. detected  Indeed, i n some preparations, channel openings are not  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 N M D A receptor activation. Glycine also reduces desensitization of the N M D A 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 N M D A receptor complex may contribute to the slow decay constants observed for N M D A 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 N M D A responses (Westbrook and Mayer, 1987).  At the same concentration required to antagonize N M D A  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 E P S P and the somatic action potential were not altered by Zn2+ but the slow N M D A E P S P was indicating that the antagonism of the slow E P S P by Zn2+ was selective and did not reflect a failure of synaptic transmission. So far the role of endogenous zinc i n modulating N M D A receptor activity remains unclear. However, evidence for its role as a n endogenous modulator of synaptic transmission is supported by it being presence i n the nerve terminals of mossy fibers (Haug,  1967) where it can be co-released with glutamate  (Aniksztejn et. al.. 1987).  However, because few N M D A 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 N M D A receptors have been demonstrated to participate in synaptic transmission (Westbrook and Mayer, 1987). 11.1.3. N M D A antagonists.  A  large  number  of  compounds  possess activity as N M D A antagonists now exist (for reviews see,  that  CoUingridge  and Lester, 1989; Watkins and Olverman, 1987; Watkins et. al.. 1990).  The  role of N M D A receptors i n neurotransmission has been elucidated primarily on the bases of the use of 2-£imino-5-phosphonovalerate (APV). APV is a potent competitive antagonist of N M D A 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 N M D A responses and, i n turn, be rapidly removed upon washout (a valuble asset for demonstrating the degree of contribution of N M D A receptors to synaptic transmission). Consequently, it is the N M D A antagonist of choice unless penetration into the brain is desired.  In i n 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 N M D A receptor-mediated responses by entering the open channel i n a highly voltage- and use-dependent manner (Hicks and Guedes, 1981; MacDonald et. al.. 1987). 11.1.4. Glycine antagonists. Recently, compounds including, 7-chlorokyneurenic 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 N M D A receptor-channel complex. These compounds have been primarily used to demonstrate that glycine fully occupies the allosteric site i n physiological preparations of brain slices (Fletcher and Lodge, 1988; Kemp e t 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  selectively activate them.  (all structural analogues  of glutamate)  which  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 A C P D receptor is a metabotropic receptor linked to inostitol triphosphate (IP) turnover and the L-AP4 receptor is a recently discovered n o n 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 N M D A  receptors play a critical role i n synaptic function, their voltage-dependent block by magnesium limits their participation i n mediating the E P S P resulting from a  unitary synaptic activation.  Instead, the non-NMDA kainate and A M P A  ionotropic receptors are responsible for the voltage-independent portion of the EPSP which underlies the fast synaptic response i n many hippocampal pathways. The ion channels linked to the kainate and A M P A receptors equipermeant to Na+ and Cs+ ions (Ascher and Nowak,  are  1988; J a h r and  Stevens, 1987; Vyklicky et. al.. 1988) and are therefore believed to be relatively non-selective between Na+ and K+ (or Cs+).  Unlike N M D A channels, the  kainate and A M P A 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 A M P A exhibit relatively linear negative slope behaviours when examined over membrane potentials between -90 m V and +30 m V (Mayer and Westbrook, 1984).  The reversal potential for both kainate and quisqualate induced  currents occurs around 0 m V (Mayer and Westbrook, 1984), Such properties have also been demonstrated for the non-NMDA E P S C (Hestrin et. al.. 1990). Kainate and A M P A 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  Westbrook, 1987).  to  kainate's  affinity  for A M P A receptors  (Mayer  and  Channels activated by either kainate or quisqualate (an  agonist at A M P A 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; J a h r and Stevens, 1987).  These events have also been observed i n 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 E P S P . Kainate has also been reported to activate a high conductance channel (30 to 50 pS) (Cull-Candy and Usowicz. 1987; J a h r and Stevens, 1987). however, this may reflect a nonspecific  action of kainate at the N M D A receptor channel  complex. The non-NMDA component of the E P S C 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 n o n - N M D A component of the E P S C may actually reflect the mean open-time of A M P A channels. enough  Since the concentration of glutamate i n the synapse remains high to  persistently  activate  the  NMDA  receptor-channel  complex,  desensitization at A M P A receptors may account for the fast decay time of the non-NMDA E P S C .  Indeed, desensitization of the A M P A receptor is rapid to  exogenous application of glutamate (Kishkin et. al.. 1986), a property w h i c h appears to be determine by the kinetics of the receptor channel complex (Tang et. al.. 1991).  Alterations i n the desensitization process would,  therefore,  subserve as a mechanism by which fast excitatory synaptic transmission could be modified. 11.2.2. The non-NMDA A C P D receptor.  Quisqualate activates another  non-NMDA receptor, however, rather than being associated with an ionotropic receptor, it is linked to a metabotropic one. 1,3-dicarboxylate  Since trans-1-amino-cyclopentyl-  (ACPD) is the most potent agonist for this receptor, it is  termed the A C P D receptor.  In hippocampal neurons, activation of the A C P D  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 N M D A receptor activation (Palmer et. al.. 1988).  Such negative  regulation by N M D A 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 A C P D 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 i n the  E P S P due to a reduction i n 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 Lglutamate and they are, therefore,  thought to promote  glutamatergic synapses (Forsythe and Clements, 1988).  autoinhibition at 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 N M D A receptors as well. TTius, their use i n delineating the n o n - N M D A from the N M D A 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 C N Q X and D N Q X have been reported to block N M D A responses by acting at the strychnine-insensitive glycine receptor (Birch et. a l . . 1988) . In the C A l region of the hippocampus, the use of C N Q X and D N Q X to block non-NMDA component of synaptic transmission, has demonstrated that the non-NMDA receptors but not the N M D A receptors predominantly mediate EPSPs during low frequency stimulation under "normal" conditions (conditions where Mg2+ is i n the perfusate) (Andreasen et. al.. 1988; Blake et. al.. 1988; Collingridge et. al.. 1988; Neuman et. a l . . 1988).  The finding that excitatory  synaptic transmission can be completely blocked by C N Q X when elicited at a low intensity stimulation suggests that synaptic activation of the N M D A receptor need not be required to mediate the E P S P (Collingridge and Lester, 1989) . The quinoxaline derivatives have also been used to demonstrate the N M D A component of sjmaptic transmission. Since Mg2+ is a potent cintagonist of channels coupled to the N M D A receptor, under conditions where Mg2+ i n the perfusate is omitted, N M D A EPSPs can be evoked by low frequency stimulation when non-NMDA EPSPs are blocked (Andreasen et. al.. 1988; Collingridge e t aL, 1988; Hestrin e t al.. 1990; Lambert and Jones, 1990). The quinoxaline derivatives have proven to be valuble pharmacological tools i n studying excitatory neurotransmission. However, because they exert poor selectivity towards antagonizing either kainate or A M P A 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 A M P A ionotropic receptors will be developed  i n the near future and  questions pertaining to the relative contributions of either receptor to the fast EPSP will 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 G A B A activates at least three types of G A B A receptors.  They include two  subtypes of G A B A ^ 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 G A B A ^ receptors while the slow IPSP by activation of G A B A g receptors. Though IPSPs are displayed i n both granule and pyramidal cells, m u c h of the work done to characterize the G A B A 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 i n 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 feedforward 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 i n the strata along the oriens/alveus boarder or i n 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 G A B A ^ binding sites i n C A l to CA3 are moderate where as G A B A g binding sites i n the same subfields are primairily observed i n strata oriens and radiatum but not i n stratum p5n'amidale (Bowerv et. al.. 1987). 12.2. Properties of the G A B A ^ 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 m V (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 i n 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 G A B A ^ 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 i n sequence with the E P S P 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 m V (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 G A B A ^ receptor-mediated potential.  Recently, Davies et. al. (1990) demonstrated that the orthodromic  fast IPSP could be pharmacologically separated from the E P S P 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 G A B A mediated CI" channel exhibits multiple conductance  states  i n 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 G A B A 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 G A B A mediated chloride chginnel it appears that for a unitary IPSC, G A B A 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 Langmoen, 1980).  and  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 G A B A but rather from a sequential release of quanta. Indeed, the h)^erpolarization i n 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 G A B A g 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 i n 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 m V (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 i n p5nramidal  neurons after a brief train of action potentials which are dependent u p o n 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 E G T A fails to alter it (Knowles et. al.. 1984). Furthermore, the slow IPSP can be elicited without an E P S P or action potentials (Knowles e t al.. 1984). G A B A ^ receptors appear not to participate i n 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 e t al.. 1985; Newberry and Nicoll, 1985) .  This led to the hypothesis that the slow IPSP was mediated by  activation of G A B A g receptors and was later confirmed by the development of specific centrally acting  GABAQ  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 G A B A g receptor mediated IPSP i n 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 C G P 35348 (Olpe e t al.. 1990).  There is groAving evidence which suggesting that the G A B A g 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) i n mediating G A B A g 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 G A B A 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 G A B A g 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 i n a variety of physiological and pathophysiological states. For example,  because  the potential produces a decrease i n 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 i n 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 i n immature animals supports  this view (Xie and Smart, 1991),  Since the slow IPSP appears to regulate the  activation of the N M D A receptor it may also play an important role i n regulating synaptic plasticity (Morrisett et, al,. 1991) or other events w h i c h require the participation of N M D A receptors. 12.3.1, Presynaptic G A B A g receptors. GABAg  receptors  function  in  reducing  There is growing evidence that s)niaptic  potentials  through  a  mechanism independent of its postsynaptic effects. This type of inhibition is thought to involve activation of presynaptically located G A B A g (Bowery et. al.. 1981).  receptors  Initial experiments suggested that only EPSPs were  blocked by the pres)niaptic actions of baclofen  (Ault and Nadler,  Lanthorn and Cotman, 1981; Olpe et. al.. 1982), but later demonstrated IPSPs were affected  1982;  experiments  as well (Misgeld et. al.. 1989; Peet and  McLennan, 1986). Recent experiments have further supported the existence of presynaptic G A B A g 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 G A B A g receptors then an increase i n K"*" conductance leading to a hyperpolarization i n 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 G A B A g receptor-mediated  K+  conductance is supported by the observation that the presynaptic actions of baclofen are blocked by barium (Misgeld et. al.. 1989), a n ion which has been demonstrated conductance  to  block  (Gahwiler  the  postsynaptic  and Brown,  GABAg  receptor-mediated  1985; Newberry  and Nicoll,  K"*"  1985).  However, Lambert et. aL (1991) did not observe an antagonism of the baclofeninduced  pres3niaptic  GABAg  receptor-mediated  responses  to  barium.  Therefore, the existence of a pres5niaptic G A B A g receptor-mediated K+-current remains to be determined. Several indirect lines of evidence suggest that pre- and postsynaptic G A B A g 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 G A B A g responses are more resistant than postS)maptic G A B A g responses to G A B A g antagonists (Dutar and Nicoll, 1988; Davies et. aL. 1990; Davies et. al.. 1991; Gage, 1992). The observation that the postsynaptic effects mediated by G A B A g 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 G A B A ^ 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). and Nicoll,  1982a) or 4-AP  depolarizing  potential.  This  Application of either pentobarbitone (Alger  (Avoli and Perreault, pharmacologically  1987) potentiates  induced  potentiation  the 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 currentsource density (CSD) analysis of field potentials which reveal that during maximal activation of the potential the active sink occurs i n the middle portion of the stratum radiatum (Perreault and Avoli, 1989).  The depolarizing IPSP is  mediated by activation of G A B A ^ receptors since it is mimicked by dendritic applications of G A B A (Alger and Nicoll, 1982b; Andersen et. al.. 1980b; Thalmann et. al.. 1981) and is abolished by either bicuculline or picrotoxin (Alger and Nicoll, 1982a). pres)maptic  It appears that this potential is generated by  release of G A B A since spontaneous  GABA  receptor-mediated  depolarizations have been observed (Alger and Nicoll, 1980) and depolarizing responses to iontophoretically applied G A B A are not significantly altered when 4-AP is applied (Perreault and Avoli, 1991).  Though not conclusive, the  depolarizing response to G A B A is thought to be mediated by activation of extrasynaptic G A B A 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 i n 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 i n pyramidal cells while having little effect on the increased conductance  (Perreault  and Avoli,  1987),  If different  CI" concentration  gradients exist i n the soma and the dendrites of pyramidal cells then this would explain the opposing effects of G A B A when applied to the dendrites and soma of the pyramidal cells. The physiological significance for a G A B A 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 G A B A ^ mediated  depolarizing potentials  are  responsible for the recruitment and synchronus activation of GABAergic  interneurons which generate high amplitude IPSPs i n neighboring granule and pyramidal cells. 13.0. L O N G - T E R M POTENTIATION IN T H E HIPPOCAMPUS Long-term  potentiation  (LTP)  of  S3maptic  transmission was  first  demonstrated by Lomo i n 1966 and later analyzed by Bliss and Lomo; Bliss and Gardner-Medwin i n 1973. LTP has since been a topic of great interest to many neurobiologists because  it displays use-dependent  properties  resemble certain physiological processes such as learning and memory.  that No  where else i n the mammalian central nervous system has LTP been as intensely studied as i n the hippocampus, the presumed locus for information storage.  Consequently, m u c h 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 i n 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 a n d 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 i n 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 i n their anaethestized preparations while i n 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 i n 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 i n the slope of the field EPSP measured i n the dendrites.  Following a high frequency  tetanic  stimulation of the hippocampal afférents, the slope of the field E P S P 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 longlasting increase i n the amplitudes of the population spike and population EPSP, a prolonged decrease i n population spike latency, the production of E - S potentiation and an increase i n 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 i n the height and slope of the intraceUular EPSP (Andersen et. al.. 1977; Schwartzkroin, 1975) with no detectable changes i n 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 w h i c h supports the E-S potentiation observed i n 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 i n synaptic conductance that is not associated with a shift i n the E P S P reversal potential (Barrionuevo et. al.. 1986).  13.2. Projections i n 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 i n other afferent pathways i n the hippocampus including the lateral perforant  pathway  (McNaughton  et.  al.. 1978). mossy  fiber  projection to C A 3 (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 feedforward inhibitory synapses i n 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  Schwartzkroin and Wester,  et.  al.. 1979; Lynch et.  al.. 1977;  1975) and dentate gyrus (Bliss et. al.. 1973;  McNaugton and Barnes, 1977).  A n exception to this generalzed observation  occurs i n C A 3 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 N M D A 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 i n 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). threshold for  LTP induction has been  reported  to require  The  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 i n the strong input but also i n the weak input, which, when tetanized alone does not display LTP (Barrionuevo and Brown, 1983; Levy and Steward,  1979; McNaughton et. al.. 1978).  "associative" nature of LTP.  This property describes  Associative LTP can occur when the  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 N M D A E P S P (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  hyperpolarized  if afferent  (Mallnow  activation  and Miller,  is paired  when  1986) demonstrates  the cell is the voltage-  dependence of associativity and further supports a role for N M D A receptors i n associative LTP. 13.6. Requirement for Calcium The perfusate  observations  that hippocampal  were incapable  slices exposed  to a Ca2+-free  of supporting S3niaptic transmission and, not  suprisingly, LTP suggest an essentlEil role for Ca2+ i n 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 i n 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 E G T A (Lynch et. a l . . 1983) and BAPTA (Morishita and Sastry, 1991) block the induction of LTP i n 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 N M D A receptor. B u t what about S3mapses that do not display N M D A receptor-dependent LTP?  Do  they require an posts)maptic increase intracellular Ca2+? The apparent answer to this question is yes!  In fact, N M D A 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 voltagedependent 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 i n 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  i n 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 i n synaptic efficacy and 2) those events that transpire following the tetanus producing a stable non-decremental increase i n 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 i n 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,  A t the Schaffer coUateral/commissural-CAl  pyramidal cell sjmapse, one factor which determines the expression of these events is the degree of N M D A 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 G A B A receptor-  mediated neurotransmission. It is widely accepted that N M D A receptors play a paramount role i n the induction of LTP (CoUingridge et, al.. 1983; CoUingridge and BUss, 1987; Wigstrom and Gustaffson, 1988). dependence,  Because of its voltage-  however, the degree of N M D A 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 G A B A 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  h5^erpolarization receptors.  of the  neuron  to  depolarize,  as a result  of  the  and shunting effect produced by activation of G A B A ^  The finding by Wigstrom and Gustaffson (1983) that picrotoxin  greatly facUitates the induction of LTP further emphasizes the importance of inhibition i n regulating the induction of LTP. Recently, presynaptic G A B A g receptors have been demonstrated to play an important modulatory role i n the induction of LTP i n 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 G A B A g receptor  antagonists  2-hydro3^-saclofen  and C G P 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 G A B A g receptormediated 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 i n synaptic efficacy lasting hours in vitro and days i n vivo.  Recently, m u c h 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 a n increase i n 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 i n 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 i n turn degraded a cytoskeletal protein (fodrin) resulting i n unmasking a subpopulation of dendritically localized glutamate receptors.  However, the strength of this  hypothesis was questioned when two independent groups separately reported no increase i n glutamate binding i n potentiated hippocampal tissue (Goh e t 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 i n 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 i n hippocampal LTP stems from the observations that phosphorylation of protein F l by membrane bound P K C 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 P K C including B 5 0 and GAP 43 are phosphorylated during LTP. Furthermore, translocation of cytosolic P K C to the membrane bound form has been reported to be enhanced i n potentiated tissue (Akers et. al.. 1986) and phorbol esters, activators of PKC (Malenka e t aL. 1986; Gustaffson e t al.. 1988) as well as injection of PKC (Hu e t al.. 1987) induce an LTP-like potentiation.  In addition to P K C , activation of CaMKII  kinase also appears to be activated following the induction of LTP. CaMKII kinase has been detected postsynaptically particularly i n the dendritic spine (Kennedy e t 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 i n 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 C a M K l l activity i n the maintenance of LTP was reported by Malinow et. al. (1988). They demonstrated that sphingosine, an inhibitor of diacylglycerol and calmodulin (both activators of P K C and C a M K l 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 C a M K l l kinase can be activated by auto-  phosphorylation (Saitoh and Schwartz, 1985) and reports suggesting that P K M , 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 i n 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 P K C and C a M K l 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 P K C and C a M K l l kinase in the expression of LTP of excitatory synaptic transmission, participation of postsynaptic protein kinases appears to modulate long-term changes i n 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 a n increase i n the non-NMDA but not the N M D A component of the E P S P i n C A l neurons.  Similar results were also  reported by MuUer et. al. (1988) (but see Bashir et. al.. 1989).  Davies et. a l .  (1989) reported that the sensitivity of C A l neurons to iontophoretically applied quisqualate and A M P A 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 i n presynaptic terminal excitability has been reported i n 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 i n a greater facilitation of neurotransmitter release.  S u c h a presynaptic  mechanism would be consistent with the finding of Baimbridge and Miller (1981) where the uptake and retention of labelled calcium i n 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 K u h n t (1986) reported increased Ca^"^ uptake i n 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 E G T A 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 i n 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 i n 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 accumulation  produced  by  an  increase  i n pres5maptic  employed,  Ca2+  Ca2+ could  go  undetected. Further evidence for a presynaptic involvement i n the expression of LTP arises from observations that tetanus-induced as well as Ca2+-induced LTP results i n a sustained increase i n the release of glutamate and aspartate i n the dentate gyrus (Bliss et. al.. 1986, but see Aniksztejn et. al.. 1989). A similar long-lasting increase for aspartate has been observed i n 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 E P S P (presumably due to an asynchronous release of transmitter) was increased i n hippocampal slices incubated i n 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. accurately  determine  whether  neurotransmitter release, synapse i n question.  maintenance  involves  an  In order to increase  in  a quantal analysis could be conducted on the  It should be noted, however, that this technique when  applied i n 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  i n these  studies some quantal events may have gone undetected due to the poor signal to  noise  ratio  microelectrode  of  this recording  technique.  Recent  advancements  recording techniques, particularly the "on-slice" whole  voltage clamp recording technique  (Blanton et. al.. 1989) have  investigators  expression  to  assess  whether  of  LTP  resides  in cell  enabled 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 nonlinear 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  primarily mediated by a presynaptic mechanism.  that associative  LTP  was  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 i n 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 i n 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 changes  i n synaptic efficacy.  for  Indirect evidence for such interactions i n  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  depolarization. independently  afferent  input  is  paired  with  a  sustciined  postsynaptic  The fact that no LTP occurs when the two events occur suggests  Induction of LTP.  a  postS3niaptic  contribution  to  the  associative  Furthermore, the observation that associative LTP is  primarily presynaptic i n 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 i n 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 i n 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 i n 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 i n 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 i n 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 i n C A l p5n:amidal neurons (Bredt et. al.. 1992; Vincent and  Kimura,  1992)  contradicts  an  involvement  collateral/commissural-CAl pyramidal cell LTP.  of  NO  in  Schaffer  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  confounded by the strict requirements they must possess.  is  Recently, Ballyk  and Goh (1991) adveinced the hypothesis by Goh and Sastry (1985) that potassium ions (thought to be involved i n 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 cooperativity between presynaptic involvement retrograde  flbers.  enhance  Future studies investigating the  of K+ i n LTP may substantiate the monovalent  cation as a  messenger.  14. SACCHARIN A S A P H A R M A C O L O G I C A L TOOL O F INVESTIGATION 14.1. History Since its discovery by Remsen and Fahlberg i n 1879, saccharin has been used as a non-caloric sweetner i n nearly all countries of the world.  Its use  alone or i n combination with cyclamate was so popular i n the food industry that by 1967, it was estimated that nearly 7 5 % 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. i n 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 i n the study and the evidence for saccharin as a potential carcinogen i n m a n 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 until 1970 that B i y a n et. al. confirmed the initial finding of Allen and coworkers.  Their results were supplemented with histological evidence that so  lacked i n the 1957 study.  In the next 7 years evidence was put forth by a  variety of investigators varifying the carcinogenic effects of saccharin i n other mammalian  species.  Though  by  this time,  conclusive  evidence for  its  carcinogenicity i n humans was still lacking, results from the animal studies prompted the Canadian government to ban the use of saccharin i n the food, drug and cosmetic industries i n M a r c h 1977.  Because of the controversy  surrounding saccharin as a potential agent for carcinogenicity i n humans, other counties have yet to follow Canada's suit. 14.2. Chemistry and Physical Properties Saccharin  is  a  2,3-dihydro-3-oxobenzisosulfonazole  commercially synthesized from 0-sulfanoylbenzoic acid.  which  is  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 i n 290 m L of H2O), however, its sodium salt is not (1 g dissolves i n 1.2 m L of H2O). Because the p K a of saccharin is approximately 1.8, almost all of the drug is ionized at neutral p H . 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 i n humans and is excreted unchanged i n the urine (Lethco and Wallace, 1975).  essentially  It has been detected i n  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 i n 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 i n 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-Nnitrosourea  (MNU)  when  combined with saccharin produced  a  greater  incidence of bladder tumors than when M N U 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 w i t h 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 D N A (Lutz and Schlatter, 1977) nor is it metabolized to a reactive  electrophile  (Sweatman  and  Renwick,  1979).  Furthermore,  at  a  pH  approximating the p H 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  monophosphate (cGMP).  triphosphate  (GTP)  to  guanosine  3',  5'-cyclic  This is particularly interesting since changes i n the  intracellular levels of c G M P have been implicated i n normal and abnormal cell growth (Kram and Tompkins, 1973; K i m u r a and Murad, 1975; Vesely et. al.. 1976).  The actions of saccharin are, however, not confined to one enzyme  system, infact other systems not implicated i n tumerogenesis are also altered by saccharin.  For example, changes i n intracellular adenosine 3', 5'-cyclic  monophosphate (cAMP) concentration i n 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 i n rat musle and liver membrane preparations suggests that the taste transduction process may be affected i n 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 K o h n . 1984).  Saccharin has also been found to  stimulate protein kinase C activity i n 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 i n 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 i n 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 N G F induced neurite out-growth i n the D R G 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 i n 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 i n  guinea-pig hippocampal slices raised the prospect for a possible involvement of endogenous growth factors i n the development of LTP (Chirwa, 1988; Sastry e t aL, 1988a; Sastry et. aL. 1988b; Xie et. al.. 1991) 14.6. Involvement i n 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 N M D LA (Chirwa,  1988).  Furthermore, the observation that saccharin blocked LTP induced by applied N G F further supported the involvement of growth factors i n LTP.  15. MATERIALS A N D M E T H O D S The following pages are organized i n sections describing i n 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 i n 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 British 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 i n a communal wire "housing" cage (dimensions:  59 x 54 x 23 cm).  Animals arriving the following week were housed i n 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 i n the room were on between 6 am to 6 pm. The  guinea-pigs  had free access  to  Guinea-pig Chow contained i n a  compartment located on the side of the cage and water dispensed through a small drinking tube connected to an inverted 250 m l 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 guineapig 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 i n 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 until  surgical anaesthesia was achieved.  This was  established if the animal  appeared unconscious, respiration was predominantly thoracic i n 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 skull. This enabled a pair of scissors to be inserted under the skull 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 skull case and was immediately placed on dissecting paper (ventral sidedown) 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  hippocampus was perpendicular to the blade of the tissue chopper.  of  the  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 j u s t 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 m l 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 i n 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 i n 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 i n 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 i n 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 u n t i l it entered the base of the circular chamber.  The  main inlet line was inserted through this hole. The inlet line fed through a n 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 i n the heating block.  Because the  temperature sensing device fed-back to a heater control panel, the superfusate i n the chamber could be monitored and maintained at a constant temperature, A gold "female" grounding p i n was inserted into the bottom of the circular chamber though a hole drilled j u s t below the inlet line. The m a i n inlet line was connected to a manifold which was fed, i n t u r n , by eight lines each connected to a separate superfusion barrel (60 m l syringe tube).  The superfusion barrels were mounted on an adjustable a l u m i n u m  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 a l u m i n u m bracket. 15.4. Perfusion Media The physiological normal media was prepared daily and contained (in mM):  N a C l 120, K C l 3.1, NaHCOa 26. N a H 2 P 0 4 1.8. MgCl2 2, C a C l 2 2,  and  dextrose 10. The p H of the medium was 7.4 when bubbled with carbogen.  In  some experiments, saccharin (sodium salt; Sigma), 6-cyano-7-nitroquinoxallne2,3-dione Sigma),  (CNQX;  Tocris  Neuramin),  2-amino-5-phosphonovalerate  D(-)2-amino-5-phosphonovalerate  [(-)APV;  Sigma],  (APV;  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 i n total osmolarity.  In addition to the normal  physiological medium, two other media were used i n 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 p H 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 i n 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, u p to eight different media could be superfused i n one experiment. However, i n a typical experiment, only 3 to 4 barrels were used.  The m a i n  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 i n all the barrels as well as the reservoir were aereated with carbogen through lines that were connected to a common plexiglass tube that was, i n turn, connected to a regulator attached to a carbogen tank.  To minimize "dead space" i n 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 i n 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. made  Extracellular recording electrodes  were  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)  microelectrodes were filled with either, 4 M MQ) or a solution containing 4 M  micropipette  CH3COOK  CH3COOK  puller.  The  (tip resistances, 70 to 85  and 100 m M QX-314 (Astra  Pharmaceuticals) (tip resistance, 85 to 120 MQ). 15.5.2. Amplifiers.  Extracellular field potentials were amplified w i t h  a D A M - 5 A differential pre-amplifier (World Precision Instruments).  During  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 i n 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 m a x i m u m D C current command was ±10 nA; the maximum gain i n dSEVC was 10 nA/mV and the capacitance neutralization range was -1 to 4 pF, In the C C 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 i n 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 i n the C C mode, some were monitored i n the D C C and d S E V C modes. In these particular cases, i n 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 i n both D C C and d S E V C modes was 3 0 % current passing and 7 0 % voltage recording for each cycle.  In both D C C and d S E V C 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 i n d S E V C 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 i n 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 K H z and 1 K H z 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 i n 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, D C 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, i n 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 m m 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  i n 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 i n 1 |im steps at a rate of 200 steps/second. electrodes  were mounted on D K l electrode carriers.  micropositioner,  the  electrode carriers were  Like the Optikon  capable  stimulating electrodes i n all three coordinate planes.  Stimulating  of positioning  the  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). isolated, table.  The whole recording set-up sat on a vibration-free, air  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 i n 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 i n the stratum radiatum. The stimulation strengths were adjusted so that the height of population spikes measured 1 to 1.5 m V or the amplitude of the field E P S P s were between 0.5 to 1.0 mV. Only those responses that exhibited a 1,5 fold or greater increase i n 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 hippocampus.  were obtained  from  CAl^j neurons of  the  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. served as the control for the experiment.  These  responses  Paired-pulse facilitation (interstimulus interval, 40 msec) of the E P S P was used as a test to determine the viability of the neuron and its associated excitatory S5niapses. The neuron was considered for use i n the experiment if the second pulse generated an E P S P 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 i n  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 i n inducing LTP, the latter stimulation was employed i n some of the intracellular experiments conducted because LTP of the E P S P 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 i n individual hippocampal pyramidal neurons if the stratum radiatum was stimulated i n conjunction with sufficient postsynaptic depolarization. intracellular  The number of pairings determined whether the  E P S P exhibited  potentiation (LTP).  short-term  potentiation  (STP)  or  long-term  In the present study, LTP of the intracellular EPSP slope  was induced i n slices superfused with 20 \iM picrotoxinin by performing 25 consecutive pairings. The pairings involved injecting D C current (3 nA to 4 nA) through the recording electrode to depolarize the neuron near the reversal  potential of the E P S P 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 E P S P peak to the second E P S P 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 i n millivolts. The initial slopes of the extracellular and intracellular EPSPs were computed on-line by the DATA 6000 and, unless otherwise stated, expressed i n 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 i n m V represented  the height of the E P S P 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 i n 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,  comparisons test.  the  data  was  subjected  to  Duncans' multiple  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. E X P E R I M E N T A L PROTOCOLS Previous experiments demonstrated that 10 m M saccharin when applied for 5 or 10 minutes blocked LTP of the population spike i n 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 E P S P (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 m M , applied for 10 minutes) effects excitatory and inhibitory synaptic transmission and whether such effects could account for it blocking LTP i n guinea-pig hippocampal CAljr, neurons.  16.1. Saccharin and LTP of the Field E P S P 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 i n quantitating LTP-induced changes i n S5maptic transmission. For example, one can never observe the population spike i n isolation since it is superimposed upon the field  EPSP.  always  The fact that the population spike  undergoes changes i n 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 E P S P 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 E P S P is measured.  Thus, an increase i n the slope, as opposed to an increase i n the height, of the EPSP was taken to reflect a potentiation of relatively pure  monosynaptic  transmission. Since the present study was interested i n examining the effects of saccharin on synaptic transmission, the slopes of the fleld EPSPs were recorded. The  fleld  EPSPs were evoked  i n 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 E P S P In order to establish that saccharin blocked LTP of the field E P S P byacting on the individual CAl^j neurons, experiments identical to those used to demonstrate the effects of saccharin on the extracellular E P S P slope were also conducted on the intracellular E P S P slope.  In order to minimize the standard  error, whenever possible, the experiments were conducted on a slightly larger sample size than those used i n 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  concommitant low frequency activation of the stratum radiatum.  with  In these  experiments, the slices were equilibriated with the picrotoxinin containing medium. Intracellular EPSPs were recorded from to 0.2 Hz stimulation of the stratum radiatum.  CAly^ neurons i n response Membrane potentials of the  neurons were clamped at -80 m V so if any potentiation of the E P S P occured  after pairing, it would not result i n 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 E P S P  slopes were recorded for 10 minutes. Saccharin (10 mM) was then superfused onto the slices for 10 minutes. pairing was performed.  During the 8th minute of drug superfusion,  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 N M D A 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 N M D A 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 N M D A 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 N M D A receptor antagonist, A P V and has been interpreted as being necessary for the induction of LTP i n these neurons (CoUingridge et. al.. 1988; Haung et. al.. 1986). Whether saccharin interfered with the late N M D A 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 C N Q X or 25 |iM D(-)APV.  In  impaled  CAl-^  neurons  exposed  to  a  Mg2+-free  medium,  depolarizations induced by bath applied N M D A (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. separate  experiments,  to reduce  In  the possibility that the N M D A induced  depolarizations were not contaminated by depolarizations produced by a nonspecific action of N M D A on non-NMDA receptors, the non-NMDA receptor antagonist,  C N Q X (10 ^M) was added to the Mg2+-free media.  In this  experiment, 0.1 |J.M TTX was also present i n the media Experiments were also conducted to examine the effects of saccharin on NMDA and non-NMDA components of the intracellular E P S P slope.  In these  experiments, EPSPs were evoked by stimulating the stratum radiatum at 0.2 Hz. The  EPSPs were evoked i n either a normal medium, a normal medium  containing 40 |J.M A P V or i n a Mg2+-free medium containing 10 |iM C N Q X . Stable control responses were obtained for 10 minutes before the slices were exposed to saccharin. minute  application  of  Records were acquired at the 10th minute of a 10 10 m M  saccharin  after  resuperfused with their respective control media.  which  the  slices  were  Additional responses were  recorded after a 20 minute recovery from saccharin. 16.5.2. Saccharin and input-output (I-O) relationships of the E P S P and the IPSP.  Experiments were conducted to investigate the "input-output" (I-O)  relationship of a CAl^^ pyramidal cell E P S P and IPSP i n the presence and absence of saccharin. Control EPSPs were evoked i n 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 E P S P 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 E P S P 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 E P S P . I-O plots were made for the IPSP evoked i n 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 i n 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 C N Q X and 40 fxM APV, In order to record IPSPs i n this medium, the stimulating electrode was placed i n the stratum radiatum approximately 0.5 m m from the cell under investigation, IPSPs recorded  i n 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 G A B A ^ and G A B A g 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 C N Q X and A P V containing medium. Permanent records were taken before, during the last minute of, and following  a  experiments  15 minute  recoveiy from  saccharin application.  using  same  were  picrotoxinin was  the  added  protocol  to the modified  conducted  except  Separate 20 |iM  superfusing media i n order  to  investigate the effects of saccharin on the slow IPSP. 16.5.4.  Saccharin and QX-314 injected neurons.  lidocaine derivative,  QX-314 has has been  reported  The quartemary 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 i n 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. neuron through the microelectrode current).  AVith  QX-314 was injected into the  depolarizing current (+1 nA, D C  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  9th minute  were taken before and during the  of saccharin  application for subsequent analysis. 16.5.5. Saccharin and paired-pulse depression of the fast IPSP. d-Pulse depression of the fast IPSP has been extensively  Paire  studied i n the  hippocampus and is thought to involve a decrease i n release of G A B A due to activation of G A B A g autoreceptors on the pres5niaptic terminals of inhibitory interneurons (see chapter 12.3.1). pulse  activates  presynaptic  Presumably, G A B A released by the first  GABAg  receptors  which,  in  turn,  reduces  transmitter release evoked by the second pulse. This appears as a decrease i n amplitude of the fast IPSP arising from the second pulse when compared to the one evoked by the first. Recently,  the induction of LTP i n 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 G A B A g mediated autoinhibition of G A B A release. In view of the above, the possibility that saccharin blocked the induction of LTP through an action at the pres5niaptic G A B A g 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 G A B A g receptors.  In  these  experiments,  slices were  superfused  with  medium  containing 20 |iM C N Q X 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 i n 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 E P S P 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 E P S P 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 E P S P and is reflected as an increase i n E P S P 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 N M D A receptor is activated. In addition, since the fast IPSP has also been reported to shunt the E P S P i n 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 N M D A receptor.  To investigate this possibility, experiments were  conducted to examine the effects of the fast IPSP on paired-pulse facilitation of the EPSP, first, i n normal (control) medium and then i n medium containing saccharin. For these experiments, the EPSP-fast IPSP-slow IPSP sequence was recorded from C A l ^ neurons i n response to stimulating the stratum radiatum at 0.033 Hz. Paired-pulse facilitation of the E P S P was elicited by delivering two identical shocks (interstimulus interval, 40 msec) to the stratum radiatum. Records were obtained i n 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 i n picrotoxinin containing media. In order to determine if saccharin blocked tetanus induced LTP by augmenting the fast IPSP, the following experiments were performed. expose to the picrotoxinin containing media.  Hippocampal slices were  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. R E S U L T S 17.1. Saccharin and LTP of the Fleld E P S P 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 E P S P slope that quickly decayed within 10 minutes. Thereafter, LTP of the E P S P slope was not observed i n the 6 slices tested (Figure 17.1b).  In  separate control experiments, slices not exposed to saccharin exhibited a PTP of the E P S P 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 E P S P which was on average twice the pre-tetanic  control  responses (Figure 17,1a). These results demonstrate that saccharin blocks LTP of the field E P S P 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 E P S P 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 E P S P slope that rapidly decayed to levels approaching the pretetanic control values which persisted until the experiment was terminated (Figure 17.2b). This was observed for all 9 slices tested.  However, i n 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 i n saccharin treated slices. Following the PTP, LTP was observed i n all 9 slices tested (Figure 17.2a).  It, therefore,  appears that saccharin  blocks LTP of the population spike and field E P S P by exerting its effects on the individual CAl-^ neurons.  a  1 mV 350  T  20 ms  O 300--  CL O  m  i  o  . ^  200-  3  ^  05  °  u-  p X UJ  250-  CO  o  i  h 1  ài  1  i  i  i  i  i  i  T.  T.  ^  I  a  150-  I 100  O  8  y 0  0  20  30  40  50  Time  (min)  60  70  80  90  Figure 17.1. Saccharin blocks LTP of the field E P S P slope. The field E P S P slope was recorded prior to a n d after a tetanic stimulation (arrow) of the stratum radiatum. This procedure was executed i n 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 i n the presence of saccharin but does i n the normal medium. Superimposed traces at the top of the graph illustrate field E P S P s 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 m V 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 A N O V A with Duncan's multiple comparison test (p<0.05, n=6 sUces).  y  250  T  O 0) û. _o _ CO o û-  LxJ  o  °  13 ° u en  o  o  20 ms  b  200--  c  00 o CL CJ  T  150--  looi o î ^  •  •  O  4  T* T  * o A^* y O X* T: ? ^ i ? 9*9 g O.*, •  Q o i  5mV  y  i  1  •  T • i  O '  0  T  é y  0  10  20  30  40  50  Time  (min)  60  -H 70  h80  90  Figure 17.2. LTP of the intracellular E P S P slope is blocked bv saccharin. The slope of the intracellular E P S P 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 o n 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 E P S P s 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 m V and 20 msec. D a t a 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 A N O V A with Duncan's multiple comparison test (p<0.05, n=9 slices).  3.500 T ^  y  3.000i E  0.5 mV  o 2.500  20 ms  o ^ o o  =(t  ^  2.000-  T  O  c  o 'o  0  1  1.500--  3 ^  O  1.000 +  O  o  o y  0.500  0  20  40  60  80  100  Time (min)  Figure 17.3. Maintenance of LTP is not blocked bv saccharin. A post-tetanic application of saccharin (solid b a r o n 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 m V a n d 2 0 msec. Data are presented as meein ± S E M . Asterisks Indicate significant difierences from the control responses as calculated by a one-way A N O V A 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). significant differences  Though  were observed between control responses and post-  tetanic responses, differences between the responses obtained during the posttetanic  application of  saccharin and  withdrawal were not significant.  the  responses  recorded  after  its  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 E P S P 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 i n the E P S P slope (Figure 17.4). Similar results were observed i n two other CAlj-, neurons each obtained from different slices. 17.5. Saccharin and N M D A 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 until the end of the train upon which a slow hyperpolarization ensued.  In the presence of saccharin, a n  identical tetanus produced a depolarization that was longer i n duration t h a n  y+z  x+z 5mV 50 ms  3i 3 if) E > c  2A  2-  A  QO  (71 iX (y") CL  •  •  A y  0  10  20  30  40  —I 50  Time  1 50  1 70  1 80  1^ 90  1 100  (min)  Figure 17.4. Saccharin reversibly blocks pairing-induced LTP of the intracellular E P S P slope. The slope of the intracellular E P S P 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 i n normal medium after a 20 minute a n d 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 a n d z. Some traces are superimposed for comparisons of the E P S P slopes recorded at their respective times. The membrane potential of the C A l ^ , neuron was clamped at -80 mV.  A  Control  B  Saccharin (10 mM)  C - A P V (25 nM)  '^f/^  '-^—'^L/^'^t  C N Q X ( 10 jiM)  E 30 min Recovery  A+B 5mvL_ 400 Hz, 200 pulses  0.2 s  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 p M D(-)APV; D. 10 nM C N Q X and E , normal medlvmi after a 30 minute recovery from D. A+B are superimposed traces of the depolarizations recorded i n 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, a n identical tetanic stimulation was administered to slices superfused with media containing either 25 |J,M D()APV or 10 |iM C N Q X (Figure 17.5C and 17.5D, respectively). Compared w i t h the control response, the depolarization and post-tetanic  h5^erpolarization  produced i n A P V containing medium were slightly reduced and those i n C N Q X 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. Because  Saccharin and depolarizations produced by applied NMDA.  depolarizing  responses  in C A l  neurons  decrease to  repeated  applications of exogenously applied N M D A (a phenomenon thought to reflect desensitization),  care was taken to allow sufficient time between repeated  N M D A 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 N M D A were separated by at least 5 minutes. When slices were superfused with 10 m M saccharin for 10 minutes, no change i n membrane potential or input resistance was observed (Figure 17.6A). subsequent  application  of N M D A  during the  last minute  A  of saccharin  application produced a depolarization that did not differ significantly from the depolarization produced by N M D A prior to application of the drug (peak depolarization to N M D A i n mV, control:  16 ± 4; during the last m i n 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 N M D A depolarization was also not significantly altered by saccharin (ratio of Rj^ to applied NMDA. control: 0.6 ± 0 . 1 ; during the last m i n 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 C N Q X and 0.1 |iM TTX (peak depolarization to N M D A i n mV.  A  Mg2+-free  Sacdiafta(IOinM)  -APV(25(iM)  J*  60 mV  NMOA(IOtiM) 3min  B  Mg2+-frae and CNQX (10 MM) Saccharin (10 mM)  -APV(25MM)  NM0A(10|iM)  30 mV 3min  Figure 1 7 . 6 . Depolarizations Induced b v applied N M D A are not significantly altered to the presence of saccharin. A shows depolarizations of a C A l ^ , neuron recorded to a Mg^^-free medium to response to bath applications of 1 0 |JM N M D A superfused for 3 0 seconds (soUd b a r below the trace). N M D A was applied before, during and after a 1 0 mtoute application of 1 0 m M saccharin. The neuron was later exposed to 25 \M D(-)APV for 5 mtoutes to block the N M D A toduced depolarization. B. the depolarizations of a CAl^ neuron toduced by applied N M D A were performed to a Mg2'''-free m e d i u m contatotog 1 0 n M C N Q X and 0.1 \iM tetrodotoxto. Traces to A and B were recorded from two different cells.  control: 19 + 2; during the last m i n 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 N M D A and non-NMDA components  intracellular EPSP.  of the  Previous experiments demonstrated that saccharin did  not alter the height of the intracellular E P S P i n slices superfused with a normal media (Chirwa, 1988). The same was observed for the slope of the intracellular EPSP (slope of the E P S P i n V/sec, control: 1.94 ± 0.50; during the last m i n 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 N M D A 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 m i n application of 10 m M saccharin did not significantly alter the non-NMDA E P S P slope (slope of the E P S P i n 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 m i n recoveiy from saccharin, a subsequent application of 10 |iM C N Q X blocked the EPSP, confirming it was dependent upon S5niaptic activation of the non-NMDA receptors (last trace i n Figure 17.7B), The N M D A component of the E P S P 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 N M D A mediated E P S P was not significantly altered (slope of the N M D A E P S P i n V/sec, control: 0.52 ± 0.05; during the last m i n 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 A P V to the superfusing media completely abolished the E P S P and, therefore, demonstrated  the E P S P resulted from  activation of the N M D A receptors (last trace. Figure 17.7C).  synaptic  Saccharin (10 mM)  ±APV and C N Q X  NoftnaJ medium  B  Normal medium and±APV(40nM)  r C  Mg2+-free and C N Q X (10 |iM)  Control  10 min  20 min Recovery 10 m V 100 ms  3.000 T  Normal medium  Normal medium and ±APV  2.000 •• Q. O Mg ^ Q. U  1.000  0.000  free  and CNQX  c 2 2 Oo  o u o  1c 1o  o a U CO  Figure 17.7. The intracellular E P S P slope recorded i n the presence of excitatory amino acid antagonists is not significantly altered b y saccharin. In A, the E P S P s 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 E P S P recorded at the 10th minute of saccharin application. The last trace Illustrates the E P S P recorded i n the normal m e d i u m after a 2 0 minute recovery from the drug. EPSPs were also recorded tn normal medium and 40 |iM APV, B and i n Mg2+-free medium containing 10 p M C N Q X . C. The last trace In B a n d C Illustrate the block of synaptic responses when both A P V and C N Q X were present In their respective media. D, shows a histogram of the E P S P slopes recorded i n the different media In the absence (blank histogram) and presence (cross-hatched histogram) of saccharin. Traces i n A to C are averaged from 4 consecutive responses. D a t a are represented as m e a n ± 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 N M D A and the non-NMDA receptors. 17.6.  Saccharin and Input-Output fl-O) Relationships of the E P S P 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 i n amplitude as illustated i n Figure 17.8A. A plot of the maximal E P S P 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 E P S P 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 i n the presence of saccharin as illustrated i n 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 i n Mg^+-free Medium To further show that saccharin selectively affects the IPSP, the following experiments were performed. stimulation  of  the  stratum  In a Mg2+-free medium containing 10 |j,M CNQX, radiatum  produced  intracellularly  recorded  postsynaptic potentials consisting of an N M D A E P S P followed by an IPSP (Figure 17.9A). Application of 10 m M saccharin decreased the duration of the N M D A E P S P and increased the amplitude of the IPSP (Figure 17.9B). After a  Control  Control  Saccharin  Saccharin  10 mV  40 ms  10 mV  zooms  B  20  10  15  > J,  Q . t Q/.)  > 10  to Û4-  20  40  60  Stimulation ( ^ )  80  100  10  20  30  40  50  Stimulotion (/iA)  Figure 17.8. The effects of saccharin on the input-output (I-O) relationship of E P S P s and IPSPs recorded from C A l ^ neurons. A illustrates a series of E P S P s recorded from a C A l ^ , neuron each being evoked by stimulating the stratum radiatum with increasing stimulation strengths. Traces i n A depict E P S P s 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 E P S P s recorded i n the picrotoxinin containing medium (open circles) a n d 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) a n d saccharin contcdning medium (closed circles). Traces tn A a n d 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 i n the C N Q X containing medium, 40 ^iM APV was added. The N M D A E P S P 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 i n the shape of the N M D A E P S P by saccharin was, i n 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 A P V and 20 |iM C N Q X .  Simulation of the stratum  radiatum by an electrode positioned i n close proximity to the recording electrode evoked monosynaptic IPSPs characterized by a fast IPSP (IPSP^) slow IPSP (IPSPg) sequence as seen i n Figure 17.1 OA. D u r i n g 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 i n 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 i n the presence of saccharin (Figure 17.1 OA and 17.1 OB). Because the decay of the fast IPSP was increased i n 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, i n 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 n M C N Q X . stimulation of the stratum radiatum produced a n E P S P followed by a n IPSP In the C A l i ^ neuron, A. B shows the response In the presence of 10 m M saccharin applied for 10 minutes. After a 10 minute recovery i n the C N Q X containing Mg2"'"-free medium, 40 p M A P V was added which blocked the NMDA-dependent E P S P and revealed a n underlying IPSP, C. In D, saccharin was applied again. Note that compared to C, the ampUtude and duration of the IPSP i n D were both enhanced. These changes are clearly illustrated when the traces are superimposed In E. E a c h trace represents the average of four consecutive responses recorded from the same cell.  400 ms  B  4mV 0.2 nA  Figure 1 7 . 1 0 . 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 1 0 minute application of saccharin and following a 1 0 minute recovery In the normal medium. B shows similar effects of saccharin on the IPSC. Stimulus artifacts i n B are blanked for clarity. The normal m e d i u m and saccharin containing medium contained 4 0 yM A P V and 2 0 |JM C N Q X 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 Control Saccharin  Latencv to Peak fms) Control Saccharin  IPSP/CA  100  96 ± 2  16 ± 1  IPSP/Cg  100  57 ± 5*  245 ± 8  26 ± 4*  n 6 6  I P S P / C A and I P S P / C g were evoked In media containing 40 p M A P V a n d 20 p M C N Q X to block synaptic excitation. Control responses were taken as 100%. Responses to saccharin were expressed as a percent of the controls a n d 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 i n 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 i n Figure 17.12. In these cells, compared to the control IPSPs, application of 10 m M saccharin for 10 minutes caused a significant increase i n 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  GABAQ  responses, experiments were conducted to determine if the drug was capable of blocking responses mediated by activation of the pres3maptic G A B A g receptors. In these  experiments,  excitatory  S3niaptic transmission was  blocked  by  superfusing the slices with a normal medium containing 40 |iM A P V 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  4mV 0.2 nA 400 ms  B  Figure 17.11. The slow IPSP/C flPSP/CB) is decreased bv saccharin. A illustrates a slow IPSP recorded from a C A 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. S t i m u l u s artifacts In B were blanked for clarity. The slow IPSP/C were evoked i n normal media containing 40 n M APV, 20 ^lM C N Q X and 20 |iM picrotoxinin. Traces represent the average of four consecutive responses. Traces In A and B were recorded from two different cells.  T A B L E 17.2. Effects of saccharin on some properties of the IPSP/Cg. IP§P Hglght  Latencv to Peak fms)  D u r a t i o n at 1II  Saccharin  Control  Saccharin  n  249 ± 12  503 ± 28  503 ± 2 5  7  Control  Saccharin  Control  100  53 ± 5*  247 ± 10  max fms)  IPSP/Cg was Isolated using a media containing 20 \M. picrotoxinin. 40 p M A P V a n d 20 |iM CNQX. The height of the control responses were normalized to 100% a n d 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 Q X 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 Q X - 3 1 4 . In A, prior to a 10 minute application of 10 m M saccharin, a C A l ^ neuron injected w i t h QX-314 produced a synaptic potential consisting of a n E P S P followed by a fast IPSP w h e n the stratum radiatum was stimulated. The trace In B shows the postsynaptic potential d u r i n g 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 Control  Saccharin  100  127 ± 7*  Duration at 111 max fmsl Control 202 ± 14  Saccharin  n  235 ± 1 2 *  6  IPSP;^ was recorded from cells injected w i t h QX-314 to block postsynaptic G A B A Q 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 P I , control: 63 + 3; during the last m i n 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 G A B A g 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  contribute to a reduction of paired-pulse faciliation of the EPSP.  could  For these  experiments, an interstimulus interval of 40 msec was chosen to separate the first pulse from the second i n the stimulus pair. At this interstimulus interval, paired-pulse stimulation of the stratum radiatum produced a large facilitation of the second E P S P compared to the first i n slices exposed to a normal medium (Figure 17.15A).  However, i n 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 m i n 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 m i n 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 E P S P 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  IOOt  75-  o  à?  50--  CO O CN CL  25-  o  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 m e d i u m 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 m e d i u m (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 m e d i u m and saccharin containing medlima contained 40 [iM A P V and 20 nM C N Q X . Traces In A a n d 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 E P S P and palred-pulse depression of the fast IPSP i n neurons injected with QX-314. A illustrates both the E P S P 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) m e d i u m 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 E P S P 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 i n 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 E P S P slopes could be tetanically induced i n 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 E P S P 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, i n picrotoxinin  treated slices not exposed to saccharin, robust LTP occurred for both the extracellular and intracellular E P S P slopes (Figure 17.16a).  When comparing  the PTP of both the extracellular and intracellular E P S P slope i n control slices and slices exposed to saccharin, the PTP occuring immediately after the tetanic stimulation was decreased i n the slices treated with saccharin. It, therefore, appeared that saccharin could still block LTP i n the CAljh> region even if the fast IPSP was blocked by picrotoxinin.  a  450 1  M  Extracellular  o 350-  b -  2500 1  c o  O  o m O  O 1  O i  T* O  .a  150-  ^  i  9  1 mV 10 mV  • 20  50 J  (U Q.  300 1  CL  250-  _o CL  O i  ms  mtraceMular  200-  1  150-  100O  0  2  »  10  O  8  20  30 Time  40  50  60  (min)  Figure 17.16. LTP i n plcrotoxliiln-treated slices is not blocked bv saccharin. Simultaneous extracellular and Intracellular E P S P s were recorded i n 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 E P S P slopes (closed circles). However, In slices not exposed to saccharin, LTP of both the extracellular and intracellular E P S P 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 A N O V A 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 i n picrotoxinin containing media a and In picrotoxinin containing media with saccharin b. Calibration bar represents I mV, 20 msec for the extracellular E P S P s and 10 mV, 20 msec for the Intracellular E P S P s .  18. DISCUSSION  The results presented i n this study demonstrate that the induction of LTP i n 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 E P S P slope. The fact that LTP persisted after a post-tetanic application of saccharin suggested that the drug selectiyely interferred with some process inyolyed i n 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 i n the induction of this LTP has been attributed to the relief of the yoltage-dependent block of N M D A channels by extracellular Mg2+ and their simultaneous actiyation by stimulation of the afferent input (Gustaffson and Wigstrom,  1988; Collingridge and Singer, 1990).  postsynaptic  This results i n a rise i n  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 i n the CAlj-, neuron. Whether this effect was due to a n action of the drug on the N M D A 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 i n determining whether or not LTP is expressed i n the neuron.  Indeed, when the late  NMDA-dependent  component of the tetanus-induced depolarizing plateau is blocked by APV, subsequent development of LTP i n these neurons is not observed and Lester,  1988; Huang et. al.. 1988).  (CoUingridge  Whether saccharin blocked the  induction of LTP by interfering with the late N M D A 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 i n a prolonged decay phase of the non-NMDA E P S P (Larson and Lynch, 1986). The depolarization produced by repetitive activation of the nonN M D A receptors has been demonstrated to prime the membrane potential to the voltage range where the N M D A receptors are activated (Pacelli and Kelso, 1989). The possibility that saccharin could interfere with the activation of the N M D A receptors i n this manner was unlikely since the early component of the depolarizing plateau was not blocked. The finding that the depolarizations and associated changes i n input resistance produced by exogenously applied N M D A i n a Mg2+-free medium or a Mg2+-free medium containing C N Q X were not significantly altered i n the presence of saccharin and that the slopes of the non-NMDA E P S P or N M D A EPSP were also not significantly altered by the drug further supported the notion that saccharin did not antagonize the N M D A 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 i n the synaptic responses. For example, with respect to the IO plot for the EPSP, the I-O plot for the IPSP was proportionately upward i n the presence of saccharin.  shifted  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 N M D A E P S P by enhancing the duration of the underlying IPSP. It appeared that this effect was, i n fact, due to the IPSP and not to a depression of N M D A receptor-mediated  synaptic  transmission per se because only the duration and not the slope of the N M D A E P S P was reduced i n the presence of saccharin. It was possible that saccharin decreased the duration of the N M D A EPSP, however, i n recordings where the contribution of the IPSP  was minimal (neurons with R M P s approaching -70  m V or i n picrotoxinin-containing media), the duration of the N M D A E P S P was not reduced i n the presence of the drug. Because recent investigations have provided evidence suggesting that S)niaptic activation of G A B A ^ receptors  (Wigstrom  and Gustaffson,  1983  Wigstrom and Gustaffson, 1986) and presynaptic G A B A g receptors play an important role i n modulating the induction of LTP i n 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  i n the  same  manner  as  the  barbiturate or  benzodiazepene.  Alternatively, saccharin may have interfered with G A B A uptake.  Indeed  nipecotic acid, an inhibitor of G A B A uptake, also enhances the IPSP (Alger and Nicoll, 1982). Saccharin caused some suppression of the G A B A g receptor-mediated slow IPSP.  It was unlikely that this effect was due to a shift i n 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 G A B A g receptors rather than the  postsynaptic  GABAg  receptors  inhibits  tetanus-induced  LTP  in  hippocampal neurons through a mechanism involving autoinhibition of G A B A release. Based on these reports, it was conceivable that saccharin blocked the induction of LTP by antagonizing the presynaptic G A B A g 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 G A B A g receptors (Davies et. al.. 1990; Thompson and Gahwiler, 1989).  Thus, an antagonism of  presynaptic G A B A g 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 i n the ratio of responses depression.  to paired-pulse facilitation or to paired-pulse  Because paired-pulse depression of the fast IPSP was  significantly altered i n the presence  of saccharin, it appeared  not  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 G A B A receptors rather than on the presjmaptic G A B A receptors. saccharin-induced  changes  i n the  fast  IPSP  Whether or not the  could alter  paired-pulse  facilitation or paired-pulse depression was, therefore, examined. Results from paired-pulse facilitation experiments demonstrated that i n the presence  of  saccharin a slight decrease i n paired-pulse facilitation of the E P S P 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 i n pairedpulse facilitation of the E P S P was observed. A similar effect was also exhibited for the saccharin-induced increase i n paired-pulse depression of the fast IPSP. One possible explanation which may account for an increase i n paired-pulse depression of the IPSP is the enhanced fast IPSP arising from the first pulse may have exerted conductance changes i n 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 i n the stimulus pair. Paired-pulse depression was greatly reduced i n this particular experiment when compared to the experiments where an interstimulus interval of 300 msec was  employed  because, i n the latter case, the fast IPSP evoked by the first pulse had already decayed to a greater extent. The change i n conductance associated with the IPSP has also been demonstrated to decrease the amplitude of the dendritic E P S P by a shunting effect (Dingledine and Langmoen, 1980).  In the present studies maximal  shunting of the E P S P 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 i n the paired-pulse facilitation experiments it would be expected that the shunting effect be maximal i n the presence of the drug rather than i n its absence.  This effect could explain why paired-pulse facilitation of the E P S P  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 i n 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 N M D A receptoroperated 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 N M D A EPSP does provide some merit to this postulation. Furthermore, the latency to peak of the fast IPSP increased i n the presence of saccharin to a time which fell within the range of reported rise times of the N M D A 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 E P S P slope nor the  intracellular E P S P slope exhibited LTP i n 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 transmission.  and inhibitory synaptic  Moreover, based on our experiments, the blockade of the  induction of LTP appears not to depend upon an antagonism of the N M D A receptors, G A B A ^ receptors or the G A B A g receptors, all of which have been implicated i n 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 frequency activation of the stratum radiatum.  low  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 i n 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 N G F 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 i n 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 E P S P induced a long-term potentiation when nerve growth factor (NGF) was present. The effect of N G F was. however, antagonized when saccharin was present (Sastrvet. al.. 1988). Other studies conducted i n this laboratory supported the involvement of trophic factors i n the induction of LTP.  That saccharin was capable of  antagonising their LTP-inducing actions i n 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 N M D A 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 N G F 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 i n the induction of LTP then, conceivablely it could block the induction of LTP without antagonizing the N M D A or the G A B A g receptors. In summary, the present studies have demonstrated that saccharin blocks the induction of LTP i n the C A l region of the guinea-pig hippocampus through a mechanism that does not involve an interference Avith the N M D A and the non-NMDA glutamatergic receptors as well as the G A B A ^ and the G A B A g receptors.  19. CONCLUSIONS Based on the experiments conducted i n this study the major findings are summarized as follows: 1.  Saccharin blocked  stimulation  or,  (b)  the  pairing  induction of LTP a  strong  by  postsynaptic  either,  (a)  a  tetanic  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 N M D A and the non-NMDA glutamate receptors. 4.  Saccharin decreased  the slow IPSP and potentiated  the fast  IPSP,  however, these effects did not appear to be responsible for the drug's ability to block the induction of LTP. 5.  The evidence provided demonstrates that saccharin does not block the  induction of LTP by altering excitatory or inhibitory synaptic transmission.  20. R E F E R E N C E S Abraham, W. C. 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