Non-Associative Hippocampal Long-Term Depression (LTD) And

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Gerolemos Christofi

A thesis submitted for the degree of Doctor of Philosophy in the University of London Faculty of Science

Department of Physiology University College London September 1992 ProQuest Number: 10045542

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT

Long term potentiation (LTP) is an enduring, activity-dependent increase in synaptic transmission. Long term depression (LTD) is an enduring, activity-dependent decrease in synaptic transmission. Both phenomena may form the neuronal basis of learning and memory. Intracellular recording in isolated slices of rat brain was used to study (i) associative LTP in somatosensory neocortex, and (ii) non-associative LTD in hippocampus. (i) In layer V cells of neocortex, the incidence of induction of associative LTP was much lower in the slice than reported for the awake cat; this might be explained by the loss of extrinsic noradrenergic activity in the slice. To test this hypothesis, the actions of noradrenaline and the 8 ,-adrenergic receptor agonist, isoprenaline, were investigated on ( 1) membrane properties, and ( 2 ) the probability of induction of associative LTP. The drugs produced a dose-dependent, reversible reduction of spike accommodation and an increase in excitability but had no effect on the depolarising slope or peak amplitude of sub threshold excitatory postsynaptic potentials (EPSPs) evoked by white matter stimulation. The probability of induction of associative LTP was not changed by these drugs, and hence the lower probability in vitro than in vivo cannot be explained solely by the loss of extrinsic noradrenergic activity. (ii) In hippocampal CAl pyramidal neurones, postsynaptic potentials (PSPs) were evoked by test stimulation of stratum radiatum. Conditioning was non-associative and consisted of postsynaptic depolarization and firing, induced by either high frequency antidromic stimulation or postsynaptic injection of depolarizing current pulses. LTD was reliably induced postsynaptically when conditioning was applied during pharmacological block or reduction of synaptic transmission, achieved by transiently bathing slices in a bathing medium containing either a raised [Mg^^], selective glutamate antagonists, or a 7 -aminobutyric acid receptor antagonist. The postsynaptic induction of LTD required extracellular Ca^^. Measurements of resting and activity-dependent increases in cytosolic [Ca^^] were made using the fluorescent Ca^"^ indicator FURA-2 iontophoresed into the cell to elucidate the conditions required for LTD induction. Possible reasons why the pharmacological reduction of synaptic transmission during conditioning stimuli could facilitate LTD induction are discussed. DEDICATION A^IEPOEIE

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ACKNOWLEDGEMENTS

I am glad to have the opportunity to express my gratitude to my supervisor, Dr Lynn Bindman, for her patience and encouragement over the last three years. I am grateful to my buddy, Dr Alex ("cor blimey, Guv!") Nowicky for developing the computer programs which helped make the analysis of much of the work possible, and for his support throughout my time at UCL. I would like to thank Dr Stephen Bolsover for allowing me the use of the imaging equipment in his laboratory and for technical advice and help with the Ca^"^-imaging experiments. Dr Michael Duchen, Ms Beverley Clark and Professor Tim Biscoe kindly provided access to computers, when emergencies arose. Many members of Department (both past and present) have provided friendship and general support during my stay at UCL; in particular Vincent O’Connor {aka Jason Cundy), Margaret Carey, Roz Pearce, Beverley Clark, Fatwan Al-Mohanna, Alex "MC" Dougall, Louise Upton, Rob Fern, and Liza Rosser.

A special acknowledgement must be made to the Medical Research Council for the award of a Postgraduate Scholarship, without which this work could not have been pursued.

The work described in section A of this thesis was conducted during the first year of my training, in collaboration with Dr Alex Nowicky, co-worker in Dr Bindman’s group. I conducted the vast majority of the work described in Section B by myself. For technical reasons, the Ca^'^-imaging experiments (Sections 2.2 and 5.8) were conducted in collaboration with either Dr Alex Nowicky (in the majority of cases). Dr Lynn Bindman, or Dr Stephen Bolsover. TABLE OF CONTENTS

TITLE PAGE 1 ABSTRACT 2 DEDICATION AND ACKNOWLEDGEMENTS 3 LIST OF FIGURES 8 TABLES 10 ABBREVIATIONS 11

CHAPTER 1

GENERAL INTRODUCTION

PREFACE 13

1.1 Learning and memory 13 The Hebbian synapse 13 1.2 Long-term potentiation 14 Methods of LTP induction 15 LTP - A neural substrate for learning and memory? Possible behavioural correlates 15 Associative LTP and its behavioural correlate 16 1.3 Organization of the hippocampus 17 Cortical Areas 17 Main Cell Types 19 Afferent Fibres 19 Recurrent inhibitory circuits and postsynaptic inhibition 20 The neuronal (tri-synaptic) circuit 21 Lamella 21 1.4 LTP in the hippocampus 21 An induction threshold for LTP in the hippocampus 21 The NMDA receptor and the induction of hippocampal LTP 22 NMDA receptor-dependent LTP 22 NMDA-receptor-independent LTP 24 Calcium and LTP 25 The role of GAB A receptor-mediated inhibition in LTP induction 25 GABA^-mediated inhibition 25 GABAg-mediated inhibition 26 Enhancement of E-S potentiation 26 Expression of hippocampal LTP: presynaptic, postsynaptic or both? 27 Possible presynaptic mechanisms underlying LTP expression 27 Possible postsynaptic mechanisms underlying LTP expression 29 Can quantal analysis of synaptic variability resolve the LTP expression site issue? 30 1.5 LTP in the neocortex 31 Historical background 32 GABA a inhibition and neocortical LTP 32 NMDA receptors and the induction and maintenance of neocortical LTP 32 The associative, postsynaptic induction of LTP in the neocortex 33 Associative LTP in the awake animal 36 Postsynaptic and associative LTP 36 Neuronal transport 36 Postsynaptic conductance changes 37 Associative LTP - some questions 37

SECTION A: INVESTIGATION OF B-ADRENERGIC MODULATION OF SYNAPTIC TRANSMISSION AND ASSOCIATIVE LTP IN SLICES OF RAT SENSORIMOTOR CORTEX

1.6 Introduction 39 1.7 Forebrain noradrenergic connections 39 1.8 Adrenergic receptors in the neocortex 40 1.9 Effects of Noradrenergic agonists on hippocampal regions 42 Dentate gyrus 42 Area CA3 43 Area CAl 44 1.10 Effects of noradrenergic agonists on sensory neocortex 46 1.11 Aims of the investigation 48

SECTION B: LONG-TERM DEPRESSION (LTD) IN THE HIPPOCAMPUS AND NEOCORTEX

1.12 Introduction 50 1.13 Neural network models predict enduring decreases in synaptic transmission 50 1.14 Long-term depression 51 Definitions 51 Associative or homosynaptic LTD in the hippocampus 52 Heterosynaptic LTD in the hippocampus 54 The role of NMDA receptors in homo- and heterosynaptic LTD in the hippocampus 56 Prolonged low-frequency activation of NMDA receptors can induce LTD 57 NMDA-receptor-independent LTD 58 What component of the EPSP is depressed? 59 Factors necessary for the induction of LTD in vitro 59 The ACPD (metabotropic) receptor in LTD 60 1.15 Activity-dependent LTD in the neocortex 60 NMDA receptors and the postsynaptic induction of neocortical LTD 60 The role of Ca^^ in LTD 62 1.16 Aims of the present study 63 CHAPTER 2

MATERIALS AND METHODS

2.1 The postsynaptic induction of non-associative long-term depression (LTD) in rat hippocampal slices 65 Dissection and hippocampal slice preparation 65 Artificial cerebrospinal fluid 6 6 Experimental recording chamber 6 6 Recordings from the submerged hippocampal slice 67 Harp construction 6 8 Electrodes 6 8 Recording electrodes 6 8 Stimulating electrodes 69 Voltage recordings and current injection apparatus 70 Microelectrode support and drive systems 70 Prevention of electrical interference and mechanical vibration 71 Hippocampal slice evaluation 71 Hippocampal recording and stimulation 72 Characterization of passive membrane properties and synaptic responses 76 Conditioning paradigms 76 Induction of hippocampal LTP 76 Induction of hippocampal LTD 77 Drugs 78 Raised magnesium ion bathing solutions 78 Glutamate receptor antagonists 78 7 -amino butyric acid (GABA^) receptor antagonist 79 Analysis of results 79 Measurement of cell stability during and after drug perfusion 80 2.2 Measurements of intracellular Ca^^ using Fura-2 microfluorimetry 80 2.3 Methods for studying synaptic transmission and the postsynaptic induction of LTP in layer V neurones in slices of rat neocortex 83 Dissection and neocortical slice preparation 83 Neocortical recordings in the gas-liquid interface mode 83 2.4 Recording and stimulation 84 Pyramidal cell identification 84 Drugs: Noradrenergic and 6 -agonists 8 6 2.5 The conditioning paradigm 8 6 The postsynaptic induction of neocortical LTP 8 6 Analysis of Results 8 6

SECTION A

INVESTIGATION OF B-ADRENERGIC MODULATION OF SYNAPTIC TRANSMISSION AND ASSOCIATIVE LTP IN SLICES OF RAT SENSORIMOTOR CORTEX

CHAPTER 3

RESULTS

3.1 Introduction 89 3.2 Effects of 6 -adrenergic agonists on membrane properties 90 3.3 Effects of 6 -adrenergic agonists on evoked synaptic transmission 92 3.4 Effects of 6 -adrenergic agonists on E-S coupling 94 3.5 Effects of 6 -adrenergic agonists on the postsynaptic induction of associative LTP 95 CHAPTER 4

DISCUSSION

4.1 Introduction 99 4.2 Effects of 6 -adrenergic agonists on passive membrane properties 99 4.3 Effects of 6 -adrenergic agonists on evoked synaptic transmission 99 4.4 Effects of 6 -adrenergic agonists in E-S coupling 101 4.5 Effects of 6 -adrenergic agonists on the postsynaptic induction of associative LTP 101 4.6 The postsynaptic associative conditioning paradigm 103 GABA a inhibition 104 GABAg inhibition 105 4.7 Other neuromodulators 105

SECTION B

LONG-TERM DEPRESSION (LTD) IN THE HIPPOCAMPUS AND THE NEOCORTEX

CHAPTER 5

RESULTS

5.1 The postsynaptic induction of LTD of synaptic transmission in rat hippocampus 108 Passive properties of CAl pyramidal neurones 108 Responses to orthodromic stimulation 111 Effect of antidromic stimulation in normal medium 111 Control recordings 116 Effect of high frequency antidromic conditioning on test EPSPs in normal acsf 119 Effect of antidromic stimulation in high [Mg^^] solutions 119 Effect of perfusing high [Mg^^] on passive membrane properties 119 Effect of perfusing high [Mg^^] on synaptic transmission 119 Effect of antidromic conditioning in high [Mg^'*'] solutions on EPSPs 122 Reversal of LTD to LTP 130 Effect of perfusing high Mg^"^ with no added Ca^"^ on synaptic transmission 130 The effect of antidromic conditioning in high Mg^"^ with no added Ca^‘‘‘ 130 5.2 Effect of intracellular conditioning during synaptic block in high [Mg^^] 133 Postsynaptic responses during conditioning in raised [Mg^^] 142 5.3 Glutamate receptor antagonists 145 Affect of bath application of glutamate antagonists on synaptic transmission 145 Intracellular conditioning in the presence of glutamate receptor antagonists 152 Intracellular conditioning in D-AP5 156 5.4 Effect of conditioning current pulses in normal acsf 158 5.5 Effect of depolarizing current pulses on LTD in acsf containing a GABA a receptor antagonist 158 5.6 Input resistance and spike threshold during LTD 171 5.7 Activity-dependence of induction of LTD 174 5.8 Measurements of intracellular [Ca^^] 176

CHAPTER 6

DISCUSSION

6 .1 Introduction 179 6.2 Antidromic stimulation in normal acsf 179 6.3 The postsynaptic induction of non-associative LTD by antidromic conditioning 180 6.4 The postsynaptic induction of non-associative LTD requires extracellular Ca^^ 182 6.5 The postsynaptic induction of non-associative LTD by intracellular current pulses 184 The postsynaptic induction of non-associative LTD in high [Mg^^] solutions 184 The postsynaptic induction of non-associative LTD in glutamate receptor antagonists 184 6 . 6 Intracellular conditioning in acsf with or without bicuculline methiodide 185 6.7 How does block of synaptic activity facilitate the induction of LTD? 187 6 .8 Postsynaptic actions of GABA in the hippocampal CAl region 188 Hypothesis 1 189 Hypothesis 2 189 6.9 Do different threshold levels of Ca^'*' predict the direction of synaptic modification? 190 Hypothesis 3 191 6.10 The role of LTD in the hippocampus - possible behavioural correlates in vivo 194

REFERENCES 196

LIST OF FIGURES

Figure 1.1 Types of long-term synaptic modification. 18

Figure 2.1.1 Schematic representation of a hippocampal brain slice and the respective positions of recording and stimulating electrodes used in this study. 74

Figure 2.4.1 Positions of recording and stimulating electrode in a slice preparation of rat sensorimotor cortex. 85

Figure 3.2.1 Blockade of accommodation observed during 20 isoprenaline application to bathing medium. 91 Figure 3.2.2 NAd reduces the spike threshold to depolarizing current injection. 93

Figure 3.5.1 The associative induction of LTP. 96 Figure 3.5.2 Representative waveforms and time course of an experiment involving the attempted induction of associative LTD before and during 20 fiM ISO perfusion. 98 Figure 5.1.1 The postsynaptic responses of a CAl pyramidal cell to intracellular injection of current. 1 1 0 Figure 5.1.2 Postsynaptic responses of a CAl pyramidal neurone to intracellular current injections showing activation of the anomalous rectifier. 113 Figure 5.1.3 Postsynaptic response of a CAl neurone to electrical stimulation and superimposed intracellular current injection. 115 Figure 5.1.4 Postsynaptic responses of a CAl pyramidal neurone to antidromic electrical stimulation at the alveus. 117 Figure 5.1.5 The affect of prolonged electrical stimulation of the stratum radiatum on EPSP slope. 118 Figure 5.1.6 Response of a cell to slice perfusion with a raised [Mg^^]. 121 Figure 5.1.7 The affect of transiently perfusing a hippocampal slice with acsf containing 25 mM Mg^"^ and 2 mM Ca^"^ on synaptic transmission in the CAl region of the hippocampus. 124 Figure 5.1.8 Transient perfusion of acsf containing 25 mM Mg^'^ and 2 mM Ca^"^ does not induce LTD of synaptic transmission in the CAl region of the hippocampus. 125 Figure 5.1.9 Antidromic conditioning in 25 mM Mg^"^ and 2 mM Ca^^ induces non-associative LTD. 128 Figure 5.1.10 The postsynaptic induction of non-associative LTD by antidromic conditioning during synaptic block. 129 Figure 5.1.11 The affect of transiently perfusing acsf containing 15 mM Mg^"^ and no added Ca^"^ on synaptic transmission in the CAl region of the hippocampus. 132 Figure 5.1.12 No LTD induced by antidromic conditioning during block of synaptic transmission when Ca^"^ omitted from bathing solution. 135 Figure 5.1.13 The postsynaptic induction of LTD by antidromic conditioning. Bar chart summarizing the results obtained in section 5.1. 138

Figure 5.2.1 LTD produced by repeated intracellular conditioning pulses applied during synaptic block by 25 mM Mg^"^, 2 mM Ca^^. 140 Figure 5.2.2 Graph showing normalized EPSP slope for 7 experiments in which intracellular conditioning during synaptic block during slice perfusion with 25 mM Mg^'^ and 2 mM Ca^'^. 141 Figure 5.2.3 Examples of spike trains and membrane potential changes in 3 sets of experiments during synaptic block. 144

Figure 5.3.1 The affect of perfusing different concentrations of CNQX on synaptic transmission in a hippocampal slice. 147 Figure 5.3.2 The affect of perfusing 10 fiM CNQX on synaptic transmission in a CAl hippocampal neurone. 149 Figure 5.3.3 Glutamate and GABA^ receptor antagonists block PSPs evoked in CAl. 151 Figure 5.3.4 Normalized plot of the time course of recovery of EPSP slope following transient slice perfusion with CNQX with or without AP5. 153 Figure 5.3.5 The postsynaptic induction of LTD in CNQX. 155 Figure 5.3.6 The postsynaptic induction of LTD by intracellular conditioning pulses in glutamate receptor antagonists. 157

Figure 5.4.1 Failure to induce LTD postsynaptically in normal acsf. 159

Figure 5.5.1 The postsynaptic induction of LTD by intracellular conditioning pulses in slices bathed with the GABA^ receptor antagonist, bicuculline methiodide (1 (iM). 161 Figure 5.5.2 Normalized plots for the experiments in which intracellular conditioning was applied to postsynaptically induce non-associative LTD in a GABA^ antagonist. 164 Figure 5.5.3 LTD repeatedly induced postsynaptically in the same cell, in the presence of bicuculline methiodide. 167 Figure 5.5.4 The postsynaptic induction of LTD induced by intracellular conditioning. 173

Figure 5.8.1 Measurements of intracellular Ca^"^ during intracellular conditioning using the Ca^"^ indicator FURA-2 iontophoresed postsynaptically. 178

Figure 6.1 A model of how postsynaptic dendritic levels of intracellular Ca^'^ may determine changes in the efficacy of synaptic transmission. 193

LIST OF TABLES

Table 3.1 Summary of the layer V cell characteristics. 89 Table 3.2.1 Dose-dependence of blockade of spike accommodation. 90 Table 3.2.2 showing effect of 20 //M NAd and ISO on cell input resistance measured 20 min after onset of drug perfusion. 92 Table 3.3 Effect of drugs on the EPSP slope (initial 1-2 msec) 30 min after onset of drug perfusion. 94 Table 5.1.1 CAl pyramidal cell characteristics from which intracellular recordings were made. 108 Table 5.1.2 summarizing the data obtained from the experiments in which antidromic conditioning was applied to induce LTD postsynaptically during block of synaptic transmission. 126 Table 5.2.1 summarizing the data obtained from the experiments in which intracellular conditioning was applied to postsynaptically induce LTD during synaptic block. 142 Table 5.3.1 The postsynaptic induction of LTD in glutamate antagonists 156 Table 5.4.1 Intracellular conditioning in normal acsf does not induce LTD of PSP slope. 158 Table 5.5.1 The postsynaptic induction of non-associative LTD in the presence of bicuculline methiodide. 171 Table 5.7.1 Postsynaptic responses produced by the conditioning paradigms used in this study. 175

10 LIST OF ABBREVIATIONS

4-AP 4-aminopyridine ACPD 1-amoni-cyclopentane-1,3-dicarboxylate acsf Artificial cerebrospinal fluid AD Antidromic Ag/AgCl Silver/silver chloride AHP Afterhyperpolarization Alv Alveus ANOVA Analysis of Variance AP3 D,L-2-amino-3 -phosphonopropionate AP5 2-amino-5-phosphonopentanoate BAP 6 -adrenergic potentiation BAPTA l,2-bis(2-Aminophenoxy)ethane A/,A/,A/\V-tetraacetic acid °C Degrees Celsius C/A Commissural/associational CAl comu ammonis area 1 CA3 cornu ammonis area 3 Ca"+ Calcium [Ca'+]i Intracellular free calcium ion concentration F Potassium current Cl Chloride cm Centimetres CNQX 6-cyano-2,3-dihydroxy-7-nitroquinoxaline CNS Central nervous system CO2 Carbon dioxide Cond. Conditioning DAP Depolarizing afterpotential dc Direct current df Degrees of freedom DG Dentate gyrus DHP Dihydropyridine DMSO Dimethylsulphoxide EC Entorhinal cortical EGTA Ethylene glycol-bis-(^-amino-ethyl ether) N,N,N\N- tetraacetic acid EPSC Excitatory postsynaptic current E-S EPSP - Spike F Fisher (/^-distribution g Conductance GABA 7 -amino-butyric acid HVA High-voltage activated Hz Hertz IC Intracellular conditioning IPSP Inhibitory postsynaptic potential I-V Current to voltage relation K+ Potassium Kd Dissociation constant X Wavelength Lat Lateral ipp Lateral perforant path

11 LTD Long-term depression LTP Long-term potentiation LVA Low-voltage activated fiM Micromolar MO Megaohms max Maximum Med Medial mf Mossy fibre Mg2-^ Magnesium min Minimum min. Minutes mM Millimolar ml Millilitres mm Millimetres mpp Medial perforant path msec Milliseconds mV Millivolt n Sample number n Number of release sites Na+ Sodium nA Nanoampere nm Nanometres NMDA V-metbyl-D-aspartate P Probability value for Fisher’s Exact Test of Independence P Probability P Probability of release PP Perforant path pH -logio hydrogen ion concentration PSP Postsynaptic potential PT Pyramidal tract Q Quantal content QX-314 N- (2,6-Dimethylphenylcarbamoylmethyl) triethylammonium bromide Rin Apparent input resistance Sch/com Schaffer collateral-commissural SD Standard deviation sec Seconds S.E.M Standard error of the mean TEA T etraethy lammonium TTX Tetrodotoxin UV Ultraviolet V Volt VL Ventro-lateral VIP Vasoactive intestinal peptide Vm Resting cell membrane potential VOCC(s) Voltage-operated calcium channel(s)

12 CHAPTER 1

GENERAL INTRODUCTION

PREFACE

1.1 Learning and memory

The neocortex and the hippocampus are areas of the brain known to be involved in learning and memory in mammals. Yet our understanding of the cellular mechanisms underlying these phenomena remains rudimentary. This study investigates the conditions and cellular processes associated with the induction of long-term potentiation (LTP) and long term depression (LTD) of synaptic transmission, events proposed as mechanisms underlying learning and memory. The thesis is divided into 2 sections: Section A investigates the neuromodulatory actions of noradrenaline (NAd) and the fi- adrenergic agonist, isoprenaline (ISO) on synaptic transmission and the induction of associative LTP in layer V pyramidal cells in rat sensorimotor cortex. Section B investigates the postsynaptic induction of non-associative, heterosynaptic LTD in the rat hippocampus.

The Hebbian synapse By the late nineteenth century, biologists had discovered that most mature neurones lose their capacity to divide, consequently models of learning and memory that evoked de novo production of brain cells were abandoned. In the closing decade of the nineteenth century, Tanzi (1893) and Ramon y Cajal (1894) independently proposed the connectionist view of learning and memory, i.e. learning involved the strengthening of connections amongst neurones, whilst memory was the product of such modification. However, there was no direct evidence to support this concept at that time. The connectionist view has been restated several times during the past century, evolving the hypothesis that the synapse is the critical site of plastic change (Konorski, 1948; Hebb, 1949; Eccles, 1953 and Kandel, 1977). Psychologist, Donald Hebb (1949) suggested that a synaptic modification for learning occurs as a consequence of coincidence between presynaptic and postsynaptic activity. Hebb originally stated what

13 today has become known as Hebb’s postulate that: "When an axon of cell A is near enough to excite cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells such that A's efficiency, as one o f the cells firing B, is increased. " (Hebb, 1949) In modern terminology: the conjunction of excitatory activity in both pre- and postsynaptic neurones, in some way strengthens their synaptic connection, such that the presynaptic cell can thereafter more readily excite the postsynaptic cell. Such a theoretical connection is known as a Hebbian synapse.

1.2 Long-term potentiation

The discovery of long-term potentiation (LTP) in the late 1960’s (Lomo, 1966) and early 1970’s (Bliss and Lomo, 1970) and its first full-length description (Bliss and Lomo, 1973; Bliss and Gardner-Medwin, 1973) provided biologists with a electrophysiological model of learning and memory. LTP has since been used to test Hebbian ideas, and has been shown to behave like a Hebbian synapse (for reviews see Bliss and Lynch, 1988; Brown et al., 1988a,b.; Wigstrom and Gustafsson, 1988). LTP describes an activity-dependent, enduring increase in synaptic transmission, which can be produced by brief high frequency tetanic stimulation of an excitatory monosynaptic pathway (Bliss and Lomo, 1973). The enhancement of transmission is manifest by an increased depolarizing slope and an increased amplitude of the excitatory mass waves (postsynaptic potentials) evoked by test stimuli in that pathway. Bliss and Lomo (1973) originally reported that in the hippocampus of anaesthetized rabbits, a brief period of high frequency stimulation applied to the afferent nerve fibres of the perforant path, induced an enduring increase in the amplitude of extracellular field potentials recorded in the granule cells of the dentate gyrus which could last for hours. The increase could last for days or weeks in unanaesthetized rabbits with chronically implanted electrodes (Bliss and Gardner-Medwin., 1973). LTP has now been demonstrated at all the major synaptic connections of the hippocampal trisynaptic circuit (Schwartzkroin and Wester, 1975; Lynch et al., 1977; Andersen et ah, 1977; Alger and Tyler, 1976; Yamamoto and Chujo, 1978) and other regions of the brain: subiculum and septum (Racine et ah, 1983), medial geniculate (Gerren and Weinberger, 1983), visual cortex (Tsumoto and Suda, 1979; Komatsu et

14 a l, 1981; Lee, 1982; Artola and Singer, 1987), somatosensory cortex (Brons and Woody, 1980; Voronin, 1985; Bindman et a l, 1988a,b), frontal cortex (Sutor and Hablitz, 1989a), prefrontal cortex (Hirsch and Crepel, 1990) and piriform cortex (Kanter and Haberly, 1990).

Methods of LTP induction Variations of two conditioning paradigms have been used to induce LTP, both fulfilling the Hebbian requirement for the conjunction of pre- and postsynaptic activity. The first is high frequency and high intensity stimulation of an afferent pathway, typically consisting of electrical shocks delivered at 100 Hz for 1 sec (see Bliss and Lynch, 1988; also Teyler and DiScenna, 1987) resulting in homosynaptic LTP. The second is the conjunction of presynaptic release of neurotransmitter with depolarization of the postsynaptic neurone (Kelso et a l , 1986; Wigstrom and Gustafsson, 1986) giving rise to associative LTP. Both paradigms are highly effective as methods of inducing of hippocampal LTP, achieving success rates of >80% (Gustafsson and Wigstrom, 1988; Bliss and Lynch, 1988).

LTP - A neural substrate for learning and memory? Possible behavioural correlates The fact that LTP occurs in both the hippocampus and neocortex, and the possibility that a connection might exist between hippocampal and neocortical learning and memory and LTP has generated considerable interest in the phenomenon as a synaptic model of memory in the last two decades. What is the basis for this interest? The importance of the hippocampus for memory processing has been evident since the discovery that bilateral removal or lesion of the hippocampus in Man profoundly impairs the ability to lay down new memories (Scoville and Milner, 1957; Milner, 1972; Zola- Morgan ef a/. ,1986; Squire and Zola-Morgan, 1988; Squire, 1989). LTP shares many features characteristic of learning and memory. Representational and declarative memory is known to be rapidly induced, long-lasting, associative and contextually specific. Likewise LTP is rapidly induced (McNaughton, 1983), long-lasting (Bliss and Gardner-Medwin, 1973; Racine et a l, 1983), synapse- specific (Andersen et a l, 1977), and associative (Levy and Steward, 1979; Barrionuevo and Brown, 1983; Gustafsson and Wigstrom, 1986a,b; Kelso et a l, 1986, Bindman et

15 a l, 1988). Spatial learning is thought to be an important function of the rat hippocampus (O’Keefe and Nadel, 1978). In behavioural studies, McNaughton et al. (1986) showed that blockade of spatial memory acquisition occurred when LTP was maximal. Maximal bilateral induction of LTP in rats led to impaired acquisition of spatial learning in a circular maze, though previously learned spatial tasks were unaffected. These authors suggested that saturation of LTP can be regarded as a functional lesion of synaptic plasticity, and that the impaired ability to learn new spatial tasks can be attributed to a consequent loss of short-term memory in the hippocampus. Foster et al. (1991) extended this work by showing that LTP induced in the hippocampal perforant path is not permanent, and rats can still learn when LTP has faded. Other behavioural experiments have revealed parallels between aspects of LTP and learning and memory (see Bliss and Lynch, 1988). Morris et al. (1986) and Morris (1989) showed that in rats, intraventricular infusion of the selective glutamate receptor antagonist of the N-methyl-D-aspartate (NMDA) receptor, D-L-2-amino- phosphonopentanoic acid (D-AP5) which blocks LTP induction in the hippocampus, without otherwise affecting synaptic transmission, impaired spatial learning tasks in a water maze. More recently, Davis et al. (1992) reported that D-AP5 impairs spatial learning in a manner highly correlated with its corresponding impairment of hippocampal LTP in vivo and in vitro. The parallels are compelling, though the link between LTP and learning and memory remains mostly correlative.

Associative LTP and its behavioural correlate The suggestion that activity-dependent, long-lasting changes in the efficacy of synaptic transmission could provide a neural substrate for learning and the memory trace has become more compelling since the demonstration that associative LTP could be induced in both hippocampus and neocortex. Further experiments revealed that the process of induction of associative LTP conformed to a Hebbian model of learning (Hebb, 1949): only those synaptic inputs that were associated in time with depolarization and firing of the postsynaptic cell were potentiated, while those inputs that occurred hundreds of milliseconds or seconds apart from cell depolarization were not potentiated. The induction of LTP was thereby demonstrated to be postsynaptic, input-specific and associative (Wigstrom and Gustafsson, 1986; Kelso et al. , 1986, in hippocampus; but

16 see also Bonhoeffer et a l, 1989; Baranyi anÜ Feher, 198la,b; Baranyi and Szente, 1987; Bindman et al., 1988, in neocortex). Associative conditioning paradigms used to induce LTP are thought to mimic at cellular level that of classical, associative or Pavlovian conditioning: Pavlov’s dogs, which naturally salivated at the taste or smell of food, were conditioned to salivate (when hungry) in response to previously insignificant sensory stimuli, such as the sight of a food bowl or the sound of the footsteps of the man bringing their food, or, in experiments, to musical sounds. The conditioned salivation only occurred after the conditioning stimulus had been repeatedly presented in temporal association with the arrival of food. The conditioning was dependent on the cerebral cortex for the analysis of the sensory inputs (Pavlov, 1928). In neocortex or in hippocampus, a weak test input (analogous to the conditioning sound) in one afferent pathway to a single neurone was repetitively associated with a strong input (analogous to the smell or taste of the food) in another pathway to the cell; after tens of pairings, the weak input elicited a persistently potentiated synaptic response - LTP. Figure 1.1 summarizes the types of long-term synaptic modification that may occur in the hippocampus (and neocortex). For a comprehensive review on hippocampal LTP see Bliss and Lynch (1988).

1.3 Organization of the hippocampus

The major part of the work described in this study was performed on slices of rat hippocampus where LTP has been best characterized. The hippocampal cortex, with its relatively simple neuronal circuitry (in comparison to the neocortex), offers distinct advantages for the study of synaptic activation of cortical neurones. The cell body layers of the pyramidal and granule cells can be easily discerned under the microscope, and it is possible to stimulate a known afferent pathway and record monosynaptic events in a known population of postsynaptic hippocampal neurones. Its regular anatomy and possible involvement in learning and spatial memory have excited an increasingly large number of investigators. |

Cortical Areas In comparison to other cortical areas with their six layers, the hippocampus or archicortex appears relatively simple. The hippocampus is composed of two longitudinal

17 A Homosynaptic LTP

B Heterosynaptic LTP

C Associative LTP

D Homosynaptic LTD

E Heterosynaptic LTD

F Associative LTD

Figure 1.1. Types of long-term synaptic modification. For the sake of simplicity, each postsynaptic neurone is shown receiving only two afferents. Vertical lines at presynaptic sites indicate action potentials arriving at afferent terminals. Those at the postsynaptic neurone do not necessarily indicate action potentials, but indicate depolarization beyond some threshold. Changes in synaptic efficacy are indicated by the size of the circles. P and D indicate potentiated and depressed synapses, respectively. Heterosynaptic LTP has rarely been observed in the neocortex. (Adapted from Tsumoto, 1992).

18 strips named regio inferior and regie superior by Ramôn y Cajal (1893). Lorente de N 6 (1934) renamed these areas CAl and CA3-CA4 respectively (CA for comu ammonis). Today, the latter terms are widely used (see Figure 2.1.1A, Chapter 2, page 74). Next to the CA3-CA4 areas, is found the dentate fascia. On the other side, CAl borders the I (CAl and CA4, the subiculum, parasubiculum and dentate fascia)^ subiculum. In combination,j[the tour regions make up the hippocampal formation. Further laterally is found the parahippocampal gyrus, including the entorhinal cortex.

Main Cell Types In rats, the CAl cortex contains a set of relatively small pyramidal cells, whereas CA3 and CA4 have larger pyramidal cells. In CAl and CA3, the pyramidal cell bodies are packed together in a dense band, being about three to four cell bodies high and 80- 100 jim wide. All cells send their apical dendrites off in one direction. In CAl, the initial parts of these are relatively straight, giving a striped appearance when sections are viewed under the light microscope. This region is referred to as stratum radiatum. The apical dendrites arborize in the outer part of CAl, in the stratum moleculare. In the other direction, the basal dendrites form a dense network in a zone called stratum oriens. In all cell types, the dendritic trees are heavily covered with spines, in pyramidal cells nearly exclusively on the thinner branches.

Afferent Fibres In contrast to the situation in the neocortex, the afferent fibres run horizontally, that is parallel to the cortical surface, and will, therefore, cross the main dendritic axis orthogonally. The fibres make synaptic contacts with boutons en passage. An afferent fibre will therefore activate a row of cells through the synapses lying at a relatively constant distance from their somata. Conversely, different parts of the dendritic tree of a given cell will be contacted by fibres coming from different sources. This arrangement allows the experimenter to selectively activate a population of synapses at a given distance from the soma (Lorente de N 6 , 1934) or make lesions of pathways synapsing on a given part of the dendritic tree. By combining histological, physiological and chemical techniques, it has been possible to establish that excitatory synaptic connections on pyramidal cells are made by synapses of the asymmetric type on the dendritic spines (Andersen et al., 1966; Storm-Mathisen et al., 1983). Excitatory synapses on interneurones are distributed on the non-spiny bulbous dendrites of such

19 neurones as well as on their somata. Interneurones probably receive less than 10% of the total number of excitatory synapses, although there is relatively little evidence that this is so (Lorente de No, 1934; Andersen et al., 1964; Schwartzkroin and Mathers,

1978; Lacailleer <3/., 1987). |The polymorphic layer of dentate gyrus contains I mossy cells which are excitatory onto | granule cells and onto inhibitory intemeurones. Recurrent inhibitory circuits and postsynaptic inhibition A number of interneurones exist in the hippocampal cortex, some of which have been identified as being inhibitory on the basis that they contain the enzyme glutamic acid decarboxylase (GAD), the enzyme that is expressed in neurones that use GABA as a neurotransmitter, and therefore probably release GABA (Ribak et at., 1978; Somogyi et al, 1983). GAD-positive synapses have been found to contact shafts of middle-sized dendrites in the stratum radiatum and stratum moleculare (Storm-Mathisen et al., 1983) and are probably inhibitory in these dendritic territories (Alger and Nicoll, 1982a). One type of interneurone is the basket cell; these are located at the stratum pyramidal/stratum oriens border (Ramon y Cajal., 1894, 1911; Lorente de No, 1934; Schwartzkroin and Mathers, 1978; Schwartzkroin and Kunkel, 1985). Basket cells have axon collaterals ramifying in a dense basket-like plexus around the soma of pyramidal and dentate granule cell bodies where they also form synapses (Ramdn y Cajal., 1893; Schwartzkroin and Kunkel, 1985). They are strongly excited by pyramidal cells, artd in turn, they directly inhibit pyramidal cells (Knowles and Schwartzkroin, 1981). Andersen et al (1964) proposed that the basket cells were most likely to be responsible for recurrent inhibition of these neurones. In addition, they receive direct excitatory input from afferents coursing through strata radiatum and oriens (Frotscher et al., 1984; Schwartzkroin and Kunkel, 1985) and it has been suggested that the basket interneurones mediate feedforward and feedback inhibition of pyramidal cells (Knowles and Schwartzkroin, 1981; Schwartzkroin and Kunkel, 1985). Other inhibitory interneurones include a distinct group of cells that are localized at the stratum oriens/alveus border (Ramon y Cajal, 1911; Lorente de No, 1934), referred to as chandelier cells, which terminate on the initial axon segment in the CAl region. A third group of interneurones referred to as stellate cells, are found in the apical and basal dendritic territories in CA3 and CAl, in the hilus, and, to a smaller extent, in the molecular layer of the dentate fascia. The majority of stellate cells appear to terminate on the shafts of middle-sized and small dendrites, but do not seem to be common on the thinner spine-bearing

20 dendrites (Gottlieb and Cowan, 1972).

The neuronal (tri-synaptic) circuit The major inputs to the hippocampus come from the adjacent entorhinal cortex and the septum in the fibres of the perforant path. This is an excitatory pathway whose main input is onto the dentate granule cells, although an input also goes to the distal dendrites of the pyramidal cells. The dentate cells in turn send an excitatory input through the mossy fibres to the proximal dendrites of CA3 pyramidal cells. These in turn send an excitatory input to the CAl pyramidal cells through the fibres of the stratum radiatum, as well as an output to through the fimbria. The CAl cells are the major output cells of the structure, sending fibres through the alveus and fimbria into the other parts of the brain. In vivo, recurrent inhibitory circuits operate in all three major cell populations (Andersen et al., 1964a; Andersen et al., 1966).

Lamella The axons of the cells in one of the cortical strips in the hippocampal formation cross the border to the neighbouring area orthogonally. The thin strip of tissue with this orientation, as shown in Figure 2.1.1A (Chapter 2, page 74), has been named a lamella (Andersen et al., 1971). A section along such a plane, normal to the hippocampal surface, will therefore be parallel to both the main dendritic axis of the cells and to the axons between them. This arrangement allows a thin transverse slice (Skrede and Westgaard, 1971), with its neuronal circuit maintained, to be used for in vitro studies.

1.4 LTP in the hippocampus

An induction threshold for LTP in the hippocampus An important feature of hippocampal LTP is that it exhibits an intensity threshold for induction (Bliss and Lomo, 1973, Bliss and Gardner-Medwin, 1973; Schwartzkroin and Wester, 1975; McNaughton et al., 1978; Yamamoto and Sawada, 1981 and Lee, 1983) such that an increase in the intensity of the conditioning stimuli increases (i) the probability of inducing LTP, and (ii) its magnitude. This property is believed to reflect the requirement for co-activation or co-operativity between afferent nerve fibres and postsynaptic assemblies of neurones (McNaughton et al., 1978; Fredlander et al., 1990;

21 For the Schaffer collateral/commissural input onto CAl pyramidal neurones (Collingridge et a l, 1983 Wigstrom and Gustafsson, 1985a,b) and for the perforant path input onto the granule cells of the dentate gyrus (Harris et al, 1984; Errington et al, 1987), the induction of LTP is, under some experimental conditions, dependent on the activation of postsynaptic NMDA receptors, since its induction can be blocked by AP5. Malinow, 1991a). Co-operativity between afferent fibres determines the level of depolarization achieved in the postsynaptic neurones.

The NMDA receptor and the induction of hippocampal LTP NMDA receptor-dependent and independent forms of hippocampal LTP have been shown to exist. Both forms are briefly reviewed below.

NMDA receptor-dependent LTP (Please see inset on opposite page). Interestingly, the density of the NMDA binding sites is greater in the hippocampus than anywhere else in the brain (Monaghan and Cotman, 1985). Strata radiata and oriens of the CAl area display the highest density of sites in the brain while levels in the dentate gyrus and molecular layer are slightly lower. Binding within the CA3 region is moderately high except within the stratum lucidum which has low binding levels (Monaghan and Cotman, 1989). The mechanism by which LTP is initiated has been deduced from studies designed to determine the conditions under which NMDA receptors/channels contribute to synaptic transmission in the hippocampal Schaffer collateral-commissural pathway. Following synaptic release, glutamate binds to both postsynaptic NMDA and non-NMDA receptors (Collingridge et al. , 1983) but the EPSP during low frequency stimulation is primarily supported by current flow through the non-NMDA channels (Collingridge, 1985; Herron et al. 1985). Though the NMDA receptor binds glutamate, the associated channel is largely prevented from passing current into the postsynaptic cell because the channel pore is immediately blocked by extracellular magnesium (Mg^"^) ions (Nowak et al. , 1984; Mayer et al. , 1984; Ascher and Nowak, 1986,1988; Mayer and Westbrook, 1987). The NMDA receptor channel exhibits a strong voltage-dependence that allows current to pass only when the postsynaptic cells are depolarized (Mayer e ra/.,1984; Mayer and Westbrook, 1987; Nowak et al. , 1984). At resting potential when the postsynaptic cell is hyperpolarized, electrical forces are favourable for the divalent Mg^"^ to bind in the channel. When the postsynaptic cell is depolarized, these forces decrease and Mg^"^ is less likely to occupy its binding site in the channel, and other ions can flow into the cell. The severity of the blockade decreases with depolarization and finally vanishes around zero mV resting potential (Mayer et a/. ,1984, Nowak et al. , 1984). The apparent voltage-sensitivity of the NMDA channel is secondary to the voltage-dependence of the Mg^"^ block.

22 Two factors are necessary for the induction of LTP in the hippocampus: (i) The NMDA ionophore must open (in response to glutamate binding); (ii) the postsynaptic ceil must be depolarized (to allow current flow through the NMDA ionophore). Both of these requirements can be summarized to a single condition: LTP induction will only occur when a critical level of depolarization flows through the NMDA receptor channel in the postsynaptic cell (Collingridge and Bliss, 1987; Wigstrom and Gustafsson, 1987). This hypothesis is probably the single most influential idea in understanding LTP induction. Examination of its correlates helps to explain most of the induction properties of LTP. Stimulus threshold, frequency requirements, and associative LTP are explained by the need to provide sufficient depolarization to remove the Mg^'*’ blockade from the NMDA channels. Synapse specificity could result because synapses not active during tetanic stimulation have no glutamate bound to their NMDA receptors (see Brown et ah, 1988 and Wigstrom and Gustafsson, 1988). In addition, this suggests that the induction mechanisms of LTP reside, at least in part, in the postsynaptic cell. It is the entry of Ca^"^ ions into the postsynaptic neurone via the NMDA channel that is thought to trigger the biophysical and/or structural changes that manifest LTP. Pharmacological blockade of the NMDA receptors, postsynaptic hyperpolarization, or the injection of Ca^"^ ion buffers into the postsynaptic neurone, prevent the induction of LTP in the dentate gyrus and CAl region of the hippocampus (Collingridge et ah, 1983; Lynch et ah, 1983; Harris et ah, 1984; Malinow and Miller, 1986; Errington et ah, 1987; Malenka et ah, 1988; Malenka, 1991; Colino et ah,\992\ Huang et ah 1992; Hanse and Gustafsson, 1992). Malenka (1991) and co-workers (Colino et ah, 1992; Huang et ah, 1992) have suggested that the threshold for LTP induction in hippocampal area CAl may be continually adjusted according to the recent history of NMDA receptor activation in terms of postsynaptic receptor activation such as the magnitude and timing of postsynaptic depolarization during synaptic transmission. These authors postulated that this may provide a physiological mechanism by which LTP can be transiently inhibited. Similar suggestions have recently been made for LTP induction in the dentate gyrus (Hanse and Gustafsson, 1992). Interestingly, a similar suggestion has also been made for the induction of hippocampal long-term depression (LTD) (Dudek and Bear, 1992) (see Section 1.4.1, page 57). Studies of how the reversal potential of the NMDA channel varies as the ratio of Ca^^ to Na"^ is changed, suggest that the NMDA channel is about 7-10 times more

23 permeable to than Na"^ (Ascher and Nowak, 1988; Mayer and Westbrook, 1988). The NMDA channel is also more permeable to Ca^"^ than other transmitter-gated cationic channels (Mayer and Westbrook, 1988). However when compared to the voltage-selective Ca^^ channels, which have a selectivity ratio for Ca^'^ versus caesium in excess of 1000, NMDA receptors are much less Ca^'*’ selective (Lee and Tsien, 1984).

NMDA-receptor-independent LTP It has been known for some time that the induction of LTP at a number of synapses is independent of the activation of NMDA receptors (for review, see Johnston et a t, 1992). Grover and Teyler (1990) demonstrated a non-NMDA dependent form of LTP induced by high frequency (200 Hz) tetanization of the Schaffer col lateral/commissural input onto the CAl region of the hippocampus. This component of LTP was resistant to D,L-AP5 (up to 200 /iM) but blocked or substantially reduced by nifedipine, an antagonist of dihydropyridine-sensitive Ca^'^ channels, suggesting that a route for Ca^^ entry postsynaptically could be through voltage-operated Ca^"^ channels (VOCCs). Injection of cells with the Ca^^ chelator BAPTA ( 1,2-bis(2-aminophenoxy)ethane %W-tetraacetic acid) before tetanization also blocked this form of LTP. Recently, Aniksztejn and Ben-Ari (1991) described, in the hippocampal CAl region, a Ca^"*"-dependent, NMDA-independent form of LTP produced by blockade of channels. Indeed, other studies in the hippocampal CAl region suggest that VOCCs may play a role in the induction of LTP (Regehr, et a l, 1989; Regehr and Tank, 1990; Westenbroek et al. , 1990). Bramham et al. (1991) investigated whether LTP in the medial (MPP) and lateral (LPP) perforant path (MPP) of anaesthetized rats was dependent on NMDA receptor activation. They reported that in the MPP, LTP was blocked by perfusion of AP5

24 across the perforant path synaptic (terminal) zone via an implanted push-pull cannula. Surprisingly however, these authors found that LTP induction in the LPP was not block by AP5. Harris and Cotman (1984) and Williams and Johnston (1989) reported that the induction of LTP at commissural/recurrent/associational (C/A) synapses required NMDA receptor activation, but that mossy fibre LTP was not blocked by AP5. They attributed this to the lack of NMDA receptor binding in area stratum lucidum of CA3, the termination of area CA3.

Calcium and LTP The recurring theme in both NMDA receptor dependent and independent LTP induction is the involvement of Ca^^. More direct evidence of the importance of postsynaptic Ca^"^ entry was provided by two experiments. First, it was shown that injection of Ca^'^ chelators such as EGTA (potassium ethylene glycol-bis-N,N,N’,N’- tetracetic acid) into postsynaptic neurons prevented the induction of LTP (Lynch et al. , 1983). Second, release of Ca^"^ into postsynaptic neurones by the caged Ca^^ compound Nitr-5, a photolabile nitrobenzhyldrol tetracarboxylate Ca^"^ chelator, produced a potentiation of synaptic transmission (Malenka et al. , 1988). These two experiments taken together, strongly support the idea that an elevation of postsynaptic Ca^"^ is the critical triggering factor in LTP. Given that a rise in Ca^"^ within the dendritic spine is an essential step in the induction of LTP, the question of great interest is: what are the biochemical processes activated by Ca^"^ that are responsible for LTP? (For reviews see Bliss and Lynch., 1988; Linden and Routenberg., 1989; Madison e ta l, 1991).

The role of G ABA receptor-mediated inhibition in LTP induction GABAy^-mediated inhibition. Inhibitory mechanisms have been shown to suppress the induction of LTP, as first identified by Douglas (1982) in rat dentate gyrus in vivo. Postsynaptic inhibition in the hippocampus is mediated by the inhibitory neurotransmitter 7 -aminobutyric acid (GABA, Anderson et al. , 1964; Anderson et al., 1980; Alger and Nicoll, 1982) via action at GABA^ receptors, evoking a somatic hyperpolarization mediated by an increased chloride conductance. Moreover, this observation provided evidence for a postsynaptic role in the induction of LTP.

25 Wigstrom and Gustafsson (1983) demonstrated that LTP in vitro could be facilitated in the presence of the GABA^ antagonist, picrotoxin, which lowered the induction threshold for eliciting LTP. In support of this, Scharfman and Sarvey (1985) showed that procedures which reduce or prevent postsynaptic depolarization during conditioning, such as co-application of GABA, blocked LTP induction in vitro. GABAs-mediated inhibition. Not all actions of GABA are mediated by picrotoxin/bicuculline-sensitive GABA receptors. GABA has been shown to inhibit the presynaptic release of other neurotransmitters, an action which was bicuculline-resistant (Hill and Bowery, 1981; Spencer et al., 1986). This action was mimicked by baclofen, which had no action on GABA^ receptors. The receptors that mediate this effect have been termed GABAg. In hippocampal pyramidal cells, GABAg receptor activation results in a hyperpolarization of membrane potential due to an increase membrane conductance to (Blaxter and Carlen, 1985; Gahwiler and Brown, 1985; Newberry and Nicoll, 1985). Blockade of GABAg, like GABA^ receptor mediated IPSPs (Wigstrom and Gustafsson, 1983; Mott et al., 1990; Olpe, 1990) can facilitate the induction of LTP, because elimination of the hyperpolarizing action enhances the expression of the NMDA receptor-mediated conductance (Collingridge a/., 1983; Dingledine etal., 1986; Davies et a l, 1991).

Enhancement of E-S potentiation LTP is often accompanied by an increase in the probability of spiking to an EPSP of fixed stimulus strength. This phenomenon is termed EPSP - spike coupling (E-S coupling) or simply E-S potentiation and represents a change in the responsiveness of the cell to the EPSP, apparently independent of the change in the EPSP itself (for review, see Chavez-Noriega and Bliss, 1991). In many experiments there is more potentiation of the population spike than can be accounted for by the potentiation of the EPSP. This is mostly seen in the curve relating population spike size to EPSP slope. Typically the effect of LTP is to shift the curve to the left, which indicates that the cell is more likely to fire in response to an EPSP of fixed size (Anderson et al., 1980; Bliss et al., 1983; Chavez-Noriega et al., 1989). Evidence for enhanced E-S coupling was observed in the earliest extracellular studies of hippocampal LTP (Bliss and Lomo, 1973). These observations have been extended by Taube and Schwartzkroin (1988),

26 who showed with intradendritic recordings that tetanization can increase the probability of firing without significant change in the size or shape of the EPSP. The mechanism that have been proposed to account for E-S potentiation fall into two categories: (1) a postsynaptic change in cell excitability, or (2) an increase in the ratio of synaptic excitation to inhibition (Chavez-Noriega and Bliss 1991; Wathey et ah, 1992). Some authors have suggested that an increase in postsynaptic excitability would degrade the specificity of LTP and is therefore unlikely to occur in CAl pyramidal cells (Wilson et ah, 1981; Abraham et ah, 1987). However, increased cell excitability would also be manifest as an increase in postsynaptic inhibition.

Expression of hippocampal LTP: presynaptic, postsynaptic or both? The expression of hippocampal LTP is not associated with lasting changes in resting membrane potential or apparent input resistance of the postsynaptic neurones (Andersen et ah, 1980; Barrionuevo and Brown, 1983). Neither is it due to a reduction in synaptic inhibition (Haas and Rose, 1984; Wigstrom and Gustafsson, 1985a; Abraham et ah, 1987; Taube and Schwartzkroin, 1987; Bliss et ah, 1987). There is much debate as to the site of origin of LTP expression. Is expression presynaptic, postsynaptic, or both? A number of mechanisms have been suggested to mediate the maintenance and expression of hippocampal LTP.

Possible presynaptic mechanisms underlying LTP expression (1) Enhanced release of neurotransmitter - the case for a retrograde messenger. By monitoring the levels of neurotransmitter in the extracellular fluid, prior to and after conditioning, it has been shown that LTP is associated with an increase in the absolute levels of neurotransmitter in the extracellular fluid, suggesting that potentiation is maintained by an increase in the presynaptic release of neurotransmitter (Skrede and Malthe-Sorenssen, 1981; Dolphin et ah, 1982; Bliss et ah, 1986; Lynch and Bliss, 1986; Errington et ah, 1987). Increased amounts of transmitter collected from the tissue during LTP may not be entirely due to increased release from each axon terminal, because an increased quantity of transmitter may be due to decreased uptake into neighbouring axon terminals and glial cells (discussed below). If LTP is induced postsynaptically but maintained by increased release of transmitter following each presynaptic action potential, there has to be a retrograde messenger released from the

27 postsynaptic cell which causes the presynaptic cell to increase the release of transmitter. Arachidonic acid, an unsaturated fatty acid released during LTP, is one candidate for a retrograde messenger (see Bliss and Lynch, 1988; Lynch et al. , 1989; Williams et a l, 1989; Bliss et al.y 1991). Additional direct evidence for the potential role of arachidonic acid in LTP comes from the findings that an increase in the extracellular arachidonic acid can be measured in vivo from the dentate gyrus during LTP (Lynch et al. , 1989) and that application of arachidonic acid can enhance synaptic transmission both in vivo and in vitro (Williams, et al., 1989). Interestingly, application of arachidonic acid does enhance low frequency synaptic transmission by itself, but requires concomitant activation of the presynaptic fibres by a (weak) tetanus. Even then, synaptic transmission only increases slowly over the time course of 1-3 hours, at which point tetanus induced LTP was occluded. The slow time-course of the arachidonic acid effect was taken to suggest that arachidonic acid may act as a retrograde messenger on the later phases of LTP (Williams et al. , 1989). Important questions that remain are: (i) what concentration of arachidonic acid is released at synapses and (ii) how fast and at what concentration applied arachidonic acid reached synapses within the slice? Recently, Miller et al. (1992) showed that arachidonic acid potentiated NMDA receptor currents in dissociated cerebellar granule cells from postnatal rats and pyramidal cells from slices of adult rat hippocampus. These authors suggested that arachidonic acid could increase Ca^*^ currents through the NMDA channel and thus augment increases in intracellular Ca^^ caused by glutamate. If presynaptic transmitter release is enhanced in LTP, one might expect to find an increase in both the AMPA-sensitive and NMDA-sensitive components of the EPSP. The available evidence is not consistent. On one hand, Kauer and coworkers (1988) and Muller and Lynch (1988) found that the NMDA component did not significantly increase under conditions that normally produce LTP. They concluded that only the AMPA- sensitive component of the EPSP was capable of being potentiated and that LTP expression was purely postsynaptic. On the other hand, Bashir et al. (1991) and Malinow and Tsien (1991b) reported a significant potentiation of NMDA-mediated transmission under similar conditions. Interestingly, Xie et al. (1992) recently reported that in dentate gyrus granule cells, both LTP and LTD (page 59) of the NMDA receptor-mediated synaptic response

28 could be induced. LTP of the NMDA receptor-mediated synaptic transmission was induced by high frequency (50 or 100 Hz) afferent stimulation, which could be blocked by postsynaptic hyperpolarization (to -100 mV) during the tetanus. However when lower frequency (10 Hz) afferent stimulation were applied during the hyperpolarization, LTD of the NMDA receptor-mediated response was induced. (2) Reduced uptake of neurotransmitter. One possible site at which arachidonic acid could act is on a glial cell glutamate uptake mechanism (Barbour et al. , 1989). These authors reported that arachidonic acid inhibited the uptake of glutamate into glial cells in the salamander retina (Barbour et al. , 1989) for tens of minutes. Reduced uptake could act to increase the amount of glutamate in the extracellular fluid and perhaps also in the synaptic cleft. This would not be a strictly presynaptic mechanism but would result in an increase in extracellular glutamate levels. Recently, nitric oxide has been implicated as a retrograde signal (Schuman and Madison, 1991; O’Dell era/., 1991; Haley era/., 1992; Gribkoff and Lum-Ragan, 1992; Izumi era/., 1992; for reviews see Bredt and Snyder, 1992; Garthwaite, 1989, 1991).

Possible postsynaptic mechanisms underlying LTP expression (1) Increased number of postsynaptic receptors. Baudry et al. (1980) reported that there was an increase in the level of glutamate binding in potentiated tissue; these workers proposed that LTP was maintained by an increase in the number of postsynaptic glutamate receptors. However, Sastry and Gob (1984) and Lynch et al. (1985a,b) failed to observe similar findings. The assay used by Baudry et al. (1990) for measuring the density of glutamate receptors was criticized as being inappropriate (Sastry and Goh, 1984; Lynch et a l, 1985b). The physiological relevance of the binding site measured in the experiments of Baudry et al. (1980) is unclear, and may have been extrasynaptic; Pin et al. (1984) suggested that this site may represent a vesicular-uptake site. Lynch et al. (1976) reported that LTP is associated with a reduced dendritic responsiveness to iontophoretically applied glutamate in the CAl region of the hippocampus. In contrast, Schwartzkroin and Taube (1986) and Taube and Schwartzkroin (1988) reported that following LTP in CAl, there was no corresponding

29 change in neuronal sensitivity to iontophoresed glutamate. However, an increase in the number or an increase in the responsiveness of postsynaptic receptors may still play a role in the maintenance of LTP. Davies et al. (1989) reported an increase in the responsiveness of hippocampal neurones to iontophoretically applied AMPA. The effect was not immediate, and took at least one hour to reach its maximum, suggesting that there is a succession of underlying processes responsible for the maintenance of LTP; the earliest phase may be mediated by presynaptic mechanisms, such as the increased release of neurotransmitter, and the latter by postsynaptic modification. (2) Morphological modification. LTP has been associated with morphological changes at the level of the synapse and synaptic spine that are believed to result in the enhancement of synaptic current and improved synapse-dendritic coupling (Lee et al. , 1980; Chang and Greenough, 1984; Brown et al., 1988; Desmond and Levy, 1988; Andersen, 1989; Schottler, 1990). The structure of the synaptic region has been compared in control tissue with that in which LTP has been induced, and correlated changes in synaptic structure have been noted (Lee et al., 1980; see Desmond and Levy, 1988).

I issue of Can quanta! analysis of synaptic variability resolve th^LTP expression ? Statistical analysis of synaptic variability (quantal analysis) of synaptic transmission has been proposed as a means of settling the debate of whether the expression of LTP is pre- or postsynaptic (Brown et al., 1988; Bekkers and Stevens, 1989). Implicit in the quantal hypothesis of transmitter release is that transmitter is released in multimolecular packets and normally release consists of an integral number of these units. Evidence for this account comes from studies of the vertebrate neuromuscular junction (Fatt and Katz, 1952, del Castillo and Katz, 1954; Boyd and Martin, 1956; for review see Martin, 1977; Redman, 1990). Since the development of whole cell voltage-clamp recording techniques in the in vitro brain slice (Edwards et al. 1989; Blanton et al., 1989) with its improved signal to noise ratio compared to conventional intracellular recording, it has become possible to resolve small, unitary excitatory postsynaptic currents (EPSCs). Using the latter technique, various groups (Malinow and Tsien 1990; Bekkers and Stevens, 1990; Malinow, 1991a) analyzed the variability of EPSCs recorded prior to and after the induction of synapse specific LTP in the hippocampus. These groups estimated the

30 numbers of failures and changes in the number of release sites (n) and/or the probability of release from each site ip) before and after the induction of LTP, and applied the assumptions used in quantal analysis of neuromuscular transmission. They concluded that the changes measured were presynaptic in origin and could not be explained by an increased postsynaptic responsiveness; potentiation was also associated with a decrease in the number of synaptic failures (Malinow and Tsien, 1990, Malinow, 1991a; but see Friedlander et al. , 1990). This observation cannot be explained by a decrease in the rate of removal of neurotransmitter as suggested by Barbour et al. (1989). Inhibition of glial glutamate uptake is would be seen as increased quantal size, not increased probability of transmitter release. However, other workers (Larkman et al. 1991; Stratford et al., 1992) have suggested that fitting the data with a simple binomial distribution, and assuming a constant postsynaptic responsiveness (Jack et al., 1981) may not be justified. Without these assumptions the interpretation of the number of failures, of the origin of changes in the variables n and p and related statistics is certainly open to question. Interestingly, Kullman and Nicoll (1992) have recently proposed evidence for both pre- and postsynaptic sites of LTP expression. Analysis of the amplitude of spontaneous miniature synaptic events (minis or mepps - miniature end plate potentials) thought to be synaptic responses to single packets of transmitter (del Castillo and Katz, 1954; Martin, 1977; Edwards et al., 1990) following LTP induction in hippocampal slices or cultures, has failed to resolve this issue any further (see Manabe et ah, 1992; Malgaroli and Tsien, 1992).

1.5 LTP in the neocortex

LTP of synaptic transmission in the neocortex is poorly characterized, compared with that in the hippocampus. The complex neuronal circuitry and heterogeneity of neurones make it extremely difficult to record reliably from a known population of cortical neurones and stimulate an identifiable, homogeneous afferent pathway in the in vitro neocortex. It is not surprising then that the vast majority of research has focused on hippocampal LTP. However, there is no reason to suppose that neocortical LTP is vastly different from LTP in the hippocampus (for reviews see Bindman et al. 1991; Tsumoto, 1992). Figure 1.1 summarizes the various forms of synaptic modification that

31 can occur in the neocortex.

Historical background Long-lasting, activity-dependent changes in the efficacy of synaptic transmission in the neocortex in vivo were demonstrated in the 1960s. Bindman et al. (1962, 1964) reported that field potentials recorded in the somatosensory cortex of anaesthetized cats, evoked by sensory stimulation of the contralateral forepaw, could be potentiated by the application of polarizing current to the surface of, or locally within, the cortex (also see Voronin, 1971). Later, Bliss et al. (1968) demonstrated that activity-dependent persistent increases and decreases in synaptic transmission could be produced in undercut slabs of neocortex in the anaesthetized cat. In the 1970s, Pavlovian conditioning of eye-blink responses was found to be associated with increased excitability of neurones in the motor cortex of awake cats, selectively within the region projecting to muscles involved in the conditioned response (Brons and Woody, 1980; Woody, 1984). However, progress on LTP as such in the neocortex was delayed until the 1980s when isolated slice preparations were developed, and intracellular recording and induction of LTP were used.

GAB A A inhibition and neocortical LTP block of GABAy^-mediated inhibition In neocortex, ^ is necessary for the induction of LTP in visual cortex after the critical period (Teyler et al., 1990; Artola and Singer, 1987, 1990) but in sensorimotor cortex it does not improve the probability of inducing LTP in layer V neurones, speculatively because there is also potassium-mediated (GABAg) inhibition (Bindman et al., 1988b; Bindman and Murphy, 1990). In frontal cortex also, Sutor and Hablitz (1989a,b) found that block of GABA^ inhibition was not required for the induction of LTP. Importantly, associative LTP can be produced with an incidence of 50-60% in awake cats, using no drugs (Baranyi et al., 1991 and see below). The role of GABAg inhibition in the induction of neocortical LTP remains to be investigated.

NMDA receptors and the induction and maintenance of neocortical LTP As in the hippocampus (Collingridge et al., 1983), antagonists of NMDA receptor-mediated activity block the induction of LTP (Artola and Singer, 1987, 1990; Bindman and Murphy, 1988b; Sutor and Hablitz, 1989b) (But see Aroniadou and

32 Teyler, 1992, for NMDA receptor-independent LTP in the visual cortex). However, unlike in the hippocampus, in the neocortex (visual, sensorimotor, frontal and preffontal) the NMDA receptor-mediated component of the EPSPs can be seen in normal [Mg^*^] (1 mM as in cerebrospinal fluid) at low frequency stimulation and at membrane potentials only a few mV depolarized above the resting membrane potential (about -80 mV). Moreover, it was not always necessary to use a GABA^ blocker to uncover the NMDA receptor-mediated component (Artola and Singer, 1987, 1990; Aram et al., 1987; Sutor and Hablitz, 1989b; Hirsch and Crepel, 1990). In some experimental preparations the NMDA receptor-mediated component of the EPSP had the same latency of onset as a non-NMDA receptor mediated component (Artola and Singer, 1990; Hirsch and Crepel, 1990), while in others it started from milliseconds to tens of milliseconds after the onset of the non-NMDA receptor-mediated EPSP (Aram et al. , 1987; Sutor and Hablitz, 1989b). The non-NMDA receptor-mediated component of neocortical EPSP may be mediated largely by glutamate as transmitter since it was blocked by kynurenate (a non-selective glutamate antagonist; Aram et al., 1987; Jones and Baughman, 1988) or by 6,cyano-7-nitroquinoxaline-3,3-dione (CNQX) (Artola and Singer, 1990; Hirsch and Crepel, 1990). In view of the presence of an NMDA receptor-mediated component in neocortical EPSPs prior to potentiation, it is not surprising that, unlike in the hippocampus (Gustafsson et ah, 1987), NMDA receptor-mediated activity participated in the expression of LTP (Bindman and Murphy, 1988a; Artola and Singer, 1990). Following application of AP5, it was seen that in some cells the LTP was manifest as both an enhanced non-NMDA receptor-mediated component and a later, enhanced NMDA receptor-mediated component (Bindman and Murphy, 1990 and Artola and Singer, 1990), while in other cells the LTP was expressed only as an NMDA receptor-mediated enhancement (Artola and Singer, 1990).

The associative, postsynaptic induction of LTP in the neocortex Baranyi and Feher (1978) carried out the first intracellular study of LTP of neocortical EPSPs. Antidromic action potentials elicited by electrical stimulation of the pyramidal tract and orthodromic EPSPs evoked by stimulation of the ventro-lateral (VL) nucleus (VL-EPSPs) were recorded in single pyramidal neurones in the motor cortex of anaesthetized cats. One of two paradigms was used to condition the cells: the first was

33 antidromic stimulation at 15-60 Hz for 5-30 seconds, and the second was 150 repetitive pairings of the VL-EPSPs with a preceding antidromic action potential (10-300 ms). Potentiation was judged to have been induced if there was (i) an increase in the magnitude and/or decrease in the onset latency of the VL-EPSPs or (ii) an increase in neuronal excitability. Plastic modifications were observed in 14% of the conditioned neurones, lasting from a few seconds to 27 minutes post-conditioning. It was found that the pairing conditioning paradigm was more effective as an inducer of LTP than high frequency antidromic stimulation. Since GABA^ receptors regulate intracortical inhibition, it is conceivable that feedback inhibition may have prevented LTP induction by antidromic conditioning alone, by preventing sufficient depolarization of NMDA antidromic conditioning receptor channels, since the \ would be expected to simultaneously excite a population of both excitatory and inhibitory inputs. These authors’ observation implied that in the neocortex pairing of presynaptic and postsynaptic activity was more effective as an inducer of LTP than postsynaptic depolarization alone, possibly because depolarizing current injection would be expected to bypass the inhibitory circuits. Later Baranyi and Feher (1981) proposed that the convergence of excitatory input onto a neocortical neurone could create the conditions that lead to enduring changes in synaptic efficacy (heterosynaptic facilitation, Kandel and Tauc, 1965). Intracellularly recorded EPSPs were evoked from separate afferent pathways by stimulation of the VL- nucleus (VL-EPSPs), the cortical surface (Call-EPSPs), and the contralateral radial nerve (SS-EPSPs: somatosensory). In each experiment, one input was selected as the control with the stimulus parameters set to evoke sub threshold EPSPs. A second input received the conditioning stimuli, with the shock parameters set to evoke one or more orthodromic action potentials. These authors found that the time interval between pairing of stimuli had to be short: control EPSPs were repetitively paired 150 times with the conditioning stimuli that preceded the former by 0-100 ms. Potentiated EPSPs were those judged to show an increase in amplitude and/or a decrease in onset latency. Conditioning of SS-EPSPs with orthodromic action potentials evoked by VL-stimulation induced potentiation in 54% of trials. VL-EPSPs conditioning of the Call-EPSPs induced potentiation in 36% of trials. Call-EPSPs conditioning of the VL-EPSPs induced potentiation in 21% of trials. In most cases the potentiation was short-lived, lasting for only a few min. However in three cases it endured for 18-55 minutes. In 30% of neurones, potentiation of an EPSP was associated with an increase in input

34 resistance and a 4-6 mV depolarization of the resting membrane potential. The significance of the increased membrane resistance is that the length or space constant would thereby be increased, synaptic potentials at the soma would be larger and E-S coupling enhanced. However, changes in input resistance and membrane potential were also observed for neurones that failed to support potentiation of an input. Baranyi and Feher (1981) ruled out the possibility that concomitant changes in input resistance and/or membrane potential were responsible for the underlying potentiation of synaptic potentials, i.e. enhancement of synaptic potentials was not due to a non-specific increase in the membrane excitability of the postsynaptic neurone as had been subsequently suggested by Bindman and Prince (1986). Further evidence for a postsynaptic locus for the mechanisms that underlie changes in the potentiation of synaptic transmission was reported by Baranyi and Szente (1987). These authors made intracellular recordings from 533 neurones in the motor cortex of anaesthetized cats. Repeated pairing of mono- or oligosynaptic EPSPs with injections of depolarizing current into the postsynaptic neurone or with antidromic action potentials evoked by stimulation of the pyramidal tract, successfully induced LTP, lasting 40-60 minutes, in 28% of conditioned neurones. The average duration of the enhancement was 49 minutes and extinction of the enhancement was often seen. However, in 43 cells the EPSPs did not decrement until the end of the recording (mean duration of LTP 116 minutes). The onset of the conditioning stimuli followed the test shock by 0 - 2 0 0 ms, allowing a degree of temporal conjunction between presynaptic activity and postsynaptic depolarization. If the neurone was hyperpolarized by either an intracellular injection of hyperpolarizing current or exposure to the inhibitory neurotransmitter, GABA, LTP was not induced. Recently, Baranyi et al. (1991) blocked postsynaptic Na"*” conductance and firing activity by intracellularly injecting QX-314 (A-(2,6-dimethyl-phenyl- carbamoylmethyl)triethyl-ammonium bromide) postsynaptically. Injection of QX-314 did not prevent the induction of LTP. They reported that postsynaptically injected current induced membrane depolarizations of >30 mV, could substitute for Na"*”- dependent spiking in stimulus pairs in eliciting LTP. Their observations suggested that neither the Na^-mediated part of the postsynaptic action potential nor the persistent Na"*' conductance (Stafstrom et al. , 1985) per se is necessary for the induction of LTP if adequate amount of postsynaptic depolarization are in temporal conjunction with

35 synaptic activation. This is similar to reports in the hippocampus in vitro (Gustafsson and Wigstrom (1986; Kelso et a/. ,1986). However, it should be noted that QX-314 can also block the GABAg-mediated slow IPSP in the hippocampal CAl region (Nathan and Lambert, 1990, 1991). It is therefore unclear whether block of GABAg-inhibition was an important factor in these experiments.

Associative LTP in the awake animal An important finding that added strength to the proposal that associative LTP could underlie the neural basis of learning was reported by Baranyi et al. (1991), who were able to induce input-specific, associative LTP in the motor cortex of conscious cats. Pairing of test trans-synaptic stimulation with current injection-evoked action potentials induced LTP of test responses in 58% of the cells tested, whereas in anaesthetized cats it did so in just 28% of the cells. Thus postsynaptically induced LTP occurred with a higher probability (0*5 - 0*6) in unanaesthetized, awake cats than in anaesthetized cats. This difference in probability of induction may be due to the reduction on tonically active inhibitory influences in the awake preparation, because GABA^ receptor-mediated inhibition prevents NMDA receptors from being fully activated. I Alternatively, other neuromodulators which affect the NMDA receptor (eg. noradrenaline) may behave 1 differently in the awake and sleeping state.

Postsynaptic Ca^^ and associative LTP An important finding by Baranyi and Szente (1987) was that intracellular injection of the Ca^^ chelator EGTA blocked the induction of LTP following pairing in 53 cells. EGTA was no longer effective after 30 minutes, and then LTP was successfully induced in 10 out of 24 of these cells. A rise in postsynaptic [Ca^^] was presumably needed for the induction of LTP, as previously shown by Lynch et al. (1983) and later by Malenka et al. (1988) in hippocampus.

Neuronal transport Effects of the neuronal transport blocker colchicine were also examined in cortical cells in vivo by Baranyi and Szente (1987). Colchicine was injected intracellularly; it caused a gradual increase in neuronal excitability after about 15 minutes. Its effects on induction of LTP was studied therefore in the first 10 minutes. In 44 cells, colchicine was injected before pairing and in only three EPSPs was LTP induced (the expected

36 number would be 12). The drug effects were not reversible in two to three hours. This interesting observation suggests that postsynaptic tubular transport or de novo synthesis of protein may play a part in the induction of LTP. Baranyi and Szente (1987) concluded that the site for the mechanisms that underlie the induction of LTP was the postsynaptic membrane.

Postsynaptic conductance changes It has been suggested that the maintenance of LTP in neocortical cells may involve changes in postsynaptic conductances. In awake cats, Baranyi et a l (1991) have also reported that postsynaptic induction of synapse-specific LTP is associated with a reduction in outward currents in the potentiated but not the unpaired EPSPs. The current was identified as the potassium-mediated A current (IJ as LTP induction was blocked with intracellular Cs^ or aminopyridine, but not by apamin, a blocker of Ca^'*’- activated currents, or TEA (tetraethylammonium) (see also Woody et al. , 1989). In their whole-cell patch clamp experiments in hippocampal slices (page 30), Malinow and Tsien (1990) used Cs^ in the recording electrodes to block postsynaptic channels; this would also be likely to block the postsynaptic modifications in currents (Ik) seen in LTP by Baranyi et a l (1990). It could be argued that the only effect of blocking a Ik would be manifest as an enhanced E-S coupling. However, the increased membrane resistance due to block of Ik would increase the cell’s space constant, and this might be observed as an increase in the EPSP slope or amplitude^ but does not explain the input specificity

Associative LTP - some questions In slice preparations of adult rat sensorimotor cortex, Bindman et a l (1988a; Bindman and Murphy, 1990) observed an associative induction of LTP of EPSPs recorded from deep-layer (V) cortical cells, through pairing of white matter afferent fibre activation with depolarization induced by intracellular current injection. The associative enhancement of EPSPs lasted throughout the duration of the recording period (15-50 min) without any significant decay. However, a lower percentage of EPSPs, about 10-14%, were potentiated in the slice in vitro than in the in vivo cat studies of Baranyi and colleagues cited above. What reasons might account for this relatively low incidence of associative LTP observed in the cortical slice? Possibilities are discussed below:

37 (1) Differences in cell and/or species type. One possible explanation might be due to Bindman and colleagues sampling from layer V and VI of the cortex. These deep layer cells in visual, somatosensory and motor cortices have been reported to have less capacity for plasticity than superficial layer cells (Komatsu et al., 1981; Keller et al. 1990a,b). However, Baranyi and Feher (1981), Baranyi and Szente (1987) and Baranyi et al. (1991) also recorded from deep layer V motor cortical cells and reported a much higher incidence of associative LTP in both the anaesthetized (28%), and awake (55%) cat in vivo. The possibility does however exist that a species difference between rat and cat may account for the different results reported by these groups. (2) Differences in resting membrane potential (V J. Examination of the V^s reported in the cat and rat studies above may account for the difference: in the studies of Bindman et al. (1988a) and Bindman and Murphy (1990), the resting potential was on average 12 mV more negative than reported by Baranyi and colleagues (1987). The more negative resting potential of the in vitro rat neurones may mean that the voltage-dependent block by Mg^"^ of the NMDA receptor channel is more powerful than in vivo. This would mean that during conditioning in vitro, a greater level of depolarization of the rat neurones would be required to activate the NMDA receptors and alleviate the Mg^"^ block of the channel to allow the critical entry of Ca^"^ required for the induction of LTP. (3) The loss of neuromodulatory influences in the slice preparation. One explanation for the lower incidence of associative LTP observed in the slice could be that the cortical slices are isolated from extrinsic noradrenergic activity which could be involved in the induction of associative LTP in the awake animal. Electrophysiological investigations have suggested that the noradrenergic projections to the forebrain may play an important role in modulating synaptic transmission in mammalian cortical and hippocampal circuits, particularly with respect to developmental and synaptic plasticity (Section A, page 39; Kasamatsu, 1983; Bear and Singer, 1986; Harley, 1988; Lehmenkuhler gf a/., 1991; L^Vm et al., 1988; McCormick, 1989). Intracellular recordings in the in vitro slice preparation allows a direct test of this hypothesis. Therefore, the actions of one such possible neuromodulator, noradrenaline (NAd) and the 6 -adrenergic agonist, isoprenaline (ISO), were investigated on both synaptic transmission and on the induction of associative LTP in layer V

38 pyramidal cells in rat sensorimotor cortex. This investigation forms the first part of this study and is introduced in section A.

SECTION A: INVESTIGATION OF B-ADRENERGIC MODULATION OF SYNAPTIC TRANSMISSION AND ASSOCIATIVE LTP IN SLICES OF RAT SENSORIMOTOR CORTEX

1.6 Introduction

LTP of synaptic transmission is a neuronal model of learning and memory in mammals. As mentioned in the Preface , isolated brain slices play a key role in the analysis of the phenomenon. Recently, the probability of induction of associative LTP in layer V cells in sensorimotor neocortex was shown to be much higher in the awake cat than in the slice. Electrophysiological investigations have suggested that in mammals, noradrenergic projections to the forebrain may play an important role in modulating synaptic transmission in cortical and hippocampal circuits, particularly with respect to developmental and synaptic plasticity (Kasamatsu, 1983; Bear and Singer, 1986; Harley, 1988; Lehmenkuhler gf a/., 1991; Levin et al., 1988; McCormick, 1989). d However it is clear that noradrenaline (NAjJ produces different effects in different regions of the cortex. The following sections briefly examine the distribution of noradrenergic fibres in the rat forebrain, and the localization of noradrenergic receptors in the rat neocortex. Subsequent sections survey the actions of NAd and noradrenergic agonists and antagonists observed in various cortical regions, such as the hippocampus and neocortex. The effects of NAd on the hippocampus are dealt with in greater detail since the vast majority of the work has been carried out in this structure. The final section presents the reasons for undertaking this investigation.

1.7 Forebrain noradrenergic connections

Anatomically the development of fluorescent histochemistry has made possible the detailed visualization of forebrain NAd connections. The NAd axons innervating neocortex, hippocampus and cerebellum originate from a small group of NAd cells

39 clustered in the dorsal pons, the locus coeruleus, which provides a dramatically widespread direct input to many structures of the central nervous system. In the rat it has been estimated that each of the 1500 neurons per locus coeruleus nucleus must give rise to 500,000 terminals to account for the divergent and comprehensive innervation observed. In the forebrain the locus coeruleus provides the sole NAd innervation for extensive regions including neocortex, olfactory bulbs, limbic cortex and sensory thalamus. In the hindbrain, the cerebellum and specific brainstem and spinal cord targets are also supplied by NAd primarily or exclusively from locus coeruleus (Moore noradrenergic and Bloom, 1979). Other NAd nuclei of the brainstem have been described, but their projections are more limited than those of the locus coeruleus. These other nuclei target regions of hypothalamus, certain limbic nuclei and the spinal cord, but the terminal fields do not overlap those of the locus coeruleus (LC) cells and their functional role may not be linked to that of the locus coeruleus. Retrograde tracer studies have indicated extensive collateralization of locus coeruleus neurones such that the same neurone can project to widely separated structures (Room et al., 1981; Steindler, 1981). The highly clustered appearance of this group within the pons might reinforce the idea that locus coeruleus would be activated in concert and locus coeruleus output would be relatively synchronous (Aston-Jones and Bloom, 1981a,b). Kety (1970) suggested that a widespread release of NAd occurred in all innervated areas when LC cells are active, and that the diffuse and direct nature of the LC projections is consistent with a system capable of an enhancement and "consolidation" signal to many areas of the CNS. However, research employing neurotoxins, such as 6 -hydroxytryptamine ( 6 -OHDA) and N-(2-chlorethyl)-N-ethyl-2- bromobenzlamine (DSP-4), which are taken up specifically by noradrenergic axons and which produce widespread depletion of NAd, particularly from forebrain sites, was less supportive of Kety’s hypothesis. Chronic NAd depletion in animal models did not result in dramatic failure of attention and memory, although subtle effects were reported (Mason and Iverson, 1975).

1.8 Adrenergic receptors in the neocortex NAd-containing axons of the rat neocortex ascend from the locus coeruleus via the dorsal tegmental bundle through the medial forebrain bundle, anterior septal region, and the internal and external capsule. More rostrally located areas have a denser NAd

40 innervation than do more caudally located areas (Doucet et al., 1987, 1988). Measurements of NAd concentrations in the frontal, parietal, temporal, and occipital areas (Palkovits et al., 1979) corroborate these findings by a decrease of Nad concentrations along this rostrocaudal sequence. Within the neocortex,the NAd- containing axons show a layer-specific orientation with horizontal fibres in layer I, radially oriented fibres in layers II and III, short oblique or tortuous axons in layers IV and V, and strictly rostrocaudally oriented fibres in layer VI (Morrison et al. 1978). The highest density of NAd containing terminals is found in the superficial layers of frontal and parietal areas with a continuous decrease down to layer VI (Doucet et al., 1987, 1988).

«1, (%2, 6 i, and 62-adrenergic receptor subtypes are found in the rat cortex (Zilles et al., 1990), the location of which has been shown by binding studies. The highest concentration of the postsynaptic «i-receptors is located in the prefrontal cortex. The density decreases in the frontoparietal areas and reaches lowest values in the occipitotemporal region. The cK 2-receptors show the highest concentration in the temporal areas, whereas the 6 -receptors are homogeneously distributed over the whole neocortex (Diop et al., 1987). The laminar distribution of a-receptors is quite uniform over all cortical layers

(Young and Kuhar, 1980), whereas the 6 -receptors (mainly 6 ,) show a higher density in layers I through III compared with the deeper layers (Palacios and Kuhar, 1980, 1982;

Rainbow et al., 1984a). 61-receptors are highly concentrated in layers I and II, whereas

62-receptors are most densely packed in layer IV and the deeper part of layer V

(Rainbow et al., 1984a). Eighty percent of the 6 -adrenergic receptors in layers I and II are of the 6 % subtype. Equal proportions of both subtypes are found in layers IV and

IVb. The distribution of 61-receptors is therefore comparable with the laminar distribution of NAd-containing terminals.

Aoki and colleagues (1987) reported a different laminar distribution for 6 - receptors using polyclonal antibody and immunohistochemical studies. According to this study, the supra- and infragranular layers have the same 6 -receptor densities, but the density in layer IV is lowered by about 15%. It is unclear whether the quantification of optical density in immunohistochemical specimens permits a comparison with autoradiographic results. The cellular localization of 6 -receptors in perikarya, dendrites and postsynaptic densities within spines, axons, and glial processes argues for both a

41 neuronal and non-neuronal, as well as a pre- and postsynaptic localization. Recently, Jones et a l (1985) and Palacios et al. (1987) reported a very high density of the «i-subtypes in layer V. These receptors showed a very high density on the superficial and lower borders of this layer, as shown by a double band, which fused to a single band in the cingulate cortex. Generally the «i-receptor density is higher in the more rostral compared with the more caudal cortical regions. Palacios and Warmsley (1984) described a high density of (Xg-receptors in layers I and IV. Thus the

« 1 and «2-receptors display a different anatomical distribution. In the rat hippocampus,

«2-receptors are found on GABA-containing terminals, where NAd activation of « 2- receptors enhances GABA release (Pittaluga and Raiteri, 1987). It is currently unknown whether such a mechanism is also valid in the neocortex, but GABA acting at GABA^ receptors on NAd terminals increases NAd release both in the hippocampus and cortex (Bonanno and Raiteri, 1987). Furthermore, in the hippocampus, NAd may have excitatory actions on GABAergic interneurones (Andreason and Lambert, 1991). This is supported by immunocytochemical studies showing that noradrenergic fibres form asymmetric (excitatory) synapses on the somata and dendrites of GABAergic interneurones in the hippocampus (Miller and Bacon, 1989). NAd has also been shown to have an excitatory effect on interneurones in the hippocampus (Pang and Rose, 1987; Otomakhov, 1988).

1.9 Effects of Noradrenergic agonists on hippocampal regions

Long lasting changes in synaptic transmission have been produced experimentally with NAd in the hippocampus, where the actions of NAd have been most extensively studied, which could mediate NAd modulation of selective synapses. The following sections examine the various actions of NAd and noradrenergic agonists and antagonists on various hippocampal structures.

Dentate gyrus Lynch and Bliss (1986) reported that in the dentate gyrus, B-adrenergic activity increased presynaptic glutamate release and enhanced the field EPSP (Lynch and Bliss, 1986). In the hippocampal dentate gyrus, cell firing to perforant path input was enhanced, as measured by an increased population spike, by electrical stimulation of the

42 locus coeruleus (Bliss and Wendlandt, 1977; Assaf et al., 1979; Harley et al., 1982; Dahl and Winson, 1985; Harley et a/., 1989; Sato et a/., 1989), or by glutamate activation of locus coeruleus (Harley and Mil way, 1986), or by NAd iontophoresed directly in the cell body layer (Neuman and Harley, 1983), as well as by bath applied NAd to hippocampal slices (Lacaille and Harley, 1985; Stanton and Sarvey, 1985b and 1986). When changes did occur in the dentate field EPSP, they were of insufficient magnitude to account for the increase in the population spike according to Lacaille and Harley (1985). However, in the CAl region synaptic responses were unaffected (Sarvey, 1988). In the dentate gyrus, bath-applied NAd acting via Ô- adrenergic receptors, produced furthermore, a persistent increase in population EPSPs and in population spikes elicited by medial perforant path stimulation (Lacaille and Harley, 1985; Stanton and Sarvey, 1985c; Stanton and Sarvey, 1987). The duration of exposure to NAd was an important

parameter in the production of long-lasting changes, as a 1 0 min bath application produced long-lasting effects in only 25% of the slices tested (Lacaille and Harley, 1985), while 30 min applications (Stanton and Sarvey, 1985b) induced long-lasting enhancement of the population spike which occurred in all the experiments reported and persisted for up to 5 hours after NAd wash-out. This phenomenon was referred to as norepinepherine-induced long-lasting potentiation (NE-LLP) (Norepinepherine is the American name for NAd). However, a concomitant long-lasting decrease was observed in field potentials elicited by stimulation of the lateral perforant path (Dahl and Sarvey, 1989). It has been suggested in a number of studies that the persistent effects of NAd or ISO in the dentate gyrus depend on the activation of NMDA receptors, because they can be blocked by superfusion with the NMDA antagonist AP5 (Burgard et al., 1989; Stanton et a/., 1989). In addition, B-adrenergic agonists produced an increase in calcium currents in granule cells of the dentate gyrus (Gray and Johnston, 1987).

Area CA3 Hopkins and Johnston (1984) examined NAd-induced long-lasting enhancement in the mossy fibre input to CA3. They first perfused a hippocampal slice with a low concentration of NAd while activating mossy fibre input to the pyramidal cells at a low rate. They selected stimulus levels which produced only field EPSPs. No enhancements were observed. They also stimulated the mossy fibres at a high rate for a

43 brief period without NAd superfusion. They intentionally selected high rate conditioning stimulus parameters which did not produce LTP of responses to subsequent low rate test stimulation. Finally they combined NAd superfusion with the brief high frequency stimulation. NAd superfusion paired with the high frequency input did result in LTP of test EPSPs to the low rate mossy fibre stimulus. Using a stronger mossy fibre tetanus, which could produce long-lasting effects on its own, Hopkins and Johnston also showed that NAd significantly prolonged the normal high-frequency induced LTP. They suggested that NAd may be released with the high-rate stimuli used to produce long-term changes in response in the hippocampus since NAd fibres are present in the same areas as the input fibres. Furthermore, in the mossy fibre input to the CA3 region, NAd and B-adrenergic agonists produced a long-lasting, frequency-dependent increase in population EPSPs, spikes, and excitatory postsynaptic currents (EPSCs), which could be mimicked by cAMP analogues (Hopkins and Johnston, 1988).

Area CAl Until recently, evidence has suggested that NAd does not play a role in long-term plasticity in the CAl region. Lesions of noradrenergic inputs to CAl, depletions of NAd, and noradrenergic agonists and antagonists do not appear to antagonize LTP in the CAl region (Dunwiddie et al. 1982; Stanton and Sarvey, 1985b). NAd modulation of selective synapses was observed by Segal (1982), who reported a greater postsynaptic depolarization to iontophoresed glutamate in the presence of NAd, but not to serotonin (5-HT). Previous studies established that the short term postsynaptic effects of NAd in CAl pyramidal cells mediated by B-adrenergic receptors, occur as a result of increased intracellular cAMP, which depolarized pyramidal neurones and reduced the Ca^"^- activated potassium conductance (Madison and Nicoll, 1982, 1986a,b; Haas and Konnerth, 1983). Hegginbotham and Dunwiddie (1991) studied both acute long-term effects of B-adrenergic activation in CAl, and observed a persistent increase in extracellularly recorded population spikes which were not accompanied by changes in the preceding field EPSP. Heginbotham and Dunwiddie (1991) and Dunwiddie et al (1992) demonstrated that NAd and ISO could induce long-term increases in the excitability of pyramidal neurones in the CAl region where they produced a persistent enhancement of population EPSP to spike (E-S) coupling, i.e. in the population spikes

44 evoked by a given stimulus, but not in the preceding field iEPSPs. These authors termed this phenomenon 6 -adrenergic potentiation (BAP) and suggested that enhanced evoked activity, both excitatory and inhibitory, could occur if the increased membrane resistance induced by NAd or ISO improved the coupling of, or the spread of, excitatory and inhibitory potentials between dendritic sites and the axon hillock. Unlike in the hippocampal dentate gyrus, Heginbotham and Dunwiddie (1991) found that the enhancing action of ISO could not be blocked by AP5, although AP5 did block the induction of LTP following afferent tetanic stimulation. Furthermore, unlike LTP, BAP was found not to be Ca^^-dependent. However, the effect of ISO was blocked by the

6 i-adrenergic receptor antagonist timolol (Heginbotham and Dunwiddie 1991). The persistent increase in population spike induced by ISO could be mimicked by superfusion with several cAMP analogues and by forskolin (which directly activates adenylate cyclase) but not by the inactive form of forskolin, dideoxyforskolin. Forskolin and cAMP analogues also induced decreases in AHPs, but did not depolarize pyramidal neurones as consistently as ISO (Hegginbotham and Dunwiddie, 1991; Dunwiddie et aL,1992). These authors hypothesized that if NAd produced a persistent depolarization of the pyramidal neurones, then it would facilitate the induction of LTP in this region by helping to remove the voltage-dependent block of the NMDA receptor- operated channel that is critical to the induction of LTP in CAl. Thus, though the changes induced by NAd, which they observed, may only have been relatively persistent (ie., hours, rather than days or weeks), they proposed that NAd may have produced a transitory state in which more enduring changes in function could be facilitated by intermediate-term changes induced by 6 -adrenoreceptor activation. That is, NAd and ISO could increase the likelihood of triggering LTP in the hippocampal CAl region. However, they did not report whether in presence of NAd of ISO the LTP occurred with an increased probability, or whether the amplitude of the LTP induced in the presence of drugs was greater than in controls, like the mossy fibre experiments in CA3 of Hopkins and Jons ton (1984).

While both the a- and 6 -adrenergic subtypes of receptors are prevalent in the hippocampus, the excitatory actions of NAd in CAl appear to be mediated by the 6 - adrenergic system, and involve the adenylate cyclase second messenger system (Haas and Konnerth, 1983; Segal, 1982). Intracellular studies in CAl (Haas and Konnerth, 1983; Madison and Nicoll,

45 1982, 1986a,b; Segal, 1982; Dunnwiddie et al., 1992) demonstrated that postsynaptic 6 - adrenergic receptors mediate a reduction of potassium currents underlying slow afterhyperpolarizations (AHPs). The slow AH? is Ca^'^-dependent, but NAd does not block the entry of Ca^"^ postsynaptically. Madison and Nicoll (1986a,b) proposed that NAd may enhance the intracellular sequestration of Ca^"^ or specifically block the channel. Reduction of the slow AH? increases the likelihood of multiple action potentials to a suprathreshold depolarizing stimulus. The observation of longer bursts of action potentials to depolarizing input and of a higher frequency of firing within a burst following NAd application (Otmakhov and Bragin, 1982, Madison and Nicoll, 1982, 1986a,b) is readily explained by the reduced AHP. NAd has also been shown to increase the amplitude and frequency of spontaneously occurring IPSPs in area CAl (Madison and Nicoll, 1988) although it has no effect on GABA-mediated responses in this area. Andreason and Lambert (1991) have proposed that noradrenergic fii-receptors may play a role in modulating the presynaptic mechanisms underlying feed-forward and feed-back inhibition in CAl, whereby NAd may have excitatory actions on GABAergic interneurones.

1.10 Effects of noradrenergic agonists on sensory neocortex

It has been postulated that NAd plays a role in arousal in mammals. What is known about noradrenergic actions on the neocortex? Some studies have shown that the spontaneous activity of cortical neurones may be inhibited by 6 - adrenergic receptors and excited by a-adrenergic receptors (Bevan et at., 1977; Olpe et at., 1980). lontophoretic application of NAd changes neuronal sensitivity to sensory stimuli in some primary areas of cortex by increasing the response to the excitatory effects of acetylcholine and to the inhibitory effects of GABA (Sullivan et <2/., 1989; Waterhouse et a/., 1980). Moreover, NAd can have long-lasting effects on spontaneous and evoked spike activity in somatosensory cortex (Armstrong-James and Fox, 1983; Waterhouse et a/,1980; Sullivan et al., 1989). Both a- and B-adrenergic receptor subtypes are found in the rat cortex and both systems are involved in modulating neocortical activity (Mori-Okamoto era/., 1991; Zilles et al., 1990). The prolonged actions of NAd provide support for its postulated role in the influence of an animal’s state of arousal on the neuronal activity underlying learning and memory (Harley, 1988; Sullivan et a/., 1989). Indeed, a persistent facilitatory effect of NAd on inputs to cortical

46 and limbic areas could form a link between an aroused state of an animal and the

formation of memory traces for important events. Both a- and 8 -subtypes are involved in the modulation of neocortical spike and field potential activity (Bindman et al., 1990; McCormick, 1989; Nowicky et al. 1990; Mouradian et al.,\99\\ Sato gf a/., 1989; Waterhouse, 1982). NAd increases the response of neurones to excitatory or inhibitory input (Waterhouse et al., 1981, 1982) and the concentration of cyclic-3’,5’- monophosphate (cAMP) in the brain by activation of adrenergic receptors, lontophoretic application of NAd changes neuronal sensitivity to sensory stimuli in some primary areas of cortex by increasing the response to the excitatory effects of acetylcholine and to the inhibitory effects of GABA (Sullivan et a/., 1989; Waterhouse et a/., 1980; Waterhouse et al., 1982) and potentiates glutamate excitation in cerebellar Purkinje cells (Marshall and Tsai, 1988). Armstrong-James and Fox (1983) studied the effects of iontophoresed NAd on the spontaneous activity of somatosensory layer I cells in the anaesthetized rat, whilst continuously monitoring the extracellular NAd levels electrochemically at the carbon fibre recording tip of their multi-barrelled microelectrode. In the absence of NAd there were clear differences in spike amplitude, firing rate and firing pattern of deep (800- 1400 ^m) and superficial (0-800 ^m) cells. They reported that low levels of iontophoretically ejected NAd enhanced neocortical unit firing, lasting for hours in some cells. However, they emphasized that induction of enhanced responsiveness by NAd in their study occurred only in the deeper layers of somatosensory cortex and required very low concentrations (<0*01 fiM in the electrode tip). NAd at higher concentrations (O'05 to 0 '5 (jlM ) or iontophoresed in more superficial layers was invariably inhibitory. Recovery from inhibition was quickly reversible, occurring within about 1 minute of extracellular NAd concentrations falling below detectable (0*01 /xM) levels. The changes they observed in the deeper layers altered the firing pattern in the anaesthetized rat to mimic that of the awake, aroused rat. Since locus coeruleus cells increase their firing rate at the sleep/wake transition (Aston-Jones and Bloom, 1981), these results suggest that locus coeruleus activity may mediate the cortical waking activity pattern. This pattern could continue for hours following a 5 minute period of NAd iontophoresis. Although they were observing spontaneous activity in the anaesthetized animals, they suggest that the increased firing rate in these cells represents increased | level of I synaptic input.

47 Intracellular studies in cat cortical slice preparations (Foehring et al., 1989; Schwindt et at., 1992) have demonstrated that postsynaptic Bj-adrenergic receptors mediate a reduction of a slow calcium-activated potassium conductance (gKc^+), underlying the corresponding slow AHPs and thereby reducing spike frequency adaption, or accommodation. The slow AHP is caused by the decay of two separate

currents: a slower, Ca^"^-mediated current (decay time constant, ~ 1 sec), which is insensitive to the Ca^'^-dependent channel blocker, apamin, and a Na^-dependent current (decay time constant, —5 sec) (Schwindt et at., 1989; Schwindt ef a/.,1988a,b). The slow Ca^^-mediated current contributes to the first few seconds of the slow AHP, and the Na"^-mediated K'*’ current is solely responsible for the remainder of the slow AHP. In layer V (Betz) cells of the cat somatosensory cortex, Foehring et al (1989) examined the effect of NAd and noradrenergic agonists and antagonists on membrane currents, on the repetitive firing behaviour, and the ionic basis of this behaviour. However, they did not investigate the effect of NAd during evoked synaptic activity in their experiments. They reported that NAd application reversibly reduced rheobase and both the Ca^"^- and Na'^-dependent portions of the slow AHPs that followed sustained firing evoked by constant current injection. NAd did not alter Ca^'*’ spikes in the presence of tetrodotoxin (TTX) and tetraethylammonium (TEA) and inward Ca^^ currents were slightly increased. This is similar to observations in the hippocampal dentate gyrus where B-adrenergic agonists produced an increase in calcium currents (Gray and Johnston, 1987). Foehring et al., 1989 concluded that reduction of the slow Ca^"^-mediated current was not caused by reduction of Ca^'*’ influx. NAd also produced an increase in the persistent Na"^-current (Foehring et al., 1989). The faster Ca^^-dependent medium AHP, the fast AHP, the action potential, and the input resistance were unaffected by NAd application.

1.11 Aims of the investigation

Although there is evidence that in the cortex, a-adrenergic agonists enhance the excitatory actions of glutamate application recorded extracellularly (Mouradian et al., 1991), no detailed intracellular analysis of immediate or long-term actions of NAd or ISO has been carried out in the layer V somatosensory cortical neurones (but see Ifor voltage-clamp analysis in cat layer V cells Foehring et al., 198^. Analysis reported in this thesis was carried out on the synaptic

48 response to white matter stimulation in layer V neurones of rat sensorimotor cortex for two reasons. The first is that preliminary studies in slices showed that NAd and ISO reversibly enhanced the amplitude and/or late components of some extracellular field potentials and intracellular EPSPs in this region (Bindman et al., 1990), but the complexity of neocortical circuitry makes it difficult to interpret the extracellular field potentials. The changes observed in the study of Bindman et al. (1990) could reflect changes in the resting membrane potential, changes in the threshold for activation of the sodium spike, or perhaps other changes such as the coupling between the dendritic site at which the EPSP is generated and the soma. The second reason is that our research group is interested in the mechanisms involved in the postsynaptic induction of associative LTP in the neocortex, in particular in layer V cells of the somatosensory cortex, using a similar pairing paradigm to that used in the neocortex and hippocampus (Baranyi and Szente, 1987; Bindman gr a/., 1988; Gustafsson gf a/., 1987; Kelso and Brown, 1986). Associative LTP can be induced in layer V cells in the rat somatosensory cortical slice by repetitive pairing of weak subthreshold EPSPs evoked by stimulation of the subcortical white matter with postsynaptic depolarizing current

injection, but with very low incidence, i.e. in only about 1 0 % of the inputs tested (Bindman et al., 1988, Bindman and Murphy, 1990). In contrast, post synaptic induction of associative LTP of EPSPs evoked by thalamocortical or pyramidal tract stimulation in layer V cells of motor cortex in vivo has a much higher incidence, of about 30% in the anaesthetized cat (Baranyi and Szente, 1987) and 55% in the awake cat (Baranyi et al., 1991). One explanation for the lower incidence of associative LTP observed in the slice could be that the cortical slices are isolated from extrinsic noradrenergic activity which could be involved in the induction of associative LTP in the awake animal. The actions of NAd and the fi-adrenergic agonist, ISO were investigated on both synaptic transmission and on the induction of associative LTP in layer V pyramidal cells in rat sensorimotor cortex. In the present study, a postsynaptic associative protocol for the induction of LTP was used instead of tetanic stimulus trains for two reasons. Firstly, this was the experimental paradigm producing LTP in 50 to 60% of conditioned EPSPs in the awake cat (Baranyi et al., 1991). Secondly, postsynaptic injection of EGTA blocks the postsynaptic induction of LTP (Baranyi and Szente, 1987), suggesting that LTP recorded in the injected cell is a localized effect of postsynaptic depolarization on the potentiated synapses. The spike firing produced by

49 the conditioning pulses may well induce LTP in synapses on other cells, but not in synapses of recurrent circuits. Postsynaptic conditioning allows a more selective analysis of neocortical phenomena than tetanic stimulation of afferents.

SECTION B: LONG-TERM DEPRESSION (LTD) IN THE HIPPOCAMPUS AND NEOCORTEX

1.12 Introduction

The concept that changes in synaptic efficacy are the way in which the brain stores information has been the | impetus for a great deal of work on LTP. However, if synaptic efficacy were only to increase as a result of LTP, saturation of learning would occur, limiting the capacity to form new associations when the relationship between events changes (Wilshaw and Dayan, 1990). In terms of modifiable synaptic efficacy, the time-honoured principle of "what goes up must go down" would not be obeyed (Sejnowski, 1977; Wilshaw and Dayan, 1990). Hebb (1949) did not discuss the consequence of uncorrelated or negatively correlated pre- and postsynaptic activity. Are we to assume that uncorrelated activity reduces synaptic efficacy? Or that it has no effect on efficacy? The idea that positively correlated activity causes synaptic strengthening and that either uncorrelated or negatively correlated activity causes synaptic weakening was in fact an essential part of Stent’s (1973) extension of the Hebbian idea to the problem of understanding experience-dependent development of striate cortex (cf. Bear, 1987; Singer, 1988; Frégnac, 1991; Frégnac and Shultz, 1991). Sejnowski (1977a,b) proposed a "covariance" (modification) rule in which changes in synaptic strength are proportional to the covariance between pre- and postsynaptic spiking.

1.13 Neural network models predict enduring decreases in synaptic transmission

Neural network models that store information at synapses rely on mechanisms for increasing and decreasing synaptic strength, such that modification of the simple Hebb rule to include an enduring depression of synaptic strength can improve the efficiency and performance of associative recall (Sejnowski, 1977; Wilshaw and Dayan, 1990).

50 Since forgetting is such a conspicuous counterpart to learning, one might expect to find the cellular analogue of forgetting as the obverse of LTP: neuronal forgetfulness might be an enduring decrease in synaptic strength that follows the heavy use of a specific synapse under certain conditions. Alternatively, such a decrease in synaptic strength might represent enhancement of the inhibition of memory mechanisms and could prevent information storage. LTP in the hippocampus does not last indefinitely (Barnes, 1979; Racine et al., 1983) and behaviourally, habituation and extinction could be correlates of decreased responsiveness of synapses (Morris and Wilshaw, 1989).

1.14 Long-term depression

Definitions Long-term depression (LTD) of synaptic transmission is defined as an enduring !but not in the decrease in synaptic transmission. LTD in the neocortex hippocampus can be produced by seemingly identical afferent tetani to those inducing LTP; LTD can be homosynaptic, affecting those synapses in the stimulated pathway. Homosynaptic LTD can occur when the input pathway produces postsynaptic depolarization which is above a certain threshold but inadequate to give rise to LTP (Artola et al., 1990). However, when the strength of transmission at some synapses is increased by the conjunction of postsynaptic depolarization with the synaptic activity, there is often concomitant depression at other inactive synapses - heterosymptic LTD; this affects synapses on the postsynaptic cells that are inactive at the time of the afferent stimulation. Heterosynaptic LTD can occur when postsynaptic depolarization and firing are not associated with evoked synaptic activity in a test pathway (Lynch et a l, 1977; Dunwiddie and Lynch, 1978). The former paradigm is associative, and the latter is non-associative; both paradigms have also been termed anti-Hebbian (Lisman, 1989) but it should be noted that the term has not been used consistently in the literature (see Brown et al., 1990). Figure 1.1 summarizes the various forms of LTD that can occur. Both hetero- and homosynaptic LTD have been studied in the hippocampus (Lynch et al., 1977; Dunwiddie and Lynch, 1978; Levy and Steward, 1979; Stanton and Sejnowski, 1989), cerebellum (for review see Ito, 1989; Kano and Kato, 1987; Crepel and Jaillard, 1991), and cerebral cortex (Bindman et a l, 1964; Bliss et al., 1968; Bindman et al, 1988; Artola et a l, 1990; Hirsch and Crepel, 1990). However, unlike

51 LTP, LTD has been an elusive phenomenon, and little is yet known of the underlying mechanisms. Indeed, Bear (1991) suggested that "progress in this area has been hampered by the fact that extant models of synaptic weakening in cortex have not as yet proven to be very reliable in the hands of others... " The physiological rules for hetero- and homosynaptic LTD are not entirely clear at present, but like the induction of LTP, the induction of LTD is postsynaptic (see Kano and Kato, 1987, Stanton and Sejnowski, 1989, and Pockett et al., 1990 for different experimental conditions under which LTD can be produced in the cerebellum and hippocampus, and Bindman et al., 1988b, Berry et al., 1989, and particularly Artola et a l, 1990, for neocortex).

Associative or homosynaptic LTD in the hippocampus Levy and Steward (1979) reported that successive associative potentiation (LTP) and "depotentiation" (LTD) could be induced in vivo in the hippocampal dentate gyrus (DG) of anaesthetized rats using paired and non-paired stimulation procedures. High frequency conditioning stimuli to one of the entorhinal cortical (EC) pathways to the dentate gyrus elicited LTP at the ipsilateral synapse, but not at synapses of the collateral, crossed pathway to the contralateral DG. Associative LTP of the crossed EC- DG projection could be induced following paired conditioning of ipsi- and contralataral inputs by conjoint stimulation of the EC bilaterally. Furthermore, LTP could be reversed by subsequent conditioning of the ipsilateral system alone. Successive potentiation and "depotentiation" was possible using paired and non-paired stimulation paradigms, even after lesions which prevented neural loops through the EC. These authors interpreted their results as evidence for a ‘Hebb’ type synapse which has the ability for "erasure". Conditions in which stimulation of a synaptic pathway rather than its inactivity might lead to synapse-specific changes in strength are crucial for producing homosynaptic LTD. Stanton and Sejnowski (1989) explored conditions under which different patterns of stimulation of a hippocampal pathway, rather than its inactivity, could produce either LTP or LTD of synaptic strength. Such conditions are thought to be critical because presynaptic stimulation is an ideal way of achieving a synapse specific change, and could mimic natural conditions under which LTD might occur. These authors demonstrated that an AP5-resistant, associative homosynaptic LTD could

52 be induced in vitro in the hippocampal CAl region using an experimental paradigm that paired a low-frequency (5 Hz) test input to the subiculum, negatively correlated in time with a high-frequency (100 Hz) conditioning input to the Schaffer col lateral / commissural fibres. Homosynaptic LTP could be induced by stimulating the conditioning side alone; when test and conditioning inputs were activated in phase, associative LTP of the extracellular EPSP and population spike in the test input synapse was induced. However, when test and conditioning stimuli were applied out of phase, an associative LTD of both population spike and field EPSP was produced (see also Figure LIE). The depression in the field EPSP slope was smaller than in the population spike, raising the possibility that the LTD observed by these authors was restricted to the soma and the spike-generating mechanism rather than a true localized decrease in synaptic weight. A low frequency train of test stimuli produced no long-lasting changes in synaptic transmission. It is unclear, however, whether Stanton and Sejnowski (1989) fully explored the effects of omitting the single test pulses in the out-of-phase conditioning paradigm. They reported that activation of the conditioning site alone produced homosynaptic LTP of the conditioning pathway, and stated that this " did not significantly alter the amplitude of responses to the test input", although they presented no relevant data. Omitting the single test pulses in the out-of-phase conditioning paradigm would restrict the inducing stimulation to the conditioning pathway and might fulfil the conditions for producing heterosynaptic LTD at inactivated synapses. Stanton and Sejnowski (1989) suggested that postsynaptic hyperpolarization coupled to presynaptic activation may trigger LTD. They inferred that a test stimulus that is out of phase with a conditioning stimulus arrives when the postsynaptic neurone is hyperpolarized, as a consequence of IPSPs and the afterhyperpolarization from mechanisms intrinsic to pyramidal neurone membranes. They tested this hypothesis in another set of intracellular experiments by injecting current intracellularly through their recording electrode in order to hyperpolarize (or depolarize) the postsynaptic cell while stimulating a synaptic input. Pairing the injection of depolarizing current with the test input led to associative LTP of the test synapse, confirming previous results (Kelso et a l, 1986; Gustafsson et a l, 1987), and not in the subicular pathway. Conversely, prolonged hyperpolarization of the cell soma paired with the same test stimuli induced ! inactive LTD in the stimulated pathway, but not in thej^control subicular pathway. The application of either depolarizing current, hyperpolarizing current, or the test 5 Hz

53 synaptic stimulation alone did not induce LTD in synaptic strength. Thus pairing of afferent input stimulation with depolarization or hyperpolarization of the postsynaptic cell could lead to LTP and LTD respectively. Although hyperpolarization did not lead to LTD per se, it may have prevented the occurrence of LTP thus permitting the expression of the underlying LTD. Stanton and Sejnowski (1989) concluded that the form of synaptic plasticity they observed confirmed the predictions of a covariance rule k (Sejnowsi, 1977a,b; Bienenstock et al., 1982; Frégnac, 1991; Frégnac and Shultz, 1991). Interestingly, Malinow and Miller (1986) previously found that postsynaptic hyperpolarization blocked LTP induction, but did not induce LTD. However, these authors used higher frequency test stimulation in conjunction with hyperpolarization. This may have raised the intracellular dendritic voltage to a level which was above that sufficient for LTD induction, and below the level for inducing LTP. Alternatively, the intracellular dendritic voltage may have been increased to a level to where both LTP and LTD occur, but the two phenomena may have cancelled each other out. However this theory has yet to be confirmed experimentally. Indeed the theory that LTD depends on a discorrelation between presynaptic and postsynaptic activity (Stanton and Sejnowski, 1989), or a partial depolarization within a narrow depolarization range (Artola etal., 1990), or both has been criticized as being too demanding energetically on the cell (Routtenberg, 1991). Using the same conditioning paradigms as Stanton and Sejnowski (1989), Chattarji et al. (1989) reported that both associative LTP and LTD could be induced in the commissural/associational (C/A) inputs, but not in the mossy fibre inputs to CA3 pyramidal neurones. They suggested that associative LTP and LTD may permit storage at C/A synapses of covariance information between C/A and mossy fibre inputs, and proposed that the differences between synapses in CA3 pyramidal cell neurones are likely to reflect their different roles in processing the information flow from the dentate granule cells to CAl pyramidal neurones. Chattarji et al. (1989) proposed that the mossy fibres could to be providing a ‘teaching’ signal that ‘instructs’ the C/A inputs through associative interactions, since they can influence, but cannot be influenced, by signals arriving from other pathways.

Heterosynaptic LTD in the hippocampus Heterosynaptic LTD in the hippocampal area CAl was first described by Lynch et

54 al. (1977) who found a depression of field EPSPs evoked by test stratum radiatum shocks, following tetanic stimulation of commissural afferents terminating on the basal dendrites. But in the same issue of Nature, Andersen et al. (1977), using the same experimental procedure, did not observe LTD. Dunwiddie and Lynch (1978) found that trains of antidromic stimuli applied to the CAl cell population (exciting axons but also interneurones) resulted in depression lasting at least 15 minutes in test field EPSPs in some, but not all slices. These authors had earlier reported that LTP was accompanied by a persistent decrease in cell firing in response to glutamate applied by iontophoresis to the dendrites of CAl pyramidal cells (Lynch et al., 1976). They proposed that repetitive stimulation and postsynaptic firing gave rise to a persistent decrease in synaptic transmission generalized to the whole neurone, while LTP was confined to the terminals activated during the conditioning train. In more recent studies, Bradler and Barrionuevo (1989) showed that tetanization of hippocampal mossy fibres induced LTP of non-tetanized (heterosynaptic), non-mossy fibre afferents (Schaffer collateral/commissural and fimbrial fibres). In contrast, tetanization of non-mossy fibre afferents, did not induce LTP but induced LTD of mossy fibre responses. Pharmacological manipulations have helped experimenters to produce LTD: Pockett and Lippold (1986) were successful in inducing LTD (up to 2h) in field potentials when they bathed hippocampal slices in acsf containing a high [Mg^^] during antidromic conditioning, thereby eliminating synaptic transmission via recurrent pathways and via orthodromic pathways excited by stimulus spread. Pockett et al. (1990), using intracellular recording, found that high frequency antidromic stimulation of the alveus gave rise to LTD (>30 min) in all experiments in CAl when synaptic transmission was blocked in a high [Mg^^] solution during conditioning. When conditioning was applied to slices bathed in acsf, conditioning produced LTD in two thirds of experiments and non-associative LTP in 20%. These authors also noted that PSP depression could not be explained by a fall in input resistance. Abraham and Wickens (1991) found that the GABA^ antagonist, picrotoxin facilitated the induction of heterosynaptic LTD of the population spike (by 21%) in CAl, but they found a

depression of only a few percent in population EPSPs (4% or 8 %) following conditioning applied to the alveus, or oriens, respectively. Indeed, the depression observed by these authors may represent a reduction in E-S coupling, rather than

55 synaptic strength. The ability to induce LTD and LTP in separate, but converging neighbouring, synaptic populations provides for a range of complex adaptive responses that a neurone may exhibit to significant patterns of activity. Further complexity is introduced when both associative and non-associative mechanisms can be engaged, as has been shown for LTP in CA3 neurones (Bradler and Barrionuevo, 1990) and when there exists regional variability in the expression of associative and non-associative LTD. Both forms of LTD have been described for areas CAl and CA3 (Pockett et al. , 1990; Abraham and Wickens, 1991; Bradler and Barrionuevo, 1990; Chattarji et al., 1989; Stanton and Sejnowski, 1989), while only associative LTD has been observed in the cerebellum (Ito, 1989). Recently, Christie and Abraham (1992a) reported that the associative and non- associative protocols of Stanton and Sejnowski (1989) produced NMDA-dependent heterosynaptic LTD in the dentate gyrus (LPP) of anaesthetized rats, following conditioning of the medial perforant path (MPP) (see also Christie and Abraham, 1992b; and below). These authors found that the ipsilateral perforant path synapses in the dentate gyrus sustained only non-associative LTD. Christie and Abraham (1992a) suggested that such regional variation in the ability to generate different forms of synaptic plasticity could be due to these brain areas differing in their information storage and processing capabilities, perhaps reflecting differential contributions to learning and memory.

The role of NMDA receptors in homo- and heterosynaptic LTD in the hippocampus

The increased intracellular Ca^'^ level that results from its influx through the NMDA receptor channel is the first step in LTP induction (Lynch et al., 1983, Malenka et a/. ,1988). Ca^'^ levels then, can be thought to represent the degree of correlated synaptic activity, and this information is used by the LTP induction mechanisms. According to the results of Stanton and Sejnowski (1989), LTD results when a synapse is active, when its use is uncorrelated with activity in other synapses. Goldman et al. (1990) reasoned that the signal for lack of correlation might well be the lower Ca^’*' levels in dendritic spines. According to this view, synaptic activity with increased spine Ca^^ levels would trigger increased synaptic strength (LTP), whereas the same activity with lower spine Ca^^ levels would produce synaptic weakening (LTD). Goldman et al.

56 (1990) suggested that simply blocking influx through the NMDA channel with AP5 would provide a test for this hypothesis. During NMDA channel block, any synaptic activity in the presence of AP5 would appear to the neurone as uncorrelated synaptic use. Although AP5 blocked LTP induction, but not induction of LTD in the experiments of Stanton and Sejnowski (1989), Goldman et al. (1990) suggested that continued stimulation in the presence of the antagonist should, according to the above hypothesis, produce LTD and reverse pre-existing LTP. Using multiple tetani (prolonged tetanization from 50-100 Hz) and several stimulation patterns in various concentrations of AP5, Goldman et al. (1990) failed to reverse LTP to LTD in region CAl of rat hippocampal slices. These authors concluded that presynaptic activity uncoupled from postsynaptic NMDA receptor activation does not reduce synaptic strength, and suggested that some unanticipated mechanism underlies LTD in the hippocampus. Stevens (1990), writing in the "News and views" column of Nature, commented that "three respected groups [including his own] have been unable to find LTD using what they believe to be the conditions reported by Stanton and Sejnowski (1989)" \ Indeed, the reliability of the associative LTD effect of Stanton and Sejnowski (1989) still remains controversial (see also Christie and Abrahams, 1992b).

Prolonged low-frequency activation of NMDA receptors can induce LTD Dudek and Bear (1992) suggested that synaptic depression can be triggered by prolonged NMDA receptor activation that is below the frequency threshold for inducing LTP. It has been demonstrated in the hippocampus that homosynaptic LTP can be reversed (or "depotentiated") by prolonged 1 Hz stimulation applied within several hours of LTP induction. Such a pattern of stimulation could substantially reduce the potentiated response (Barrionuevo et al., 1980; Bramham and Srebro, 1987; Staubli and Lynch, 1990; Fujii et a l, 1991) and may be behaviourally equivalent to the extinction of salivatory responses seen in the Pavlovian conditioning experiments: dogs conditioned to salivate (when hungry) in response to musical sounds alone (previously repeatedly presented in temporal association with the arrival of food) eventually no longer salivated following the repeated presentation of the musical sounds in the absence of food (Pavlov, 1928). Dudek and Bear (1992) demonstrated that homosynaptic LTD could also be induced in a "naive", previously unpotentiated hippocampus. These workers were able to induce AP5-sensitive homosynaptic LTD of population EPSP test responses

57 to stimulation of the Schaffer collateral projection to area CAl in rat hippocampus, by applying low-frequency stimuli at 1-3 Hz. They ruled out generalized changes in postsynaptic responsiveness of excitability, damage, or fatigue of the stimulated inputs as a reason for the depression. Since both the reversal of LTP (depotentiation) described by Fujii et al. (1991), as well as the depression observed by Dudek and Bear (1992) were blocked by AP5, the latter authors suggested a common mechanism for both phenomena which differed only in degree. They suggested that the effectiveness of the low-ffequency stimulation in producing LTD may be dependent on the recent history of activation, or strength, of the synapse. Dudek and Bear (1992) also found that the magnitude of the LTD was greatly enhanced after the induction of LTP. On the basis of a model developed for visual cortex (Cooper et al. , 1979; Bienenstock et al. , 1982; Clothiaux et al., 1991), they inferred that an adjustable ("sliding") threshold for postsynaptic responsiveness may have a plausible physiological basis in the hippocampus. Interestingly, other authors (Malenka, 1991; Colino e ra/.,1992; Huang et al. , 1992) have suggested that the threshold for LTP induction may be continually adjusted according to the recent history of NMDA receptor activation and this may provide a physiological mechanism by which LTP can be transiently inhibited.

NMDA-receptor-independent LTD Bradler and Barrionuevo (1990) demonstrated that heterosynaptic modifications of efficacy in the rat did not require the activation of NMDA receptors. Mossy fibres supported NMDA receptor-independent heterosynaptic LTD, and NMDA receptor- independent LTP. In contrast, non-mossy fibre afferents expressed NMDA receptor- independent heterosynaptic LTP induced by mossy fibre tetanus, and an NMDA receptor-independent LTD, in addition to NMDA receptor-dependent homosynaptic LTP. Since heterosynaptic LTD was induced in the absence of homosynaptic LTP, these authors suggested that these concomitant forms of synaptic plasticity rely on different mechanisms. Interestingly, Christie and Abraham (1992b) reported that associative LTD in the lateral perforant path inputs to the dentate gyrus in anaesthetized rats could be readily

induced after being "primed" by a brief period of lateral path activity at a 0 rhythm frequency (5 Hz). Furthermore, although NMDA-receptor activation was necessary for establishing the priming effect, the subsequent induction of the associative LTD was not.

58 What component of the EPSP is depressed? A currently unresolved debate in hippocampal LTP expression concerns the conflicting evidence of whether the enhancement of synaptic transmission is accompanied by an increase in the NMDA component of the EPSP as well as the AMPA-sensitive, non-NMDA component (Malinow & Tsien, 1991b). This issue was recently examined in hippocampal LTD by Xie et al. (1992), who reported that in dentate granule cells maintained in vitro, LTP and LTD of the NMDA component of synaptic responses can be induced. In the presence of CNQX to block AMPA receptors, LTP of synaptic responses was induced by brief, high (50 Hz) and lower (10 Hz) frequency tetanic stimuli of afferents. In contrast, hyperpolarization of the granule cell membrane potential to -100 mV during 10 Hz tetanic stimulation resulted in LTD of NMDA receptor-mediated synaptic potentials. Intracellular injection of BABTA into the postsynaptic cell before tetanization blocked the induction of LTP and partially blocked LTD. The results of Xie et al. (1992) provide evidence for the induction of both LTP and LTD of NMDA receptor-mediated synaptic transmission and demonstrate that the level of postsynaptic depolarization can determine which of the two forms of synaptic plasticity is expressed in response to an identical input. Furthermore, these authors’ results suggest that postsynaptic Ca^"^ influx is essential not only for the induction of LTP but also for induction of LTD of NMDA receptor/channel function.

Factors necessary for the induction of LTD in vitro Pharmacological manipulations that have facilitated the induction of LTD have been described on page 55.

59 The ACPD (metabotropic) receptor in LTD The ACPD (fr

1.15 Activity-dependent LTD in the neocortex

Homosynaptic, activity-dependent LTD has been seen in neocortex, in neuronally- isolated slabs in vivo (Bliss et a l , 1968), in slices of sensorimotor cortex in vitro (Bindman et a l, 1988b), in prefrontal cortex (Hirsch & Crepel, 1990), and in visual cortex (Berry et al., 1989; Artola et a l , 1990). No difference was detected in the stimulus parameters inducing LTP from those eliciting LTD.

NMDA receptors and the postsynaptic induction of neocortical LTD Artola et a l (1990) analyzed the postsynaptic action of impulses resulting from afferent tetani and proposed that LTP was elicited when the postsynaptic depolarization was strong enough to activate NMDA channels, but LTD was evoked when the postsynaptic depolarization was not as great, and did not reach the threshold for opening

60 the NMDA receptor-activated channels. They tested their hypothesis in two ways: (1) they modified the postsynaptic depolarizing response by reducing inhibition pharmacologically (using bicuculline) or by applying postsynaptic depolarizing current, or by altering the tetanic stimulus intensity; (2) they blocked NMDA receptor-mediated activity with AP5. i Tetanic stimulation in adult visual cortex in normal medium produced neither LTP nor LTD, while in 0.1 - 0.2 /iM bicuculline, tetanic stimulation gave rise to pathway-specific LTD and in 0.3 fiM bicuculline, post-tetanic LTP resulted. In contrast, tetanic stimulation produced LTD in cells treated with 0.3 ^M bicuculline and AP5. After the AP5 was washed out, a second tetanic stimulus, > 1 hour after the first, induced LTP (Artola et al. , 1990). Teyler et al. (1990) also reported that tetanic afferent stimulation in the presence of AP5 gave rise to LTD. The results of the postsynaptic current application during the tetanus showed that the induction of the homosynaptic LTD depended on the level of depolarization and was postsynaptic (Artola et al., 1990). They concluded that LTD had a different (lower) depolarization threshold from that required for induction of LTP. The results of the experiments with bicuculline and AP5 suggested that postsynaptic depolarization together with reduction of Ca^^ entry leads to LTD. Hirsch and Crepel (1990) reported that in slices of ratjprefrontalcortex, tetanization of the superficial layers induced either LTP or LTD in layer V pyramidal cells. These authors impaled layer V pyramidal neurones and evoked EPSPs by stimulation of superficial layers I-II. They reported that tetanic stimulation (at 50 of 100 Hz) of the superficial layers, in the presence of AP5 resulted in LTD of EPSPs suggesting that the rise in intracellular Ca^'^ is not a consequence of its entry through NMDA receptor-activated channels (but see Goldman et al., 1990 and page 57 of this thesis). The percentage of cells showing LTD when NMDA receptors were blocked was significantly higher than in the control medium. They suggested that in the absence of AP5, tetanic stimulation resulted in both LTP and LTD, with LTP masking the occurrence of LTD, depending on the activation of NMDA receptors. Hirsch and Crepel (1991) concluded that the mechanisms leading to LTP and LTD are co-localized in the same cell, and that their data supported the covariance theory (Sejnowski, 1977a,b; Bienenstock gf a/., 1982; Frégnac, 1991; Frégnac and Shultz, 1991).

61 The role of in LTD In contrast to the numerous investigations into the mechanisms underlying LTP, those underlying LTD have only recently been explored. A necessary condition for induction of LTP in the hippocampus and neocortex is an increase of intracellular [Ca^^] (Lynch et ah, 1983; Baranyi and Szente, 1987; Malenka et ah, 1988). Is this true for LTD also? Sastry et ah (1983) reported that tetanic (20 Hz) stimulation of the Schaffer collateral or commissural inputs in the rat hippocampal CAl region produced parallel homo- and heterosynaptic LTD of the population spike (of 20 min duration). Since both forms of LTD could be blocked by the Ca^"^ channel antagonist verapamil, these authors concluded that the depressions were due to postsynaptic Ca^"^ accumulation resulting from tetanization of the input. The role of Ca^"^ in the induction of homosynaptic LTD has recently been examined in the cerebral cortex, using afferent tetani as conditioning stimuli. Two apparently contradictory results have been obtained using intracellular injection of calcium chelators EGTA or BAPTA into cells of rat visual or frontal cortex. On the one hand, in layer II/III cells, Kimura et ah (1990) and Yoshimura et ah (1991) found LTD of EPSPs in cells injected with Ca^^-chelators, following tetanic stimulation of white matter that elicited LTP in the simultaneously recorded extracellular field response, reflecting the synaptic activity of cells with no Ca^"^ chelator. These results suggest that there are postsynaptic mechanisms through which tetanic inputs may produce LTD if these inputs are associated with no increase, or only a subtle increase, in the concentration of postsynaptic free Ca^^. On the other hand, Hirsch and Crepel (1992, in frontal cortex) and Brocher et ah (1992, visual cortex) found that intracellular BAPTA (or EGTA in the latter study) prevented induction of LTD. The latter results suggest that a rise in intracellular [Ca^^] is needed for induction of LTD, and whether it is LTP or LTD that is induced is dependent on the prevailing concentrations of postsynaptic Ca^"^ during the tetanic stimulation; the former results imply th^t although Ca^"^ is also required for LTD, the rise in intracellular [Ca^^] is less than that causing LTP. Note that the same experimental paradigms were used previously in hippocampus and neocortex to deduce that Ca^^ was needed for LTP, but no LTD was observed! Presumably the precise [Ca^^] is the critical factor in determining whether LTP, LTD or no change ensues.

62 The use of nimodipine, an antagonist of dihydropyridine-sensitive voltage- dependent channels (Wickens and Abrahams, 1991) was shown to prevent LTD induced by high-frequency antidromic tetanization of the alveus in slices of rat hippocampus in vitro, but this was LTD of the population spike in CAl and not of the field EPSP, so different mechanisms could be involved, (such as a decreased E-S coupling, rather than a decrease in synaptic strength; see page 26 for enhanced E-S coupling in LTP). LTD could also be produced in the absence of picrotoxin when the L-type Ca^^ agonist BAY- K8644 was applied to the slice surface. Block of the NMDA receptor did not prevent induction of homosynaptic LTD in visual or frontal cortex (Teyler et ah, 1990; Hirsch and Crepel, 1991; Artola et ah, 1990), so the rise in intracellular Ca^"^ is not a consequence of its entry via NMDA-receptor activated channels.

1.16 Aims of the present study

Conditions necessary for the postsynaptic induction of LTD in the CAl region of the rat hippocampus were explored. It was investigated whether LTD could be induced postsynaptically by bathing slices transiently in a high [Mg^^] solution to block transmitter release during antidromic conditioning. In the intracellular recordings of Pockett et ah (1990), the measure of synaptic response was peak amplitude, which could be an EPSP or a reversed IPSP. By measuring the initial depolarizing slope of EPSP, it was examined whether LTD occurs in the monosynaptic EPSP. The dependence of the postsynaptic induction of hippocampal LTD on extracellular Ca^"^ was examined by omitting it from the bathing medium. As a more discrete conditioning procedure than antidromic trains, repeated intracellular depolarizing pulses were applied to the cell. Direct measurements of the rise of cytosolic [Ca^^] in the soma during intracellular conditioning were also made. Instead of blocking transmitter release it was investigated whether the presence of selective glutamate ionotropic receptor antagonists during conditioning would be effective as a means of reliably producing LTD. CNQX was used as an antagonist of AMPA receptors; it is also a weak antagonist of the kainate receptor (Young et ah, 1991). In some.experiments D-AP5 was additionally used to block NMDA channels. I The first set of experiments involving antidromic or intracellular stimulation in |high [Mg^"""] suffered from the disadvantage that perfusion of slices with high [Mg^"^] was

63 sometimes found to be toxic. Therefore, to avoid the use of high slices were transiently bathed with CNQX with or without D-AP5 in order to (1) reduce the likelihood of inducing non-associative LTP, as observed by Pockett et al. (1990); (2) allow any glutamate released during conditioning to act on the metabotropic glutamate receptor (Bear, 1991; Stanton et al, 1991) implicated in the induction of LTD; and (3) to block recurrent inhibition because glutamate excitation of the inhibitory intemeurones in CAl is reduced or blocked by these antagonists (Andreason and Lambert, 1991). Finally, the dependence of the postsynaptic induction of LTD on blocking GABA^-mediated IPSPs was examined by applying intracellular conditioning in the presence of bicuculine methiodide. This drug was reported to facilitate the induction of LTD (Wickens and Abraham, 1991). Preliminary reports of this work have been published (Christofi et al., 1991a,b; Christofi et al., 1992; Christofi e ta l, 1993).

64 CHAPTER 2

MATERIALS AND METHODS

2.1 The postsynaptic induction of non-associative long-term depression (LTD) in rat hippocampal slices

Dissection and hippocampal slice preparation Adult male Sprague-Dawley rats (160-180g) were killed by cervical dislocation and the spinal cord was cut at Cl. The skull was exposed by cutting the skin along the midline with a scalpel blade, separating the pelt and then scraping away the underlying soft tissue. The point of a pair of dissecting scissors was very carefully inserted into the magnum foramen just beneath the skull at the opening of the spinal cord and the skull cut along the midline caudo-rostrally, just above the cerebellum. Rongeurs were used to carefully peel away the bony plates of the skull together with the dura matter, leaving the pia matter. Following exposure, cold (0 to 3°) oxygenated acsf was immediately poured over the brain in order to wash away any blood and chill the tissue. The brain was removed by lifting it from underneath with a small spatula. The brain was then immediately submerged in oxygenated cold acsf (0 to 3°). The procedure, including killing, removal of the brain and submersion in acsf took about 30-45 sec. To dissect out the hippocampus, the cold brain was laid dorsally on filter paper moistened with cold acsf and the cerebellum and brainstem were cut away. The brain was then bisected along the midline and one half was re-submerged in fresh cold acsf. The thalamic regions were pushed medially with the spatula so that the fimbria could be seen. The small spatula was inserted into the third ventricle under the fimbria and moved caudally and rostral ly to cut the commissural fibres at the hippocampal poles. The hippocampus was then flipped out laterally and the final connections with the entorhinal cortex were cut with the spatula so that it was completely freed from the rest of the brain. Further trimming of any remaining connective tissue was carried out before the hippocampus was rinsed quickly with cold acsf, and then laid dorsal side up, with the long axis at right angles to the blade, on filter paper moistened with the cold ascf, and placed on the chilled nylon stage of a tissue chopper (Sorvall TC-2). The chopper blade was

moistened with cold acsf and transverse slices (nominally 2 0 0 ^m) were cut from the

65 middle third of the hippocampus. Individual slices were carefully removed from the cutting blade with a fine sable hair brush (size number 4, TVB) and laid carefully on filter paper moistened with cold acsf in a chilled petri dish (resting on ice). Successive slices were prepared in the above way resulting in a total of about 12 slices from both hippocampi which were transferred to an incubation chamber (Alger et a l, 1984), consisting of a perspex box with a hinged lid. The bottom of the incubation chamber was covered with about 500 ml tap water (3cm deep) which covered a scintered gas bubbler through which 95% 0 2 /5 % CO2 was continually bubbled. The slice dish was placed on a central pedestal 6 cm above the water level. A perspex sheet covered the gas bubbler and acted as a protective baffle on which condensed water droplets formed, rather than on the underside of the chamber lid, thus preventing droplets from falling onto the slices. The slices remained in this humidified atmosphere for at least 1 hour at room temperature before being transferred to the recording chamber. The whole procedure including removal of the brain, both hippocampi and slicing usually took 8-9 min.

Artificial cerebrospinal fluid The standard artificial cerebrospinal fluid (acsf) solution used for all experiments (both hippocampal and neocortical) was freshly prepared for each experiment and had

the following composition (mM): NaCl 125; KCl 3*2; NaHCOs 19; NaH 2P0 4 1*2;

MgCl2 1; CaCl2 2; D-glucose 10 mM (all compounds were obtained from BDH). The osmolarity was 295-300 mOsmL \ The pH was 7*25 after 10 min of bubbling at room

temperature (~ 23°C) with 95% O 2 and 5% CO 2, and 7*3 when continuously bubbled

with 95% O 2 and 5% CO 2 at a temperature of 30-32°C. The acsf was bath applied at a

flow rate of 1 -2 ml min ^

Experimental recording chamber All the experiments reported in this study were performed in a standard slice interface chamber, to which minor modifications were made to allow recording from submerged hippocampal slices. Intracellular recordings from sensorimotor cortical slices were made in the interface mode as in Bindman et al (1988). All experiments were performed at a temperature of 32-32 *5°C, maintained by a heating element and regulated by a thermistor-current feedback circuit (designed and built in the electronics

66 workshop, UCL). Acsf at room temperature was delivered smoothly via a variable speed peristaltic pump (Watson- Marlow, 5025 pump with a 508MC2 pump head; flow rate, 1*3-2 ml min'*). The acsf was held in a 250 ml conical flask and was continously bubbled with 9 5 %0 2 /5 %C0 2 . The reference electrode was fixed in the central well of the recording chamber where it was in continuity with the acsf bathing the slice. Surplus acsf from the central well of the slice chamber was sucked away by a 21 guage hypodermic needle attached to a 1 ml syringe and to a three-gully peristaltic pump (Crouzet) running at 60 rpm which provided the negative pressure required for suction. The dead space volume of the acsf delivery system, between the acsf holding flask and the underside of the slice, was 3-4 ml.

Recordings from the submerged hippocampal slice The hippocampal experiments required the more rapid exchange of drug solutions in and out of the slices compared to the neocortical interface experiments. Consequently, to achieve rapid exchange, intracellular recordings were made from submerged slices, with the fluid level in the central well of the recording chamber maintained at 2 mm above the slice. A relatively low fluid level was used to keep electrode capacitance low. Preliminary experiments revealed that the flow of bathing medium in the central slice well was too turbulent to allow long term stable recordings. Furthermore, dead spaces created by eddy currents at the edge of the circular slice well could prolong the time taken to wash a drug out of the tissue. Simple modifications were made to the slice well to reduce the turbulence and make the flow more laminar: 1 D-shaped segment of pre-set Dow Corning Sylgard plasticiser (10% w/v, BDH), was positioned on each side of the central well (dimensions: length 2 cm, maximum width 7

mm, thickness 6 mm). Subsequently, the Sylgard D’s were replaced with ones made from medical grade perspex. A round glass coverslip (diameter 19 mm, BDH) was also coated with a thin film of Sylgard; once set, this was secured beneath the D’s, to the bottom of the central well with a smear of silicon grease (Dow Corning). The "sticky" surface texture of the film helped prevent the movement of the slice. To prevent the flow of acsf from washing the slice away, a platinum frame, across which nylon threads were stretched (termed a "harp"), was used to hold down the slice (Edwards et al., 1989. These minor modifications made it easier to maintain stable recordings for periods of typically 2-4 hours, and up to 7 *5 hours. The Sylgard D’s and the coverslip

67 were extensively washed and rinsed after each experiment, and used for a maximum of

1 0 experiments, after which they were replaced with new ones.

Harp construction Fine nylon stockings or tights provide a convenient material for making the threads. A hole was made in the stockings so that a "ladder" formed. The parallel array of compound threads (consisting of three finer individual threads) were then tightly stretched over a ring (about 2 cm diameter, eg, the mouth of a test tube) and held in place with an elastic band. Under a dissecting microscope (Prior S2000) a few of these compound threads were then separated into individual fine fibres which were arranged across the mouth of a test tube to form a parallel array of threads about 0*4-0 * 6 mm

apart. A U-shaped Ft frame was made by bending a 2 • 8 cm length of 0 • 5 mm diameter Ft (Goodfellow Metals) wire into a U-shape, and then flattening it in a vice. The dimensions of the Ft frame were 0*9cmxlcmx0*9 cm. The two parallel arms of the frame were coated with a fine smear of cyanoacrylate adhesive (RS Components), and the frame was then balanced across the threads so that the side arms were perpendicular to the them. The Ft frame was weighted with a £1 coin and left overnight until the adhesive dried. It then washed in distilled water. Harps were re-used for about 25 experiments. They were carefully washed in 10% alcohol and then rinsed with distilled water after each experiment.

Electrodes

Recording electrodes Glass microelectrodes were pulled from borosilicate glass (o.d 1*2 mm., Clark electromedical GC120F-10) on a horizontal one-stage puller (designed by Livingstone and Duggar, 1934, and built in the Department’s electronics workshop). They were filled with 4 M potassium acetate. Before filling, microelectrode tips were examined under a light microscope (Watson Barnet) using a xlO phase objective at a final magnification of x225, but could not be adequately resolved. Only electrodes with fine tapers and d.c series resistances of 60-100 MÎÎ when electrodes were filled with 4M potassium acetate were used. Experimentally it was found that electrodes with series resistances of lower than 60 MQ made poor neuronal penetrations, whereas those with

68 resistances of greater than 120 MO often became electrically "noisy" with time, and resisted the passage of applied current following penetration. Electrodes were tested electrically in situ before attempting to impale cells: when lowered into the bathing medium or onto the slice surface, rectangular current pulses, 40-50 ms duration, -0*6 to -hi *0 nA were passed through the electrode, and the resulting voltage displacements inspected. Electrodes were discarded if (i) they showed rectification on the passage of applied current, (ii) a significant capacitative component in their response, or (iii) if the resistance could not be balanced out with the bridge circuit of the preamplifier (Axoxlamp 2A; Axon Instruments).

Stimulating electrodes Two types of stimulating electrodes were employed in the experiments reported in this study: (i). For the neocortical experiments, bipolar stimulating electrodes were made from a pair of electrolytically sharpened tungsten microelectrode wires (Clarke Electrochemical Instruments, TM50-5) which were insulated except for their tips. The distance between their tips was fixed to 0*5 mm with epoxy-resin (Araldite, 50% wt/wt, Ciba-Geigy), and their ends connected to an isolated stimulator (Devices, MK. IV) with light-weight sleeved wires. (ii). Subsequent experiments on hippocampal slices were performed with stimulators constructed in the laboratory, as described below: Two 15 cm lengths of 55 fim diameter varnish-insulated nickel-chromium ("nichrome") wire (a gift from G.L Collingridge, University of Birmingham) were passed through a 10 cm length glass capillary tube (o.d 1*2 mm, Clark Electromedical Instruments) so that each wire protruded from both ends of the capillary by 2*0 cm. The gaps between the glass and wires were sealed with araldite. The wires protruding from one end of the capillary tube, formed the tips of the stimulating electrodes, were twisted along their lengths and thinly smeared with araldite in order to maintain the distance between them at 0*5 mm. Once set, they were then squarely cut so that they were of equal length (1 *50-2*00 cm). The insulation covering each protruding wire from the remaining end of the capillary tube was scraped away and light-weight sleeved wires were soldered to them for later connection to an stimulator (Devices, MK. IV). The whole assembly was tested for electrical continuity throughout construction and before use.

69 Each pair of stimulators was secured in a micromanipulator (Prior) with coarse and fine control. The stimulation parameters for all experiments comprised of square wave pulses (0*05-0*5 ms duration and 0-30 Volts in strength) delivered via the stimulator. A digitimer (Digitimer D4043) was used to control the rate of stimulation and also to generate a trigger pulse (±12 or 5 V) which preceded each shock. The trigger pulses were used to activate the oscilloscope beam and stored on FM tape to aid later analysis.

Voltage recordings and current injection apparatus Intracellular microelectrodes were connected to the headstage (HxO • 1) of a Axoclamp 2A pre-amplifier (Axon Instruments) via a 1 *5 cm silver/silver chloride (Ag/AgCl) wire. The reference electrode was formed by another piece of Ag/AgCl wire inserted into a 1 *5 cm length of Portex tubing (i.d 1-2 mm) containing pre-set agar (BDH) made up with physiological (0-9%) saline. It was secured in the bubble trap where it was in electrical contact with the acsf bathing the slice. The agar interface between the silver and the acsf prevented any noxious interaction between the slice and AgCl. A fresh reference electrode was used for each experiment. The Axoclamp pre amplifier had bridge balance (0-1000 MO) and current injection (0-10 nA) facilities. The voltage signal was amplified ten times by the Axoclamp before being passed to a d.c. input of a Tektronix 5A18N dual trace amplifier. The output was displayed on a Tektronox 511 storage oscilloscope and passed to an FM channel amplifier of a four- channel tape recorder (Racal 4DS; 3 FM channels and 1 audio channel) and stored on FM tape (BASF or Maxell; 120 min recording time). The frequency bandwidth used to record the voltage signal was 0-1250 Hz or 0-2500Hz for action potentials as at the lower frequency bandwidth led spike amplitude attenuation. The input current signal from the Axoclamp was treated in a similar manner and also stored on FM tape. The Axoclamp pre-amplifier had an internal voltage calibration and an input current measurement facility. Calibration signals (1*0 nA at 20 mV and 10 mV per division) were routinely recorded and stored on FM tape either before or at the end an experiment.

Microelectrode support and drive systems Intracellular electrodes were held in a nylon holder and clamped with a light

70 weight nylon screw. The holder was connected to a digital microelectrode drive (Clark Electromedical Instruments). The drive unit was supported by a mild steel arm clamped to steel bar which was firmly secured to a lathe-carriage secured to the mild steel base plate. The lathe-carriage allowed the microelectrode to be moved to any position within the slice chamber. The drive unit was driven by remote control, allowing advancement or withdrawal of the microelectrode in controlled steps of 2 iim or 1 0 fim per step at choice.

Prevention of electrical interference and mechanical vibration The recording chamber (supported on four rubber bungs) recording and stimulating electrode assemblies, drive units and headstages were secured to a heavy mild steel base plate resting on three inflated car inner tubes. The whole assembly was surrounded from beneath, and above and all three sides by a shield of aluminium sheeting. The shield, base plate, perfusion system, water bath and electrical equipment were earthed through the common ground connection on the Tektronix amplifier. These precautions ensured that the recordings were relatively free from artifactual voltage distortions. The base plate, supported by the inflated inner tubes, was mounted on a wooden table standing on rubber blocks. This allowed the whole assembly to vibrate together at

6 Hz and minimised the effects of incidental vibration at the slice-recording and stimulation interface.

Hippocampal slice evaluation Following a recovery period of 1-2 hours, a single slice was transferred with the fine sable hair brush to the central well of the recording chamber, submerged in acsf and positioned on the Sylgarded cover slip, and held down by the harp. The slice was continually perfused with warm (30-32°C), oxygenated acsf at a flow rate of 1*3-2 ml min ^ Slices were illuminated from below by direct light from a fibre-optic source (Micro Instruments, KL1500-T). Slice viability could be judged crudely by appearance: healthy slices were translucent and the cell body layers were clearly defined when viewed under a dissecting microscope (x2*5 phase objective, final magnification x25; W. Watson & Sons, Ltd ). Dead or hypoxic slices had a characteristically granular appearance, and no attempt was made to record from them.

71 Hippocampal recording and stimulation The stimulating electrodes were positioned on the slice between two strands of the harp before cell penetration. In some experiments, two pairs of stimulating electrodes were positioned on the slice: one was positioned in the stratum radiatum to orthodromically stimulate CAl, and the other placed at the alveus to antidromically activate CAl neurones. Figure 2.1.1 A shows a schematic representation of a hippocampal slice and Figure 2.1. IB shows the respective positions of the recording and stimulating electrodes. An intracellular microelectrode was positioned over area CAl, above the slice surface using the lathe-carriage. The electrode was advanced perpendicular to the slice plane, or at an angle of 20-30° using the microdrive, until it touched the surface of the acsf and completed the recording circuit. Hyperpolarizing current pulses (-0 • 2 nA of 30-40 msec) were passed through the electrode at 10 Hz while it was advanced further until it touched the surface of the slice, manifest as a sudden increase in electrode resistance. The Axoclamp bridge was then balanced, the maximum capacitance neutralization applied without positive feedback and the voltage offset to 4-40 mV. Hyperpolarizing current pulses (-0*2 nA, 30-40 ms in duration) were applied through the recording electrode at 10 Hz. The microelectrode was then advanced through the slice in steps of 2 fim followed by the application of a brief passage of depolarizing current (—60 nA) evoked by tweaking the "clear " switch on the Axoclamp. Indication of the close proximity of an electrode tip to a cell membrane was suggested by an increase in electrode resistance; this was followed the application of positive current through the electrode to enable cell penetration. If a cell was not successfully impaled the "clear" button on the Axoclamp was tweaked to unclog the electrode tip of tissue debris. A sudden large drop in resistance often accompanied breaking of the electrode tip. A maximum of three to four micro-electrode passes were attempted with each unbroken electrode. During a microelectrode electrode pass, negative shifts in membrane potential were monitored on the oscilloscope. Alternatively (but invariably far less successfully) cell penetration was attempted by depression of the Axoclamp "buzz" button after each electrode step, or the capacitance neutralization was used to prompt brief oscillation of the capacity compensation. Occasionally, glial cells were impaled having stable membrane potentials of about -85 to -100 mV, but not showing membrane charging during the passage of hyperpolarizing or depolarizing current (-f/-0*6-l *0 nA). On penetration of a neurone there was a sudden negative

72 Figure 2.1.1. Schematic representation of a hippocampal brain slice and the respective positions of recording and stimulating electrodes used in this study.

A. Schematic illustration of a hippocampal brain slice showing its lamella organization. Abbreviations: Alv, alveus; Sch/com, Schaffer/commissural pathway; Sch/col, Schaffer collateral; DG, dentate gyrus; mf, mossy fibres; pp, perforant path. (Adapted from Brown et al., 1990).

B. Schematic illustration of the experimental arrangement used to record from the CAl region of the hippocampus. An intracellular recording electrode was positioned in the cell body layer of CAl and test shocks were delivered via a bipolar ("Test") stimulating electrode positioned in stratum radiatum. Conditioning stimuli were delivered either (i) intracellularly through the recording electrode, or (ii) orthodromically, by stimulation of the Schaffer collateral inputs via the test electrode, or (iii) antidromically, by application of stimuli via a second stimulating ("condit.") electrode positioned in the alveus.

73 B Cond

Alveus

Stratum Oriens

Test Stratum Pyramidale

Stratum Radlatum

CA1

74 shift in the potential which was often accompanied by the appearance of large, fast action potentials (1-2 ms duration). The cell was immediately hyperpolarized to prevent firing, by up to 30 mV by application of up to -1 *0 nA of current through the electrode.

In the absence of action potentials, membrane charging was observed over the first 8 msec of the current pulse. If a stable impalement was to be achieved, there followed a spontaneous hyperpolarization to a steady level. Hyperpolarization was maintained for

1 0 min during which time the electrode resistance was balanced out through the bridge circuit of the Axoclamp. About five min after impalement and still during applied hyperpolarizing current to the cell, ortho- and antidromic stimulation were applied to evoke EPSPs or reversed IPSPs and fast (2-3 msec) action potentials (100-120 mV). The stimulation parameters used to evoke PSPs comprised square wave pulses 0 05-0 1 ms in duration and 0-30 Volts in strength delivered at 0 • 1 Hz. The membrane potential was altered during stimulation in order to examine the voltage dependency of the PSPs. Hyperpolarizing and depolarizing currrent pulses were applied through the recording electrode which preceded and continued during stimulation. Stimulation to evoke PSPs was applied once the membrane had become fully charged, usually 30-50 msec after the onset of the current pulse. This procedure was used to ascertain whether the PSP was an EPSP, reversed IPSP, or an EPSP-IPSP sequence. In cases where an EPSP could not be evoked (e.g., where an orthodromically evoked pure or predominantly IPSP was obtained, or if an antidromic action potential could not be evoked), it was often possible to move one or both stimulating electrodes during recording without losing the impaled cell. In order to unmask a synaptic response as a result of antidromic stimulation at the

alveus, a hyperpolarizing current pulse (2 0 0 msec in duration) and of sufficient intensity (-O'2 to -1 '0 nA) to block the antidromic action potential, was superimposed on it. Having successfully evoked synaptic responses, stimulation was then stopped and hyperpolarization was continued for a further 3 or 4 min, after which time the d.c current was gradually reduced to zero and the control period commenced. If the cell !at > I Hz was not stable after this time, i.e., if it spontaneously fired action potential^ only suppressed by hyperpolarizing current, then it was unlikely that stable penetration could be maintained, and the cell was abandoned.

75 Characterization of passive membrane properties and synaptic responses Orthodromic and antidromic stimuli used to evoke PSPs (or action potentials) were separated by five seconds. During this interval, the apparent input resistance of the cell was continually monitored throughout the experiment with the passage of hyperpolarizing and depolarizing current pulses injected through the recording electrode (150-200 ms duration, -0*6 to -1-0 *4 nA) and the resultant voltage measured at the plateau of the response (usually 140 or 180 msec). The application of hyperpolarizing and depolarizing current of different magnitudes were used to examine the current- voltage (I-V) relationship of the neurone. This produced displacements of membrane potential (V^) within the linear voltage range for most hippocampal pyramidal cells (Connors, Gutnick & Prince 1982). Throughout the experiment at 25-30 min intervals, the neurone was made to fire action potentials, either synaptically, or by injection of depolarizing current pulses (150-200 ms duration). This enabled measurement of the action potential amplitude and the firing threshold (membrane potential). The bridge balance of the electrode was continually monitored and adjusted when necessary, and achieved by balancing out the fast transients of the electrode voltage response, thus leaving the voltage response of the cell. Cells accepted for experimentation had a stable membrane potential of more than -65 mV, action potentials of more than 65 mV (threshold to peak), and apparent input resistances of >20 MÎÎ, Neurones failing to meet any of these criteria were abandoned. The neurone’s was monitored throughout an experiment via the digital voltmeter supplied with the Axoclamp pre-amplifier, as well as a Gould chart recorder (Gould RS 3400) which also recorded PSPs and current injection. Occasionally there was a small d.c drift (1-5 mV) over about 30-60 min between the Ag/AgCl wires. In order to separate the d.c drift from the V^, the cell firing threshold was monitored over the course of the experiment, and the membrane potential was also measured at the end of an experiment. The latter was achieved by stepping out of the cell and observing the change in potential on both the oscilloscope and Axoclamp.

Conditioning paradigms

Induction of hippocampal LTP Two types of conditioning paradigm were used in this study, and each is briefly

76 described below: (a) High frequency stimulation of the Schaffer collateral afferent input to CAl was used to induce LTP (as developed by Bliss and Lomo, 1973; Bliss and Gardner-Medwin,

1973) and consisted of 6 , 0*5 sec trains of shocks delivered at 100 Hzlat 10 sec intervals. (b) Low frequency weak afferent stimulation paired with strong postsynaptic depolarization. This pairing paradigm was described by Gustafsson and Wigstrom (1986) Kelso et al. (1986) and Bindman et at. (1988). It consisted of pairing PSPs evoked by weak stimulation of stratum radiatum 40-50 times with intracellular depolarizing current pulses (100-300 msec in duration; -Hi *0 to 2*5 nA, sufficient to elicit > 8 action potential per pulse) at 0 • 1 Hz. The onset of the depolarising pulse was timed to correspond within ± 20 msec of the onset of the onset of the PSP.

Induction of hippocampal LTD The induction of hippocampal LTD involved one of three conditioning paradigms: (a) The postsynaptic, non-associative induction of LTD of test EPSPs to stimulation of the stratum radiatum by antidromic high frequency tetanic stimulation. The antidromic stimulating electrode at the alveus was used to deliver conditioning trains consisting of 6-9 bursts of 50, 0*1 ms bipolar pulses at 100-120 Hz, at 10 second intervals. Test stimulation was switched off during conditioning. (b) Postsynaptic induction of LTD by postsynaptic depolarizing current injection. In thes^^xperiments, the recording electrode was used to apply the conditioning stimuli which comprised 6-9 depolarising current pulses (500 ms in duration, +1 *5 to 3 *5 nA injection). In some experiments the cell’s was depolarized by 10-17 mV, by injection of steady dc ("holding") current through the recording electrode and held at the depolarized level during conditioning. Test stimulation was switched off during conditioning after which the cell was repolarized by removal of the depolarizing current and test stimulation was resumed. (c) Homosynaptic LTD. In other experiments high frequency orthodromic conditioning

of the afferent inputs in stratum radiatum was applied. The paradigm consisted of 6 bursts of 50, O' 1 ms bipolar pulses at 100-120 HZ, at 10 second intervals. In experiments involving the tétanisation of inputs, the polarity of the stimulus was alternated between trains in order to reduce the likelihood of polarizing the electrodes. The voltage strength and/or the stimulus duration was doubled (0*05 to 0* 1 msec).

77 Drugs In order to assess the pharmacological effects and time-course of action on synaptic transmission and the postsynaptic induction of LTD, slices were bathed with acsf containing either: (i) raised concentrations of Mg^"^ with or without added Ca^^; (ii)

glutamate receptor antagonists CNQX with or without D-AP5; and (iii) the 7 -aminobutyric acid (GABAa) receptor antagonist bicuculline methiodide.

(:). Raised magnesium ion bathing solutions

Standard acsf was altered when MgCl 2 was increased to 25 mM by reducing NaCl

to 8 8 mM to maintain osmolarity. In some experiments 25 mM MgCl 2 and no added

CaCl2, with NaCl 2 at 91 mM was used. Other solutions contained 15 mM Mg^"^ and no added Ca^^, with NaCl reduced to 107 mM. Trace Ca^"^ contamination from the other compounds making the acsf was calculated using the manufacturer’s assay to be 2 • 7- 3 • 1 fiM. Further contamination due to the glassware, would increase the level to about

10 fjM. The solutions were also continuously bubbled with 95% O 2 and 5% CO 2. All solutions were freshly prepared on the day of the experiment and bath applied at a flow rate of 1 • 3-2 ml min 'K , the effects / Preliminary experiments (n=7) were performed in order to examinej(pn /I synaptic transmission of transiently bath perfusing the slices for 5 min with acsf containing a raised Mg^"*" concentration (see Section B Results).

(ii). Glutamate receptor antagonists The drugs used were the selective N-methyl-D-aspartate (NMDA) receptor antagonist D-2 amino-5-phosphonovalerate (D-APV, 5-20 fiM., Watkins and Evans, 1981; Sigma) and the selective antagonist for the a-Amino-3-hydroxy-5-methy 1-4- isoxazolepropionoic acid (AMPA) receptor sub-type, 6-cyano-7-nitroquinoxaline-2,3- dione (CNQX, 5-20 fiM., Tocris Neuramin). Aliquots stock solutions of both APV (1 mM in distilled water) and CNQX (10 mM in 0* 1 % DMSO) were prepared and frozen for subsequent use. The required concentration of each drug was prepared by thawing out the aliquot and then diluting the stock with acsf. Since CNQX is light sensitive, the cylinder containing the drug solution was wrapped in aluminium foil once prepared and remained so throughout the experiment. As a further precaution, perfusion of the slice with CNQX, was performed with the light source illuminating the slice switched off.

78 These solutions were also continuously bubbled with 95 % 0% and 5 % CO2.

(iii). 7-amino butyric acid (GABAJ receptor antagonist Hippocampal slices were continously perfused with the (GABAJ receptor antagonist bicuculline methiodide (1-2 ^M, Sigma) in acsf for at least 40 min prior to the commencement of recording and throughout the remaining recording period. In some experiments, the effect on synaptic transmission was observed by applying the drug after an initial period of recording in standard acsf alone during which intracellular conditioning was applied. The required concentration of drug was prepared by thawing a pre-ffozen 1 mM stock aliquot and diluting it in acsf.

Analysis of results Recorded potentials and currents were stored on FM tape (Racal 4D) for subsequent off-line analysis. Voltage records were digitized (lOkHz) using a Gould 1604 digital storage oscilloscope with a GPIB interface to a personal computer. Records were analyzed with programmes using Lab Windows (National Instruments) software.

EPSPs were averaged (n = 8 ) and the initial depolarising EPSP slope (over 1 ms) and peak amplitude were measured at fixed latencies defined by user-designated cursor positions. Graphs of averaged, normalized EPSP slopes were obtained as follows: for each cell, the 100% level was set at the mean EPSP slope in the 5 min prior to the diffusion of drug into the slice. The averaged slopes for the rest of the time course of the experiment were expressed as a percentage of that 5 min mean. The data for each cell in an experimental group was aligned at the start of the 5 min period prior to the diffusion of solution or drug into the slice (or in ascf alone) and the mean ± SEM calculated across the cells for the averaged slopes at each time. Statistical comparisons were made (paired or unpaired Student’s r-test, 2-tailed) between this control and the mean EPSP slope of measurements taken from 30 to 35 min (high Mg^"^, bicuculline methiodide, or acsf alone) or 40-45 min (CNQX experiments) after conditioning stimulation or the reperfusion of the slice with acsf when no conditioning stimuli were applied. Statistical and graphical representation of data were assisted by the use of the computer (Del Corporation AT) and various software packages including Lotus 123

(version 2*01), Freelance Graphics, and Sigmaplot (version 4* 1).

79 Measurement of cell stability during and after drug perfusion The following criteria were applied to check the stability of the EPSP recording during the control, and the condition of the neurone after experimental procedures. The control period (15- 20 min) started after the peak amplitude had stabilized. If on analysis the depolarizing slope was found nevertheless to decline steadily during the control, so that a decrease in slope or amplitude after an experimental procedure could be accounted for by extrapolation of the declining slope in the control, the experiment was discarded. The firing threshold in acsf was used as a second indicator of changes in membrane potential. Membrane potential, firing threshold and spike amplitude were used to monitor the recovery of the cell from a high [Mg^^] bathing solution. If the membrane potential did not return to control levels within 15 min of reperfusion with acsf (n=3) or if firing threshold fell by >3 mV or spike amplitude by > 4 mV between the control and the after effect of a bathing solution or stimulation, the experiment was discounted (n=2). Rejected cells are not included in the numbers given in the Results of Section B.

2.2 Measurements of intracellular Ca^^ using Fura-2 microfluorimetry

Changes in somatic and dentritic cytosolic Ca^"^ concentration during the conditioning paradigm were measured using Ca^^ microfluorimetry. A hippocampal slice was placed in a modified chamber on the stage of a Zeiss IM microscope and continuously perfused with standard ascf as described above. The ratiometric fluorescent Ca^^ indicator, Fura-2 (Grynkiewicz et al., 1985) (obtained from Molecular Probes) was injected into the postsynaptic cell. The tip of the pipette was filled with a solution of 10 or 20 mM Fura-2, 75 mM KCl and 2 M acetate. The shaft was then back-filled with 4 M KAc. The series resistances of the Fura-2-filled electrodes were between 110 and 145 MO. After impalement of a CAl pyramidal neurone situated less than 100 fim from the bottom of the slice, FURA-2 was iontophoresed into the cell with -0*5 to -1 *0 nA for about 10 min. The slice was illuminated with ultraviolet light at 350 or 380 nm to excite the FURA-2 and emitted (green) light was passed through a dichroic mirror (DC400, Glen Spectra) and broad band green filter (DF 510/30, Glen Spectra) and was imaged by an intensified CCD camera (Extended ISIS, Photonic Science). After 10 min of

80 iontophoresis, the slice was viewed under ultraviolet light at 380 nm in order to check the level of Fura-2 filling. If the cell soma could not be adequately resolved after this time, filling was continued for a further 10 or 15 min in order to also allow diffusion of Fura-2 into the cell. Only cells in which the soma could be resolved were accepted for further study. The cell’s and firing threshold was checked continuously throughout the experiment. In order to measure resting levels of cytosolic [Ca^^] the slice was successively illuminated with light at 350 and 380 nm, using narrow band filters (DF series, Glen Spectra). Cytosolic [Ca^^] was calculated from ratio of 350 and 380 nm fluorescences as described by (Grynkiewicz et al., 1985 and below) using a Kj of 224 nM. In order to measure cytosolic Ca^^ concentration during electrical stimulation the slice was illuminated with steady 380 nm excitation light for 2 min; cytosolic Ca^^ concentration was then calculated from the fluorescence at this single wavelength as described below and by Silver et al. (1992). (/) In order to quantitatively relate the fall of fluorescence at 380 nm to a Ca^"^ concentration, the basic calibration equation for an indicator:

[Ca^^l = /ird(/max-/m!n) / (^ssQ-^in) (Grynkicwicz era/., 1985) 380

where 7 ^ and are at zero [Ca^^] and saturating [Ca^^] respectively. Although 7„,x and depend on the path length and the concentration of Fura-2, the ratio

(4 iax/ 4 im) is an easily measured constant for the particular fluorescence microscope used. We can therefore write:

[Ca^"^] = ^d(^max"^38o) / [^380~(^min/^max)Anax] ( 2 . 2 . 1 )

Rearranging gives:

[Ca^^] X [Zjgo - ( 7niin/fmax) 4 iax] ~ Kj(I„jax “ I380) ( 2. 2 . 2) So,

/38o([Ca^+]+% = /max([Ca:+](C//mJ + % (2.2.3)

or, solving for in terms of [Ca^+] and

81 C x = /38o([Ca'+] +K,) / {(W W [C a:+ ] +K,} (2.2.4)

As /max depends on path length and the concentration of Fura-2, it will be different in each experiment and at each pixel. The following protocol was used to estimate of a particular cell. First, the resting level of [Ca^^] was measured using the ratio method. was then calculated by entering the values of resting [Ca^^] and the /jgo value at resting [Ca^^] in Equation 2.2.4. Recording was then switched to the single wavelength method, with constant illumination at 380 nm. The value of [Ca^^] corresponding to any particular during the depolarization could now be calculated using Equation 2.2.1. Orthodromic PSPs were evoked by stimulation of the stratum radiatum (0 • 1 Hz) and monitored during a control period of 10-15 min following injection of FURA-2 (ie., about 25 min following impalement). Orthodromic stimulation was then ceased and 8 depolarizing current pulses of 500 ms duration and 4-0*5 to 4-2*5 nA were injected through the recording electrode in order to induce postsynaptic depolarization and firing in standard acsf. PSP stimulation was resumed for a control period 10-15 min and the bathing medium was then switched to one containing either raised [Mg^^] (25 or 15 mM) with or without added Ca^"*" (2fiM when added) or 10 fiM CNQX. Following 5 min of perfusion with the high Mg^"^ or the glutamate receptor antagonist by which time synaptic transmission had been blocked (with Mg^'^) or maximally reduced (with CNQX) stimulation of the stratum radiatum was switched off, and injection of depolarizing current pulses was repeated to evoke the same number of spikes as seen in acsf. In some experiments postsynaptic depolarization was applied in acsf containing 1 tiM bicuculline methiodide. In one experiment, following a control period of depolarization in acsf, the slice was bathed in acsf containing 10 ixM tetrodotoxin (TTX, Sigma) to block sodium channels, and the depolarizing current pulse injection was repeated. The fluorescence of dye in the cell body was estimated by subtracting the background fluorescence recorded from regions of area CAl adjacent to the impaled cell from the fluorescence in the cell body. At the end of an experiment, contaminating fluorescence from the microelectrode tip was measured by withdrawal of the electrode from the slice, which was then re illuminated with ultraviolet light at 350 and 380nm and the cyotosolic [Ca^'^J was then

82 re-calculated taking into account this value.

2.3 Methods for studying synaptic transmission and the postsynaptic induction of LTP in layer V neurones in slices of rat neocortex

Dissection and neocortical slice preparation A coronal block of brain containing the sensorimotor cortex was cut with a scalpel blade, and chilled by immediately submerging it in bubbled cold (0-3 °C) acsf for 1 min. The block was then placed in a petri dish also containing cold acsf, and trimmed to include the dorsal aspect of the cerebral cortex and the underlying white matter together with the hippocampus and some thalamus. After trimming, the block was lifted free of the acsf with filter paper and the caudal side dried. The caudal side was then stuck to the silicone cutting block of a Vibroslice tissue slicer (Campden Instruments Ltd) with a thin smear of cyanoacrylate adhesive (RS Components). When the adhesive had dried

(about 2 0 sec) the block and tissue were secured in the cutting chamber of the Vibroslice, fixed onto its cutting carriage and submerged in bubbled ice-cold acsf. Slices were prepared by slowly advancing the cutting carriage while the blade was vibrating. The first slice was always discarded as it was of unknown thickness. Five or six coronal slices were prepared, each 400 /xm thick. The whole procedure took 8-10 min. After cutting, each slice was transferred with the fine sable hair brush onto filter paper moistened with acsf in a petri (slice) dish resting on ice before transfer to the incubation chamber (page 6 6 ). The were incubated for at least 1 hour before a singe slice was transferred to the recording chamber.

Neocortical recordings in the gas-liquid interface mode A slice of sensorimotor cortex was transferred with the fine sable hair brush to the slice interface chamber where they were suspended on lens tissue (length, 6 cm; width,

1 cm) supported by a nylon mesh (mesh width, about 1 mm) stretched across a perspex ring (height: 10 mm; diameter: 25 mm). The underside of the slice was continuously perfused with warm oxygenated acsf with the same composition as used for the hippocampal experiments. Slices were left for a further hour to recover from transfer. They were illuminated from above with light the fibre-optic source. Healthy slices had a shiny, translucent, quality when viewed under the dissecting microscope. Slices that

83 appeared granular were either hypoxic or dead and were discarded.

2.4 Recording and stimulation

Intracellular microelectrodes were placed in layer V in a zone spanning 40-60% of the total depth of the cortical grey matter below the pial surface (Paxinos and Watson, 1982) estimated to be layer V. At the end of each experiment, the location of the recording site was always measured in order to confirm that the recording electrode had been in the correct layer. Stimulating electrodes were positioned on the subcortical white matter of the corpus callosum, 0*5 to 1 mm laterally from the radial column in which the recording electrode was to be placed to reduce the likelihood of eliciting antidromic action potentials (Figure 2.4.1). The stimulation parameters comprised square wave pulses (0*05-0*1 ms duration, 0-30 Volts) delivered via the stimulator (page 70). In some experiments, the stimuli were increased in duration (0*2 to 0*5 msec) to elicit suprathreshold PSPs. Depolarizing current pulses (100 msec in duration) were applied throughout the experiments to test whether they were EPSPs, reversed IPSPs or EPSP/IPSP sequences.

Pyramidal cell identification Neocortical cells can be divided into two groups: (i) interneurones which are confined to the neocortical grey area and (ii) pyramidal cells which have axons that project into the underlying white matter, although within each division there are a number of sub-types of neurone in terms of morphology, connectivity, neurotransmitter and electrophysiology (McCormick et al. , 1986; Mason and Larkman, 1990). Neuronal responses were tested by applying suprathreshold shocks to the white matter underlying the grey. If an antidromic action potential could be elicited, then the neurones were classified as being pyramidal. Neurones that failed to elicit antidromic action potentials may still have been pyramidal but their axon may have not been in the vicinity of the stimulating electrodes, consequently, it may have not been possible to directly stimulate their axons.

84 lateral stim recording medial stim

cerebralfi;;);^ cortex'2i8|

corp

Figure 2.4.1. Positions of recording and stimulating electrode in a slice preparation of rat sensorimotor cortex. Stimulating electrodes were positioned either laterally or medially to the recording electrode in the corpus callosum (From Bindman et a l (1988)).

85 Drugs: Noradrenergic and B-agonists Slices were bathed in either noradrenaline (5-20 fiM', Sigma) or the B-adrenergic receptor agonist isoprenaline (10-20 ^M; Sigma). Final drug concentrations were prepared by thawing out stock aliquots a few minutes before perfusion, and then diluting them with acsf. They were then applied to the bathing medium and perfused for up to 30 min followed by re-perfusion with normal acsf. Accommodation of action potential firing, already well characterised in layer V neurons (Foehring et al., 1989) was tested with 500 msec depolarizing current pulses periodically throughout each experiment. Cell excitability was also measured periodically as the current needed to evoke an action potential (100 msec, 0*1 nA to 2 nA). Blockade of accommodation was used to test whether the drug had reached the recorded cell; the criterion of accommodation block was >50% increase in the number of action potentials elicited by the depolarizing pulse as a result of drug perfusion.

2.5 The conditioning paradigm

The postsynaptic induction of neocortical LTP The protocol for the associative induction of LTP was as described by Bindman et al. (1988): PSPs evoked by weak stimulation of stratum radiatum were paired 40-50 times with intracellular depolarizing current pulses (100-300 msec in duration; 4-1 «0 to

2 • 5 nA) at O' 1 Hz, sufficient to elicit > 8 action potential per pulse. The onset of the depolarising pulse was timed to correspond within ± 20 msec of the onset of the PSP. LTP was defined as a significant increase in the initial EPSP slope and/or the peak amplitude at 30 min post-pairing.

Analysis of Results Recorded neocortical potentials and currents were stored on FM tape (Racal 4D) for subsequent off-line analysis. Voltage records were digitized (lOkHz) using a Gould 1604 digital storage oscilloscope with a GPIB interface to a personal computer. Records were analyzed with programmes using LabWindows (National Instruments) software.

EPSPs were averaged (n= 8 ) and the initial depolarising EPSP slope (over 1 ms) and peak amplitude were measured at fixed latencies defined by user-designated cursor positions. The amplitude and threshold of action potentials, and voltage deflections

86 produced by intracellular current injection were measured from averages (4 to 16 sweep) or single records. Statistical and graphical representation of data were assisted by the use of the computer (DellCorporation AT) and various software packages including Lotus 123 (version 2*01), Freelance Graphics, and Sigmaplot (version 4*1). The particular statistical test performed for each set of experiments is reported in Section A: Results.

87 SECTION A

INVESTIGATION OF B-ADRENERGIC MODULATION OF SYNAPTIC TRANSMISSION AND ASSOCIATIVE LTP IN SLICES OF RAT SENSORIMOTOR CORTEX

RESULTS - CHAPTER 3 DISCUSSION - CHAPTER 4

88 CHAPTER 3

SECTION A: RESULTS

3.1 Introduction

Within each division of the neocortex there are a number of sub-types of neurone which have been identified on the basis of their morphology, connectivity, neurotransmitters and electrophysiology (McCormick et al., 1985; Mason and Larkman, 1990; Larkman and Mason 1990). Neurones that produced antidromic action potentials on direct stimulation of their axons in the white matter had an upshoot directly from the resting membrane potential, ie no EPSP) along with a depolarizing after potential (DAP) on the repolarizing phase of the action potential. The results of the experiments are based on stable intracellular recordings from 41 layer V neurons, from 41 slices, of which 40 were exposed to drugs.

Table 3.1 Summary of the layer V cell characteristics (percent ± 1 S.D, n=41)

Cell characteristic Value ± 1 S.D

Cell location (% depth of grey matter below pia) 54 ± 5%

Mean membrane potential (V^, mV) -75 ± 4 -5

Total spike amplitude (baseline to peak, mV) 93 + 6*2

Firing threshold (mV) 15 ± 4 -4

Spike amplitude (threshold to peak, mV) 78 ± 5 -4

Input resistance (Mfi) 23 ± 5 -8

Percent cells showing accommodation block 901-2%

Mean EPSP amplitude (mV, control) 8*6 ± 3*3

Mean EPSP onset latency (msec) 2-8 ± 1-1

Mean EPSP peak latency (msec) 8*7 ± 2-7

The EPSPs were often followed by IPSPs, revealed by depolarizing current but often reversed at normal resting potential. Rarely, a late hyperpolarizing IPSP was observed

at latencies greater than 1 1 0 msec.

89 3.2 Effects of lî-adrenergic agonists on membrane properties

Blockade of accommodation occurred within 10 min of drug perfusion, and was dose-dependent. Of the 40 cells exposed to drugs, the number of cells reaching the criterion of >50% increase in the number of spikes elicited by the depolarizing current pulse is shown in Table 3.2.1.

Table 3.2.1 Dose-dependence of blockade of spike accommodation.

Percent cells showing Fraction Drug block of accommodation of cells (1 0 min post perfusion)

NAd 5 fiM 14 (1/7)

NAd 1 0 fiM 25 (2 /8 )

NAd 2 0 fiU 75 (9/12)

ISO 1 0 fxM 71 (5/7)

ISO 2 0 fiM 83 (5/6)

Figure 3.2.1 demonstrates the blockade of accommodation observed after application of 20 fiM ISO. In this experiment, a 500 msec depolarizing current pulse was applied through the recording electrode in order to make the cell fire action potentials during perfusion with normal acsf. The cell fired 7 action potentials within the first 350 msec, during which accommodation can be seen as a lower frequency of firing during the pulse. Following 10 min of slice perfusion with 20 fiM ISO, the cell’s resting potential depolarized by 2 mV (from -75 mV in acsf to -73 mV in ISO) and the same current pulse produced 20 spikes with accommodation greatly reduced. The blockade of accommodation was most pronounced in cells which depolarised 2-5 mV following NAd or ISO perfusion (42*5%), but was still evident in cells which the resting potential did not change following drug perfusion. Furthermore, when cells in which drug perfusion induced depolarization of resting potential were repolarized to resting levels with steady dc current injection through the recording electrode, blockade of accommodation was still evident. The NAd induced blockade of accommodation

90 CONTROL

20 uM ISO

25mV 1.5nA 1 00ms

Figure 3.2.1. Blockade of accommodation observed during 20 /xM isoprenaline application to bathing mediumJin this and subsequent figures, "n" refers to the number of traces averaged. A. Control: +0*7 nA, 500 msec depolarizing pulse (lower trace) to the cell produced 7 spikes (upper trace, voltage).

B. After 10 of 20 ISO perfusion the same current produced 20 spikes. Cell resting potential was -75 before and -73 mV in ISO. Mean 1^ 23 MO (n=16). The blockade of accommodation was most pronounced in cells which depolarized 2-5 mV with NAd or ISO, although it was still evident in cells in which resting potential did not change.

^1 was reversible within 15 to 20 min of reperfusion with normal acsf. However, the time required for reversal of the ISO effects was > 45 min. In addition to depolarization, small, transient changes hyperpolarizations (2-4 mV) were observed with NAd and ISO, usually lasting for a few minutes following onset of drug application in 25% (10/40) of cells. The remaining 13 cells (32*5%) remained at the same potential. The most consistent depolarizations occurred with the 20 /xM ISO, but were not long-lasting, following drug washout. No significant effects on cell input resistance were observed after exposure to 20 fiM NAd and 20 /xM ISO, as shown in Table 3.2.2.

Table 3.2.2 showing effect of 20 /xM NAd and ISO on cell input resistance measured 20 min after onset of drug perfusion (F 2 22=0* 65, n=25).

Mean input resistance (R^n, MO ± 1 S.E.M)

control 18-6 ± 1-6 MO

2 0 min post-onset of 20*0 ± 1-8 MO drug perfusion

However, despite the lack of effect on a consistent increase in excitability was observed with 20 /xM NAd and 20 /xM ISO, observed as a decrease in current for spike threshold (Foehring et al., 1989), i.e., a reduction in the threshold for activation for the sodium spike. Figure 3.2.2 shows such an example during perfusion of 20 |xM NAd. In this experiment, NAd did not induce a depolarization of the V^, but did reduce the threshold to depolarizing current injection (from -1-0 * 6 to 0*57 nA).

3.3 Effects of B-adrenergic agonists on evoked synaptic transmission

No significant effects of the drugs on EPSP slope were observed 30 min after

onset of drug perfusion (F 4 26=1 *08) or at 45 min washout, even at doses producing marked block of accommodation. The mean EPSP slopes (initial 1-2 msec, as a percentage of control values ± 1 S.E.M.%) for the various drug doses are shown in Table 3.3.

92 B

0.60 nA j10 mV 20 msec

Figure 3.2.2 NAd reduces the spike threshold to depolarizing current injection. Upper 3 traces, voltage; lower 2 traces, current. Action potentials attenuated by digitization. Only part of current pulse is shown.

A. Control, +0*6 nA (200 msec) depolarizes the cell to spike threshold. B. 15 min post perfusion of 20 /xM NAd; 0*57 nA depolarizes the cell to spike threshold. C. Superimposed depolarizing responses reveals that following NAd, spike threshold is reached sooner with lower current. Resting potential (-72 mV) was the same in A and B. Mean 25 MQ (n=16).

93 Table 3.3 Effect of drugs on the EPSP slope (initial 1-2 msec) 30 min after onset of drug perfusion. Control values taken as 100% (no. of cells in parentheses).

Mean initial EPSP slope as Drug/concentration percentage of control (± 1 S.E.M)

NAd 5 /xM 95-6% ± 3-7% (5)

10 /xM 89% ± 3 . 4 % (5) 20 /xM 104-0% ± 11-9% (10) ISO 10 /xM 92-3% ± 3-8% (5) 20 /xM 115-6% ± 6-4% (6)

Similarly, no significant effects were observed on peak amplitudes during drug perfusion compared to control (1-way ANOVA, F 4 26 = 0.33. In addition, following 45 min of reperfusion with acsf there were no significant differences in EPSP slope or amplitude compared to the original control values (for slope, F 3 21 = 0*31; for peak amplitude, F 3 22 = 0.93). The modest reduction (less than 15%) observed in the group EPSP peak amplitudes during drug treatments could be accounted for by the depolarisation induced by the drugs. In some experiments there were drug-induced effects on some EPSPs, namely on the duration of sub threshold EPSPs or on the spike latency in suprathreshold responses. For example, in a number of cells tested, there was an increase in the probability of firing to synaptic stimulation after application of the higher drug concentrations, indicating that the spike threshold may have been affected by the drugs, but this was not always a consistent effect. In 4 cells, (1 in 20 fjM NAd and 3 in 20 juM ISO) drug perfusion produced an increase in EPSP duration, accompanied by a depolarization if 1-4 mV in 2 of the cells and no change in the other 2. The increase in EPSP duration was manifest as an enhanced later component of the EPSP response. The enhanced duration could be secondary to noradrenergic effects on membrane currents.

3.4 Effects of ft-adrenergic agonists on E-S coupling

The B-adrenergic actions of the drugs were assessed on E-S coupling on sub threshold and suprathreshold EPSPs in 6 neurones (3 in 20 fiM NA, and 3 in 10 fxM ISO). A decreased spike latency was observed in 1 cell following application of 10 fiM ISO, but this was accompanied by depolarization of the resting potential by 5

94 mV. In 2 other neurones, there was a reduced spike latency or increased probability of firing after exposure 20 fiM NAd accompanied by hyperpolarization, but no change in initial depolarizing slope. The remaining 3 cells showed no drug effect on the spike latency, the probability of firing, or on the depolarizing slope of suprathreshold EPSPs although accommodation was blocked in each case.

3.5 Effects of B-adrenergic agonists on the postsynaptic induction of associative LTP The probability of postsynaptic induction of LTP was assessed in 15 cells bathed in normal acsf: LTP was induced in 2 out of 15 cells (13%) (Figure 3.5.1 (1) upper waveforms). Possible drug actions were examined in two series of experiments. In the first, 20 fiM NAd (n=2) or 20 iiM ISO (n=2) was perfused for 30 min and then one or more successive conditioning attempts with increasing current strength were carried out at 30 min intervals. In 1 out of 4 neurones, an increase in depolarizing slope and peak amplitude was produced by the third pairing in 20 fiM NAd (Figure 3.5.1 (2) lower waveforms). The increase in the depolarizing slope of the EPSP was delayed by 6 msec after the onset of the potentiated EPSP. In 10 cells, the probability of induction of associative LTP was compared before and after continuous drug perfusion (see Figure 3.5.2 for example). A doubling in the mean number of spikes was elicited per pairing train in the presence of the drugs (with 20 fiM NAd, 220 of the control firing rate, n=7; and with 20 fiM ISO, 205 % of the control, n=7. Associative LTP was not induced following the first pairing attempt (PI) in normal acsf: at 30 min, the mean peak amplitudes were 94*2 ± 3*5% of the control values (n=10). After 30 min perfusion of either 20 fxM Nad (n=5) or 20 fiM ISO (n=5) a second pairing attempt (PII) was undertaken using the same pairing parameters. In 20 NAd, the mean peak EPSP amplitudes were 97*7 ± 6*7%, and in 20 fiM ISO, the values were 101 *4 + 3*9%. Similarly, no associative LTP was observed at 30 min after the second set pairings (ie., post PII). A comparison of the mean peak values at 30 min after PI and PII for the two groups revealed no significant differences (Fj 16=0*25). Thus the likelihood of postsynaptic induction of LTP was not increased in the presence of either drug (1 out 14, 7%) compared with the incidence in acsf (2 out of 15, 13%).

95 PRE + POST PAIRING IN ACSF

5 mV

1 0 ms

PRE + POST PAIRING IN 20 /J.M NAd

10 mV

1 0 ms

Figure 3.5.1. The associative induction of LTP. In both 1 and 2, the control responses are superimposed as dashed lines and the potentiated responses are shown by the solid lines 30 min post conditioning.

1. Waveforms from an experiment illustrating associative LTP in acsf. Averaged EPSP (n=8) before and after successful LTP induced by associative pairing in acsf. The conditioning paradigm consisted of pairing the test shock with +1 • 2 nA current pulses (150 msec duration) 50 times. The current pulse was applied 5 msg:the^nset of the test shock. Each pairing elicited 8 spikes/pulse. Note the 30%\increa’se in peak EPSP amplitude but not in the initial depolarizing slope, followed by reduction of early (17 msec peak latency) hyperpolarizing IPSP. The late IPSP (110 msec peak latency) was also reduced but not seen on this timebase. The cell was repolarized in LTP to control resting potential of -70 mV for comparison. Mean 50 MO (n=16).

2. Averaged EPSPs (n=8) from another experiment illustrating associative LTP induced in the presence of 20 fiM NAd. Conditioning paradigm: +1 «5 nA current pulses (300 msec in duration) were paired with test shocks 50 times. Each current was applied 20 msec prior to the test shock pulse and dicited 2(J-|^spikes/pulse. Note the increase of 21% in the peak amplitude. The 75% increase m depolarizing slope was seen from 6 msec after the onset of the EPSP. Membrane potential unchanged after LTP induction, at -68 mV. 15 MO (n=16).

96 Figure 3.5.2. Representative waveforms and time course of an experiment involving the attempted induction of associative LTD before and during 20 fiM ISO perfusion.

A. Superimposed averaged EPSP (n=8) at (1) pre-pairing period PI and 25 min post- PI; (2) Pre-and 25 min post-20 iiM ISO perfusion; (3) pre-second pairing period PII, and 35 min post-PII. Dashed lines indicate per-treatment, solid lines indicate post treatment. Mean 20 MO (n=16).

B. Plot of experimental time course of averaged (n=8) peak amplitudes of EPSP ( # ) and initial depolarizing slope (over 1-2 msec) (▼). Vertical lines indicate PI and PII, and horizontal bar the duration of 20 /iM ISO perfusion.

97 A 1 . pre and post P

pre and post 20/xM ISO

3 pre and post P II

5 mV 1 0 ms

B 25 r 1 25

20 20 IJ.U ISO 2 0 Ë > P I 15 15 E < LÜ 0_ • • CL a . 10 1 0 3 CO ## (/) o_ CL

5 ^ tV ÜJ

0 ------L_i------1------1------1------Li------1— :— I------I 0 0 15 30 45 60 75 90 105 120 TIME (min.)

98 CHAPTER 4

SECTION A: DISCUSSION

4.1 Introduction

This investigation suggests that although the postsynaptic 6-adrenergic actions of NAd and ISO on layer V cells are similar to those found in hippocampus, and can probably be attributed to effects on some and Na'^ currents, the enhancement of synaptic transmission and the induction of LTP observed in the dentate gyrus and CA3 was not seen in layer V sensorimotor cortical cells.

4.2 Effects of B-adrenergic agonists on passive membrane properties

The most striking effect of bath application both of NAd and ISO was seen in the blockade of accommodation, thought to be due to reduction of Ca^^-activated K'*’ conductances. The blockade was most pronounced in cells which depolarized following drug perfusion but was still evident in cells which the resting potential did not change as a result of drug perfusion or cells in which the membrane was voltage-clamped. Blockade was dose-dependent and reversible. There was therefore no long-term action of the drugs on layer V cells.

4.3 Effects of B-adrenergic agonists on evoked synaptic transmission

The consistent lack of effect of NAd or ISO on the depolarizing slope and peak amplitude of EPSPs evoked by white matter stimulation in 40 layer V cells suggests there is no presynaptic B-adrenergic action on transmitter release at these synapses on layer V cells in sensorimotor cortex in contrast to synapses in the dentate gyrus where 6-adrenergic activity increases presynaptic glutamate release, and enhances field EPSPs (Lynch and Bliss., 1986). A similar lack of effect of NAd and ISO was observed on field EPSPs in the hippocampal CAl region (Lacaille and Harley, 1985; Heginbotham and Dunwiddie, 1991, Dunwiddie et al., 1992), and in layer III cells of rat visual cortex, where bath application of NAd had no enhancing effect on intracellularly

99 recorded PSP amplitude (Brocher et al., 1992) (but see Nowicky et al., 1990 and below). Thus, synapses in rat layer V sensorimotor cortex, layer III visual cortex, and hippocampal CAl, differ from those in the hippocampal CA3 region where U-adrenergic actions enhance the field EPSPs and population spikes (Hopkins and Johnston, 1988) and to synapses in the dentate gyrus where 6-adrenergic activity increases presynaptic glutamate release, and enhances the field EPSP (Lynch and Bliss, 1986; Harley and Milway, 1986; Harley et al., 1989; Sato et al., 1989). Previous studies using the cortical slice preparation have demonstrated reversible enhancement by NAd and ISO of some component of extracellularly recorded field potentials observed in both rat layer V somatosensory cortex (Bindman et al., 1990) and visual cortex (Nowicky et al., 1990). In rat layer V cells, Bindman et al. (1990) reported a 6-adrenergic enhancement of late components of field potentials, but only with strong stimulation, which would evoke suprathreshold intracellular responses. This could be explained by polysynaptic actions of NAd, perhaps as evidenced by occasional of late EPSPs, or as an increase in the numberj^action potentials evoked by stimulation. The action potentials would then contribute to a larger field potential amplitude observed. No 6-adrenergic actions on the early components of the field EPSPs were observed by these workers, and so presynaptic actions of the drugs can be ruled out, unlike that demonstrated in the dentate with an increased release of glutamate (Lynch and Bliss, 1986). However, the lack of a 6-adrenergic effect on synaptic transmission observed in this study is in contrast to layer II/III of rat visual cortex, where a reversible 6-adrenergic enhancement of EPSP amplitude was observed (Nowicky et al., 1990). In this study an increase in EPSP duration could be secondary to noradrenergic effects on the membrane potential in 2 of the cells which depolarized. However, in the 2 cells that did not depolarize, the enhanced duration could be due to changes in membrane currents enhancing di- or polysynaptic transmission in other cells. The enhanced EPSP duration was reversible and probably similar to that intra- and extracellularly recorded reversible enhancement observed in rat visual cortical layer II/III cells with NAd (Nowicky et al., 1990). A similar action on PSP duration was recently reported in layer III cells in slices of rat visual cortex, where bath application of NAd reversibly reduced the slope of the falling phase of PSPs, in a non voltage- dependent manner, i.e. it increased PSP duration (Brocher et al., 1992). Interestingly, however, as observed in the present study in layer V somatosensory cells, but unlike the

100 observations of Nowicky et al (1990), Brocher et al. (1992) reported that NAd had no enhancing effect on intracellularly recorded PSP amplitude. This further suggests that the enhanced EPSP duration was possibly due to the effect of NAd on membrane currents which enhance poly-synaptic transmission. Layer II/III pyramidal neurons may be different from the layer V cells with respect to distribution of noradrenergic fibres and intracortical circuits and receptor densities (Mori-Okamoto et a/., 1991; Zilles et al., 1990). It has been reported that it is the a-adrenergic agonists that are capable of enhancing spike discharges excited by iontophoretic application of glutamate to several cortical layers, including layer V (Waterhouse et a/., 1980). Even if the a-adrenergic system enhances excitatory processes, then application of NAd, which would activate both receptor systems might be expected to produce some enhancement unless the two receptor systems are antagonistic to each other. However, the results for NAd and ISO were very similar.

4.4 Effects of B-adrenergic agonists in E-S coupling

In half of the sample of suprathreshold responses (3 out of 6 cells), NAd or ISO enhanced E-S coupling, but the reduced spike latency or increased excitability could be caused by the membrane depolarization or di- or polysynaptic effects secondary to the 15- adrenergic action on the postsynaptic membrane currents, which may be similar to the observed increase in the duration of some EPSPs, as mentioned above.

4.5 Effects of B-adrenergic agonists on the postsynaptic induction of associative LTP

Reduction of the slow AHPs observed in the presence of the drugs increases the likelihood of multiple action potentials to a suprathreshold depolarizing input. Thus one might expect that in the presence of the drug, the increase in depolarizing activity during the trains would have produced a larger inward calcium current entry via NMDA-gated channels and/or voltage operated calcium channels (VOCCS) necessary for induction of LTP. This has been proposed for the effect of 6-adrenergic agonists in hippocampal dentate gyrus (Gray and Johnston, 1987) and CA3 (Hopkins and Johnston, 1984).

101 In the hippocampal CAl region, Heginbotham and Dunwiddie (1991) and Dunwiddie et al. (1992) proposed that NAd may produce a transitory state in which enduring changes in function can be facilitated by intermediate-term changes induced by 6-adrenoceptor activation by producing a persistent depolarization of pyramidal cells, thereby facilitating LTP induction by helping to remove the voltage-dependent Mg^"^ block of the NMDA channel. In the present study, however, despite a doubling in the mean number of spikes elicited per pairing train in the presence of the drugs, which one would expect to facilitate the entry of a larger Ca^"^ inward current via NMDA receptor channels and/or VOCCs necessary for the postsynaptic induction of associative LTP, the likelihood of LTP was not increased compared with the incidence in acsf. Thus, the 6- adrenergic action of the drugs did not increase the probability of producing associative LTP or appear to modulate the induction of associative LTP in layer V cells of the somatosensory cortical slice preparation. The short-term effects of NAd and ISO, such as depolarization or hyperpolarization, and block of accommodation, on the layer V cell synaptic responses lin these conditions resembled those observed in the hippocampal CAl region. However, in hippocampu^ a long lasting enhancement of the population spike but not the field EPSP can be produced in CAl by afferent tetanic stimulation (Heginbotham and Dunwiddie, 1991; Dunwiddie et al., 1992). Detailed examination of the enhanced EPSPs during associative LTP in cortex has shown differences from those previously described in the hippocampus (Gustafsson et al., 1987; Kelso and Brown, 1986). In vivo, the monosynaptic component of the EPSPs in layer V cells evoked by pyramidal tract (PT) stimulation was potentiated after associative conditioning, but that evoked by stimulation of thalamic nucleus VL was not (Baranyi etal., 1991). In slices, white matter stimulation is not selective for input pathways (neither is stimulation of VL or PT in vivo, although more selective) and may have excited rapidly conducting axons of thalamo-cortical afferents that cannot be modified by conditioning, as well as more slowly conducting afferents that can be potentiated, thereby leading to the delayed delay in onset of the potentiated component of the EPSP during LTP (Bindman et al., 1988; Bindman and Murphy, 1990) and the present study.

102 4.6 The postsynaptic associative conditioning paradigm

In the present study, a postsynaptic associative protocol for the induction of LTP was used instead of high frequency stimulation of afferent fibres for two reasons: Firstly, this was the experimental paradigm producing LTP in 50 to 60% of conditioned EPSPs in the awake cat (Baranyi et al., 1991). Secondly, postsynaptic injection of EGTA blocks the postsynaptic induction of LTP (Baranyi and Szente, 1987), suggesting that LTP recorded in the injected cell is a localized effect of postsynaptic depolarization on the potentiated synapses. The spike firing may well induce LTP at synapses on other cells, but not in synapses of recurrent circuits. Postsynaptic conditioning allows a more selective analysis of neocortical phenomena than tetanic stimulation of afferents. However, the lack of immediate effects of NAd and ISO in the slice on early components of EPSPs and on the induction of associative LTP in layer V cells should not be extrapolated to other neocortical synapses. One possibility that may account for the low incidence of associative LTP observed in this study, could be that an insufficient level of current injection was used during the conditioning paradigm (Gustafsson et al., 1987). However, similar current strengths reliably produced LTP of conditioned EPSPs in layer V cells in the awake cat (Baranyi et al., 1991). The possibility cannot be ruled out that although the pairing paradigm used in this study produced a similar degree of excitation in response to both test and conditioning stimulation to that in vivo (Baranyi et al., 1991), it may not always have sufficiently depolarized the dendrites to facilitate the inward Ca^^ currents responsible for LTP, even in the presence of the B-adrenergic agonists. This is unlikely, however, since in two additional experiments a third attempt at LTP was conducted with even stronger depolarizing current without any success (not shown). Furthermore, using a similar pairing paradigm, in slices of rat anterior cingulate cortex, Sah and Nicoll (1991) reported that LTP did not occur even when using depolarizing currents sufficient to reverse the EPSP. In summary, the present study demonstrates that although NAd or ISO have marked postsynaptic effects on excitability, neither on its own enhances layer V excitatory synaptic transmission, nor increases the incidence of induction of associative LTP. The low incidence of neocortical associative LTP observed in this study is perplexing because in layer V cells in vivo, a similar pairing paradigm produced

103 associative LTP in 50-60% of conditioned EPSPs (Baranyi et al,, 1991). Neither NAd nor ISO increased the probability of inducing associative LTP in layer V cells of rat sensorimotor cortex. It is concluded that the incidence of induction of LTP found to be lower in vitro than in vivo (Baranyi and Szente., 1987; Baranyi et at., 1991) is not due simply to a lack of noradrenergic activity in the slice. The layer V cells recorded in vivo are obviously not isolated from their extrinsic neuromodulatory influences which may be important in the induction of associative neocortical LTP. However, in vitro it may be that additional neuromodulators, present in vivo, are required for the induction of associative LTP in layer V cells in rat somatosensory cortex (Section 4.7, page 105). Factors which may be important in influencing the probability of inducing associative LTP in the neocortical slice are discussed below.

GABA^ inhibition In the hippocampus and in layer II to IV cells of the neocortex it has been shown that addition of GABA^ antagonists (e.g. picrotoxin or bicuculline methiodide) to the bathing medium to block IPSPs facilitates the induction of LTP (Wigstrom and Gustafsson, 1985; Artola and Singer, 1987 Artola etal., 1990a,b). In the present study, bicuculline methiodide was not added to the bathing medium in any of the experiments. A block of GABA^ receptors may not be an important factor in the present study, since in rat layer V cells of the somatosensory cortex, Bindman et al. (1988) showed that 2 out of 6 cells in which LTP was induced (in one cell by pairing and in the other by high frequency afferent stimulation) were not exposed to bicuculline methiodide. Conversely 19 out of 22 cells in which LTP was not obtained by either pairing or tetanization of afferents were bathed in bicuculline methiodide. Furthermore, a later study by Bindman and Murphy (1990) reported that, in layer V cells, LTP elicited by pairing or afferent tetanization was produced in only 11/53 layer V neocortical neurones. Of these successful experiments, 6/53 were carried out in the presence of bicuculline methiodide. Of the 42 cells which failed to produce LTP, bicuculline methiodide was present in two-thirds of the experiments. Bindman et al. (1988) and Bindman and Murphy (1990) concluded that the Hack of GABA^ inhibition was not essential for the induction of LTP in layer V cells of rat somatosensory neocortex.

104 GABAg inhibition In contrast to the effects of bicuculline, the effects of GABAg receptor antagonists and agonists on the induction and expression on LTP are likely to be complex. Late IPSPs (latency >110 msec) thought to be GABAg-receptor mediated (Connors et al., 1988; Karlson et al. 1988) were rarely observed in this study. Activation of GABAg receptors both inhibit and hyperpolarize neocortical neurones (Connors et al. , 1988; Howe et al. , 1987). Inhibition is probably mediated by release of GAB A onto GABAg autoreceptors on the terminals of GABAergic neurones, which may also contribute to frequency depression of neocortical IPSPs (Deisz and Prince, 1989). These receptors, as well as GABAg receptors mediating slow IPSPs in neocortex, are likely to be activated during a tetanus, and activation of both sets of receptors mediating IPSPs could influence (possibly in an opposing manner) the induction of neocortical LTP. If this is the case then it may explain the apparent lack of effect of bicuculline in the experiments of Bindman et al. (1988) and Bindman and Murphy (1990) on the induction of LTP. It would be interesting to investigate the effect of blocking both GABA^ and GABAg receptors in the neocortex; this might allow the unopposed expression of LTP.

4.7 Other neuromodulators

In the isolated slice preparation, it may be that other neuromodulators must be present to enhance the probability of the induction of LTP. Since the completion of the present study, a requirement for the synergistic action of NAd and ACh has been reported in layer II/III cells of rat layer visual cortex in vitro (Brocher et al., 1992). They reported that activation of both noradrenergic and cholinergic receptors facilitated the induction of LTP of peak PSP amplitude in layer III cells in slices of rat visual cortex following tetanic stimulation of white matter. Both noradrenergic (ISO) and cholinergic (carbachol and muscarine) agonists raised the probability that tetanic stimulation induced LTP in this area of the cortex, and suggested that their observations were comparable to those seen in vivo, where noradrenergic and cholinergic receptor activation has previously been proposed to synergistically facilitate the induction of LTP when combined with tetanic stimulation (Artola and Singer, 1989). Brocher et al (1992) proposed that application of both agonists, either separately or additively, enhanced the depolarizing response to the applied tetanus and increased NMDA receptor-gated

105 conductances during the response. Application of AP5 in the presence of the agonists blocked the induction of LTP. Furthermore, they reported that when cells were hyperpolarized by 20 mV below resting potential during the tetanus, LTP failed to occur, even in the presence of muscarine and NAd, suggesting that these neuromodulators do not facilitate LTP induction per se if they are prevented from enhancing the depolarizing response to the tetanus. However, Brocher et al (1992) did not attempt to induce LTP using a postsynaptic pairing paradigm in their study. Additionally, vasointestinal peptide (VIP) may play a role in modulating the induction of LTP in the neocortex. VIP and VIP receptors have particularly high concentrations in the rat neocortex (Lorén et al., 1979; Staun-Olsen et al., 1985). Bath perfusion of low concentrations of VIP (0*1 to 1 /xM) has been shown to have long lasting effects (> 1 hour after application) on membrane properties in layer II/III neocortical cells in vitro (Dodt et al., 1990). VIP slightly hyperpolarized the membrane potential and increased excitability to current injection of these neurones. VIP also significantly increased orthodromically evoked EPSPs, and revealed a late NMDA- mediated component which also persisted after wash-out (Dodt et al., 1990). It has also been shown that NAd and vasointestinal peptide (VIP) interact synaptically to increase the cortical cAMP levels (Schaad et al., 1987). Both NAd and VIP are found lin terminals projecting, to the (same dendrites in layer VI. Magistretti and Morrison (1988) have suggested that an interaction between NAd and VIP in neocortex might act to markedly increase functional enhancement of specific cell columns in the neocortex. In the neocortex VIP locally modulates cAMP in vertical columns. NAd fibres are distributed along broad transverse strips of cortex running from the frontal poles back to occipital cortex. Concurrent release of NAd along a cortical strip and of VIP within a vertical column could mediate long-lasting changes in the response of that columnar group. In the hippocampus, Prospero-Garcia et al (1991) reported that iontophoresis of VIP in area CAl increased the spontaneous firing rate of pyramidal neurones and induced a regular, 0-like pattern of discharge of about 4 Hz. Evoked activity of CAl interneurones was also enhanced by VIP. In dentate gyrus, granule cell spontaneous activity only slightly increased, while interneurone activity was reduced by VIP application. A similar synergistic effect has been noted for NAd and VIP in hippocampus (Church, 1983). •

106 SECTION B

LONG-TERM DEPRESSION (LTD) IN THE HIPPOCAMPUS AND THE NEOCORTEX

RESULTS - CHAPTER 5 DISCUSSION - CHAPTER 6

107 CHAPTER 5

SECTION Bi RESULTS

5.1 The postsynaptic induction of LTD of synaptic transmission in rat hippocampus

Experiments were carried out on a total of 89 submerged hippocampal slices from 79 rats. Intracellular recordings (lasting for at least 50 min and up to 7 *5 hrs) were made from 96 pyramidal CAl neurones with electrophysiological characteristics summarized in Table 5.1.1.

Table 5.1.1 CAl pyramidal cell characteristics from which intracellular recordings were made.

Cell characteristic Value (± 1 S.D)

Membrane potential (V^,, mV) -73*1 ± 4-9 Total spike (baseline to peak, mV) 98 *2 + 8*6 Firing threshold (mV) 16-1 ± 3-7 Spike (threshold-peak, mV) 82-1 ± 3 '4 Input resistance (R^,, MQ) 31-0 ± 1*9

Passive properties of CAl pyramidal neurones The postsynaptic responses of CAl neurones following injection of hyperpolarizing and depolarizing current evoked characteristic pyramidal cell responses. Injection of current pulses, subthreshold for eliciting action potentials (spikes), invariably produced non-rectifying linear current-voltage responses in neurones examined over a 15-25 mV range negative to the resting potential (see Figure 5.1.1 for example). All the cells showed a maximum charge of the membrane within 40 msec of the onset of the current pulse, which was maintained without decrease for the duration of the current pulse (150-200 msec). In most cells, current-voltage relations showed a degree of rectification such that the chord resistance increased with depolarization and decreased with hyperpolarization of the cells from the resting

108 Figure 5.1.1. The postsynaptic responses of a CAl pyramidal cell to intracellular injection of current.

A. Eight superimposed averaged (n=4) postsynaptic responses of CAl pyramidal neurone to intracellular injection of current. Upper traces, voltage; lower traces, current. Injection of a 4-0*4 nA current pulse evoked a spike followed by a hyperpolarizing afterpotential (AHP). The asterisk (*) at 180 msec marks the measurement latency used to construct the I-V relations shown in B below.

B. Current-voltage relations for the CAl pyramidal cell shown in A. Plot of change of voltage measured at 180 msec (* in A) after the onset of current injection. Each point is the mean of 4 consecutive records evoked by the same current intensity. The I-V relation shows measurements before ( # ) and 45 min after (O) the postsynaptic induction of LTD by antidromic conditioning.

Before LTD: mean 40 MO (n=16); mean spike threshold 14*8 mV (n=8); mean spike amplitude (threshold to peak) 72*6 mV (n=8). After LTD: mean 41 MO (n=16); mean spike threshold 14*7 mV (n=8). -68 mV.

109 o il

VU 9 0 Z'O 9*0-

OS AUi g O0SUJ 09

Auu OS V U 1. potential. This anomalous rectification, so called because the current-voltage relation deviates from that predicted from the Goldman constant field equations (Goldman, 1943), is thought to be due to activation of time- and voltage-dependent (or mixed Na"^ and K^) conductances responsible for the rectification seen at hyperpolarized membrane potentials (Hotson et al., 1979; Halliwell and Adams, 1982; Brown et ah, 1990), and was seen as sag on the voltage response to hyperpolarizing current pulses (Figure 5.1.2). Responses to the passage of suprathreshold depolarizing current pulses (150-200 msec) evoked action potentials in the current pulse. Increasing the current intensity and/or the pulse duration, resulted in earlier spike generation, producing a train of spikes that was maintained for the duration of the current pulse, but which showed spike-ffequency adaption or accommodation. Spike accommodation was poor in cells that had resting potentials close to firing threshold. Action potentials triggered by the anodal break on switching off a hyperpolarizing pulse were seen in cells that had resting potentials close to firing threshold. Such cells generally had high input resistances and were discarded.!

Responses to orthodromic stimulation CAl cells responded to sub threshold stimulation of the stratum radiatum with an EPSP often followed by a hyperpolarization lasting up to 400 msec. Generation of an action potential by the EPSP was seen following suprathreshold stimulation which produced a hyperpolarizing PSP consisting of an early and late phase. The early component reversed between -75 and -65 mV. The later component was not easily reversed, partly because it was difficult to pass enough current to hyperpolarize the cells below -90 mV, but was markedly attenuated between -85 and -95 mV (not shown). Superimposition of hyperpolarizing or depolarizing current pulses on the EPSP evoked conventional pyramidal cell responses (Figure 5.1.3). Subthreshold stimulation evoked a conventional EPSP, which increased in amplitude with hyperpolarization and decreased with depolarization. Suprathreshold depolarization evoked a spike and unmasked the IPSP component of the response.

Effect of antidromic stimulation in normal medium Hippocampal CAl cells showed a characteristic response to antidromic

111 Figure 5.1.2. Postsynaptic responses of a CAl pyramidal neurone to intracellular current injections showing activation of the anomalous rectifier.

A. Postsynaptic responses of a CAl pyramidal neurone to intracellular current injections. Six averaged (n=4) superimposed records. Upper traces, voltage; lower traces current. Note the time- and voltage activated inward rectification after 40 msec (sag, # ) activated by the highest hyperpolarizing current intensity. Closed (# ) and open circles (O) mark the measurement latency (40 and 140 msec, respectively) used to construct the current-voltage relations shown in B.

B. Current-voltage relations for the CAl pyramidal cell shown in A. Plot of change of voltage measured at 40 msec ( # ) and 140 msec (O) after the onset of current injection. Each point is the mean of 4 consecutive records evoked by the same current intensity.

Mean 36 MO (n=16); mean spike threshold 15 mV (n=8); mean spike amplitude (threshold to peak) 87 mV (n=8). V„ -70 mV.

112 20 mV 0.9 nA B 50 msec mV 2 0 -1

15 -

10 -

- 0.6 -0.4 - 0.2 0.2 -5 -

-10 -

-15 -

-20 -

-25 J

113 Figure 5.1.3

A. Five superimposed intracellular responses of a CAl neurone to constant electrical stimulation of the stratum radiatum (at time indicated by filled arrow head) superimposed on intracellularly injected current pulses. Upper traces, voltage; lower traces current. Stimulation of the stratum radiatum sub threshold for eliciting a spike, evoked a conventional EPSP which increased in amplitude with hyperpolarization and decreased with depolarization. Increasing depolarization evoked a spike, demonstrating that the initial response is an EPSP, and not a reversed IPSP. The asterisk (*) marks the measurement latency used to plot the I-V relation below in B. V„ -71 mV; 30 MO.

B. Current voltage relation for the CAl neurone in A. Graph of change in membrane potential measured at 180 msec (* in A) following current injection for the cell in A. Each data point is the mean of 4 consecutive records evoked by the same current intensity.

114 0.7 nA B 20 mV mV 60 msec

nA - 0.6 - 0.2-0.4 0.2

-10 -12 -14 -16 -18

-20

115 stimulation: stimulation that was subthreshold for evoking an antidromic spike when applied at the alveus, often elicited a synaptic response which appeared either as a depolarizing (EPSP) or hyperpolarizing (IPSP) event, or a combination of both (EPSP- IPSP sequence). Single suprathreshold shocks at the alveus elicited a single action potential which took off from the baseline level and therefore showed a considerably shorter latency than orthodromic action potentials, a higher degree of regularity and followed high frequency stimulation more easily than the orthodromic response. Responses to threshold stimulation characteristically showed an all-or-nothing behaviour. Depolarizing after potentials (DAPs) which appeared as a "hump" were always observed on the repolarizing phase of single antidromic action potentials after the spike. DAPs reversed between about -45 and -52 mV when superimposed on depolarizing current pulses, and -85 and 105 mV on hyperpolarizing pulses. They were variably deformed by the presence of an underlying IPSP whose magnitude depended on the membrane potential. Superimposition of hyperpolarizing current pulses under an antidromic spike unmasked any contribution due to a synaptic response, increased the spike amplitude and reduced or reversed the DAP. Further hyperpolarization abolished the antidromic spike completely (Figure 5.1.4). Superimposition of depolarizing current pulses also resulted in reduction of DAPs (not illustrated).

Control recordings In order to investigate whether any progressive depression of synaptic transmission occurred, manifest as depression of test EPSP slope over a lengthy recording period, orthodromic EPSPs elicited by stratum radiatum stimulation at 0 • 1 Hz were recorded from 3 CAl neurones, together with cell input resistance and resting potential, for periods of 50-105 min without any experimental manipulation. In 2 neurones the EPSP slope, input resistance and membrane potential remained stable throughout the recording period. In the third neurone the EPSP slope increased by 25- 30% of the original slope over a period of 66 min accompanied by a decrease in input resistance of 9 • 8 Mfl but with no significant change in resting membrane potential. Figure 5.1.5 shows a normalized plot of EPSP slope against the time-course for these 3 experiments.

116 control DAP -0.2 nA -0.4 nA

40 mV 0.8 nA

50 msec

Figure 5.1.4. Postsynaptic responses of a CAl pyramidal neurone to antidromic electrical stimulation at the alveus (filled arrow head). Upper traces, voltage; lower traces, current. Each trace is the mean of 8 consecutive responses. Subthreshold stimulation (control) at resting potential evoked an antidromic spike of 114 mV amplitude (baseline-peak). Note the depolarizing after-potential (DAP) on repolarization of the action potential. Superimposition of a -0*2 nA hyperpolarizing current pulse under the spike substantially reduced the DAP. Further hyperpolarization (-0*4 nA) abolished the spike completely. Mean 28*6 MO (n=16); -68 mV.

117 140 -I Q) Q. 0 (O CL 120 - g LU ■ j f * 1 100 - ir# --# (0 E

80 -

60 -

40 -

20 -

1 r~ "T" “T“ “T" I I 20 40 60 80 100 Time (min)

Figure 5.1.5. The affect of prolonged electrical stimulation of the stratum radiatum on EPSP slope. Normalized plot of EPSP slope (black squares) versus time for 3 CAl pyramidal cells in which EPSPs were evoked at 0 • 1 Hz throughout the duration of the experiment. Broken lines indicate ± 1 SEM of the means. Broken lines join ± 1 S.E.M values about each normalized mean.

118 Effect of high frequency antidromic conditioning on test EPSPs in normal acsf The effect of high frequency antidromic stimulation on PSP slope during perfusion of slices with normal bathing medium was examined in 3 cells: antidromic conditioning trains delivered induced LTD in 1 cell, but LTP in the other 2 (not shown). In hippocampal area CAl, Pockett et al (1990) reported a similar mixture of effects when antidromic conditioning trains were delivered in normal bathing medium.

Effect of antidromic stimulation in high [Mgf'^] solutions Single antidromic action potentials were examined in acsf containing high [Mg^^] with or without added Ca^^. Antidromic action potentials obtained in high Mg^"*" solutions had an expected reduction in both spike amplitude and DAP (presumably due to the "stabilizing" effect of the cation on the neuronal membrane and/or a reduction in membrane excitability) (Kato et al., 1968; Kelly era/., 1969; Krnjevik, 1965; Erulkar et al., 1974). DAPs were reduced in the presence of acsf containing a raised [Mg^^] without added Ca^^ (not illustrated). Perfusion of the slices with 25 mM Mg^"*" and 2 mM Ca^^ did not significantly reduce the DAP suggesting that Mg^"^ does not completely block Ca^^ channels.

Effect of perfusing high [Mg^^] on passive membrane properties Following the exposure of the slice to high Mg^"^ solutions, the membrane potential depolarized (mean 5*7 mV ± 3 *7 SD n=32, range 0 to 21 mV). On reperfusion with acsf, the membrane typically repolarized within 10-15 min (Figure 5.1.6A). The Rÿ, increased during the depolarization. In 3 cells, the V„ was held at a steady level ("clamped") with applied hyperpolarizing current through the recording electrode during perfusion with acsf containing high [Mg^^]: the increased, by 19*2% ± 6*9% (mean + 1 S.D), showing that a resting conductance was reduced in high [Mg^^]. Figure 5.1.6B shows a copy of the current-voltage trace obtained during one such experiment in which steady dc hyperpolarizing current was injected through the recording electrode to maintain the somatic Vg, to the pre-Mg^"^ perfusion level, and illustrates the increase in R^, induced by perfusion with the high [Mg^^] solution.

Effect of perfusing high [Mg^^] on synaptic transmission Preliminary experiments (n=7) were performed in order to examine the effect

119 Figure 5.1.6. Response of a cell to slice perfusion with a raised

A. Upper trace: 200 msec hyperpolarizing current pulses (0*2 nA) delivered through the recording electrode at 0 • 1 Hz. Lower trace: voltage; responses to current pulses and to stratum radiatum stimulation to evoke EPSPs at 0 • 1 Hz. Downward deflections are hyperpolarizing membrane voltage responses to current pulse injections. Upward deflections are the synaptic responses. Perfusion with acsf containing 25 mM Mg^"^ and 2 mM Ca^^ (upward arrows) abolished the EPSP and induced a gradual depolarization of the cell resting potential (6 mV) accompanied by an increase in R^,. and R^, recovered within 15 min following reperfusion with normal acsf (downward arrows). Note the delay in the voltage response (3 min) in this cell (and in B below) due the dead space of the perfusion system and the time taken for the raised [Mg^^] solution to diffuse through the slice.

B. Raised Mg^"^ solution reduces a resting conductance in "voltage-clamped" CAl cells. Response of a cell to slice perfusion with a raised [Mg^^]. Upper trace: downward deflections produced by 200 msec hyperpolarizing current pulses (-O'2 nA) delivered through the recording electrode at 0* 1 Hz. Lower trace: voltage; responses to current pulses and to stratum radiatum stimulation to evoke EPSPs at 0 • 1 Hz. Downward deflections are hyperpolarizing membrane voltage responses to current pulse injections. Upward deflections are the synaptic responses. Perfusion with a solution containing 25 mM Mg^'^ and 2 mM Ca^^ (upward arrows) abolished the EPSP as in A; injection of steady hyperpolarizing current through the recording electrode in order to "clamp" the somatic resting potential at -73mV did not prevent the increase in Rjn, which recovered to control values following reperfusion with acsf (downward arrows).

120 t

0.2 nA 10 mV 2 min

121 on synaptic transmission of transiently perfusing the slices with acsf containing 25 mM Mg^"^ and 2 mM Ca^^. Following a stable control period of 10-15 min during which orthodromic stimulation of stratum radiatum was delivered at 0 • 1 Hz, with antidromic stimulation at the alveus 5 sec later, the bathing solution was switched to one containing the raised Mg^"^ concentration. In these experiments it was determined that a 5 min exposure of slices to 25 mM Mg^"^ 2 Ca^^ resulted in block of both orthodromic and antidromic responses at all depths within the slice in 2 to 5 min. Recovery of both synaptic and antidromic responses subsequent to replacing the high Mg^"^ with acsf took between 20 and 30 min. Therefore a 5 min perfusion period was used throughout all subsequent experiments. Figure 5.1.7B shows a plot from a single experiment of the orthodromically evoked EPSP slope against the time-course of the experiment, and illustrates the effect of bathing the slice with a high Mg^"^ containing acsf for exactly 5 min, on the initial depolarising slope and consequent block of the stratum radiatum EPSP and the antidromic spike within that period. Figure 5.1.7A shows representative averaged (n = 8) waveforms at the times shown during the experiment. On reperfusion of the slice with normal acsf, the EPSP depolarising slope has recovered within 20 min, although there is a slight reduction in the peak amplitude of the EPSP, representing a di- or polysynaptic component of the response. The results were normalised for 5 experiments and are shown in Figure 5.1.8. Comparing the mean EPSP slope before the high Mg^'^ solution reached the slice with the mean EPSP slope from 30 to 35 min after reperfusion with acsf it was found that there was no significant difference (mean EPSP slope recovered to 109% ± 8 % SE, 18 SD of its control).

Effect of antidromic conditioning in high [Mg^^] solutions on EPSPs The effect of high frequency antidromic stimulation applied during synaptic block was examined on EPSP slope. When high frequency antidromic conditioning trains (6- 9, 0*5 sec trains at 100-120 Hz) were applied during synaptic block, LTD was produced in 7/8 experiments. LTD was also recorded in an 8th neurone where PSP amplitude but not slope was analyzed. The EPSP slope remained depressed for longer than 30 min after reperfusion with acsf, by which time full recovery from the high [Mg^^] would be expected (see above). The time course of the depolarising slope of successive PSPs from a single experiment showing a marked EPSP depression

122 Figure 5.1.7. The affect of transiently perfusing a hippocampal slice with acsf containing 25 mM Mg^"^ and 2 mM Ca^'^ on synaptic transmission in the CAl region of the hippocampus. EPSPs were evoked by electrical stimulation of the stratum radiatum at 0 • 1 Hz throughout the experiment.

A. Representative averaged (n=8) waveforms from the experiment shown in B. From above downwards: during control; during synaptic block; 20 min after wash out of 25 mM Mg^'^ and 2 mM Ca^'*'; and 1st and 3rd averages superimposed.

B. The affect of perfusing acsf containing 25 mM Mg^"^ and 2 mM Ca^"^ for 5 min (indicated by the 2 vertical lines) on EPSP slope in a single experiment. Note the delay in the voltage response (2-3 min) in this cell due the dead space of the perfusion system and the time taken for the raised [Mg^^] solution to diffuse through the slice.

Before perfusion: mean 40 MO (n=16); mean spike threshold 15 mV (n = 8); mean spike amplitude 85 mV (70 mV threshold to peak) (n=8). Post washout (30-35 min): mean Rj^ 41 MO (n=16) mean spike threshold 15 mV (n = 8) and spike amplitude 84 mV (69 mV threshold to peak) (n = 8). -70 mV.

123 CONTROL

++ ++

20 MIN IN ACSF *~irrf-TnwniiriHi.i.

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124 140 -I

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Figure 5.1.8. Transient perfusion of acsf containing 25 mM and 2 mM Ca^* does not induce LTD of synaptic transmission in the CAl region of the hippocampus.

Graph showing normalized EPSP slope for control experiments (n=5) in which the time course of recovery from block of synaptic transmission following 5 min perfusion of 25 mM Mg^"^ 2 mM Ca^"^ with no antidromic conditioning. Each point is the average of 8 responses. Broken horizontal line gives 100% value over 5 min at end of control. Experiments are aligned at vertical lines which bound 5 min bath perfusion of high Mg^"*" solution. Dotted lines join ± 1 S.E.M values about each normalized mean (■ ).

125 following antidromic conditioning is shown in Figure 5.1.9B. Figure 5.1.9A shows averaged (n=8) representative PSPs taken from the experiment (see Figure legend). Once evoked transmitter release had been blocked, 8 antidromic conditioning trains were applied at 120 Hz via the stimulating electrode at the alveus, for 0 » 5 sec at 10 sec intervals whilst test stimulation was switched off. Note the partial recovery of late depolarizing and hyperpolarizing components in the response at 35 min post-conditioning but no recovery of the initial slope. The LTD was undiminished at 55 min post conditioning. The normalized, averaged results for the slices which received conditioning are shown in Figure 5.1.10 which shows the normalized plot of mean EPSP slope versus the time course, and illustrates the decline in initial slope in LTD to half the control value (mean EPSP slope at 30 to 35 min post-conditioning, 52*6% ± 11*4% 1 S.E.M. of its control, n=7). The difference between conditioned (Figure 5.1.10) and control experiments (Figure 5.1.8) was significant at p <0*01 (unpaired f-test, 2 tailed). The depression of the depolarizing slope was seen from the onset (0-1 ms) of the EPSP in 5 cells, but was more obvious in the slope from 1-2 ms in 2 cells. There was typically no recovery of the depression by the end of the recording (up to 115 min after conditioning); however the EPSP slope did recover to control values by 40 min after conditioning in 1 cell.

Table 5.1.2 summarizing the data obtained from the experiments in which antidromic conditioning was applied to induce LTD postsynaptically during block of synaptic transmission. ‘Post conditioning’ values were obtained 30-35 after the conditioning procedure. In this and subsequent tables, the value for the is given I at the end of the experiment. '

Control (n=7) Post conditioning (n=7) Cell characteristic Mean ±1 SEM ±1 SD Mean ±1 SEM ±1 SD

Total spike (mV) 97*6 2.8 6-7 99-6 3-4 7-6 Firing threshold (mV) 15-7 1-1 2-8 15-5 1-9 4-2 Spike (threshold-peak, mV) 81-9 2.8 6.6 84*1 3-5 7.8 Input resistance (R^,, MO) 28*6 3-3 9*3 29-1 3-2 9-1 Membrane potential (V„, mV) -73-4 0-8 2-3

126 Figure 5.1.9. Antidromic conditioning in 25 mM and 2 mM induces non- associative LTD.

A. Representative averages of test EPSPs (n=8) evoked by stratum radiatum stimulation. From top down: during control; in 25 mM M^"^ and 2 mM Ca^^; 35 min after antidromic conditioning trains were applied during block of synaptic transmission; and first and third averages superimposed. Six 500 msec antidromic trains at 100 Hz evoked an average of 24 action potentials per train; 1.5 nA depolarizing pulses applied via recording pipette during conditioning. In this and subsequent figures the fast vertical deflection of the EPSP response is the stimulus artefact.

B. Graph showing averaged EPSP slopes throughout experiment. Synaptic transmission was blocked by high Mg^'^ solution bath perfused for 5 min (indicated by the 2 vertical lines). Note the perfusion of dead space, and time for diffusion into slice, delays block of transmitter release by 2-4 min. Arrow indicates onset of conditioning. Reperfusion with acsf revealed LTD which showed no sign of recovery 55 min after conditioning.

Control: mean Rj^ 22 MO (n=16); spike threshold 14 mV (n=8); mean spike amplitude 83 mV (threshold to peak). At 65 min post conditioning: 1^ 23 MO (n = 16); mean spike threshold 12 mV (n=8); mean spike amplitude 79 mV (n=8). -71 mV.

127 CONTROL

25 Mg++ 2 Co+ +

35 MIN POST-CONDITIONING , iii.-wwr’

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128 140

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Figure 5.1.10. The postsynaptic induction of non-associative LTD by antidromic conditioning during synaptic block.

Graph showing normalized EPSP slope for 7 experiments in which LTD was produced by antidromic conditioning during block of synaptic transmission by perfusion of 25 mM Mg^"^ 2mM Ca^^. Each point is the average of 8 responses. Broken horizontal line gives 100% value over 5 min at end of control. Experiments are aligned at vertical lines which bound 5 min bath perfusion of high Mg^^ solution. Vertical arrow shows time of first conditioning train. Dotted lines join ± 1 S.E.M values about each normalized mean (■ ).

129 Reversal of LTD to LTP To test whether LTD of synaptic transmission could be reversed to LTP, afferent tetanic stimulation of stratum radiatum was applied after LTD had been established postsynaptically in 2 cells (not shown). LTP of EPSP slope of >30 min was produced by tetanic stimulation on both. Thus, synaptic transmission could be modulated in both directions.

Effect of perfusing high Mg^^ with no added Ca^^ on synaptic transmission The time-course and action on synaptic transmission of perfusing slices with media containing high Mg^"^ but no added Ca^"^, were examined. When acsf containing 25 mM Mg^"^ with no added Ca^"*" was bath applied for 5 min (n=2), the time course of recovery of the EPSP on reperfusion with acsf was slower (40-45 min) than for 25 mM Mg^^ and 2 mM Ca^'*’. Consequently, in order to obtain a similar wash in-wash out profile to that observed with perfusion with 25 mM Mg^'^ and 2 mM Ca^"^, slices were bathed for 5 min in acsf with 15 mM Mg^^ but no added Ca^^, which blocked synaptic transmission within 3-5 min. Experiments in which the membrane potential failed to repolarize to control levels within 10-15 min of reperfusion with normal acsf did not show recovery of EPSP slope. Figure 5.1.11A shows the effect of perfusing the slice with acsf containing 15 mM Mg^"^ and no added Ca^^ in a single experiment. In five out of five experiments in which slices were perfused for 5 min with acsf containing 15 mM Mg^"^ and no added Ca^^, there was complete recovery of the EPSP slope, in the absence of antidromic conditioning stimulation, by 30 to 35 min post reperfusion with acsf. The mean EPSP slope was 96.7% ± 7*7% of its control at 30 to 35 min post reperfusion for the 5 experiments. Figure 5.1.1 IB shows the normalised EPSP slope for all 5 experiments plotted against the time-course of the experiments.

The effect of antidromic conditioning in high Mg^^ with no added Ca^^ It is known that high Mg^"^ can weakly block postsynaptic voltage-sensitive Ca^^ channels (Aimers & Palade, 1981). Since perfusion of acsf containing raised [Mg^^] blocked synaptic transmission, it was hypothesized that the Mg^"^ blocked the entry of Ca^"^ both pre- and postsynaptically, and therefore the induction of LTD did not require the entry of Ca^^ into the postsynaptic cell. The Ca^^-dependency of the LTD

130 Figure 5.1.11. The affect of transiently perfusing acsf containing 15 mM Mg^"^ and no added Ca^"^ on synaptic transmission in the CAl region of the hippocampus.

A. Graph showing EPSP slope for a single experiment in which the slice was perfused with acsf containing 15 mM Mg^"*" and no added Ca^'^ with no antidromic conditioning. Control: mean 36 MO (n=16); mean spike threshold 14 mV (n=8); mean spike amplitude 83 mV (69 mV threshold to peak) (n=8). After wash out (30-35 min): mean Rin 35 MO (n=16) mean spike threshold 14 mV (n=8) and spike amplitude 84 mV (70 mV threshold to peak) (n=8). V„ -71 mV.

B. Graph of normalized EPSP slope for control experiments (n=5) in which the slice was perfused with acsf containing 15 mM Mg^'*' and no added Ca^"*" with no antidromic conditioning. Broken horizontal line gives 100% value over 5 min at end of control. Experiments are aligned at vertical lines which bound 5 min bath perfusion of high Mÿ'*' solution. Dotted lines join ± 1 S.E.M values about each normalized mean (■ ). Note a similar time course of recovery from synaptic block in this experiment and the normalized plot shown in Figure 5.8 of wash in/out with 25 mM Mg^"^ and 2 mM Ca^^.

131 Normalized EPSP slope (%) ® EPSP slope (mV/msec) o o d" N ê g g 8 8 ê g Ü1 Ü1 ui to ai 0) o J I L _J

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d 3 (D f 3 O) J produced by postsynaptic conditioning during synaptic block was investigated. When antidromic conditioning was applied during synaptic block in the presence of high Mg^"^ acsf with no added Ca^^, LTD of the PSPs was not induced. Figure 5.1.12B illustrates such an experiment. In this experiment antidromic conditioning applied during synaptic block failed to induce LTD after reperfusion with acsf; the initial depolarising slope of the EPSP recovered within 31 min of reperfusion with acsf as can be seen from the averaged representative waveforms in Figure 5.1.12A. In 6 out of 6 experiments it was not possible to induce LTD of EPSP slope when antidromic conditioning was applied during synaptic block by bath perfusion of the slice with acsf containing a high Mg^'*' concentration but no added Ca^^. The EPSP slope at 30 to 35 min post-conditioning was not significantly different from its control (mean EPSP slope 102*7% ± 2*8% of its control, n=6). Figure 5.1.12B shows the data normalized for the 6 experiments. Similarly, no LTD was produce in a seventh neurone in which PSP amplitude but not slope was analyzed (data not shown). The bar chart in Figure 5.1.13 summarizes the results of the experiments obtained in section 5.1.

5.2 Effect of intracellular conditioning during synaptic block in high [Mg^^]

Discrete conditioning stimuli were applied through the recording electrode during synaptic block to see whether LTD could be produced. Repeated application of depolarizing current pulses through the recording electrode produced firing of the neurones, and induced LTD as shown by Figure 5.2. IB which plots the initial depolarizing slope of successive averaged waveforms against the time course of an experiment; sample representative average PSP waveforms at various times during the experiment are shown in Figure 5.2.1 A. The normalized averaged data are shown for 7 experiments in Figure 5.2.2. A significant LTD at 30-35 min after intracellular conditioning was obtained in 6 out of the 7 slices; in the 7th slice the EPSP slope was not depressed at 30-35 min but was depressed after that time. The mean EPSP slope at 30-35 min post-conditioning was 57 *2% + 11*6% of its control (n=7). The depression was significantly different from the control (i.e. the group of cells in which synaptic block was not followed by conditioning, Figure 5.1.7) at p<0*01 (unpaired t- test, 2-tailed). Table 5.2.1 summarizes the data obtained in this series of experiments.

133 Figure 5.1.12,LTD is nptlinduced by antidromic conditioning during block of synaptic transmission when Ca^^ omitted from bathing solution.

A. Averaged (n=8) EPSP waveforms from a typical experiment. From top down: during the control; synaptic block; 31 min after conditioning trains; and first and third averages superimposed. Conditioning consisted of 9 trains eliciting a mean of 42 spikes/train (see Figure for firing and depolarization in one such train).

B. Graph showing averaged (n=8) EPSP slopes throughout the experiment shown in A. Synaptic transmission was blocked by perfusing the slice with acsf containing 15 mM Mg^"*' and no added Ca^^ for 5 min (indicated by the vertical lines). Arrow indicates onset of conditioning. Reperfusion with acsf revealed no LTD.

Control: mean 30 MO (n=16); mean spike threshold 16 mV (n=8); mean spike amplitude 95 mV (79 mV threshold to peak) (n=8). After LTD: mean R^^ 28 MO (n=16); mean spike threshold 16 mV (n=8); mean spike amplitude 96 mV (80 mV threshold to peak) (n=8) V„ -77 mV.

C. (Overleaf). Graph showing normalized EPSP slope for 6 experiments in which antidromic conditioning during synaptic block during slice perfusion with 15 mM Mg^"^ and no added Ca^"^ failed to induce LTD.

134 CONTROL

+ + + + 15 mM Mg 0 mM Ca

31 MIN POST CONDITIONING

1 0 mV

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C. Graph showing normalized EPSP slope for 6 experiments in which antidromic conditioning during synaptic block during slice perfusion with 15 mM Mg^'^ and no added Ca^"^ failed to induce LTD. Broken horizontal line gives 100% value over 5 min at end of control. Experiments are aligned at vertical lines which bound 5 min bath perfusion of high Mg^"^ solution. Vertical arrow shows time of first antidromic conditioning train. Broken horizontal line gives 100% value over 5 min at end of control. Experiments are aligned at vertical lines which bound 5 min bath perfusion of high Mg^"*" solution. Dotted lines join ± 1 S.E.M values about each normalized mean (solid squares).

136 Figure 5.1.13. The postsynaptic induction of LTD by antidromic conditioning. Bar chart summarizing the results obtained in section 5.1, including the experiments in which antidromic conditioning was applied to postsynaptically induce LTD. The EPSP slope (as a percentage of the baseline) is plotted against the groups means at 30-35 min post conditioning.

137 1 40 r Control

Zj 120 ü j 2 + 2 + co 25mM Mg 2mM Ca plus AD conditioning (n=7) m 100

o 8 0 2 + 2 + 15mM Mg OmM Ca plus AD conditioning (n = 6) oo 60 o_ o 2 + 2 + 15mM Mg OmM Ca plus _J 40 co no conditioning (n = 7) CL (/) CL 20

0 GROUP MEANS + S.E.M. POST 30-35 MIN Figure 5.2.1. LTD produced by repeated intracellular conditioning pulses applied during synaptic block by 25 mM 2 mM Ca^"*^.

A. Averaged (n=8) representative waveforms from a single experiment in which LTD was induced postsynaptically by intracellular conditioning pulses. From above downwards: during control, during synaptic block, 65 min after conditioning pulses, and 1st and 3rd averages superimposed.

B. Plot of EPSP slope versus time for the experiment shown in A. The arrow marks the point at which intracellular conditioning pulses were applied. The LTD was induced by applying 6 intracellular 500 msec pulses of 2 nA through the recording electrode, eliciting on average 13 spikes/pulse. The arrow indicates time of the first intracellular pulse.

Control: mean 40 MO (n=16); mean spike threshold 22 mV (n=8); mean spike amplitude 89 mV (67 mV threshold to peak) (n=8). After LTD (55 min post conditioning): mean unchanged at 40 MO (n=16) mean spike threshold 21 mV (n=8) and spike amplitude 88 mV (67 mV threshold to peak) (n=8). -70 mV.

139 CONTROL

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Figure 5.2.2. Graph showing normalized EPSP slope for 7 experiments in which intracellular conditioning during synaptic block during slice perfusion with 25 mM Mg^*^ and 2 mM Ca^^. Broken horizontal line gives 100% value over 5 min at end of control. Experiments are aligned at vertical lines which bound 5 min bath perfusion of high Mg^"*" solution. Vertical arrow shows time of first intracellular conditioning pulse. Broken horizontal line gives 100% value over 5 min at end of control. Experiments are aligned at vertical lines which bound 5 min bath perfusion of high Mg^"*" solution. Dotted lines join ± 1 S.E.M values about each normalized mean (solid squares).

141 Table 5.2.1 summarizing the data obtained from the experiments in which intracellular conditioning was applied to postsynaptically induce LTD during synaptic block. ‘Post cond’ values were obtained 30-35 after the conditioning procedure.

Control (n=7) Post cond (n=7) Cell characteristic Mean + ISEM ±1SD Mean ±1SEM ±1SD

Total spike (mV) 91-6 2-3 6-2 89-8 2-1 5-2 Spike threshold (mV) 15-7 1-1 2-9 15-4 1-0 2-8 Spike (threshold-peak, mV) 75-9 2-6 6.8 74-4 2-2 5*7 Input resistance (R^,, MO) 26*8 2-7 7-3 26-0 2*5 6-8 Membrane potential (V^,, mV) -68-4 1-3 3-3

Postsynaptic responses during conditioning in raised [Mg^^] Postsynaptic responses were examined during conditioning procedures. This included examining the conditioning trains during the various conditioning procedures. For each experiment, measurements were made of the number of spikes produced per antidromic train or intracellular depolarizing pulse, and the depolarization induced. These data are summarized in Table 5.7.1 (page 175). Early and late hyperpolarizing potentials were observed. When the slice was bathed in the high Mg^"^ solutions with no added Ca^^ there was summation of DAPs, and a greater probability of invasion of the soma by the antidromic spike was found (Figure 5.2.3B). Following the trains (Figure 5.2.3B), a small hyperpolarization was still seen which could have been due to some residual extracellular Ca^'^ or to a Na'^-activated rise in intracellular Ca^"^ (Gustafsson & Wigstrom, 1983), i.e. release of Ca^"^ from intracellular stores. In normal acsf these AHPs often interrupted the trains, for example in Figure 5.2.3A, which shows a postsynaptic response to high frequency antidromic stimulation during synaptic block in a slice bathed with 25 mM Mg^"^ and 2 mM Ca^^. It shows that the second and later antidromic spikes invade soma during the DAP of the previous spike, but steady depolarization does not increase (c.f. Figure 5.2.3B) and several initial segment spikes fail to invade soma. Soma membrane hyperpolarization supervened in the second half of train and prevented further firing. Note also the AHP after the train. The presence of an AHP following the train was also observed, but these were rare and consistently much smaller than in cells where antidromic conditioning was

142 Figure 5.2.3. Examples of spike trains and membrane potential changes in three sets of experiments during synaptic block. Records are digitized and artifacts partially suppressed for clarity. Arrowheads beneath voltage records in A and B indicate each shock in train.

A. Response to antidromic stimulation at 119 Hz in acsf with 25 mM Mg^"^ 2 mM Ca^^. Note second and later antidromic spikes invade soma during depolarizing afterpotential (DAP) of previous spike, but steady depolarization does not increase (c.f. B. and several initial segment spikes fail to invade soma. Soma membrane hyperpolarization supervenes in second half of train. The mean EPSP slope showed 23 % depression at 30-35 min following 7 antidromic trains with average of 25 spikes/train.

Before conditioning: mean R^, 45 MO (n=16); mean spike threshold 20 mV (n=8); mean total spike amplitude 91 mV (71 mV threshold to peak) (n=8). After LTD: mean spike threshold 21 mV (n=8); mean total spike amplitude 90 mV (70 mV threshold to peak); mean R^, 45 MO (n=16). V„ -71 mV.

B. Response of another cell to antidromic train in 15 mM Mg^"^ but no added Ca^^. During the train at 104 Hz, the DAPs summate and only 5 spikes fail to invade soma. (Same cell as in Figure 5.1.12).

C. Example of response of cell bathed in* acsf containing 25 mM Mg^"^ 2 mM Ca^'*' to intracellularly applied current pulse of 2*4 nA, at time indicated by line beneath voltage record. Average depolarization of 10 mV and mean of 15 spikes per 0.5 s pulse, 8 current pulses. Depression of mean EPSP slope to 65 % of its control value, at 30-35 min after conditioning. Before: mean Rj„ 21 MO (n=16); mean spike threshold 13 mV (n=8); mean spike amplitude 86 mV (73 mV threshold to peak). After LTD: mean Rj^ 22 MO (n=16); mean spike threshold 13 mV (n=8); mean spike amplitude 82 mV (69 mV threshold to peak)(n = 8). V„ -65 mV.

143 A

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144 applied in slices bathed with 25 mM Mg^'^ and 2 mM Ca^"^. Figure 5.2.3C shows an example of response of cell bathed in acsf containing 25 mM Mg^"*" 2 mM Ca^^ to a 500 msec intracellularly applied depolarizing current pulse. The cell shows accommodation of firing during the pulse and a reduction in action potential amplitude as well as an AHP after the pulse. In Figure 5.2.3A there is a hyperpolarization of > 6 mV after 19 spikes, while in Figure 5.2.3B it is <4mV after 49 spikes (Vm -71 in A and -70 in B). Thus the AHP is <60% in Figure 5.2.3B compared to A while the 2*5 times increase in firing would lead one to expect a larger AHP.

5.3 Glutamate receptor antagonists

Affect of bath application of glutamate antagonists on synaptic transmission Control experiments were carried out to determine the action of 5 min perfusion of CNQX (2-10 fiM) with or without D-AP5 (5-10 fxM) on synaptic transmission during stimulation of the stratum radiatum at 0* 1 Hz to evoke orthodromic PSPs. Figure 5.3.1 shows the results of a single experiment in which CNQX at 2, 5 and 10 /zM concentration was consecutively bath-applied for 5 min periods before reperfusion with acsf. The 2 jtiM CNQX concentration produced a 50% reduction in PSPs (n=2) and was not used further. Bath-application of 5 ptM CNQX gave approximately 80% reduction in EPSP slope, while 10 jxM CNQX perfusion produced >95% reduction. Recovery of EPSP slope from CNQX perfusion took longest after 10 ^M (up to 40 min Figure 5.3.ID) but in another experiment, recovery of EPSP slope following perfusion with 10 fiM CNQX occurred within 30 min (Figure 5.3.2). Figure 5.3.2 shows representative averaged (n=8) waveforms from an experiment in which perfusion of the slice with 10 ^M CNQX for 5 min resulted in a marked reduction of the EPSP slope (second averaged waveform). However, at 30 min post washout the EPSP slope recovered (third waveform). The bottom waveform shows the control and recovered waveform superimposed. A residual small EPSP after 10 /xM CNQX perfusion (Figure 5.3.1 and 5.3.2) (n = 6) was resistant to bicuculline methiodide at 1 /xM (n=3) and was abolished by subsequent application of 10/xM D-AP5 (n=6). Experiments were performed to expose the NMDA-receptor component of the synaptic response (Figure 5.3.3); Figure 5.3.3A(ii) shows EPSPs elicited in an

145 Figure 5.3.1. The affect of perfusing different concentrations of CNQX on synaptic transmission in a hippocampal slice.

A. Plot of EPSP slope (■ ) versus time course for an experiment in which CNQX (2-10 fiM) was bath applied for 5 min (indicated by the 2 vertical lines) and then washed out. Numbers between 2 vertical lines indicate the CNQX concentration in nM.

B. Representative averaged (n=8) waveforms from the experiment shown in A. (i) control; (ii) maximum synaptic block achieved by perfusion of 2 fiM CNQX; (iii) 30 min washout; (iv) shows waveforms (i) (ii) and (iii) superimposed.

C. Representative averaged (n=8) waveforms from the experiment shown in A. (i) control; (ii) maximum synaptic block achieved by perfusion of 5 /iM CNQX; (iii) 34 min washout; (iv) shows waveforms (i) (ii) and (iii) superimposed.

D. Representative averaged (n=8) waveforms from the experiment shown in A. (i) control; (ii) maximum synaptic block achieved by perfusion of 10 fiM CNQX; (iii) 40 min washout; (iv) shows waveforms (i) (ii) and (iii) superimposed.

146 B

control ii

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30 min post washout

10 mV 0 msec 10 6 n

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147 control

34 min post washout

control

^0^M CNQX

40 min post washout iv

10 mV 10 msec

148 CONTROL

10 uM CNQX

30 MIN POST WASH OUT

1 0 mV 1 0 ms

Figure 5.3.2. The affect of perfusing 10 fiM CNQX on synaptic transmission in a CAl hippocampal neurone.

Representative averaged (n=8) waveforms from an experiment in which a hippocampal slice was perfused with 10 /xM CNQX for 5 min. From top down: during the control; reduced EPSP slope (second averaged waveform). However, at 30 min post washout the EPSP slope recovered (third waveform). Bottom waveform shows the control and recovered waveform superimposed.

149 Figure 5.3.3. Glutamate and GABA^ receptor antagonists block PSPs evoked in CAl neurones. The affect of slice perfusion of CNQX and D-AP5 on evoked synaptic responses. Figures A and B show responses from the same cell in the presence of 1 fiM bicuculline methiodide to block GABA^-mediated IPSPs.

A. Representative averaged (n=8) EPSPs following stimulation (filled arrow head) of stratum radiatum. (i) Control; (ii) during perfusion with 10 fiM CNQX; (iii) during perfusion with 10 CNQX with 10 D-AP5.

B. Postsynaptic responses of a CAl neurone to electrical stimulation of the stratum radiatum (same cell as in A). Upper traces, voltage; lower trace, current. Each trace is the average of 8 consecutive responses was superimposed on an injected depolarizing current pulse. Stimulation in the presence of 10 /xM CNQX to block AMPA receptor- mediated transmission evoked a small synaptic response which increased in amplitude on depolarization (CNQX; c.f. A(ii)). Addition of 10 /xM D-AP5 to the bathing medium blocked this component of the response (CNQX + D-AP5 and A(iii)). Cell V„, -70 mV; Ri., 39-5 Mfi.

150 A (i)

CNQX

CNQX + D-AP5

20mV

lOmsec

B CNQX

CNQX + D-AP5

20mV 0.3nA

50msec

151 experiment in which synaptic stimulation in the presence of 10 fiM CNQX (and 1 fiM bicuculline methiodide to block GABAA-mediated IPSPs) resulted in a residual small EPSP, which was blocked by addition of 10 /xM D-AP5 (Figure 5.3.3A(iii)). Addition of 10 D-AP5 to the bathing medium resulted in block of this component see also Figure 5.3.3A(iii). Figure 5.3.3B shows EPSPs (corresponding to those in A) superimposed on depolarizing current pulses. The residual EPSP (corresponding to Figure 5.3.3A (ii)) observed in the presence of 10 fiM CNQX increased on depolarization ("CNQX" in Figure 5.3.3B). Addition of 10 jiM D-AP5 to the bathing medium blocked the residual component of the response ("CNQX + D-AP5" in Figure 5.3.3B), suggesting that this component was mediated by NMD A receptors. Figure 5.3.4 shows the normalized plots of averaged EPSP slope versus time for a range of CNQX and D-AP5 concentrations (n=10). Synaptic transmission was reduced by bathing slices for 5 min in CNQX with or without D-AP5 (5 fiM CNQX with D- AP5, n=5; 10/iM CNQX, n=2; 5 fiM CNQX, n=3). Recovery from the drug effects took longer than from the high [Mg^^] solutions: in the absence of intracellular conditioning, the mean EPSP slope recovered to 101 *3% ± 5*5% of its control at 40- 45 min after re-perfusion with normal medium (n=10). Thus, a period of 40-45 min after reperfusion with acsf was used as the time of complete recovery of PSPs from the effect of all the various drug doses. The slowest recovery was obtained from bath application of 10 /liM CNQX; this concentration was used in 1/5 of control experiments. When the 5 results with CNQX and D-AP5 were separated from the 5 with CNQX but no D-AP5, it was seen that the recovery of the initial slope was complete by 40-45 min in both groups.

Intracellular conditioning in the presence of glutamate receptor antagonists In the next set of experiments, intracellular conditioning was applied when maximum reduction of PSPs had been obtained with the 5 min CNQX perfusion. Orthodromic stimulation was then stopped and intracellular conditioning consisting of 6 to 9, 0 '5 s depolarizing pulses of 1*5-3 *5 nA at 10s intervals were applied through the recording electrode. Orthodromic stimulation was then resumed, and recording was continued for as long as a stable penetration could be maintained. A significant LTD was produced by intracellular conditioning in the presence of CNQX, with or without added D-AP5. Figure 5.3.5 illustrates the results from one experiment in

152 160

a 140 o w œ 120 Q. LÜ ■§ 100 N (T3 g O z

0 20 40 60 80 Time (min)

Figure 5.3.4. Normalized plot of the time course of recovery of EPSP slope following transient slice perfusion with CNQX with or without AP5.

The graph illustrates time course of recovery from drugs alone (10 /iM alone, n=2; 5 fiM CNQX alone, n=3; 5 fiM CNQX with 10-20 fiM D-AP5, n=5). The slowest recovery was obtained from bath application of 10 fiM CNQX; this concentration was used in 1/5 of control experiments and 1/5 experiments in which conditioning was applied.

153 Figure 5.3.5. The postsynaptic induction of LTD in CNQX.

A. Averaged (n=8) representative EPSPs from a single experiment. From above downwards: during the control; in 10 CNQX which reduced EPSP by about 95%; at 88 min after conditioning; and 1st and 3rd averages superimposed.

B. Plot of EPSP slope versus time for the experiment shown in A illustrating the LTD produced by intracellular conditioning pulses (indicated by the arrow) during synaptic block in the presence of 10 CNQX.

In control: mean spike amplitude (threshold to peak) 81 mV (n=8); mean spike threshold 14 mV (n=8); mean 24 DM (n=16). After recovery from drug: mean spike amplitude (threshold to peak) 82 mV (n=8); mean spike threshold 14 mV (n=8); mean 24 MO (n= 16). V„ -64 mV.

154 CONTROL

10 uM CNQX + I.e. CONDITIONING

88 MIN POST CONDITIONING

5 mV 10 ms

B o (O0)

V t T SL ■i 0_ ÿ.

1 ■ _ ■ ■

2 -

■

. 1 1 , ...... — . . — 1 1 1 1 ' 1 1 1 1 0 20 40 60 80 100 120 140 160 180 Time (min)

155 which LTD was produced following conditioning during synaptic reduction in 10 fiM C QX. Representative averaged EPSPs are shown in Figure 5.3.5A, and the time course of the experiment is shown in Figure 5.3.5B. Perfusion with 10 CNQX for 5 min reduced the EPSP amplitude and slope by about 95% (second from top waveform) compared to the control (top waveform). The third waveform shows the EPSP at 88 min after conditioning; superimposition of the control and third waveform shows more clearly the reduction in the EPSP slope. Figure 5.3.6 shows the normalized EPSP slope responses over the experiment time course for 5 experiments in which LTD was produced by intracellular conditioning during maximum synaptic reduction in CNQX, with or without D-AP5 (10 /iM CNQX alone, n = l; 5 juM CNQX with 10-20 fiM D- AP5, n=4). The normalized EPSP slope was 70% ± 5*7% of its control at 40-45 min after conditioning (n=5). The difference between the normalized control and conditioned groups (Figure 5.3.4 and 5.3.6) was significant at p <0*01 (unpaired Student’s r-test, 2-tailed). Table 5.3.1 summarizes the data obtained in this set of experiments.

Table 5.3.1 The postsynaptic induction of LTD in glutamate antagonists. Summary of the data obtained from the experiments in which intracellular conditioning was applied to induce LTD postsynaptically during reduction of synaptic transmission in the presence of glutamate receptor antagonists. ‘Post conditioning’ values were obtained 30- 35 after the conditioning procedure.

Control (n=5) Post cond (n=5) Cell characteristic Mean + 1SEM ±1SD Mean + 1SEM ±1SD

Total spike (mV) 106 1-8 4-1 105 2-3 5-2 Spike threshold (mV) 16-8 1-3 2*9 17-4 1-4 3-2 Spike (threshold-peak, mV) 88*9 1-4 3-1 87-2 1*1 2-5 Input resistance (R^,, MO) 33-7 2-0 4*4 31-6 2-4 5-3 Membrane potential (V„j, mV) -71 2-8 6-3

Intracellular conditioning in D-AP5 In two experiments intracellular conditioning took place during slice perfusion with acsf containing 10/^M D-AP5 alone. No effect of conditioning on EPSP slope was observed in these experiments (not illustrated).

156 _ 160 & g. 140

W 120 0. UJ •g 100 N 15 E 80

0 20 40 60 80 100 Time (min)

Figure 5.3.6. The postsynaptic induction of LTD by intracellular conditioning pulses in glutamate receptor antagonists.

Normalized plot of averaged (n=8) EPSP slope for 5 experiments in which intracellular conditioning was applied during reduction of synaptic transmission by bathing slices for 5 min (indicated by the 2 vertical lines) in CNQX with or without AP5 (10 CNQX alone, n = l; 5 /xM CNQX with 10-20 fiM D-AP5, n=4). The arrow marks the time at which the first intracellular conditioning pulse was applied. The dotted lines join ± 1 S.E.M values about each normalized mean (solid squares).

157 5.4 Effect of conditioning current pulses in normal acsf

Attempts to postsynaptically induce LTD in normal bathing medium by application of intracellular depolarizing current pulses were unsuccessful in 5/5 cells. Figure 5.4.1 shows a normalized plot of EPSP slope versus time for 5 experiments. The mean EPSP slope at 30-35 min post-conditioning was 103% (±1% SEM) of the control mean. The means were not significantly different (paired Student’s f-test, p < 0 '0 5 , n=5). Table 5.4.1 summarizes the data for this set of experiments.

Table 5.4.1 Intracellular conditioning in normal acsf does not induce LTD of PSP slope. Summary of the data obtained from the experiments in which intracellular conditioning was applied in normal acsf. ‘Post cond’ values were obtained 30-35 after the conditioning procedure.

Control (n=5) Post cond (n=5) Cell characteristic Mean ±1SEM ±1SD Mean ±1SEM + 1SD

Total spike (mV) 101 2-7 4 • 6 99-4 3-5 6-0 Spike threshold (mV) 12*6 0-2 0*1 12-5 0-3 0 • 6 Spike (threshold-peak, mV) 88-1 2-6 4-5 86*9 3-5 6*0 Input resistance (R^^, MO) 31-3 4-0 8*9 31*4 4*1 9-1 Membrane potential (V^, mV) -68 2*0 3-4

5.5 Effect of depolarizing current pulses on LTD in acsf containing a GABA^ receptor antagonist

GABAa antagonists (e.g. bicuculline methiodide or picrotoxin) in the bathing medium can increase the probability of inducing LTP and LTD in both neocortical and hippocampal slice preparations (Wigstrom and Gustaffson, 1985; Artola & Singer, 1987; Artola et ah, 1990; Collingridge and Davies, 1990, Wickens and Abraham, 1991). It was examined whether LTD could be postsynaptically induced in the presence of bicuculline methiodide and, if so, did the drug increase the probability of LTD induction compared with glutamate antagonists. Intracellular conditioning in the presence of the drug resulted in LTD of the test EPSP slope in 3 out of 9 slices. Figure 5.5.1 shows the results from one such

158 140 -1 0) CL I■i 120 g 100 j j v j ||------**r ca OE Z 80 -

60 -

40 -

20 -

—|— —|— 20 40 60 Time (min)

Figure 5.4.1. Failure to induce LTD postsynaptically in normal acsf. Normalized plot of EPSP slope (■ ) versus time for 5 cells in which intracellular conditioning pulses (arrow) were applied during slice perfusion with normal medium. Broken lines indicate ± 1 SEM of the means. Broken lines join ± 1 S.E.M values about each normalized mean.

159 Figure 5.5.1. The postsynaptic induction of LTD by intracellular conditioning pulses in slices bathed with the GABA^ receptor antagonist, bicuculline methiodide (1 fiM).

A. Representative averaged waveforms (n=8) from the experiment. From the top down: control; 34 min post conditioning; first and second waveforms superimposed.

B. Plot of EPSP slope versus time for the experiment shown in A. The conditioning paradigm (arrow) consisted of depolarizing the membrane potential by 14 mV by injection of steady current through the recording electrode followed by the injection of 8 intracellular depolarizing current pulses (2 • 1 nA for 500 msec at 10 sec intervals) each pulse eliciting 22 spikes.

Before LTD: mean 47*6 MO (n=16); mean spike threshold 15*8 mV (n=8); mean spike amplitude (threshold to peak) 89 mV (n=8). During LTD: mean spike threshold 15*2 mV (n=8); mean spike amplitude (threshold to peak) 88*4 mV (n=8); mean R^, 48 M 0(n=16). -72 mV.

160 control

34 min post conditioning

10 mV

5 msec B

3 ‘ 1 . 4 n 0) t I. s. ■i 1 Q. £ UJ 0.8

0.6 -

0.4 -

0.2 -

I 20 40 60 Time (min)

161 experiment in which the slice was perfused with 1 bicuculline methiodide for a least 1 hour prior to the commencement of recording (see Figure legend for details). The EPSP slope remained depressed for the remainder of the recording period (> 30 min post conditioning). There was no significant difference between the mean spike threshold, and spike amplitude compared both before and at 30-35 min post conditioning. In a second cell (not shown), intracellular conditioning during steady depolarization of the cell’s membrane potential by 10 mV failed to induce LTD on 4 occasions, each with increasing injection current levels in normal acsf. The slice was then perfused with 1 bicuculline methiodide for at least 30 min before repeating intracellular conditioning. No affect on or was observed following drug perfusion. A further 3 attempts at intracellular conditioning failed to induce LTD on each occasion, even though an increased level of current injection was used during the conditioning pulses and the cell’s membrane potential was also depolarized by up to 16 mV. However LTP of EPSP slope could be induced in this cell by high frequency orthodromic stimulation of the stratum radiatum. LTD was induced in a third cell (not shown) in a slice perfused with 1 /xM of the

GABA a antagonist using the similar conditioning stimuli. In the 6 remaining cells intracellular conditioning in the presence of the GABA^ antagonist failed to induce LTD of the test EPSP slope. Figure 5.5.2A shows a normalized plot of EPSP slope versus the time-course of these 3 experiments in which LTD was induced postsynaptically. At 30-35 min post conditioning the mean EPSP slope for the 3 cells was 56*46% (± 2*7% SEM, n=3) of the control value. The slope depression seen in these 3 cells was comparable to that obtained when LTD was induced with antidromic and intracellular conditioning during perfusion in the raised [Mg^^]. There was no significant change in spike threshold, spike amplitude, or input resistance before or after LTD induction in this group, or between this group and any of the other groups (high [Mg^^] and glutamate antagonist experiments) in which LTD was induced. However, when the results for all 9 cells were normalized, the depression of EPSP slope was much smaller. Figure 5.5.2B shows a normalized plot of EPSP slope versus time-course for all 9 cells. Comparing the means at 30-35 min post-conditioning in bicuculline methiodide with the control, the mean EPSP slope was

162 Figure 5.5.2. Normalized plots for the experiments in conditioning was applied to postsynaptically induce non-associative LTD in a^CiAfiA^^antagonist. Normalized plots of EPSP slope illustrating the affect of intracellular conditioning in the presence of bicuculline methiodide (1-2 /^M).

A. The postsynaptic induction of LTD by intracellular conditioning pulses in slices bathed continuously bicuculline methiodide. Normalized plot of EPSP slope (■ ) versus time for 3 experiments. The arrow indicates the time at which conditioning pulses were applied. Broken lines join ± 1 S.E.M values about each normalized mean.

B. The postsynaptic induction of LTD by intracellular conditioning pulses in slices bathed continuously bicuculline methiodide. Normalized plot of EPSP slope (black squares) versus time for 9 cells in which intracellular conditioning was applied during slice perfusion with bicuculline methiodide. The arrow marks the point at which intracellular conditioning pulses were applied. Broken lines indicate ± 1 SEM of the means. Broken lines join ± 1 S.E.M values about each normalized mean.

163 CD Normalized EPSP slope (%) Normalized EPSP slope (%) ê g s s ê ê 8 8 8 8 ê

£ O

O - 8 84*78% (± 3.3% SEM, n=9) of the control value. The difference between the means was significant at p<0*05 (Student’s paired r-test, 2 tailed). The mean EPSP slope at 30-35 min post-conditioning in bicuculline methiodide was also significantly different from the means at 30-35 min post-conditioning in acsf alone (Student’s unpaired r-test, p<0*05. The slope depression seen in the overall group was smaller than that observed following antidromic and intracellular conditioning during perfusion in the raised [Mg^^] and the glutamate antagonists. It was tested whether LTD could be induced repeatedly in the same cell. Figure 5.5.3 shows the results obtained from an experiment in which intracellular current conditioning was carried out in acsf containing IfiM bicuculline methiodide following an initial failure in acsf alone (Figure 5.5.3 A1 and Bl, see Figure legend for details). The slice was then perfused with acsf containing 1 fiM bicuculline methiodide for 43 min before the conditioning was repeated. Perfusion of bicuculline methiodide had no effect on Rin or although it did result in a gradual and reversible growth of the test EPSP slope, observed over the first 1-2 msec (Figure 5.5.3 A2 and B2), which appeared to reverse and then stabilize at a higher level than pre-perfusion values during continued low frequency test stimulation. Since the increase in PSP slope could be due to a reversal of the IPSP component, rather than the EPSP, test EPSPs were superimposed on 200 msec depolarizing current pulses. This confirmed that the drug did act on the early monosynaptic component of the test response. The second set of conditioning pulses with the same conditioning parameters as in acsf also failed to induce LTD, although conditioning did slightly increase the latency of spike firing (Figure 5.5.3 A3 and B3). However, a third attempt with an increased level of current injection did induce LTD of the test responses (Figure 5.5.3 A4 and B4). Subsequent intracellular conditioning pulses using the same conditioning current levels produced further "stepwise" LTD (Figure 5.5.3 A5 and B5, and A6 and B6). To test whether LTP could also be induced in this cell, 6 tetanic trains were applied to the stratum radiatum (see legend of Figure 5.5.3B for details). Tetanic stimulation resulted in LTP of EPSP slope for 50 min (Figure 5.5.3 A8 and B8) following a 20 min period of post tetanic potentiation (PTP) (Figure 5.5.3 A7 and B7). There was no significant change in cell input resistance (R J, spike threshold, or spike amplitude as a result of drug perfusion or accompanying LTD or LTP in this

165 Figure 5.5.3. LTD repeatedly induced postsynaptically in the same cell, in the presence of bicuculline methiodide. Repeated intracellular conditioning was applied (at arrows) during perfusion of the slice first with normal acsf and then after 28 min with 1/iM bicuculline methiodide.

A. After 1 (at the first arrow) the cell membrane potential was depolarized by 11 mV by steady injection of current followed by the injection of 8 depolarizing pulses, each of + 1 '5 nA, eliciting 22 spikes per 500 msec pulse delivered at 10 sec intervals. This failed to induce LTD (Bl). The slice was then perfused with acsf containing 1 nM bicuculline methiodide (horizonal black bar). Perfusion of bicuculline methiodide resulted in a gradual and reversible growth of the test EPSP slope (shown at 2 and in B2) which stabilized to a higher level than pre-perfusion values. A second set of conditioning pulses using the same conditioning parameters as in the first attempt also failed to induce LTD in bicuculline (illustrated at 3 and in B3). A third attempt (3rd arrow) with an increased level of current injection (steady depolarization of 13 mV plus 8 pulses, +3*0 nA eliciting 27 spikes per 500 msec pulse, delivered 58 min after the onset of perfusion of bicuculline methiodide did induce LTD (shown at 4 and in B4). Subsequent intracellular conditioning pulses produced further LTD (illustrated at 5 and in B5, 6 and B6). LTP was also induced in this cell by application of 6 tetanic trains delivered to the stratum radiatum (100 Hz, 500 msec, at 10 sec intervals). Tetanic stimulation produced PTP of test EPSP slope (shown at 7 and in B7) which declined within 20 min to a slope greater than the pre-stimulation control for a further 50 min (shown at 8 and in B8).

B. Representative averaged (n=8) waveforms at the times shown in graph A. Bl: from the top down: control; 15 min post-conditioning; first and second waveforms superimposed. Subsequent waveforms (B2 to B8) show the pre- and post-conditioning responses superimposed. Waveforms B5, B6 and B8 are shown at an increased gain in order to show more clearly the effect of conditioning on the depolarizing EPSP.

At start: mean 39 MO (n = 16); mean spike threshold 13 mV (n = 8); mean spike amplitude (threshold to peak) 86 mV (n=8). At end: mean R^^ 39 *5 MO (n=16); mean spike threshold 13 mV (n=8); mean spike amplitude 83 *0 mV (n=8). V„, -67 mV.

166 -ü 5 n 0 CO E i t t t & g. 4 H O CO 0_ 2 3 8 CO Û_ LU 3 - L ■ 1 ■ J kf Vf S 3 A 2

2 - y

1 -

1---- 1---- 1---- 1---- 1---- 1---- 1---- r" —,------1 , 1 1 40 80 120 160 200 240 280 Time (min) control 15 min post conditioning

bic methiodide

control

3 pre conditioning

20 mV

4#10 m sec

168 pre

20 mV

10 msec post

pre

post

pre

post

10 mV

10 msec

169 10 min post

pre

20 mV

10 msec

38 min post

pre

10 mV

10 m se c

170 cell. Table 5.4.2 summarizes the characteristics of all 9 experiments cells including the 3 cells in which LTD was induced. Table 5.5.1 The postsynaptic induction of non-associative LTD in the presence of bicuculline methiodide. Summary of the data obtained from the experiments in which intracellular conditioning was applied during slice perfusion with bicuculline methiodide. ‘Post cond’ values were obtained 30-35 after the conditioning procedure.

Control (n=9) Post cond (n=9) Cell characteristic Mean ±1SEM ±1SD Mean ±1SEM ±1SD

Total spike amp (mV) 96*9 2*5 8.0 96*3 2*5 7*8 Spike threshold (mV) 14*1 0*8 2*7 14*1 0*8 2*6 Spike (threshold-peak, mV) 82*9 2*3 7*2 82*6 2*0 6*5 Input resistance (R^, MO) 37*8 3*6 11*3 38*8 11*4 3*6 Membrane potential (V^, mV) -71

Although bicuculline methiodide appeared to favour the induction of non- associative LTD in 3/9 experiments, it did not increase the probability of the postsynaptic induction of LTD when compared to conditioning in acsf alone (0/5 cells). These results were not significant at the level of 0 » 05 % (Fisher’s Exact Test of Independence, P = 0*231). Therefore bicuculline methiodide does not appear to increase the probability of the postsynaptic induction of non-associative LTD see Discussion). The bar chart in Figure 5.5.4 summarizes the results of the experiments in which intracellular conditioning was applied to postsynaptically induce LTD.

5.6 Input resistance and spike threshold during LTD

Cell input resistance (R J did not change significantly either as a result of drug perfusion or accompanying LTD. Comparing in neurones before and after drug perfusion and in normal acsf, with or without postsynaptic conditioning, the means (± SEM) were 30*6 ± 1*6 MO and 30* 1 + 1*5 MO respectively (n=44). Comparing R^ in the control with that during LTD obtained in four sets of experiments, the means (± SEM) were 31*7 ± 2*1 MO and 31*4 ± 1*2 MO respectively (n=28). There was no significant difference in before and after drug perfusion or induction of LTD within any of the groups making up the pooled data given above.

171 Figure 5.5.4. The postsynaptic induction of LTD induced by intracellular conditioning. Bar chart summarizing the results obtained in the experiments in which intracellular conditioning was used to induce LTD. The EPSP slope (as a percentage of the baseline) is plotted against the group means at 30-35 or 40-45 min post conditioning, depending on the pharmacological manipulation used.

172 'y / / \ 25 mM i.c conditioning (n=7)

2 + 120 25 mM Mg control (n = 6) 100 Bic meth i.c conditioning LÜ (n=9) CO < ACSF i.c conditioning ÛÛ 1 80 (n=5) O 1 $ $ ^ CNQX + / - AP5 i.c conditioning CO 60 (n = 5)

] CNQX + / - AR5 control CL O 40 (n = 10) _J l / l

CL CO 20 CL LÜ 0 30-35 min 30-35 min 40-45 min

GROUP MEANS ( + / - S.E.M.) There was no change in mean spike threshold, comparing the mean value during the control and during LTD (mean change 0*22 mV ±0*2 SD, 0*11 SEM; n=28), indicating that there was no change in membrane potential.

5.7 Activity-dependence of induction of LTD

A possible reason for the failure to induce LTD in the absence of Ca^'*' could be due to a difference in the amount of soma activity induced postsynaptically during conditioning. Examination of the firing and depolarization during spike trains showed that LTD could be induced by antidromic spiking where the steady baseline depolarization was only a few mV (mean 5 • 7 mV) due to the Mg^^ perfusion and there was little summation of DAPs (e.g. Figure 5.2.3A). LTD could also be obtained using depolarizing current which elicited comparatively few action potentials per train (e.g. Figure 5.2.3C). Table 5.7.1 summarizes the postsynaptic responses produced by the conditioning paradigms. In the experiments in which LTD was induced by intracellular depolarization in the presence of glutamate antagonists, the mean number of spikes per pulse was comparable to that observed when LTD was induced in raised [Mg^^] (Table 5.5.1). experiment However in the glutamate antagonists^there was a mean depolarization of 11 mV during conditioning but no steady depolarization during drug application. The smaller mean depolarization in the latter experiments could be a factor in the smaller mean decrease in EPS? slope found with glutamate antagonists in comparison with the experiments with block of transmitter release by a raised [Mg^^]. Failure to induce LTD by antidromic conditioning in the absence of added Ca^"^ to the bathing medium was due neither to a lack of postsynaptic depolarization nor to too few antidromic spikes. As shown, the group of cells in which intracellular conditioning resulted in LTD in bicuculline methiodide (3 out of 9) showed a comparable amount of depolarization (mean 12 mV) and spike firing (22 spikes per pulse) to the group in which intracellular conditioning failed to show LTD during block of GABA^-mediated IPSPs (12 mv depolarization, and 24 spikes per pulse). Furthermore, the mean net depolarization and number of spikes elicited during conditioning in bicuculline

174 Table 5.7.1 Postsynaptic responses produced by the conditioning paradigms used in this study.

Mean Mean applied Mean Spikes per Spikes per depolarization dc depolarization Mean total Method of LTD induction LTD train or conditioning produced by depolarization per train or depolarization pulse procedure solution o fV . pulse Conditioning stimulus and solution* Yes/No mV mV mV mV Mean Range

AD in 25 mM 2 mM Ca^^ Yes 6 0 0 6 37 44-486 IC in 25 mM Mg^+ 2 mM Ca^+ Yes 6 0 14 20 14 64-120 IC in CNQX + D-AP5 Yes 0 0 11 11 15 56-261 AD in 15 mM Mg^+ 0 mM Ca^+ No 4 0 14 18 29 128-378

•-J LA IC in bicuculline methiodide No 0 12 12 24 24 136-272 IC in bicuculline methiodide Yes 0 12 12 24 22 136-208 IC in acsf No 0 13 10 23 25 136-240

'AD, antidromic; IC, intracellular. were comparable to that observed during conditioning in high Mg^"^ solutions or glutamate antagonists. Failure to induce LTD in acsf does not seem to be due to eliciting as much postsynaptic firing and depolarization during the conditioning compared to the groups in which LTD was induced.

5.8 Measurements of intracellular [Ca^^]

Direct evidence that cytosolic Ca^^ could increase as a result of action potentials elicited in a high [Mg^^] bathing solution was obtained using intracellular FURA-2. Technical limitations imposed by slice thickness and light scattering properties of myelinated neurones invariably meant that somata of cells impaled close to the surface of the slice (within 100 ;^m) could not be adequately resolved to allow measurement of intracellular Ca^^ levels. Cell resolution markedly improved at depths greater than 120 fim from the top of the slice surface. Cell filling and therefore soma resolution markedly improved with time (up to 20 following penetration). Consequently, results are presented for only impaled somata situated less than 100 fim from the bottom of the slice. Occasionally, in the most deeply situated cells (<50 fim from the bottom surface), dendritic images could be resolved and imaged (results not shown). Contaminating fluorescence from the microelectrode tip was found to be negligible. Test EPSPs in CAl resulting from stratum radiatum stimulation were monitored in 200 fim thick hippocampal slices, and were blocked within 3 to 5 min of perfusion of high Mg^"^ solutions within the imaging apparatus, as in the previous experiments. During synaptic block, depolarization and firing were induced postsynaptically by injection of depolarizing current pulses through the recording electrode. Resting [Ca^^] was 55+7 nM (n = 10) with the slice bathed in acsf and did not change when the bathing medium was switched to 25 mM Mg^+ and 2 mM Ca^"^ (n=4). Figure 5.8.1 illustrates the rise in soma [Ca^^] obtained when a 126 msec intracellular current pulse (1 *4 nA) was applied to make a cell fire repetitively with the slice bathed in acsf (+ , solid line) compared with the steady level in the absence of depolarization (■ , dotted line). There was a smaller but nevertheless substantial rise in cytosolic [Ca^^] when spikes were elicited after the slice was bathed in acsf with 25 mM Mg^"*" and 2 mM Ca^"^ (v, dashed line). As seen in the previous high Mg^'^

176 experiments, the firing threshold was raised in raised Mg^'*’, so an increased current was used to elicit 6 or 7 action potentials per pulse. The slice was reperflised with acsf, and after the spike firing threshold had returned to control levels, the fluorescence measurements were repeated. They showed the same resting cytosolic [Ca^^] (a , dotted line) as at the start of the experiment. Similar increases in cytosolic [Ca^^] were observed during depolarization and firing in acsf and then in high Mg^"^ ( x and v , respectively) to those in the first run. The rise in [Ca^^] was estimated per action potential by dividing the calcium change at the end of an action potential train by the number of action potentials in that train: the mean was 13 ± 2nM with the slice bathed in acsf (n = ll). The mean rise in [Ca^^l was reduced by 52% ± 6 % during exposure of the slice to 25 mM Mg^"*" and 2mM Ca^^ (n=7). In every case a higher depolarizing current was used in high [Mg^^] to overcome the raised threshold for firing, however the number of action potentials induced in the latter manoeuvre was always comparable. Measurements of cytosolic Ca^"^ were also carried out in 9 somata when slices were bathed in 15 mM Mg^"^ but with no added Ca^"^. Resting Ca^"^ levels did not change significantly during the perfusion. The rise in [Ca^‘^1 fell by 69% ± 4% when the bathing medium was switched from acsf (n=9). The difference of 17% in the rise of [Ca^^] per action potential in the two experimental bathing media (25 mM Mg^"^ and 2 mM Ca^"^, and 15 mM Mg^"^ and no added Ca^"^) was significant at p<0*05 (Student’s f-test, 2 tailed).

177 1 2 0

100

80 ce Ü 60

0 200 400 600 time (ms)

Figure 5.8.1. Measurements of intracellular Ca^'^ during intracellular conditioning using the Ca^"^ indicator FURA-2 iontophoresed postsynaptically.

Graph of soma [Ca^'*’] at rest and during injection of depolarizing current; the numbers in brackets refer to the order in which the measurements were made. At ^est, in the absence of electrical stimulation (1;a dotted line; 4 ;B , dotted line) the ris^ p ^ u c e ü ^ ^ a 126 ms current .pulse .eliciting 8 spikes per 0.8 nA pulse in acsf (2; 4-, solid line; 5; x . . . . J s SlTOWn that was,® / _ .f i / a i - Ac IrirS+n I, solid line). The rise/produced by 6-7 spikes per 1 *4 nA pulse in 25 mM Mg^^ 2 mM, Ca^^ (3; V, dashed line; 6; 0 , dashed line). The points at time zero are resting cytosolic calcium from ratio of fluorescence of intracellular FURA2 during excitation during 350 and 380 nm light. Other points are calculated from the fluorescence during steady 380 nm excitation. For each graph the current pulse was repeated 8 times at 15 s intervals, and the fluorescence changes averaged. After measurements 1-3, the slice was reperfused with acsf, and the excitability monitored until it returned to control conditions before measurements 4-6 were begun. The resting soma [Ca^^] did not change during the experiment. -69 mV.

178 CHAPTER 6

SECTION B: DISCUSSION

6.1 Introduction

A long-lasting decrease in strength of synaptic transmission is a component of some neural network models of associative memory (Sejnowski, 1977; Willshaw and Dayan, 1990). Algorithms of synaptic plasticity use, in addition to Hebbian rules of reinforcement of active connections, rules that require depression of certain synapses. The addition of such rules increases the efficiency of information storage systems by extending the capacity for learning new associations when the relationship between events changes. If synapses were only to increase in efficacy, as a result of LTP, saturation of learning would occur. LTD in the responsiveness of synapses could be the cellular analogue of the behavioural signs of habituation, extinction and forgetting (Morris and Willshaw, 1989). Both associative and non-associative forms of LTD have been shown to occur experimentally, as described in the Introduction to Section B (page 51) but LTD has not been studied with the same attention afforded to LTP, and little is yet known of its underlying mechanisms. Novel conditions under which non-associative LTD could be reliably produced in the hippocampus by postsynaptic activation in an anti-Hebbian paradigm have been described in this study. The findings confirm and extend those of previous workers (Lynch et al., 1977; Dunwiddie and Lynch, 1978; Levy and Steward, 1979; Abraham and Goddard, 1983; Pockett and Lippold, 1986; Pockett et al.^ 1990) and are discussed in detail below. It is concluded that aspects of the experimental paradigms used in this study to induce non-associative LTD may mimic the induction of heterosynaptic LTD in vivo. The more unphysiological experimental conditions are designed deliberately to analyze the underlying phenomena.

6.2 Antidromic stimulation in normal acsf

Stimulation at the alveus that was subthreshold for eliciting an antidromic spike.

179 often elicited a synaptic response which appeared either as an EPSP or IPSP, or a combination of both (an EPSP-IPSP sequence). The EPSP was probably due to stimulation of orthodromic fibres in the stratum oriens; the IPSP was probably a GABA- mediated recurrent IPSP as described in the cat (Andersen et al. , 1964a,b; Curtis et at. , 1970). Antidromic stimulation in other experiments that was suprathreshold for eliciting antidromic action potentials also evoked chloride-mediated IPSPs, which were also probably evoked by activating recurrent pathways through interneurones (basket cells) synapsing on the somata of pyramidal cells (Lorente de No, 1934; Andersen et at., 1964a,b; Lacaille er a/., 1987).

6.3 The postsynaptic induction of non-associative LTD by antidromic conditioning

Part of this thesis extends the work of Pockett et al. (1990) who observed LTD of peak EPSP amplitude following antidromic conditioning stimuli in normal acsf, and, in some of theiir experiments, following conditioning stimuli applied during transient synaptic block produced by a high [Mg^^]. In this thesis it was shown that the slope of the monosynaptic EPSP from stratum radiatum onto pyramidal cells in CAl is subject to LTD. By superimposing orthodromic test EPSPs on 200 msec depolarizing current pulses it was confirmed that the long-lasting decrease in synaptic transmission was of the initial excitatory input and not of a reversed IPSP. The decrease in EPSP slope was not likely to be due to a generalized decline in the slice preparation since in control experiments, where no conditioning stimuli were delivered, EPSP slope either stayed constant or increased during the recording period. Neither was the LTD due to incomplete replacement of the high Mg^^ solution with one containing normal [Mg^^], since in control experiments where resting potential and EPSP slope were stable for about 20 min prior to applying a high Mg^"^ solution, washout of high [Mg^^] restored the EPSP slope and spike amplitude, provided the V„, depolarization which occurred during drug perfusion recovered within 15 min. Failure of EPSP slope and spike amplitude to recover from high [Mg^^] washout, however, was observed in a few cells whose underwent prolonged and/or intense depolarization during the high [Mg^'*'] perfusion, but these cells were not included in the analysis. Monitoring of cell firing threshold and membrane potential confirmed that the LTD could not be a consequence of membrane depolarization but was a consequence of the

180 conditioning stimuli. The occasional cell that was adversely affected by the high [Mg^^] perfusion highlights the importance of using intracellular rather than extracellular recording for such a study. The LTD was not secondary to, or accompanied by a generalized change in cell Rjn of the CAl cells, since did not change as a result of conditioning, confirming and extending the findings of Pockett et al. (1990). This result is expected from evidence that LTP (which often involves firing of postsynaptic cells with a pattern of activity similar to that induced in this study as part of its induction) does not alter input resistance of postsynaptic cells in hippocampal area CAl (Andersen et al., 1977, 1980; Barrionuevo and Brown, 1985; Bliss et at., 1987) or CA3 (Barrionuevo et at., 1986). Furthermore, the synaptic depression described in this study cannot be a particularly long-lasting form of feedback inhibition (i.e. LTP of feedback inhibitory synapses) since the conditioning works even when it is delivered in the absence of synaptic transmission. In 3 cells, high frequency antidromic stimulation in normal bathing medium induced LTD in 1 cell, but LTP in the other 2. Pockett et al. (1990) reported a similar mixture of effects in hippocampal area CAl when antidromic conditioning trains were delivered in normal bathing medium at the alveus/oriens border, suggesting that antidromic conditioning delivered in normal bathing medium can induce LTD, but the induction is less reliable than in raised [Mg^^] solutions. If activation of a lasting form of feedback inhibition can be induced by conditioning in acsf, then this could possibly be one mechanism of postsynaptic depression. It was possible that enhancement of GABAergic transmission could have accounted for the LTD observed in that 1 cell, but superimposition of orthodromic test EPSPs on 200 msec depolarizing current pulses confirmed that the depression was of the initial depolarizing slope of the EPSP and not of a reversed IPSP. In the remaining 2 cells, antidromic conditioning in acsf could have activated the test synapses (which would presumably induce LTP) by back conduction along a branch of the afferent axons which had en passage synapses on the CAl cell. In other words, orthodromic pathways via axon branches could be activated by the antidromic conditioning stimuli, resulting in associative LTP. Alternatively, efferent axon collaterals activated during antidromic conditioning could excite synapses other than the test synapse via some putative excitatory feedback pathway (which may be present) and

181 thus produce non-associative LTP. A further alternative is that antidromic conditioning may have induced both LTP and LTD of different inputs in the 2 cells, and it could be that the expression of LTP masked the expression of LTD.

6.4 The postsynaptic induction of non-associative LTD requires extracellular Ca 2+

It was shown for the first time that the postsynaptic induction of non-associative LTD was dependent on extracellular Ca^"^. When there was no Ca^"^ included in the bathing medium, high frequency antidromic conditioning failed to induce LTD, in spite of depolarization and a high frequency of firing during stimulation. The implication of these results is that Ca^"^ entry into the postsynaptic cell during the conditioning trains is required for the induction of LTD. Since Aimers and Palade (1981) had shown that high Mg^"*" solutions can block voltage sensitive Ca^'^ channels, albeit weakly, it was checked whether an activity-dependent increase in intracellular [Ca^^] in a high [Mg^^] bathing solution containing 2 mM Ca^"^ could occur. The presence of medium and early slow after hyperpolarizing potentials (AHPs), which result from Ca^^-activated 1^ during and after trains of antidromic spikes (Schwindt et aL, 1988; Schwindt et al,, 1992), indicated that Ca^^ did enter the neurone in the presence of a raised extracellular [Mg^^]. This is confirmed by the fact that AHPs were considerably reduced following antidromic trains applied when the slice was bathed in the high [Mg^^] solutions with no added Ca^"^. The small residual AHP that was still seen could be due to residual Ca^^ outside the neurone due to insufficient washout of Ca^"^, or to Na"^- or Ca^"^ release from intracellular stores (Gustafsson and Wigstrom, 1983). Given that AHPs^ were small in the absence of added Ca^"^, it is not surprising that following antidromic conditioning V. there was a larger net somatic depolarization compared to that observed in 25 mM Mg?'*' and 2 mM Ca^"^ due to summation of DAPs, and therefore a greater probability of invasion of the soma by antidromic spikes. Furthermore, measurements of cytosolic Ca^^ using Fura-2 iontophoresed intracellularly confirmed that Ca^"^ did enter the cell during postsynaptic depolarization and firing in a medium containing 25 mM Mg^"^ and 2 mM Ca^^. The rise in cytosolic Ca^"^ was always less than in acsf for an equivalent number of action potentials per conditioning pulse, in spite of the increased depolarization required to reach firing

182 threshold when in raised (mean reduction 52 ± 6%, compared to normal acsf). Moreover, exposure of slices to a bathing medium containing 15 mM Mg^"*" and no added Ca^"^ for 5 min did not wholly prevent an activity-induced rise in cytosolic Ca^"^ concentration. It is not known whether this was due to residual Ca^^ entry from outside the neurone, or to a Na"^- or Ca^"^-induced Ca^^ release from intracellular stores (Gustafsson and Wigstrom, 1983). These results suggest that the threshold level of intracellular [Ca^^] required for the postsynaptic induction of LTD is greater than the activity-dependent increase observed when no Ca^"^ was added to the bathing medium. Furthermore, they also suggest that the intracellular Ca^^ level required for the induction of LTD may be within a narrow range (see also Brocher et al., 1992 for homosynaptic LTD). However, it is unlikely that somatic Ca^^ levels are representative of those at dendrites. Regehr et at. (1989) and Jaffe et a l (1992) showed, using Ca^"^ imaging, that there is a much larger increase in cytosolic Ca^"^ in the proximal part of the apical dendrite than in the soma after either afferent stimulation which increased the field population spike, indicating soma firing had occurred (Regehr et a l, 1989) or after injection of prolonged (0 • 5 sec) depolarizing current pulses which produced trains of Na"^ spikes (Jaffe et a l, 1992). This suggests that an uneven distribution of voltage- operated Ca^"^ channels (VOCCs) may exist along dendrites. However, they did not take the surface-volume ratio into account. This is vital because Ca^^ entering the cell body is diluted into a large internal volume relative to surface area. In contrast, proximal dendrites have a much smaller internal volume relative to their surface area. For an equal rate of Ca^"^ entry, the increase in cytosolic Ca^"^ will be much larger in the proximal dendrites than in the soma. In support of Regehr et a l (1989) and Jaffe et a l (1992), using monoclonal antibody staining of rat hippocampal sections, Westenbroek et a l (1990) reported that high-voltage activated (HVA) Ca^"^ channels are located in the cell bodies and proximal dendrites of hippocampal pyramidal cells, and are clustered in high density at the base of the apical and basal dendrites. They found that immunoreactivity was always observed along the proximal region of the apical dendrites, but did not usually extend for a measurable distance along the basal dendrites. However, one should not draw conclusions too hastily from such studies as there may also be substantial spatial (and temporal) variations in intracellular [Ca^^] regulation in different parts of neurones. Thus, factors such as the distribution of cytosolic Ca^^-

183 binding proteins (e.g. inositol phosphates, calmodulin and parvalbumin) (Blaustein, 1988; Meldolesi et al., 1988) also need to be addressed before attempting to extrapolate findings from Ca^"^ imaging studies to complicated neuronal processes such as LTP and LTD of synaptic transmission. The importance of Ca^"^ is discussed further below.

6.5 The postsynaptic induction of non-associative LTD by intracellular current pulses

The postsynaptic induction of non-associative LTD in high [Mg^^] solutions It was also shown that intracellular depolarization and firing can serve as an effective postsynaptic conditioning stimuli for the induction of LTD, when applied in a non-associative paradigm. This is a more discrete form of conditioning than high frequency extracellular stimulation of bundles of axons. Using intracellular conditioning stimuli it was found that pharmacological block of synaptic transmission in raised [Mg^^] during conditioning facilitated the induction of LTD. Conditioning current levels were used which are comparable to those employed in other laboratories as conditioning stimuli for the induction of LTP, when associated in time with synaptic inputs (Kelso et al., 1986; Gustafsson et al., 1987). Using the intracellular conditioning paradigm, the long-lasting decrease in synaptic transmission was also shown to be of initial excitatory EPSP slope and not due to a reversed IPSP.

The postsynaptic induction of non-associative LTD in glutamate receptor antagonists Another new finding was that non-associative LTD could be elicited postsynaptically by intracellular current pulses when the slice was transiently exposed to ionotropic glutamate receptor antagonists (CNQX with or without AP5) during the conditioning period. Such exposure would affect the postsynaptic actions of glutamate, rather than its release. Thus pharmacological reduction of synaptic transmission, rather than block of evoked release during conditioning, also facilitated the induction of LTD. The depression was not due to incomplete washout of drugs, and was a consequence of the conditioning stimuli. The LTD observed was once again shown to be of the initial EPSP depolarizing slope that was not contaminated by a reversed IPSP.

184 6.6 Intracellular conditioning in acsf with or without bicuculline methiodide

It was shown that LTD could be induced by intracellular conditioning stimuli in the presence of a GABAa antagonist, bicuculline methiodide. However, the incidence of LTD (in 3/9 cells) was not significantly different from that observed in the absence of the drug (0/5 cells). Thus, in these 9 experiments, bicuculline methiodide did not facilitate the postsynaptic induction of non-associative LTD, in contrast to the facilitation of homosynaptic or associative LTP (Wigstrom and Gustafsson, 1985; Artola and Singer, 1987; Artola et a l, 1990; Collingridge and Davies, 1990). A simple explanation for the inconsistent effect of perfusion of bicuculline methiodide could be a failure of the drug to cause sufficient disinhibition of the slices for LTD induction. This is unlikely since of the 3 cells which showed robust LTD, slices were perfused with 1 fiM bicuculline methiodide, but of the remaining 6 cells in LTD was not induced, 3 slices were perfused with 2 fiM concentration of the drug. Where LTD was observed in bicuculline, the depression was significantly different from the pre-conditioning level, and the magnitude was similar to that seen in the presence of high [Mg^^] and larger than that observed with glutamate antagonists. Additionally, it was found that once LTD of EPSP slope had been established, it could be subsequently reversed to LTP of synaptic transmission by afferent tetanization. Thus both LTD and LTP of synaptic transmission can be induced in the same cell by different patterns of cell firing. In the absence of synaptic block, potentiation of a long lasting form of feedback inhibition might be a possible mechanism for the postsynaptic depression, e.g. by enhanced GABAergic inhibition via GABA^ and/or GABAg receptors. The fact that LTD was observed in the presence of bicuculline methiodide suggests that enhanced GABAergic inhibition via GABA^ receptor was not the mechanism for the LTD. However, one cannot not rule out the possibility that enhanced GABAg receptor- mediated transmission may be a mechanism for the LTD. Superimposition of orthodromic test EPSPs on depolarizing current pulses revealed that, once again, the depression was of the EPSP initial depolarizing slope in these 3 cells. In the remaining 6 out of 9 experiments, LTD could not be induced by intracellular conditioning during drug perfusion, suggesting that partial block of inhibitory transmission does not markedly increase the probability of the postsynaptic

185 induction of non-associative LTD, and thus is less effective than perfusion with a raised or glutamate antagonists. When the results from all 9 experiments carried out in the presence of bicuculline methiodide were averaged, the reduction in the normalized EPSP slope across the group was 15% - smaller than that observed in both high [Mg^+] and glutamate antagonists, and not much greater than observed by Abraham and Wickens (1991). These workers reported that picrotoxin facilitated the induction of heterosynaptic LTD of the population spike in CAl, but found a depression of only a few percent in field EPSPs (4% or 8%) following conditioning applied to the alveus, or oriens, respectively. Although Abraham and Wickens (1991) reported that the LTD they observed was heterosynaptic LTD, it is possible that they also induced associative LTD which might occur if the antidromic conditioning they applied at the alveus excited the test synapses by back conduction along a branch of the afferent axons. The non-associative LTD observed in bicuculline methiodide in this thesis may be heterosynaptic, induced by intracellular conditioning exciting efferent axon collaterals which then activated some excitatory feedback pathway, thereby activating synapses other than the test synapses. Using field potential recordings however, one is unable to distinguish between a small LTD in the whole cell population, or a large LTD in a few of the cells. This further highlights the advantage of intracellular over extracellular recording for such a study. Unlike perfusion of slices with high [Mg^^] (which blocks synaptic transmission) or glutamate antagonists (which affects EPSPs and IPSPs indirectly), perfusion with bicuculline methiodide only blocks some GABA-mediated IPSPs (i.e. GABA^ and not GABAg) without blocking the affects of glutamate, and is therefore less interventionist pharmacologically. The presence of bicuculline methiodide throughout the duration of the experiments allowed the time-course of LTD induction to be observed, suggesting that the depression was induced immediately following intracellular conditioning, i.e. within 2 mins. Such a conclusion cannot be drawn in experiments in which time was required for washout of drugs to occur before LTD could be observed. The affect of blocking GABAg - mediated IPSPs on the postsynaptic induction of LTD remains to be investigated. One obvious voltage-dependent mechanism that could account for the non- associative LTD observed in bicuculline methiodide is the entry of Ca^^ through

186 VOCCs during conditioning. It is possible that in the 3 cells in which LTD was successfully induced, Ca^^ entry was sufficient to trigger the postsynaptic induction of non-associative LTE^ %hereas in the remaining 6 cells, the threshold of Ca^"^ entry for LTD induction was not reached. It is known that application of GAB A in the hippocampal CAl area depolarizes dendrites and hyperpolarizes somata (both affects are accompanied by an increased conductance) (Langmoen et al., 1978; Andersen et at., 1980; Alger and Nicoll, 1982, and see below). Therefore, one would assume that in the presence of the GABA^ antagonist there would be a reduction in both dendritic depolarization and somatic hyperpolarization (or a reduction in the clamping of the cell’s resting potential). What affect would this have on Ca^'^ entry into the postsynaptic neurone during conditioning? Firstly, the reduced dendritic depolarization would be expected to cut down on dendritic Ca^'*’ entry during conditioning. Secondly, Ca^"^ entry into the soma during conditioning would be expected to increase. ^The outcome of whether conditioning induced LTD, LTP or had no effect would depend on the amount of Ca^'*' entering the postsynaptic cell. In the 3 cells in which LTD was observed, the Ca^"^ entry may have been favourable for LTD induction. On the other hand, in the 6 cells in which LTD was not induced, Ca^^ entry may have been either too low for inducing LTD, or induced both LTP and LTD of different inputs that cancelled each other out. In normal acsf, intracellular conditioning pulses failed to induce any change in PSP slope in 5 cells, suggesting that there was no potentiation of recurrent GABAergic transmission via inhibitory circuits activated by the conditioning. However, once again intracellular conditioning may have induced non-associative LTD and LTP concurrently of different inputs, which cancelled each other out, resulting in no net effect on EPSP slope. If the latter explanation was correct, then intracellular conditioning in the presence of a bicuculline methiodide might be expected to tilt the balance towards the unopposed expression of non-associative LTP. However non-associative LTP was not observed in this study following intracellular conditioning in normal acsf with or without bicuculline methiodide.

6.7 How does block of synaptic activity facilitate the induction of LTD?

The key factor in the reliable postsynaptic induction of LTD in these experiments

187 was the pharmacological block or reduction of synaptic activity during induction. How could the block or reduction of synaptic activity enhance the probability of induction of LTD? Three possibilities are: 1) that block of IPSPs enhances calcium entry into the dendrites during conditioning; 2) that LTP, produced at some synapses when the conditioning stimulation is applied in acsf is prevented by synaptic block. Thus LTD induced postsynaptically in the presence of synaptic blockers is not masked by concurrent LTP at other synapses; 3) that Ca^^ entry into the postsynaptic cell in the presence of a high [Mg^^] is reduced to a range of intracellular [Ca^^] that favours the induction of LTD. It should be noted that possibilities 2) and 3) would not have to be mutually exclusive, and that a combination of both could occur, whereas 1) and 3) would be exclusive. Before elaborating on the first two hypotheses it would be helpful to review very briefly the postsynaptic actions of GAB A, thought to be the neurotransmitter released from the inhibitory interneurones, in the CAl region of the hippocampus.

6.8 Postsynaptic actions of G ABA in the hippocampal CAl region

As mentioned in the Introduction (page 20), a number of interneurones exist in the hippocampal cortex, some of which have been identified as being inhibitory and are thought to release GABA as their neurotransmitter (Ribak et al., 1978; Somogyi et al. , 1983). Several studies (Langmoen et al, 1978; Andersen et al, 1980; Alger and Nicoll, 1982) have shown that CAl cells in the rat or guinea-pig hippocampal slice hyperpolarize in response to the application of GABA near the soma accompanied by a reduction in (increase in conductance) and depolarise when it is applied over the dendritic tree, also accompanied by a fall in R^,. The local dendritic depolarization by GABA application produces a conductance increase locally, shunting neighbouring excitatory synaptic currents to give a local inhibition. The importance of the dendritic GABA effect lies in its ability to shunt the membrane resistance locally. This leads to an efficient decoupling of local synapses of both the depolarizing and hyperpolarizing variety. In addition to a reduction of local synaptic efficacy, dendritic GABA O application could, because of its synaptic depolarizing effect, assist other excitatory synapses lying sufficiently far away to escape the shunting effect.

188 Hypothesis 1 In the present study, synaptic block was used as a manoeuvre to induce LTD. Why should block of transmitter release favour LTD induction? One possibility is as follows: in acsf the antidromic or postsynaptically induced firing during the conditioning excites recurrent inhibitory circuits from alveus/oriens neurones to the pyramidal cells (Lacaille et al., 1987). The GABA released from inhibitory interneurones increases the postsynaptic soma membrane conductance, allowing soma current to be shunted out of the cell before it reaches the dendritic region of the test synapses. Hence during conditioning in acsf, Ca^^ entry into the dendrites that was adequate for LTD induction might not occur. Synaptic block during postsynaptic induction could facilitate Ca^'*' entry into the dendrites in the following way: when synaptic transmission is blocked with a raised [Mg^^], IPSPs are also blocked, thus increasing the membrane resistance and the space constant of the cell and enabling current spread along the dendrites to be increased during antidromic or intracellular conditioning and firing. Moreover it was found that a raised [Mg^^] reduced a resting membrane conductance and hence this presumably also increased the space constant. CNQX (with or without AP5), which did not affect the resting membrane conductance, would also block recurrent inhibition because glutamate excitation of the inhibitory interneurones is reduced or blocked by these antagonists (Andreasen and Lambert, 1991).

Hypothesis 2 The experimental basis for the second hypothesis is that antidromic conditioning trains gave rise to LTP of stratum radiatum EPSPs in a small proportion of experiments in the pharmacologically untreated slice in this study and that of Pockett et al. (1990). In the present experiments, spontaneous release of glutamate from stratum radiatum terminals could have activated glutamate receptors postsynaptically, and this activation may have been "paired" with the intracellular postsynaptic depolarizing pulses, thus leading to associative LTP. By blocking AMPA receptors postsynaptically, CNQX could indirectly block the increase in intracellular [Ca^'*'] required for the induction of non-associative LTP, which might uncover LTD. Alonso et al. (1990) reported that intracellular conditioning pulses in the absence of presynaptic activation could produce LTP in layer II entorhinal cortex cells. Both the

189 induction and expression of this apparently non-Hebbian or heterosynaptic form of LTP was blocked by AP5. These workers suggested that the presence of extracellular glutamate could be sufficient to activate NMDA receptors in the absence of afferent stimulation. In support of Alonso et al. (1990), Sah et al. (1989) had previously reported that endogenous glutamate exerts a tonic action on NMDA receptors in hippocampal area CAl, such that tonic activity may enhance the excitability of pyramidal cells. Thus the LTP observed by Alonso et al. (1990) may have been Hebbian and occurred as a result of coincident activation of glutamate receptors with injection of the depolarizing pulses, although the circuitry in the entorhinal cortex is not well understood. Furthermore, D. Kullman (personal communication) reported a non- Hebbian form of LTP, similar to that described by Alonso et al. (1990) could be induced in the hippocampal CAl region by injection of depolarizing current pulses in the absence of presynaptic activation. The reduction in EPSPs produced by a raised [Mg^^] or ionotropic glutamate antagonists during conditioning would reduce the likelihood of eliciting non-associative LTP which might result from activation of glutamate receptors by extracellular glutamate, thu^/allowing the unopposed expression of LTD. X The combined effect of the reduction of PSPs by a raised [Mg^^] or ionotropic glutamate receptor antagonists, plus the membrane depolarization and reduced membrane conductance in raised Mg^"^ might account for the much greater depression of the EPSP slope observed in the present study compared with the 4% reduction of field potential slope seen following alveus tetanization by Abraham and Wickens (1991) in the presence of picrotoxin. However, since slice perfusion with CNQX (with or without AP5), or bicuculline methiodide did not (i) completely block transmitter release, (ii) depolarize the membrane potential or (iii) increase cell resting input resistance, as found with high [Mg^^] perfusion, it can be deduced that these factors are not essential in the induction of LTD, although they may influence the incidence or magnitude of the depression observed.

6.9 Do different threshold levels of predict the direction of synaptic modification?

Effective conditioning, and presumably adequate Ca^'^ entry into the dendrites.

190 V

could be brought about in these experiments either by antidromic Na^ spikes with some soma depolarization due to summation of DAPs, or mainly by soma depolarization with a lower frequency of action potentials following depolarizing current pulses. In the measurements of cytosolic [Ca^^], it was found that it increased as a result of action potentials or (in the presence of tetrodotoxin) induced by depolarizing pulses alone, above a threshold value (unpublished observations; see also Ross et al., 1991; Jaffe et a l, 1992). The intriguing question that remains is how ^/s^jthat a high [Ca^^] in dendrites can give rise to LTP and a lower [Ca^'^j to LTD? The work of Artola et al. (1990), Kimura et al. (1990), Yoshimura et a i (1991), Brocher et al. (1992) and Hirsch and Crepel (1992) in neocortex, points to the crucial role played by Ca^"^ in the induction of LTP when it is at a high level due to a high level of postsynaptic activity, and of LTD when at a lower level, but above a certain threshold. In this study, measurements of cytosolic Ca^'*' using Fura-2 showed that less Ca^"^ entered the cell soma during postsynaptic depolarization and firing in a medium containing 25 mM Mg^"'’ and 2 mM Ca^^ than in normal acsf. How do the results of Artola et al. (1990) correlate with those of this study? If we are to accept the theory of Artola et al. (1990), how can we explain the apparent lack of LTP or LTD observed in acsf, although the somatic Ca^^ entry measured during conditioning in acsf was greater than in the presence of a raised [Mg^^] which did induce LTD? If one assumes that soma Ca^^ is a reasonable representation of the dendritic Ca^"^ level, then one would expect to observe LTD or LTP following conditioning in acsf. Alternatively, the results of this study suggest that the Ca^"^ entry that occurred in normal acsf was above the threshold for producing LTD, but below that for inducing LTP. Indeed, in this study it was shown that the rise of [Ca^‘^1 per action potential in the bathing medium containing 15 mM Mg^"^ and no added Ca^"*" was 17% less than in that containing 25 mM Mg^"^ and 2 mM Ca^"^, which suggests that there is a narrow divide between the conditions for LTP, LTD and zero effect. Furthermore, the data of other workers obtained using Ca^^ chelators in visual cortex also point to there being a narrow divide (Artola et al. , 1990; Kimura et al., 1990; Yoshimura et a l, 1991; Brocher et al .,1992; Hirsch and Crepel., 1992).

Hypothesis 3 One could propose that there is an intradendritic voltage or [Ca^^] threshold level

191 (i.e. [Ca^'*’] range) above that for LTD induction and below that required for the induction of LTP. If this level were reached in conditioning, it could result in neither LTD nor LTP induction. Alternatively, a [Ca^^] threshold might exist that if reached following conditioning, favoured the induction of both LTP and LTD of different synaptic inputs. In this case both LTD and LTP might occlude each other, and appear to the experimenter as an unsuccessful attempt to induce either phenomenon. In the presence of a high [Mg^^] solution during conditioning, postsynaptic Ca^^ entry could be reduced to a dendritic level that favoured LTD induction. Thus by blocking the dendritic depolarizing effect of GABA released from interneurones activated during conditioning, CNQX could also restrict Ca^^ entry postsynaptically to a level that favoured the induction of LTD. Figure 6.1 summarizes this hypothetical situation, which may explain why LTD did not occur following conditioning in acsf, although a greater level of Ca^"^ entry occurred in acsf than in the high [Mg^‘*‘] solutions. Lisman (1989) has proposed a model for the induction of Hebbian LTP and anti- Hebbian LTD that depends on the quantitative level of the postsynaptic rise in [Ca^^]. Lisman (1989) suggested that the synaptic weight of each synapse could be stored locally by the group of Ca^"^/calmodulin kinase II (Cam-kinase II) molecules contained in the postsynaptic density (Kennedy et al., 1983; Kelly et at., 1984), a cytoplasmic structure directly abutting the postsynaptic receptors within each dendritic spine (Siekevitz, 1985). The bidirectional control of synaptic weight could be brought about by the control of different enzymes by the postsynaptic [Ca^^]. A high elevation of Ca^'^ postsynaptically could lead to phosphorylation of CaM-kinase II, an increase in synaptic weight, and LTP, while a moderate level of Ca^'*' could activate protein phosphatase 1, an enzyme directly controlled by Ca^^ through reactions with phosphatase inhibitor 1, cAMP-dependent kinase, calcineurin, and adenylate cyclase. Protein phosphatase 1 dephosphorylates CaM-kinase II, and leads to a reduction of synaptic weight, i.e., to LTD. Conclusive evidence for a common mechanism for LTP and LTD is still lacking, however, because the complete or partial blockade of LTD may reflect mechanisms with a higher sensitivity to intracellular [Ca^^] than those underlying LTP. The test of these hypotheses will depend on the ability to measure EPSPs, together with localized changes in [Ca^^] (Muller and Connor, 1991; Guthrie et at., 1991) and related biochemical events at the region of the activated synapses and the

192 i LTP LTP/LTD cancel out + CM(0 Ü LTD No change

Figure 6.1. A model of how postsynaptic dendritic levels of intracellular Ca^^ may determine changes in the efficacy of synaptic transmission. The Ca^^ levels are arbitrary, and each box is drawn the same dimensions for clarity, although this may not be the case in reality.

193 examination of whether the postsynaptic cell could behave like an analogue computer that can store a synaptic weight and modify it in accordance with the Hebbian (associative) and anti-Hebbian (non-associative) learning rules.

6.10 The role of LTD in the hippocampus - possible behavioural correlates in vivo

The question remains as to the role of LTD in the modulation of synaptic efficacy in the hippocampus. Under physiological conditions, inputs from presynaptic pathways are rarely, if ever completely null because most neurones normally operating in the brain are more or less spontaneously active. In other words, the complete absence of inputs may take place under rather artificial conditions, such as in the slice preparation. How then can the non-associative LTD observed in this study in the pharmacologically treated slice be correlated to the possible situation in vivo in the absence of pharmacological manipulation? The use of a high [Mg^^] solution to block presynaptic transmission is of course unphysiological. However, the block of transmitter release was advantageous in this study because it allows the presynaptic limb of the mechanism of LTD induction to be eliminated and therefore allows the postsynaptic component to be studied in isolation. Furthermore, antidromic or intracellular activation of CAl cells would not occur in the normal hippocampus. However, these experiments show that long-lasting depression of synaptic transmission can be induced in CAl neurones, which appears consequent upon the depolarization and firing of the postsynaptic cell. In the dentate gyrus excitatory synaptic potentiation is an associative phenomenon requiring the correlated activity of excitatory afferents and sufficient postsynaptic excitation to increase the efficacy of the particular synapses involved (McNaughton et a l, 1978; Levy and Steward, 1979; Wigstrom and Gustafsson, 1983). In contrast, when a particular presynaptic afferent is inactive concomitant with strong postsynaptic excitation produced by the activity of converging excitatory afferents, the efficacy of the synapses made by the inactive afferent upon the active postsynaptic cell decreases (Levy and Steward, 1979; Desmond and Levy, 1988). Similarly, the non-associative LTD described in this study could be equivalent to heterosynaptic LTD in vivo whereby the postsynaptic driving (depolarization and firing) is brought about by separate converging inputs.

194 The observations that bursts of postsynaptic activity in the absence of presynaptic activity induce long-term depression of synaptic inputs may be important for understanding the modulation of synaptic efficacy in the hippocampus. The LTD described in this study could have a number of effects. For example, it could serve to enhance the relative efficacy of synapses being potentiated during LTP by depressing inactive synapses on the same cell. Depression of synaptic responsiveness could be manifest behaviourally as habituation, extinction and forgetting in vivo (Morris and Willshaw, 1989). It has been shown in this study that LTD could be reversed to LTP of synaptic transmission, suggesting that synapses onto the same cell in the hippocampus could be both potentiated and depressed, which could be most economically explained if LTP and LTD both shared the same common set of regulatory mechanisms. Mechanisms that could account for LTD induction include voltage-operated regulation of intracellular levels of Ca^"*" (Goldman et aL ,1990; Lisman, 1989) and phosphoinositide hydrolysis (Schoepp and Johnson, 1988, 1989; Schoepp et al, 1990a,b). Assuming that prior LTP does not protect against LTD, LTD could also prevent LTP from saturating a synapse, by ensuring that the efficacy of a synapse is turned down every time other synaptic pathways onto the same postsynaptic cell are potentiated. This latter point could make it difficult to conceive of LTP per se as a model for permanent memory storage. However, in the absence of information about the number of synapses required to encode a given item of information, the seriousness of the saturation problem remains to be assessed. Until then, the significance of both LTD and LTP for learning and memory can be studied by incorporating them into network models of hippocampal circuitry.

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