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Serotonergic modulation of evoked responses in the hippocampal formation of the rat Klancnik, Joseph Mario 1989

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SEROTONERGIC MODULATION OF EVOKED RESPONSES IN THE HIPPOCAMPAL FORMATION OF THE RAT BY JOSEPH MARIO KLANCNIK B.A., The University of British Columbia, 1984 A THESIS SUBMITTED IN PARTIAL FULFULLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Neuroscience Program) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1989 (c) Joseph Mario Klancnik, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of T^uj Q o/o^^j ^  Q% ^ j Q C t T 7 ^ The University of British Columbia Vancouver, Canada Date OCA- to / / i n DE-6 (2/88) ABSTRACT The diffuse ascending projections of serotonin-containing nuclei in the brainstem have suggested a role for these structures in the control of forebrain systems. The hippocampal formation (HF) receives dense serotonergic projections from the dorsal and medial raphe nuclei and contains high concentrations of serotonergic binding sites, and is therefore an appropriate region of the brain in which to study serotonergic processes. The purpose of the experiments discussed in this dissertation was to investigate the influence of the serotonergic system on the electrophysiological activity of the HF, with especial emphasis on the dentate gyrus (DG). In all of the experiments, evoked population responses were used as a measure of synaptic transmission. The first two series of experiments investigated the effects of serotonin and serotonergic compounds on evoked responses, in both in vitro and in vivo preparations. The third series of experiments investigated the role of the median raphe nucleus (MRN), the major source of serotonergic afferents to the HF, on synaptic plasticity in the DG. The first series of experiments, described in Chapter 2, compared the effects of serotonin, the 5-HT^ receptor subtype agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and the 5-HT2 receptor subtype agonist l-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) on electrophysiological responses in the dentate gyrus and CA1 regions of the HF, using the in vitro hippocampal slice preparation. Bath superfusion of either serotonin or 8-OH-DPAT was found to inhibit population responses in a dose-dependent manner in both regions, with a greater effect in the CAl. The effects of 8-OH-DPAT in both regions could be attenuated significantly by the serotonergic antagonist methysergide, as could the effects of 5-HT in the CAl. The application of DOI did not produce statistically significant effects in either region, although an inhibitory effect on DG responses was observed in some preparations. These findings support an inhibitory role for the 5-HT^ receptor in both the C A l and the DG, and possibly for the 5-HT2 receptor in the DG, indicating that the endogenous serotonergic system may serve to inhibit neural transmission in both of these regions. The second series of experiments, described in Chapter 3, examined the effects of serotonergic agents on electrophysiological responses in the DG in an anaesthetized, in vivo rat preparation. In contrast to the results of the in vitro experiments, intraperitoneal injections of either 5-hydroxytryptophan (5-HTP, the metabolic precursor to 5-HT) or 8-OH-DPAT were found to facilitate the amplitude of the population spike in the DG. Intravenous (i.v.) administration of 8-OH-DPAT. was also found to increase population spike amplitude (PSA), although an attenuated effect was observed at high doses. Increases in PSA were also observed following microinjections of 8-OH-DPAT either into the lateral ventricle, or directly into the vicinity of the median raphe nucleus (MRN), suggesting that the effects of 8-OH-DPAT observed in vivo are due largely to action on presynaptic 5-HT^ receptors located on serotonergic neurons. This suggestion was supported by the finding that pre treatment with the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) attenuated the effects of i.v. 8-OH-DPAT significantly. As 5 - H T r e c e p t o r s on the 5-HT neurons are thought to be inhibitory, these experiments indicate that 8-OH-DPAT acts to attenuate a tonic inhibitory influence which the MRN neurons exert on the DG. Intravenous administration of the 5-HT-^ ligands buspirone or BMY-7378 also appeared to produce dose-dependent increases in PSA, although these effects were found not to be statistically significant; intravenous administration of the 5-HT2 agonist DOI did not produce any discernable effects on population responses, indicating that the 5-HT2 receptor may not play a functional role in the DG. The experiments in Chapter 4 examined the possible modulation of synaptic plasticity in the DG by serotonergic afferents from the MRN. High-frequency (tetanic) stimulation of the major afferent projection to the DG, the perforant path (PP), is known to produce a long-lasting increase in synaptic efficacy at the PP-DG synapse, a phenomenon known as long-term potentiation (LTP), which has been considered as a possible biological substrate of memory formation. In the experiments in this section, the magnitude of LTP was found to be enhanced significantly when tetanic stimulation was applied to the MRN and PP concurrently, as compared to when tetanic stimulation was applied to the PP alone. These results indicate a modulatory influence of ascending serotonergic projections on synaptic plasticity in the HF. Together, the pharmacological experiments performed in vitro and in vivo indicate that the endogenous serotonergic system acts to inhibit synaptic transmission in the HF, and therefore possibly information processing in this forebrain structure. Furthermore, the facilitation of LTP by MRN stimulation suggests a role for the serotonergic system in the modulation of long-lasting changes in synaptic efficacy in the DG. As the activity of serotonergic neurons in the raphe nuclei is known to vary with levels of arousal, this could provide a mechanism by which the functioning of the hippocampus may be modified in acccordance with behavioural state. TABLE OF CONTENTS ABSTRACT ii LIST OF FIGURES viii ACKNOWLEDGEMENTS x LIST OF ABBREVIATIONS xi CHAPTER 1: INTRODUCTION 1 1.1. ANATOMY OF THE HIPPOCAMPAL FORMATION 2 A. The dentate gyrus 3 B. The hippocampus proper 3 C. Intrinsic neurons 7 1) Hippocampus 7 2) Dentate Gyrus 8 D. Afferents to the hippocampal formation 10 1) Cortical afferents 10 2) Septal afferents 11 3) Brainstem afferents 11 4) Other afferents 12 E. Intrinsic pathways 12 F. Commisural pathways 13 G. Efferent projections 14 1.2. EVOKED FIELD POTENTIALS IN THE HIPPOCAMPAL FORMATION 14 1.3. ANATOMY OF THE SEROTONERGIC SYSTEM 19 A. Serotonin-containing cell groups 19 B. Serotonergic projections to the hippocampal formation 21 C. Topography of projections from the dorsal and medial raphe nuclei 23 D. Distinctions between projections from the dorsal and medial raphe nuclei 24 1.4. SEROTONIN RECEPTORS 25 A. Neurotransmitter receptor theory 25 B. Serotonergic binding sites 28 C. Serotonergic receptors 30 1) 5-HT1A receptors 31 2) 5-HT1B receptors 34 3) 5-HT1C receptors 35 4) 5-HT2 receptors 36 1.5. PHYSIOLOGICAL EFFECTS OF 5-HT IN THE HIPPOCAMPAL FORMATION 37 A. Unit activity 38 B. Intracellular measures 38 C. Evoked field potentials 41 1.6. THE PRESENT STUDY 42 CHAPTER 2: IN VITRO PHARMACOLOGICAL EXPERIMENTS 45 2.1. Introduction 45 2.2. General Methods 45 A. Preparation of hippocampal slices 45 B. The in vitro chamber 47 C. Artificial cerebro-spinal fluid 47 D. Stimulation techniques 47 E. Recording techniques 48 vi F. Quantification of electrophysiological responses 49 G. Pharmacological experiments 50 2.3. The effects of 5-HT, 8-OH-DPAT and DOI on evoked potentials in the CA1 and DG in vitro 53 A. Introduction 53 B. Methods 55 C. Data analysis 56 D. Results 56 1) Controls 56 2) 5-HT 57 3) 8-OH-DPAT 57 4) DOI 59 2.4. Blockade of serotonergic effects by methysergide and ketanserin 74 A. Introduction 74 B. Results 75 2.5. Discussion 79 CHAPTER 3: PHARMACOLOGICAL EXPERIMENTS IN VIVO 84 3.1. Introduction 84 3.2. General methods: 89 3.3. The effects of 5-hydroxytryptophan on population responses in the DG 92 Introduction 92 Methods 92 Results 93 Discussion 93 3.4. The effects of 8-OH-DPAT on population responses in the DG 98 A. Intraperitoneal administration of 8-OH-DPAT 98 Introduction 98 Methods 98 Results - 98 Discussion 101 B. Intravenous administration of 8-OH-DPAT 101 Methods 101 Surgical preparation 101 Stimulation, recording, and drug procedures 102 Results 103 Discussion 110 C. Co-administration of 8-OH-DPAT with methysergide 111 Methods 111 Results 111 Discussion ' 112 D. Intracerebroventricular administration of 8-OH-DPAT 112 Introduction 112 Methods • 112 Results 115 Discussion 115 E. Intracranial administration of 8-OH-DPAT 116 Methods 116 Results 117 Discussion 117 F. Administration of 8-OH-DPAT in 5,7-dihydroxytryptamine treated animals 124 Methods 124 Results 125 Discussion 125 3.5. Experiments with buspirone 125 Introduction: 125 Methods 126 Results 126 Discussion 129 3.6. Experiments with BMY-7378 129 Introduction 129 Methods 130 Results 130 Discussion 130 3.7. Effects of DOI and ketanserin on DG population responses 133 Introduction 133 Methods 133 Results and discussion 133 3.8. DISCUSSION OF THE IN VIVO PHARMACOLOGICAL EXPERIMENTS 134 CHAPTER 4: MODULATION OF SYNAPTIC PLASTICITY IN THE DENTATE GYRUS BY STIMULATION OF THE MEDIAN RAPHE 141 4.1. Introduction 141 4.2. Methods 142 A. General procedures 142 B. Stimulation and recording procedures 143 C. Histology: 144 4.3. Results 144 A. Paired-pulse 144 B. Tetanic stimulation 144 C. Tetanic stimulation and paired-pulse 145 4.4. Discussion 152 CHAPTER 5: GENERAL DISCUSSION 156 REFERENCES 167 LIST OF FIGURES P. 5 Figure 1.1: A) Phantom drawing of the rodent brain illustrating the position of the hippocampal formation; B) Schematic diagram of a transverse section of the hippocampal formation, illustrating the principal cell types and internal circuitry P. 18 Figure 1.2: Representative population responses evoked in the DG of an intact animal by perforant path stimulation. P.52 Figure 2.1: Schematic diagram of the hippocampal slice preparation, illustrating electrode positions and representative evoked population responses. P.61 Figure 2.2: Representative population responses recorded from the CA1 and the DG, before and after superfusion with 5-HT, 8-OH-DPAT or DOI, in vitro. P.63 Figure 2.3: Dose-response to 5-HT of population responses, in vitro. P.65 Figure 2.4: Time course of the effects of 5-HT on population responses in vitro. P.67 Figure 2.5: Dose-response to 8-OH-DPAT of population responses, in vitro. P.69 Figure 2.6: Time course of the effects of 8-OH-DPAT on population responses in vitro. P.71 Figure 2.7: Dose-response to DOI of population responses, in vitro. P.73 Figure 2.8: Time course of the effects of DOI on population responses in vitro. P.78 Figure 2.9: Antagonism of the effects of 5-HT, 8-OH-DPAT, and DOI by methysergide and ketanserin, in vitro. P.87 Figure 3.1: Evoked responses in the DG of a single urethane anesthetized preparation. P.95 Figure 3.2: Representative DG responses evoked by PP stimulation, prior to and following i.p. injection of 5-HT and 8-OH-DPAT. P.97 Figure 3.3: The effects of i.p. injection of 5-HTP on evoked population spike amplitude in the DG. P. 100 Figure 3.4: The effects of i.p. injection of 8-OH-DPAT on evoked population spike amplitude in the DG. P. 105 Figure 3.5: Mean population spike amplitudes recorded after i.v. injection of 8-OH-DPAT. P. 107 Figure 3.6: The effects of i.v. administration of 8-OH-DPAT on evoked responses in the DG. P. 109 Figure 3.7: Modulation of. late paired-pulse inhibition in the DG by i.v. administration of 8-OH-DPAT. ix P. 114 Figure 3.8: The effects of intraventricular administraion of 8-OH-DPAT on evoked responses in the DG. P. 119 Figure 3.9: Representative responses evoked in the DG before and after microinjection of 8-OH-DPAT into the median raphe nucleus. P. 121 Figure 3.10: Responses evoked in the DG of one animal after microinjection of 8-OH-DPAT into the median raphe nucleus. P. 123 Figure 3.11: Mean responses evoked in the DG after microinjection of 8-OH-DPAT into the median raphe nucleus. P 128 Figure 3.12: The effects of i.v. administration of buspirone on evoked responses in theDG. P. 132 Figure 3.13: The effects of i.v. administration of BMY-7378 on evoked responses in the DG. P. 147 Figure 4.1: The.effects of MRN stimulation on the induction of LTP in the DG: representative responses. P. 149 Figure 4.2: The effects of MRN stimulation on the induction of LTP in the DG: mean values. P. 151 Figure 4.3: Facilitation of PP-evoked population spike amplitude by prior stimulation of the MRN. ACKNOWLEDGEMENTS Much gratitude is owed to many people who provided their help and friendship over the years. First, to my thesis advisors: Ken Baimbridge, for his encouragement, hard work, and lubrication of the wheels of academia; and Tony Phillips, for his continuous support over the years. To Andy Obenaus, Lon Myronuk, and of course Stella Atmaja, technician extrordinaire, for their assistance with experiments. To Tom Richardson, for the many valuable hours we spent at the chalkboard together, and to Charles Blaha, for much discussion, and for some of the drugs used in Chapter 3. To Tim Harpur, for his statistical expertise. To Scott Mendelson, who introduced me to serotonin (maybe I'll forgive him someday). To Jim Miller, who originally talked me into neuroscience ("it's closer to the edge than racing hydroplanes.") To Mr. Yukio Kawahara and Jerry Garcia, who provided both inspiration and recreation. Of course, graduate school would not have been nearly as interesting, or bearable, without the many graduate students in psychology, physiology, and neuroscience, who provided friendship and ideas. I'll miss you all. Last and most, I'd like to thank Emma, my wife, who put up with it all. xi LIST OF ABBREVIATIONS ACh acetylcholine ACSF artificial cerebrospinal fluid AHP afterhyperpolarization ANOVA univariate analysis of variance AP anterior-posterior BSC below the surface of the cortex CCK cholecystokinin ChAT cholineacetyltransferase CNS central nervous system CSF cerebrospinal fluid C-T conditioning-test DG dentate gyrus DGC dentate granule cell DOB l-(2,5-dimethoxy-4-bromophenyl)-2-aminopropane DOI l-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane DOM 2,5 dimethoxy-4-methylamphetamine DRN dorsal raphe nucleus E E G electroencephalographic EPSP excitatory post-synaptic potential ER entorhinal cortex GABA gamma-amino butyric acid gCl chloride conductance gK potassium conductance HF hippocampal formation HPC hippocampal pyramidal cell id inside diameter IL immunolike IPSP inhibitory post-synaptic potential IP 3 inositol-l,4,5-trisphosphate IR immunoreactive i.e.v. intracerebroventricular i.p. intraperitoneal i.v. intravenous L lateral LSD lysergic acid diethylamide LTP long-term potentiation LV lateral ventricle MANOVA multivariate analysis of variance MAP mean arterial pressure mCPP metachlorophenylpiperazine MDA methylenedeoxyamphetamine MDMA methylenedeoxymethamphetamine MRN median raphe nucleus MS medial septum NCS nucleus centralis superior NE norepinephrine NELLP norepinephrine-induced long-lasting potentiation NLI nucleus linnearis intermedialis NMDA n-methyl-d-aspartate xii od outside diameter PCA parachloroamphetamine PI phosphoinositide PP perforant path PSA population spike amplitude SG stratum granulosum SL stratum lacunosum SL-M stratum lacunosum-moleculare SM stratum moleculare SO stratum oriens SP stratum pyramidale SR stratum radiatum SS somatostatin T c (test PSA/conditioning PSA) X 100% TFMPP triflouromethylpiperazine V ventral VIP vasoactive intestinal polypeptide 5,7-DHT 5,7-dihydroxytryptamine 5-HT 5-hydroxytryptamine, serotonin 5-HTP 5-hydroxytryptophan 8-OH-DPAT 8-hydroxy-2-(di-n-propylamino)tetralin CHAPTER 1: INTRODUCTION A number of influential theories of brain function have emphasized the role of brainstem nuclei in the control of forebrain systems, and in the modulation of such behavioural phenomena as consciousness, arousal and emotion. These ideas originated in studies on sleep by Mauthner (1890) and Von Economo (1929), and were crystalized into the concept of an "ascending reticular activating system" (Lindsley, 1960) following a landmark paper published by Moruzzi and Magoun in 1949. These latter authors reported that stimulation of the reticular formation, a diffuse and rather ill-defined region of the brain stem, could produce behavioural arousal and wakefulness, as well as activation and desynchronization of cortical electroencephalographic (EEG) potentials. The work of Moruzzi and Magoun was extended by Green and Arduini in 1954, who reported that brainstem stimulation could influence the EEG activity of both neocortical and archicortical structures. Advances in neuroanatomical techniques have permitted the characterization of brainstem nuclei on a neurochemical basis, one example being the identification of discrete regions containing monoamine compounds (Dahlstrom and Fuxe, 1964). Cells containing norepinephrine (NE) were found to be located principally within the locus coeruleus, those containing 5-hydroxytryptamine (5-HT, serotonin) within the raphe nuclei, and those containing dopamine (DA) within the substantia nigra and the ventral tegmental area. Neurons in these regions have been shown to project diffusely to almost the entire central nervous system (CNS), and therefore appear able to exert widespread influence on brain function (Carlson, 1987). The serotonergic system of the brain has been implicated in the regulation of a large number of physiological and behavioural processes. Research into the processes mediated by 5-HT has been facilitated greatly by the recent discovery of multiple binding sites for 5-HT in the brain. These binding sites appear to represent subtypes of neuronal receptors which may mediate the various physiological actions of 5-HT. Identification of these sites has stimulated the development of selective serotonergic ligands, and the use of these ligands as tools in the investigation of processes mediated by 5-HT. The hippocampal formation (HF) is an appropriate region of the brain in which to study serotonergic processes. The HF is a phylogenetically old region of the cortex which is innervated densely by 5-HT-containing fibres and contains very high concentrations of serotonergic binding sites (Pasquier and Reinoso-Suarez, 1978; Kohler and Steinbush, 1982). Physiological and biochemical studies indicate that some of these hippocampal binding sites serve as functional receptors. The experiments described in this thesis have examined the role of the serotonergic system in the modulation of neuronal transmission in the HF of the rat, with particular emphasis on the dentate gyrus (DG) of the HF. The primary aim of the experiments was to delineate the effects of specific serotonergic agents in both in vivo and in vitro preparations, and so to investigate the influence of serotonergic receptor subtypes on the functioning of the DG. Accordingly, this introduction will provide an overview of the HF, the central serotonergic systems, and the known effects of 5-HT on the HF, and will conclude with a description of the empirical studies undertaken in the studies in this dissertation. 1.1 ANATOMY OF THE HIPPOCAMPAL FORMATION The hippocampal formation is a phylogenetically old bilateral structure, which in the rat is situated beneath the temporal cortex, subjacent to the ventral wall of the lateral ventricle. The HF is essentially a three-layered cortical structure, composed of an intermediate cell-dense layer situated between two cell-sparse laminae. It is continuous with the five-layered neocortex via a transition zone known as the subiculum. The HF can be subdivided into two distinct, although heavily interconnected areas: the "hippocampus proper", and the dentate gyrus. These two regions are each composed of a sheet of neural tissue. During development, these sheets fold in such a way as to form, in cross-section, two interlocking "U"-shaped structures. A striking feature of these sheets of neurons is their high degree of laminar organization, not only with respect to the cell bodies of the principal neurons, but also their cellular processes, all of which constitute well defined somatic, dendritic, and projection regions (Figure 1.1). A. THE DENTATE GYRUS The dentate gyrus is organized in a distinct laminar arrangement, folded into two symmetrical regions known as the dorsal and ventral blades. The principal neurons of the DG are the dentate granule cells (DGCs). These are small (7um diameter) spherical cells, densely packed in a layer known as the stratum granulosum (SG) (Ramon y Cahal, 1898; Lorente de No, 1934; Seress and Pokorny, 1981). In the rat, these neurons extend dendritic processes only from their superior aspect. These apical dendrites extend through the stratum moleculare (SM), branching profusely as they progress toward the external surface of the DG (in primates, including man, basal dendrites are also observed on some of the DGCs (Seress and Mrzljak, 1987)). The axons of the DGCs, known as the "mossy fibres" due to the morphology of their terminal excrescences, arise from the basal pole of the soma, and project to the hippocampus proper through the hilus, which is the region enclosed by the blades of the DG (Blackstad et al., 1970; Gaarskjaer, 1978). B. THE HIPPOCAMPUS PROPER The hippocampus proper is often referred to also as "cornu Ammonis" by a fanciful morphological analogy to the horn of the Egyptian god Ammun Kneph (Duvernoy, 1988). In the literature, the use of the term "hippocampus" is somewhat inconsistent. 4 Figure 1.1 A) Phantom drawing of the rodent brain illustrating the position of the hippocampal formation (Redrawn from Andersen et al., 1971). B) Schematic diagram of a transverse section of the hippocampal formation, illustrating the principal cell types and internal circuitry (Adapted from Lorente de No, 1934). S. Ori.: Stratum Oriens S. Pyr.: Stratum Pyramidale S. Rad.: Stratum Radiatum S. Lac: Stratum Lacunosum S. Mol.: Stratum Moleculare Gran. L.: Granular Layer Mol. L.: Molecular Layer Poly. Layer: Polymorphous Layer B.C.: Basket Cell PP: Perforant Path S.C.: Schaffer Collateral Subic: Subiculum A. CA 1 (Regio Superior) Some authors use the term to refer to the cornu Ammonis, and others to the entire hippocampal formation, sometimes including the subicular region. In this thesis, the term "hippocampus" will be synonymous with "hippocampus proper". The hippocampus is organized in a lamellar fashion similar to the DG. The principal neurons in the hippocampus are large pyramidal cells, whose somata are arranged in a tier about 3 or 4 cell bodies thick in the stratum pyramidale (SP). Each pyramidal cell gives rise to two sets of dendritic processes, which project perpendicularly to the cell layer. The basal dendrites arise from the inferior aspect of the neuron in two or more primary trunks, and branch several times as they spread through the stratum oriens (SO) in a direction perpendicular to the cell field. Apical dendrites arise from a single process from each pyramidal cell, on the side of the neuron opposite to the basal dendrites. These apical dendrites form secondary branches in the stratum radiatum (SR), and tertiary branches further from the neuron, in the stratum lacunosum (SL). These processes terminate in the stratum moleculare (SM), which abuts the superior margin of the hippocampus proper, and at this point the apical dendrites branch in a lateral direction. (Lorente de No, 1934; Ramon y Cajal, 1898; Westrum and Blackstadt, 1962). The SL and SM are not well differentiated in the rodent, and are often referred to jointly as the stratum lacunosum-moleculare (SL-M). Each pyramidal cell projects an axon from the basal pole of the soma. These axons extend through the SO, then turn in a direction parallel to the cell fields to form the alveus, a sheet of fibres which extends along the ventricular surface of the hippocampus. The hippocampus also gives the appearance of being folded in half, in a direction opposite to that of the DG. Ramon y Cajal (1898) differentiated the dorsal and ventral limbs of this region into the "regio superior" and "regio inferior", respectively. Later Lorente de No (1934), subdivided the pyramidal cell fields, into regions CA (cornu Ammonis) 1 through 4, on the basis of morphological and cytoarchitectural criteria. The C A l region comprises the region closest to the subiculum, roughly equivalent to the regio superior of Lorente de No. The CA3 region includes the region in the bend of the pyramidal cell sheet, along with much of the regio inferior, while area CA4 refers to a much looser aggregate of large cells closest to, and entering the hilus. The cytoarchitecture of the pyramidal cells gradually changes along the. hippocampus from the C A l to the CA4: the somata of the pyramidal cells become larger and less densely packed, while their dendrites become shorter and thicker. C. INTRINSIC NEURONS 1) HIPPOCAMPUS Interneurons of diverse morphology are found in the hippocampus, with the majority of these being non-pyramidal cells which apparently use gamma-amino butyric acid (GABA) as an inhibitory neurotransmitter (Curtis et al., 1970). Many of these are the histologically defined "basket cells", so named because their axons form dense basket-like plexi around the somata of pyramidal cells (Ramon y Cajal, 1921; Lorente de No, 1935; Schwartzkroin and Kunkel, 1985). These interneurons maintain reciprocal connections with the pyramidal cells and appear to mediate both feed-forward and recurrent inhibition (Buzsaki, 1984; Alger and Nicoll, 1982a; Harris and Landis, 1986; Schwartzkroin and Kunkel, 1985; Schwartzkroin and Knowles, 1985). The basket cells demonstrate a considerable amount of both convergence and divergence in their synaptic connections, so that each interneuron may receive inputs from up to 500 hippocampal pyramidal cells (HPCs) and may project to many more (Andersen, et al., 1969). GABA-immunoreactive (GABA-IR) neurons are found in all strata of the hippocampus, but are particularly abundant in the SP, SR and SL-M (Sloviter and Nilaver, 1987). Populations of interneurons in the C A l have also been labelled with antisera to choline acetyltransferase (ChAT) (Frotscher et al., 1986; Geneser, 1987), somatostatin (SS), cholecystokinin (CCK) and vasoactive intestinal peptide (VIP) (Sloviter and Nilaver, 1987; Morrison et al., 1982; Kohler and Chan-Palay, 1983a). The SS-immunolike (SS-IL) neurons are localized primarily to the SO and SP. The distribution of CCK-IL and VIP-IL neurons is similar to that of the GAB A containing cells, and evidence suggests that CCK and VIP may be colocalized with GAB A (Sloviter and Nilaver, 1987). 2) DENTATE GYRUS The DG also contains several types of interneurons, both in the vicinity of the DGCs and in the hilar region. The primary interneurons in the DG are basket cells, which are situated largely within the SG or in the inner regions of the SM, and show GABA-like immunoreactivivy (Amaral, 1978; Ribak and Seress, 1983; Sloviter and Nilaver, 1987). In an analogous manner to the pyramidal cell regions, the axons of these cells form basket-like plexi terminating on the somata of the DGCs (Ramon y Cajal, 1898, Lorente de No, 1934; Kosaka et al., 1984). These basket cells receive excitatory inputs both directly from the perforant path and from recurrent collaterals of the mossy fibres (Leranth and Frotscher, 1983; Amaral, 1979; Storm-Matiesen et al., 1983), and apparently mediate both recurrent and feed-forward inhibition onto the DGCs (Buzsaki, 1984; Alger and Nicoll, 1982a; Ribak et al., 1986; Andersen et al., 1969, Lomo 1971; Lubbers and Frotscher, 1988). CCK-IL and VIP-IL neurons are also found in the DG; these CCK-IL cells may comprise a subset of the GABAergic basket cells. Up to 21 morphologically different types of intrinsic neuron have been identified in the hilus, which can be divided into the "polymorphic zone" immediately subjacent to the GL, and deep regions of the hilus (Ramon y Cajal, 1898; Lorente de No 1934; Amaral, 1979); these hilar neurons are generally larger than DGCs. As in the blades of the DG, the hilus contains basket cells (Ramon y Cajal, 1921; Lorente de No, 1934) which demonstrate immunoreactivity to GABA (Seress and Ribak, 1983). Other well-described cell types in the hilus include fusiform cells, which lie in a band subjacent to the granule cells (Ribak and Seress, 1988), and the large, acetylcholinesterase-containing mossy cells, whose dendritic arborizations are mainly contained within the hilus (Amaral, 1979; Ribak et al., 1985). Both of these neuronal types are contacted by axon collaterals from the DGCs and project largely through the hippocampal commissure (Ribak et al., 1986; Seroogy et al., 1983), with the mossy cells innervating the inner molecular layers of both hippocampi (Amaral 1978, Ribak et al., 1986). SS-IL cells are numerous in the depths of the hilus; these appear to be distinct from those containing GABA, and appear to project to the outer ML of the DG (Zimmer et al., 1983; Bakst et al., 1986; Sloviter and Nilaver, 1987) . Many of the hilar cells receive synaptic inputs not only from the DGCs, but also from the contralateral hippocampus, from cholinergic inputs, and from other GABAergic neurons (Leranth and Frotscher, 1987; Seress and Ribak, 1984; Freund and Antal, 1988) . The variety of excitatory and inhibitory inputs onto these interneurons are in a position to modulate the powerful inhibitory influences within the hippocampal formation. Non-pyramidal cells in the hippocampus and hilar cells in the DG have been classically thought of as interneurons. Recent evidence, however, contradicts this view. It appears that both GABAergic and non-GABAergic populations of these neurons project out of the HF (Shinoda et al., 1987), to the septal nuclei (Schwerdtfeger and Buhl, 1986; Lubbens and Frotscher, 1987; Chronister and DeFrance, 1979) and nucleus accumbens (Hayes and Totterdell, 1985; Totterdell and Hayes, 1987). In addition, hilar cells are known to project not only to the ipsilateral DGCs, but also to the contralateral dentate gyrus (Seress and Ribak, 1983; Ribak et al., 1985b; Alonso and Kohler, 1982). D. AFFERENTS TO THE HIPPOCAMPAL FORMATION 1) CORTICAL AFFERENTS The major source of afferents to the hippocampal formation is the entorhinal cortex (ER). The predominant projection from this region is the perforant path (PP). This fibre tract arises from layer II of the ER, travels within the angular bundle, passes through ("perforates") the subiculum and hippocampal fissure, and terminates in the DG and regio inferior (Ramon y Cajal 1898; Lorente de No, 1934; Steward and Scoville, 1972; Nafstad, 1967; Blackstad, 1956b). In the DG, the PP terminates on the outer two-thirds of the ML in a marked topographic arrangement, with fibres from the more lateral portions of the ER projecting to more distal dendritic regions (Steward and Scoville, 1972; Steward, 1976; Hjorth-Simonsen and Jeune, 1972; Hjorth-Simonsen, 1972). A smaller proportion of PP fibres enters the regio inferior, and terminates in the inner SL-M of the CA3 and CA4 regions, with a similar topographic projection to these dendritic fields as in the ML of the DG (Andersen, 1971b; Raisman, et al. 1965; Hjorth-Simonsen and Jeune, 1972; Steward, 1976). Further topographic organization of the PP is evident in that more dorsal regions of the ER project to more caudal regions of the hippocampal formation (Hjorth-Simonsen and Jeune, 1972). The PP is almost exclusively ipsilateral, but a minor crossed pathway does exist which appears to project in a manner similar to the ipsilateral fibres (Zimmer and Hjorth-Simonsen, 1975; Goldowitz et al., 1975). A second projection, the temperoammonic path, arises in layer III of the ER, and terminates in the apical dendritic fields of the C A l region (Blackstad, 1956b; Raisman et al., 1965; Steward et al., 1973, 1974; Steward, 1976). Unlike the PP, the temperoammonic path projects almost equally to both ipsilateral and contralateral targets. Both pathways from the ER to the hippocampal formation putatively use glutamate as a neurotransmitter (Storm-Matiesen and Ottersen, 1984). The lateral, but not medial, divisions of the PP and the temperoammonic path demonstrate enkephalin immunoreactivity, as do their cells of origin in layers II and III of the ER and their terminal fields (Gall et al., 1984), indicating a possible neurotransmitter role for opioid peptides in these fibres. 2) SEPTAL AFFERENTS Studies utilizing anatomical, histochemical and pharmacological techniques provide strong evidence for major projections from the medial septum and the diagonal band of Broca to the hippocampus (Lynch et al., 1972; Rose, Hattori and Fibiger, 1976, Swanson and Cowan, 1977; Meibach and Siegel, 1977a). Both cholinergic and non-cholinergic fibres from these nuclei enter the hippocampus through the fornix, and terminate on postsynaptic elements in all cell fields (Chandler and Crutcher, 1983). The cholinergic fibres appear to project most densely to the SL-M of the C A l , and the middle ML of the DG, and to areas immediately ventral to the SP in the C A l and just inferior to the SG in the DG (Nyakas et al, 1987). A recent elegant study (Freund and Antal, 1988) has demonstrated that GABA-containing afferents from the septum specifically innervate most of the GABA-containing interneurons in the C A l , CA3 and DG. 3) BRAINSTEM AFFERENTS Both the pyramidal cell regions and the DG also receive significant projections from various brainstem nuclei, including the locus coeruleus, hypothalamus, substantia nigra and the medial and dorsal raphe nuclei. Projections from the raphe nuclei will be discussed in a later section. Norepinephrine-containing fibres originate in the locus coeruleus, project through the. dorsal noradrenergic bundle, enter the HF via both the fornix and the cingulum bundle, and terminate in the HF largely in the SL-M of the CA1-CA2 region, and in the SM and granular layer of the DG (Falck et al., 1962; Fuxe, 1965; Ungerstedt, 1971; Moore, 1975, 1978; Moore and Halaris, 1975; Pasquier and Reinoso-Suarez, 1978). Projections from hypothalamic neurons to the hippocampal formation travel within the fornix and fimbria, and terminate largely in the depths of the hilar region (Pasquier and Reinoso-Suarez, 1978). The HF also receives minor dopaminergic projections from the substantia nigra pars compacta and the ventral tegmental area (Bischoff, 1986). 4) OTHER AFFERENTS Direct connections appear to exist between several other brain regions and the hippocampal formation. Afferent fibres arising from the cingulate gyrus as well as the temporal and prefrontal regions of the cortex have been described (Schwerdfeger, 1979; Duvernoy, 1988). Fibres from the nucleus reuniens of the thalamus appear to project to the hippocampus, although not to the dentate gyrus (Yanagihara et al., 1987), while neurons in the medial amygdala extend fibres to ventral regions of the hippocampal formation (Saunders et al., 1988). E. INTRINSIC PATHWAYS The axons of the DGCs, the mossy fibres, pass in a compact bundle through the CA4 and CA3 areas. These fibres then make extensive en passent synaptic connections in the SR of these regions with the apical dendrites of the pyramidal cells, as well as with basket cells and mossy cells (Ramon y Cajal 1898; Lorente de No, 1934; Blackstadt et al., 1961; Frotscher, 1985; Lubbers and Frotscher, 1987; Ribak and Seress, 1988). The pyramidal cells in the CA3 region give rise to major axon collaterals, which were first described by the Hungarian neuroanatomist K. Schaffer in 1892. These Schaffer collaterals course through the stratum radiatum of the C A l region, where they make en passent synaptic contact with C A l pyramidal cells. The major connections of the hippocampus are often considered to form a tri-synaptic loop: perforant path to dentate granule cells, mossy fibres to CA3 pyramidal cells, and Schaffer collaterals to C A l pyramidal cells. All of the synaptic contacts in this tri-synaptic circuit are excitatory, and all appear primarily to use excitatory amino acids, such as glutamate or aspartate, as neurotransmitters (Storm-Matiesen and Ottersen, 1984; White et al., 1977; Crunelli et al., 1983a; Nadler et al. 1976); these may be co-localized with opioid peptides in the PP, temperoammonic pathway, and the mossy fibres. The flow of information through the hippocampal formation is often considered as unidirectional, passing through the well-defined intrinsic circuitry from DG to CA3 to CAl; a relatively small, but significant projection extends from the cells of the CA3 and CA4 regions to the inner third of the ML of the DG (Gaarskjaer, 1978). F. COMMISSURAL PATHWAYS In addition to the intrinsic circuitry, and to the afferents from external nuclei, the hippocampi communicate with each other via the psalterium, or hippocampal commissure. As with most of the intrinsic connections of the hippocampal formation, these commissural projections terminate in discrete zones. Commissural projections from the DG terminate in the inner one-third of the ML of the contralateral DG; these fibres most likely originate from neurons in the dentate hilus (Segal and Landis, 1974; Ribak et al., 1986). Fibres from area CA3 terminate in the contralateral SO of area CA3 and SO and SR of area C A l (Blackstad, 1956a). The exact topography of the commissural projections, however, appears to vary considerably across species (Van Groen and Wyss, 1988). G. EFFERENT PROJECTIONS The majority of projections exiting the HF arise from the pyramidal cells. The pyramidal cell axons comprising the alveus project in both medial and lateral directions. The lateral projections course into the fimbria, a fibre bundle which runs along the lateral aspect of the hippocampus, and then become the pre-commissural fornix ("pre-commissural" as it projects anteriorly to the anterior commissure), and projects primarily to the lateral septal nucleus and hypothalamus (Raisman et al., 1965; Meibach and Seigal, 1977b; Swanson and Cowan, 1977). The medially directed fibres of the alveus project to the subiculum. This. structure is the source of the post-commissural fornix, which connects the hippocampus to anterior and intralaminar nuclei of the thalamus and to the mammilary bodies. Projections from the subiculum innervate virtually the entire cortical mantle (Swanson and Kohler, 1984), including a large projection to the ER, which, therefore, completes a cortico-hippocampal circuit. Additionally, some evidence indicates a direct projection from the C A l pyramidal cells to the medial prefrontal cortex (Ferino et al., 1987). 1.2. EVOKED FIELD POTENTIALS IN THE HIPPOCAMPAL FORMATION In the hippocampal formation, as in other brain regions, one can record from individual neurons, monitoring either their unit activity or membrane potentials. The relatively simple and well defined anatomical architecture and electrophysiological characteristics of the HF also lend it to the recording of interpretable field potentials, as both pyramidal and granular cells are arranged with their somata and dendritic processes in homotopic layers. In each region of the hippocampus, it is possible to stimulate afferent fibres to the cell fields and to record meaningful extracellular potentials generated by the synchronous responses of a large population of neurons. Population responses may be evoked in the DG, CA3 or CAl, by stimulation of a number of extrinsic, intrinsic, or commissural fibres. The generation and recording of field potentials evoked in the DG by stimulation of the PP is covered in detail below (see Figure 1.2); similar principles apply to the generation of field potentials in other regions of the HF. Electrical stimulation of the perforant path results in a synchronous depolarization of the dendrites of a large population of DGCs, presumably by the synaptic release of an excitatory amino acid such as glutamate or aspartate. The ionic currents which induce this intracellular depolarization also produce an extracellular hyperpolarization, which can be recorded as a slow negative potential in the extracellular space of the SM. An extracellular electrode positioned in the SG will record a concurrent slow positive potential, representing the source current for the dendritic potentials. These slow potentials, termed "population EPSPs", can be quantified to indicate the strength of synaptic transmission. At stimulus intensities high enough to generate action potentials in DGCs, a sharp negative-going potential appears, superimposed upon the slower, positive wave recorded in the SG. This negative potential, the "population spike", is the extracellular representation of ionic currents resulting from near-synchronous DGC action potentials. The intense intracellular depolarization underlying the action potential in each cell produces an extracellular hyperpolarization at the level of the cell bodies. Although action potentials are all-or-none phenomena, each discharge of a DGC evoked concurrently by the electrical stimulus contributes to the extracellular field. The amplitude of the population spike is therefore indicative of both the number of granule cells discharged by the stimulus and their degree of synchrony, and can therefore be a measure of the general level of excitability of the cell field (Lomo, 1971; Andersen et al., 1971a). As field potentials represent a composite of responses from many neurons, they are inherently far less variable than the responses from any individual cell. What is lost, with respect to unit or intracellular recording, are the patterns of ongoing neuronal activity, and the variations between individual neurons in a field; what is gained, however, is a measure of the overall state of the cell field. General measures such as population responses appear to be warranted physiologically when considering the HF, which has been likened to a series of "gates" at the levels of the DG, CA3 and C A l regions (Winson and Abzug, 1978a,b). The cell fields in the HF have been considered as "gates", as each field is in a position to regulate the bulk of impulse flow to succeeding regions in the tri-synaptic circuit. The amplitude of an evoked population spike in any of the cell regions may be thought of as representing the width of opening of such a gate, and so provides a means of quantifying the overall state of a given cell field, inasmuch as it reflects the general ability of the principal cells in the field to transmit information. Modulation of neuronal activity at the level of the DG would appear to be particularly important in the functioning of the HF, as the DG forms the first stage in the tri-synaptic loop, and so is in a position to regulate much of the information directed toward the HF from the neocortex. Doubtlessly the individual activity of specific neurons and synapses is important in the functioning of the HF. It appears, however, that groups of neurons in specific cell fields Figure 1.2 Representative population responses evoked in the DG of an intact animal by perforant path stimulation. A) Responses recorded in the molecular layer (ML): 1) low intensity stimulation; 2) high intensity stimulation. B) Response recorded in the granular layer (GL): 1) low intensity stimulation; 2) high intensity stimulation. Population spike amplitudes were quantified as the difference in potential between the first peak positivity (PI) and the first peak negativity (N), as indicated in B2. Population EPSP slopes were quantified as the maximal negative slope (dV/dT), in ML recordings, or the maximal positive slope, in GL recordings, between the baseline potential and a set latency prior to the emergence of a population spike, as illustrated by the broken lines. Scale: 5 mV, 5 msec. 18 ofthe HF may function in a common manner, under both normal and pathological conditions. In the C A l , CA3, and DG, spontaneous EEG spikes and population bursts have been recorded which appear to be qualitatively similar to evoked population EPSPs and population spikes, respectively (Buzsaki et al., 1983, Buzsaki, 1986; Suzuki and Smith, 1988a,b). In several behavioural paradigms, specific changes in both unit and EEG activity in the DG can be induced by sensory stimuli; these changes appear to affect large populations of DGCs synchronously (Berger et al., 1980; Berger, 1984), indicating a distributed form of information processing in this region of the brain. The amplitudes of evoked population responses similarly have been shown to vary with behavioural conditioning (Skelton et al., 1987) and behavioural" state (Winson and Abzug, 1978a,b). Additionally, pathophysiological epileptiform activity may be induced in the HF by many chemical or electrical stimuli, and the electrophysiological potentials which correlate with this activity often closely resemble evoked population responses. 1.3. ANATOMY OF THE SEROTONERGIC SYSTEM A. SEROTONIN-CONTAINING CELL GROUPS In 1948, Rapport and colleagues isolated a vasoconstrictor substance from blood serum which they named serotonin, and later identified as 5-hydroxytryptamine (5-HT). This compound was found to be identical with the pharmacologically-active substance "enteramine" which Erspamer had isolated from the salivary gland of the octopus in 1940. The presence of 5-HT was first detected in the brain in the early 1950's, using bioassay techniques (Twarog and Page, 1953; Amin et al., 1954). In 1962, Falck, Hillarp and co-workers developed a histochemical method for direct visualization of biogenic amines in animal tissue (Falck et al, 1962). The Falck-Hillarp process, which involved reacting freeze-dried tissue samples with formaldehyde vapour, made possible the mapping of serotonin-containing cells and their processes. With this technique, Dalstrom and Fuxe (1964) were the first to describe the groups of serotonin-containing cell bodies in the brainstem, and designated them as groups BI through B9. These groups were found to be situated largely in the midline brainstem regions which were differentiated previously by cytoarchitectonic criteria, and designated as the "raphe nuclei", "raphe" indicating a line or ridge in the mid-saggital plane (Taber et al., 1960). Some of these raphe nuclei were identified by the early anatomists (Ramon y Cajal, 1898; Winkler and Potter, 1914), and a similarity was noted in these structures between mammalian species, including humans (Olkzewski and Baxter, 1954). Until the concept of a common neurochemical system in these nuclei was introduced, there was little reason to classify the raphe nuclei together, other than on the basis of their midline locations and somewhat similar cellular morphology. The anatomical localizations of the groups differentiated by Dalstrom and Fuxe are similar to, but not completely congruent with the earlier cytoarchitectonic parcellations of the raphe nuclei. The dorsal raphe nucleus (DRN) contains groups B6 and B7, and comprises the largest aggregate of serotonin-containing neurons in the brain. Group B7, which is the largest of Dalstrom and Fuxe's parcellations, lies in the periaqueductal gray, extending from the caudal end of the oculomotor nucleus to the rostral end of the fourth ventricle. The group designated B6 lies ventral to B7, extending though the central gray along the floor of the fourth ventricle. The median raphe nucleus (MRN) is composed of the nucleus centralis superior (NCS) and the nucleus linnearis intermedialis (NLI), and corresponds to Dahlstrom and Fuxe's serotonergic cell group B8. The NCS is situated in the reticular formation, at the border of the pons and the midbrain, and extends caudally from the rostral end of the facial colliculus, where its dorsal border is separated from the NLI by the superior cerebellar peduncle, and rostrally, into the midbrain. The NLI is a narrow nucleus in the mesencephalic tegmentum which merges dorsally with the dorsal raphe nucleus. The serotonergic cell group B9 appears as a lateral extension of the nucleus centralis superior. This group does not correspond to any of the anatomically defined raphe nuclei, but to the region known as the nucleus prosupralemniscus. The dorsal and medial raphe nuclei, along with group B9, account for almost the entire ascending serotonergic projections to the forebrain. While most of the ascending fibres projecting from the DRN and MRN contain serotonin, many do not, and some may use GABA as a neurotransmitter (Kohler and Steinbusch, 1982). The more caudally located raphe nuclei, which contain the serotonergic cell groups B l through B5, give rise to serotonergic fibres whose primary projections extend either locally into the brainstem, or descend to the spinal cord. B. SEROTONERGIC PROJECTIONS TO THE HIPPOCAMPAL FORMATION From the rostrally-located raphe nuclei, the ascending serotonergic fibres radiate diffusely, reaching virtually all regions of the diencephalon and the forebrain, including the cerebral cortex (Molliver, 1987; Azmitia, 1987). Experiments with retrograde tracers have revealed that among the raphe nuclei, only the DRN and MRN project to the hippocampal formation, with a greater number of projections arising from the MRN. These two raphe nuclei are not homogeneous with respect to their projections: fibres terminating within the hippocampus originate in the more caudal regions of the DRN, and the peripheral areas of the MRN (Wyss, et al., 1979; Kohler and Steinbush, 1982). Anterograde tracing methods and immunocytochemistry have also revealed a topographic organization of the MRN projections, with the more rostro-ventral portions of the MRN terminating in the more ventro-posterior areas of the hippocampal formation (Pasquier and Reinoso-Suarez, 1978). The ascending fibres from the MRN and DRN project to cortical and subcortical structures via six tracts; two of these, the dorsal raphe forebrain tract and the medial raphe •forebrain tract, travel within the medial forebrain bundle, and contain projections to the hippocampal formation (Azmitia and Segal, 1978; Parent et al., 1981). The fibres within these two tracts reach the hippocampus via similar routes. These projections pass initially through the ventral fasciculus retroflexus, and into the rostral posterior hypothalamus and the dorsal supramammilary nuclei. From there, they travel through the medial forebrain bundle, to the diagonal band of Broca and the medial and lateral septal nuclei. (It should be noted that terminal fibres are given off in each of these brain regions as these fibre tracts course through them.) From the diagonal band and the septal nuclei, fibres from the raphe nuclei which are directed toward the hippocampal formation divide into three branches. The major branch, which appears to originate largely in the MRN, travels through the fimbria, and provides dense innervation of the stratum radiatum of areas CA2, CA3 and CA4 as well as a much weaker projection to the stratum oriens of areas CA2 to CA4 (Azmitia and Segal, 1978; Pasquier and Reinoso-Suarez, 1978). This pathway also carries fibres to the superficial polymorphic zone of the hilus, along the inferior border of the DGCs, where they terminate in a dense band, approximately 30-65um wide in the rat, forming the most concentrated region of serotonergic innervation in the hippocampal formation (Lidov et al., 1980). It is unknown whether the projections to the polymorphic zone make synaptic contact with the cell bodies of the DGCs, with hilar neurons, or with both. The C A l region is innervated primarily by a second pathway traversing the superior fornix, while a rostro-caudal pathway through the medial cingulum provides innervation to the entorhinal cortex, the anterior pole of the hippocampus, and the subiculum. Data from studies using autoradiographic (Amzitia and Segal, 1978), anterograde degeneration or retrograde tracing methods (Pasquier and Reinoso-Suarez, 1978) indicate that serotonergic fibres projecting to the CA 1 terminate most densely in the SR of that region. One histochemical study (Lidov et al., 1980), however, suggests that serotonergic innervation of area C A l may be restricted to the SL and SM layers. The largest projection from the DRN to the hippocampal formation travels within the cingulum, enters the hippocampus along the perforant path, and terminates largely in the molecular layer of the dentate gyrus. This pathway provides much greater innervation to the ventral, than to the dorsal HF (Buchan and Azmitia, 1977; Pasquier and Reinoso-Suarez, 1978). While most of the projections from the raphe nuclei to the hippocampus are ipsilateral, some fibres are crossed, although no raphe neurons appear to project bilaterally. Additionally, about 10% of the neurons projecting to the hippocampus from the DRN and MRN also innervate the entorhinal cortex, where they would be in a position to influence hippocampal function indirectly, by modifying information directed toward the hippocampal formation via the perforant path (Kohler and Steinbusch, 1982; Insausti et al., 1987). C. T O P O G R A P H Y O F P R O J E C T I O N S F R O M T H E D O R S A L A N D M E D I A L  R A P H E N U C L E I While the caudal regions of the DRN project to the hippocampal formation, the more rostral areas project to the caudate-putamen and substantia nigra. Double labelling experiments suggest that these outputs are segregated anatomically and that there is no overlap between areas of the DR which project to the striatum and to the HF. Both the DRN and the MRN also project to the amygdala. The origin of the serotonergic fibres projecting to the a m y g d a l a both overlaps w i t h , and geographically divides, those regions projecting to the s t r i a t u m and the H F . In addition to these projections to limbic structures, the D R N is also the p r i m a r y source of serotonergic innervation to the neocortex and substantia n i g r a , while the caudal region of the M R N projects heavi ly to the locus coeruleus (Imai et a l . , 1986). D. DISTINCTIONS B E T W E E N P R O J E C T I O N S F R O M T H E D O R S A L A N D  M E D I A L R A P H E N U C L E I The fibres projecting from the D R N and M R N are morphologically distinct (Kosofsky and M o l l i v e r , 1987). Projections from the M R N appear as coarse, beaded axons, containing large spherical varicosities, while those from the D R N appear as very fine axons, w i t h minute granular or pleomorphic varicosities. The coarse, beaded serotonergic axons are found to a large extent throughout entorhinal cortex and H F , and are especially abundant i n the hilus of the dentate gyrus. The diss imilar i ty between the projections from these two nuclei is underscored by recent work demonstrating a differential sensit ivity to neurotoxic agents. Large repetitive doses of the amphetamine derivatives 3,4-methylenedeoxy amphetamine ( M D A ) or 3,4-methylenedeoxymethamphetamine ( M D M A ) , or smaller doses of para-chloroamphetamine ( P C A ) appear to have a selective neurotoxic effect on the terminal regions of fine serotonergic axons, which presumably arise f rom the D R N , while the coarse, beaded axons which ostensibly originate in the M R N are spared. A s the neurotoxic effects of these drugs appear to depend on their uptake into the axon terminals, it is possible that different uptake systems i n the M R N and D R N m a y underlie the different pharmacological sensitivities. The differences i n axon type and sensitivity to neurotoxins has led to the suggestion that the D R N and M R N m a y have distinct modulatory actions i n the hippocampal formation (Moll iver , 1987). 1.4. SEROTONIN RECEPTORS The study of neurochemical systems is complicated by the fact that many of the compounds which appear to function as neurotransmitters can produce multiple effects in neural tissue. These multiple effects may be mediated by subtypes of neurotransmitter receptors. Recently, multiple subtypes of 5-HT receptors have been identified, and selective ligands have been developed as tools to investigate these receptors. This section will outline basic neurotransmitter receptor theory, and then review current concepts of 5-HT receptors. A. NEUROTRANSMITTER RECEPTOR THEORY Current concepts of neuronal communication are based on the theory of synaptic transmission between neurons, in which neurotransmitter chemicals are released from pre-synaptic elements, and interact with post-synaptic receptors, which then transduce the signal to the postsynaptic neurons. The post-synaptic receptors appear to be large surface membrane proteins. These are found on the somata, dendrites, axons and terminals of neurons, and can act to excite, inhibit, or otherwise modulate the activity of the post-synaptic cell, via the gating of membrane ion channels, or the triggering of intracellular biochemical changes. It is the interaction of the neurotransmitter and the receptor which translates the chemical message of neurotransmitter release to the electrical and biochemical messages in the post-synaptic cell. Many transmitters appear to act upon several different receptor subtypes, enabling a single neurotransmitter to elicit multiple cellular responses. It appears that the multiple neurotransmitter receptor subtypes for a particular neurotransmitter may recognize different structures on the neurotransmitter molecule. A major thrust in neuropharmacology has been the development of specific compounds which may interact selectively with specific receptor subtypes. Selective compounds have become valuable tools in differentiating the multiplicity of physiological effects often produced by many neurotransmitters. Initially, receptor subtypes were identified by the physiological processes affected by their activation. For example, multiple receptor subtypes were described for the neurotransmitter acetylcholine on the basis of functional effects, and were divided into "muscarinic" and "nicotinic" classifications, after the selective agonists found for each. Similarly, adrenoreceptors were subdivided into alpha and beta subtypes, on the basis of the effects of agonists or antagonists on vasodilation or smooth muscle contraction (Ahlqvist, 1948); further subdivisions were made subsequently on the basis of responses elicited by agonists (Lands et al., 1967) or the relative potencies of antagonists (Langer, 1974). Currently, suggestions about the existence of multiple receptor subtypes for particular neurotransmitters arise almost exclusively from studies utilizing in vitro ligand binding techniques. The principle behind these techniques is that compounds which bind to the same recognition site will tend to displace one another from that site. A compound is considered a ligand for a neurotransmitter binding site if, at a reasonable concentration, it will displace the binding of that neurotransmitter. Relatively low concentrations of ligands which exhibit high binding affinity, i.e. those which bind readily to the recognition site and do not readily dissociate, are required to displace competing compounds. Conversely, very high concentrations of ligands with low binding affinity are needed to displace competing compounds. If a ligand, at low concentration, will displace only a portion of the bound neurotransmitter, and much higher concentrations are necessary to displace significantly greater amounts, then the possibility exists that the ligand is binding selectively to a subset of the binding sites which exist for that neurotransmitter. This technique can be extended to identify specific receptor subtypes. Other, and perhaps more selective ligands can then be developed, with the use of structure-function analysis to identify the shapes and characteristics of the molecules required. Although a site may bind a neurotransmitter with high affinity, it need not be a functional receptor. A receptor may be defined as a structure, which, when activated by binding with the appropriate ligand, will transduce a signal to the postsynaptic cell, thereby transmitting information. To determine whether or not a binding site is acting as a receptor, specific electrophysiological, biochemical or behavioural correlates must be distinguished. In electrophysiological studies, direct measurement is often made of neuronal events, examining the effects of selective ligands on the gating of ion channels, or the modulation of neuronal activity or excitability. Biochemical measures are typically concerned with the modulation of biochemical processes, such as second messenger cascades, or the phosphorylation of proteins. Behavioural studies may investigate the effects of ligands on behaviours occurring either spontaneously, or in conditioning paradigms. In some cases, identification of a receptor has been made possible using molecular biological techniques, in which the genetic sequence of a receptor is identified and cloned, and the gene product expressed (Lubbert et al., 1987). To mediate the effects of an endogenously released neurotransmitter, the receptor must be situated anatomically so that it is accessible to that neurotransmitter. Interestingly, the anatomical distribution of receptors for many putative neurotransmitters does not match well with the distribution of terminals containing those compounds (Herkenham, 1987); it is possible that some of these receptors may not play a functional role in the normal brain. The serotonergic system may be considered as somewhat of an exception in that the distribution of 5-HT containing fibres does correlate well with that of 5-HT receptors (Pasquier and Reinoso-Suarez, 1978; Kohler and Steinbush, 1982). All ligands which bind to receptors do not, of course, exert the same effect. The degree to which a ligand quantitatively evokes a response by interacting with a particular receptor is termed its efficacy at that receptor. Compounds which produce qualitatively the same response as the presumed endogenous neurotransmitter are termed agonists; those which bind to the receptor without effect, and so block the effects of agonists are termed antagonists. Compounds with low intrinsic efficacy are known as partial agonists, or mixed agonist-antagonists. Additionally, antagonists may be differentiated as "competitive" or "noncompetitive": competitive antagonists bind to the same recognition site as agonists, whereas non-competitive antagonists block the effects of agonists by other means. B. SEROTONERGIC BINDING SITES On the basis of competitive binding experiments in cortical tissue, Peroutka and Snyder (1979) first proposed the existence of two serotonergic binding sites, which they designated 5-HT^ and 5-HT2- The 5-HT^ binding site was defined by a high (nM) affinity for 5-HT, while the 5-HT2 binding site was defined by a high affinity for the neuroleptic spiperone, and a low (uM) affinity for 5-HT. More recent studies examining the binding characteristics of a wide variety of compounds indicated that the 5-HT^ binding site is itself heterogeneous. Two subtypes, 5-HT^^ and 5-HT^g, have been identified on the basis of high or low affinity, respectively, for radiolabeled spiperone (Pedigo et al., 1981). A site designated as 5-HT^^ has recently been determined as having a low affinity for spiperone, but a high affinity for the compound mesulergine (Pazos et al., 1985), and recently, a 5-HTg site has also been proposed, which exhibits relatively low affinities for both 5-HT and spiperone. Distinct anatomical localizations have been found for all of these subtypes of 5-HT binding sites. The most dense concentrations of 5-HT^ binding sites in the brain are found in the dentate gyrus and lateral septal nucleus, with lesser, but still relatively high, concentrations in the raphe nuclei, C A l to CA4 fields of the hippocampus, the amygdaloid complex, and the hypothalamic nuclei (Kohler, 1984; Marcinkiewicz et al., 1984; Pazos and Palacios, 1985; Pazos et al., 1987). All of the basal ganglia are rich in 5-HTjg binding sites, while the highest concentrations of the 5-HT2 binding sites are found in the claustrum, olfactory nuclei and layers I and IV of the neocortex, with lesser concentrations in the dentate gyrus, striatum and nucleus accumbens (Pazos et al., 1985a). These patterns of distribution appear to be well preserved phylogenetically, with similar anatomical localization of serotonergic binding sites in the human and the rodent (Pazos and Palacios, 1985; Pazos et al., 1985, 1987a,b; Kohler, et al., 1986; Hoyer et al., 1986b). An exception to this is the 5-HT^g site, which is apparently not found in human brain (Hoyer et al., 1986b). The hippocampal formation, as discussed above, contains a very high concentration of 5-HT^ binding sites. These are distributed heterogeneously within this region, with especially high densities evident in the SR of the C A l region, and the SM of the DG. The 5-HT-^ binding sites appear to be located largely, if not exclusively, on postsynaptic elements in these regions (Verge et al., 1986; Hall, et al., 1985; Mura, et al., 1986; Kohler, 1984); a dense band of 5-HT-^ binding immediately subjacent to the GL in the hilus has been described in the monkey and human (Kohler, et al., 1986; Kohler, 1988), but a similar pattern has not been reported in the rodent. In the hippocampal formation 5-HTjg binding sites appear to be located on the presynaptic terminals of serotonergic and other fibres (Engel et al., 1986). The dentate gyrus, but not the hippocampus, also contains a moderate number of 5-HT2 binding sites (Kohler, 1984; Pazos et al., 1987a,b). C. SEROTONERGIC RECEPTORS The original work on 5-HT receptor classification arose from studies on the constrictive effects of 5-HT on smooth muscle, primarily in the guinea pig ilium and rat uterus. Working independently, the groups of Gaddum and Roche e Silva developed a dual receptor theory, which they published jointly (Gaddum and Picarelli, 1957). The direct effects of 5-HT on smooth muscle, which could be antagonized by dibenzylene (phenoxybenzamine), were postulated to be mediated by what they designated as the "D" receptor; the excitatory effects of 5-HT on parasympathetic cholinergic neurons, which could be blocked by morphine, were postulated to be mediated by the designated "M" receptor. • For several reasons, however, the distinction on the basis of these antagonists is no longer useful. Neither dibenzylene nor morphine are highly specific for these receptors, a fact which Gaddum and Picarelli (1957) recognized at the time. Later, it was found that morphine acted indirectly in this preparation to antagonize the actions of 5-HT by blocking the release of acetylcholine. In the literature, the names given to serotonin receptors as defined by their physiological effects vary greatly. Recently, effort has been made to standardize this nomenclature, so as to try and associate receptor subtypes with known binding sites (Bradley, et al., 1986; Glennon, 1987). To this end, emphasis has been placed on the identification of high potency agonists and antagonists, which exhibit high affinity and selectivity for specific serotonergic binding sites, and the use of these compounds to compare the pharmacological profdes of receptor-mediated effects and binding affinities. All of the serotonergic binding sites mentioned above satisfy many of the criteria which would establish them as functional receptors. The binding sites all demonstrate high affinity, saturability, and distinct anatomical localizations; in addition, all appear to mediate physiological, biochemical, and behavioural effects. Additionally, the gene sequence of the 5-HT^Q receptor has been identified, cloned, and the gene product expressed in Xenopus oocytes (Lubbert et al., 1987). The following sections will discuss the effects mediated by the various 5-HT receptor subtypes, and some of the selective pharmacological agents which have been developed for them. 1) 5-HT1A RECEPTORS The 5-HT-j^ receptor subtype has been analyzed extensively, due largely to the availability of a variety of potent and selective agents for this site. The potency of agonists in eliciting physiological, biochemical, and behavioural effects attributed to the 5-HT-^A receptor is in good accord with their affinity for the 5-HT-j^ binding site. The most selective ligand for the 5-HT-^ binding site currently available is the tritiated form of 8-hydroxy-2-(di-a-propylamino)tetralin (8-OH-DPAT) which demonstrates several thousand times the affinity for the 5-HT^ subtype than for any other known receptor (Arvidsson et al., 1981, Gozlan et.al., 1983; Peroutka, 1987). As such, this compound is considered a prototypic 5-HT^ agonist. The use of 8-OH-DPAT as a selective 5-HT^ agonist has been largely responsible for the characterization of this receptor. In addition, several arylpiperazine derivatives such as ipsaperone, buspirone, and geparone demonstrate high affinity and selectivity of the 5-HT-^ site, and have been useful in pharmacological investigations (Peroutka, 1987; Glennon, 1987). Physiological effects The majority of studies investigating the possible physiological roles of the 5-H T ^ receptor has focussed on the regions of the brain where the 5-HTj^ binding site is found in greatest density, namely the hippocampal formation and the raphe nuclei. In both of these structures the 5-HT^ receptor appears to have an inhibitory function. The physiological functions controlled by this receptor in the HF are reviewed in detail in section 1.6 below. In the raphe nuclei, the 5-HT^ receptors appear to be located on the somata and dendrites of serotonergic neurons, where they may serve as autoreceptors mediating the inhibitory feedback of 5-HT release. The inhibition of dorsal raphe cell firing by 5-HT, lysergic acid diethylamide (LSD) and related amines has been a subject of intensive study for many years (e.g., Aghajanian, 1972). Recently, it was found that the binding of the novel and highly selective 5-HT-^ ligand 8-OH-DPAT in the dorsal raphe could be reduced markedly following treatment with the serotonergic neurotoxin 5,7-dihydroxytrypamine (5,7-DHT) (Verge et al., 1985). This led to experiments which determined that the potency of serotonergic agonists in the inhibition of raphe neurons matched their affinities for the 5-HT-^, but not 5-HT-^g or 5-HT2 binding sites (Vandermaelen and Wilderman, 1984a,b; Sills et al., 1984; Sprouse and Aghajanian, 1987a; Trulson and Fredrickson, 1986). Intracellular recordings indicate that the 5-HT-^ mediated inhibition of DRN neurons is correlated with a membrane hyperpolarization and increased conductance, possible due to an increased potassium conductance (gK) (Sprouse and Aghajanian, 1987a). Biochemical effects The transduction of a signal from a ligand-activated receptor to a cellular response is often mediated by so-called "second messenger systems", in which the response is triggered by a biochemical cascade. In the adenylate cyclase second messenger system, the interaction of a ligand with a receptor activates adenylate cyclase, leading to the formation of cyclic AMP, which in turn activates specific protein kinases that mediate numerous cellular processes. Mansour et al. (1960) were the first to report 5-HT mediated stimulation of adenylate cyclase, in tissue particles of the liver fluke; since that time, 5-HT stimulated adenylate cyclase has been demonstrated in many systems (Conn and Sanders-Bush, 1987). In hippocampal tissue, 5-HT induced activation of adenylate cyclase appears to be restricted to the 5-HT^ receptor. The rank order of potency of serotonergic agonists in inducing this effect has been reported to correlate well with their affinities for the 5-H T 1 A , but not 5-HT 1 B, 5-HT l C, or 5-HT2 sites (Markstein et al., 1986; Barbaccia et al., 1983). Non-specific serotonergic antagonists inhibit this stimulation of adenylate cyclase, whereas antagonists selective for the 5-HT 2 receptor do not. Under appropriate conditions, the 5-HT^ receptor may also mediate inhibition of adenylate cyclase activity. In hippocampal membranes, 5-HT or selective 5-HT^ agonists inhibit forskolin- or VIP-induced stimulation of adenylate cyclase (De Vivo and Maayani, 1986; Bockaert, et al., 1987). The reasons underlying the apparently paradoxical ability of the 5-HT-^ receptor to mediate inhibition and stimulation of adenylate cyclase is presently unknown. Behavioural effects Increasing serotonergic tone in the brain by means of administration of metabolic precursors or uptake inhibitors results in a complex constellation of behaviours in the rat, which have been termed the "5-HT syndrome". Experiments with various 5-HT agonists and antagonists have indicated that specific components of this response may be mediated by the 5-HT-^ receptor. These include reciprocal forepaw treading, flat bpdy posture, hindlimb abduction, and Straub tail (Lucki et al., 1984; Tricklebank, 1985; Tricklebank et al., 1984, 1986ab; Middlemiss et al., 1985; Smith and Peroutka, 1986). Also in the rat, the 5-HTj^ agonist 8-OH-DPAT has been reported to facilitate male sexual behaviour (Ahlenius et al., 1981; Mendelson and Gorzalka, 1985, 1986), and to induce hyperphagia (Dourish et al., 1985; Hutson et al., 1986, 1987), indicating a role for the 5-HT 1 A receptor in these processes. 2) 5-HT1B RECEPTORS Selective agonists and antagonists for the 5-HT^-g receptor are not yet available. Recently, iodocyanopindolol (I-CYP) has been reported to label 5-HT^g sites, and may act as a 5-HT^B antagonist (Hoyer et al., 1986a). The phenylpiperazine derivatives triflouromethylpiperazine (TFMPP) and metachlorophenylpiperazine (mCPP), and the indolealkylamine RU 24969 have been proposed as selective agonists for the 5-HT-^ -g receptor, (Sills et al., 1984; Leysen, 1984) and have been treated as such in several studies (McCall et al., 1987; Kirstein and Spear, 1988). The compound RU 24969, however, has an equally high affinity for the 5-HT-^ site (Tricklebank et al., 1986b) while TFMPP and mCPP appear to be effective agonists (Kennett and Curzon, 1988) and 5-HT2 antagonists (Conn and Sanders-Bush,. 1987). The best functional correlate of the 5-HT^g receptor is as an autoreceptor on serotonergic terminals, where it appears to inhibit the release of 5-HT. This has been studied most extensively in preparations of cortex and hippocampus, in which the potencies of drugs in inhibiting the stimulation-evoked release of 3H-5-HT have been correlated with affinities at the 5-HT-^ -g binding site (Engel et al., 1986). Similarly, the 5-HT-^g receptor appears to mediate the inhibition of high K evoked release of H-5-HT from rat hippocampal and cerebellar synaptosomes (Maura et al., 1986; Raiteri et al., 1986). In hippocampal tissue, there have also been reports of 5-HT^g receptors mediating inhibition of release of acetylcholine (ACh) and NE, (Maura and Raiteri, 1986). In the basal ganglia, 5-HT-^g binding sites appear to be located postsynaptically, but functional correlates for these sites have yet to be determined. The impetus for research into 5 - H T r e c e p t o r s has declined recently following the failure to discover 5-HT IB binding sites in the human brain (Hoyer et al., 1986a; Pazos et al., 1987). 3) 5-HT 1 C RECEPTORS The 5HT^Q binding sites in the brain have been localized almost exclusively to epithelial cells of the choroid plexus, where 5 H T J C receptors may mediate inhibition of cerebro-spinal fluid production. No selective ligands exist for the 5 - H T ^ £ binding site, but the site is clearly differentiated by the displacement of 3H-5-HT, 1 2 5I-LSD, and 3H-mesulergine by serotonergic agonists and antagonists (Glennon, 1987). Biochemically, Conn et al. (1986) and Sanders-Bush et' al. (1988) recently have reported a robust stimulation of the phosphoinositide second messenger system by a receptor corresponding to the 5-HT^Q binding site (also see the section on 5-HT2 receptors below). Behaviourally, some evidence exists that the 5-HT-^ receptor may mediate hyperlocomotion in the rat (Kennett and Curzon, 1988). 4) 5-HT2 RECEPTORS The 5-HT2 binding sites demonstrate a high affinity for "classical" serotonin antagonists such as cinanserin, metergoline, and methysergide. Recently, arylpiperidine compounds such as pirenpirone, ketanserin and ritanserin have been developed as selective 5-HT2 antagonists (Leysen et al., 1981; Janssen, 1983), and 5-HT2 receptors have been defined by the blockade of the effects of non-selective agonists, such as 5-HT or LSD by these antagonists. More recently, Glennon and colleagues have investigated the use of phenylisopropylamine derivatives as selective 5-HT2 agonists (Glennon et al., 1986, 1987; Shannon et al., 1984; Titeler et al., 1987). The most selective and potent of these compounds are the bromo- and iodo- analogues of 2,5 dimethoxy-4-methylamphetamine (DOM), DOB and DOI. The physiological effects of the 5-HT2 receptor appear to be quite complex. In the facial motor nucleus, application of 5-HT results in a slow depolarization, possibly by reducing a resting membrane conductance to K"1", and a facilitation of the excitatory effects of glutamate (McCall and Aghajanian, 1979). Work with agonists and antagonists suggest that these effects are mediated by the 5-HT2 receptor. In neocortical neurons, 5-HT-induced depolarization appears to be mediated by the 5-HT2 receptor, possibly by decreasing gK (Aghajanian, et al., 1987; Davies et al., 1987). Unit activity in the locus coeruleus is decreased markedly by systemic administration of serotonin agonists, whose relative potency correlates with their affinity for the 5-HT2 binding site (Aghajanian, 1980, Rasmussen and Aghajanian, 1986; Glennon et al., 1984). These effects, however, appear to be mediated indirectly, by 5-HT2 receptors on other loci, as they are not mimicked by iontophoretic application of these agonists directly onto the locus coeruleus (Aghajanian, et al., 1987). The 5-HT2 receptor appears to be involved in the phosphoinositide (PI) second messenger system. In the PI second messenger cascade, a membrane receptor mediates the activation of phospholipase C, which catalyzes the hydrolysis of membrane phosphoinositides, leading to the production of inositol-l,4,5-trisphosphate (IP3) and diacylglycerol. IPg induces the release of calcium from intracellular stores, which then mediates numerous cellular responses. Diacylglycerol stimulates protein kinase C, which in turn phosphorylates many cellular proteins. In the mammalian cortex, the phosphoinositide-diacylglycerol system is stimulated by the 5-HT2 receptor (Sanders-Bush et al., 1988; Conn and Sanders-Bush 1984, 1985). This effect is produced by 5-HT, but not 5-HT^ selective agonists; further, the ECgg values of various 5-HT agonists in producing this effect correlate well with their affinities for the 5-HT2 binding site. Behaviourally, a common property of 5-HT2 agonists appears to be hallucinogenic activity, and, in fact, the potency of various compounds as hallucinogens in humans is correlated highly with 5-HT2 binding site affinity (Glennon et al., 1984). In cats, drug-induced limb-flicks appear to be correlated highly with 5-HT2 agonist activity, and most probably are mediated by actions at post-synaptic sites (Jacobs, 1976, 1977). 1.5. PHYSIOLOGICAL EFFECTS OF 5-HT IN THE HIPPOCAMPAL FORMATION The HF is now used routinely to study the physiological effects of 5-HT because of its dense population of serotonergic binding sites and relatively well defined electrophysiological characteristics. The majority of these studies have been confined to the C A l region of the HF. The studies reviewed in this section will be discussed according to the type of electrophysiological measure used: specific measures include extracellularly recorded unit activity, intracellular potentials, or evoked extracellular field responses. A. UNIT ACTIVITY In the hippocampus, iontophoretic application of either 5-HT or the selective 5-HT^^ agonist 8-OH-DPAT suppresses spontaneous and evoked unit activity of the majority of C A l and CA3 pyramidal cells, in both in vivo and in vitro preparations (Stefanis, 1964; Biscoe and Straghan, 1965; Jahnsen, 1980; Martin and Mason, 1987; Makhov et al., 1982; Segal, 1975). This inhibitory effect appears to be mediated by the 5-HT-^ receptor, as it can be blocked by the non-selective 5-HT antagonist methysergide (Segal, 1975), and the 5-HT^ ligand ipsapirone (Martin and Mason, 1987), but not the selective 5-HT2 receptor antagonist ketanserin (Mason, 1985). Application of TFMPP or mCPP, which demonstrate some selectivity for 5-HT^g sites, produce only weak inhibitory effects on these neurons (Sprouse and Aghajanian, 1987b). To date only one study has investigated the effects of 5-HT itself on neuronal unit activity in the in vivo DG. Stefanis (1964) reported an inhibitory effect of iontophoretically administered 5-HT; unfortunately, no assurances were provided that recordings were made from dentate granule cells. Recently, a detailed study was presented by Basse-Tomusc et al. (1986) in which was reported a decrease in unit activity in electrophysiologically identified DGCs following intravenous injections of the 5-H T j ^ partial agonist, ipsaperone. These data support an inhibitory role for the 5-HT^ receptor in modulating the function of these neurons. B. INTRACELLULAR MEASURES The majority of studies using intracellular techniques to investigate the effects of serotonergic compounds in the HF have been confined to C A l pyramidal cells, and these have all employed the in vitro hippocampal slice preparation. In these studies, 5-HT has been observed to produce three effects on C A l pyramidal cells: membrane hyperpolarization, membrane depolarization, and a reduction in afterhyperpolarization (AHP), the latter being concomitant with a decrease in the ability to accommodate to depolarizing stimuli (Jahnsen, 1980; Segal, 1980; Andrade and Nicoll, 1987; Colino and Halliwell, 1987; Wu et al., 1988). The predominant response is hyperpolarization; experiments with selective agonists and antagonists indicate this response to be mediated by a receptor corresponding to the 5-HT^A binding site. A similar hyperpolarization can be elicited by the selective 5-HT A agonist 8-OH-DPAT or the 5-HT-^  agonist 5-methoxytryptamine (Wu et al., 1988; Ropert, 1988; Andrade and Nicoll, 1987), and the hyperpolarization seen following 5-HT can be blocked by the non-selective 5-HT antagonist methysergide (Segal, 1975, 1980), but not by the selective 5 -HT2 antagonist ketanserin (Colino and Halliwell, 1987). Additional evidence for 5-HT^A receptor mediation of this hyperpolarization was suggested by Andrade and Nicoll (1987), who reported that iontophoretic application of 5-HT or 8-OH-DPAT elicited the greatest degree of hyperpolarization when applied to the SR, which is the region of the C A l with the greatest density of 5-HT-j^ binding sites. These data differ somewhat from those of Segal (1980), who reported a greater hyperpolarization when 5-HT was applied near the SP of this region. This hyperpolarizing response of the C A l pyramidal cells is sensitive to intracellular caesium, reverses at approximately the equilibrium potential for K~*~, and is unaffected by changes in CI" gradient (Segal, 1980; Segal and Gutnick, 1980; Colino and Halliwell, 1987; Ropert, 1980; Andrade and Nicoll, 1987). Experiments with K + sensitive electrodes have demonstrated a 0.5-2 mM rise in extracellular K+ following application of 5-HT, which could also be blocked by methysergide (Segal and Gutnick, 1980). These results are consistent with the hyperpolarizing action of 5-HT being mediated by an outward current. Additionally, the hyperpolarization produced by 5-HT appears to be mediated via a true postsynaptic response of the pyramidal cells, as it can also be elicited when synaptic transmission is blocked by a bath medium containing a high concentration of Mg and a low concentration of Ca (Segal, 1980). The mechanisms underlying the other responses of C A l pyramidal cells to 5-HT are less clear. The depolarization appears to be mediated by the reduction of a resting K conductance, while the reduction in AHP may be due to a decrease in a Ca dependent gK. Recent studies with serotonergic agonists and antagonists indicate that neither of these effects is mediated by known 5-HT receptor subtypes (Andrade and Nicoll, 1987; Colino and Halliwell, 1987). The intracellular responses of DGCs to 5-HT appear to be similar to those of C A l HPCs. Iontophoretic or pressure application of 5-HT has been shown to hyperpolarize DGCs, as well as to decrease AHP and decrease accommodation to depolarizing stimuli (Segal, 1981; Baskys et al., 1986, 1987). This hyperpolarization is dependent on K+ but not Ca concentrations, and is accompanied by a decreased input resistance and increased extracellular K+, suggesting that it is mediated by an increased gK (Segal and Gutnick, 1980). As in the C A l pyramidal cells, the 5-HT 1 A agonist 8-OH-DPAT also produces a hyperpolarization in DGCs, suggesting that the hyperpolarizing effects of 5-HT are mediated by the 5-HT-^ receptor. These studies are in sharp contrast to the earlier work of Assaf et al. (1981) and Crunelli et al., (1983) who reported depolarization of DGCs following iontophoretic application of 5-HT. These latter authors found that the reversal potential of this depolarization was near to the equilibrium potential for CI", and was not additive with depolarization produced by GABA; this led them to suggest that this depolarization was due to an increase in gCl", possibly due to serotonergic activation of the GABA-g receptor. The reason for the discrepancy between these studies is unclear, as the experiments resulting in hyperpolarizing and depolarizing responses were performed with similar techniques, media, electrolytes and concentrations of 5-HT. No change in AHP was observed in DGCs following 8-OH-DPAT application, as in C A l HPCs, indicating that the effect of 5-HT on this measure is probably not mediated by the 5-HT-^ receptor (Baskys et al., 1987). Interestingly, HPCs in the CA3 region appear to be relatively unresponsive to 5-HT (Segal, 1981), correlating with the relatively low concentration of 5-HT binding sites in this region of the HF. C. E V O K E D F I E L D P O T E N T I A L S To date, studies investigating the effects of serotonergic agents on evoked field potentials in the HF have all been conducted using the in vitro hippocampal slice preparation. Moreover, all of these have been confined to the C A l region. Superfusion of the in vitro slice preparation with 5-HT produces a dose-dependent decrease in the amplitude of population spikes evoked in the SP of area C A l by stimulation of the Schaffer collaterals (Olpe et al., 1984; Beck and Goldfarb, 1985), and this is preceded occaisonally by a transient increase in this measure (Beck and Goldfarb, 1985; Rowan and Anwyl, 1985). Some authors report a change in the slope of the population EPSP which is consistent both with the increase or decrease in PSA (Ropert, 1988; Rowan and Anwyl, 1985; Peroutka et al., 1986), while others report no change in the population EPSP (Beck and Goldfarb, 1985). The decrease in PSA appears to be mediated by a 5-HT-^ receptor, as a similar effect can be produced by application of either the selective 5-HT l A agonist 8-OH-DPAT or the 5HT-L agonist 5-carboxamidotryptamine. The decrease in PSA produced by these compounds can also be blocked by the non-selective serotonergic antagonists spiperone and cyproheptadiene, but not the selective 5-HT2 receptor antagonist ketanserin (Beck et al., 1985; Peroutka et al., 1986; Andrade and Nicoll, 1987; Rowan and Anwyl, 1985). The decrease in PSA following 5-HT superfusion can be reduced by pertussis toxin, and so appears to be mediated by an inhibitory guanine regulatory protein (G-protein) (Clarke et al., 1987). One study (Peroutka et al., 1987) has reported only an increase in C A l PSA after the administration of 5-HT or 8-OH-DPAT; this may be due to differing methodology, including their use of female rats. The transient increase in PSA which has been observed occasionally after application of 5-HT does not appear to be mediated by any known serotonergic receptor subtype as it can neither be produced by selective serotonergic agonists, nor blocked by antagonists (Rowan and Anwyl, 1985; Beck et al, 1985). 1.6. THE PRESENT STUDY As discussed above, the actions of serotonin in the C A l region of the HF have been the subject of intense study. Much less attention has been paid to the DG, despite the fact that it receives greater serotonergic innervation from the raphe nuclei, and also contains a greater concentration of serotonergic binding sites. Modulation of neuronal activity at the level of the DG is particularly important in the functioning of the HF, as the DG forms the first stage in the tri-synaptic loop, and so is in a position to regulate much of the information directed toward the HF from the neocortex. In addition, much of the literature concerned with the influence of serotonergic agents on the HF has been confined to in vitro preparations. Although experiments utilizing in vitro preparations are most useful for determining the responses of discrete neural tissues to pharmacological agents, the ultimate goal of most neuropharmacological research must be to ascertain the manner in which these agents influence the functioning of the organism in vivo. This is especially true in research on serotonin, which has been implicated in many behavioural and clinical phenomena, including sleep, memory, depression, anxiety, sex, feeding, and aggression. In order to make causal inferences between the physiological effects of compounds, and their effects in either behavioural experiments or in clinical settings, it is necessary to verify the results from in vitro experiments with in vivo preparations. In the whole animal, systemic administration of a drug may exert effects on a specific brain region in numerous ways. Obviously, the compound can have a direct action on the tissue in question, providing that it can permeate the blood-brain barrier. Drugs may, however, also act upon other regions of the brain, which may, in turn, modulate the activity of the region in question; they may also produce peripheral effects, such as hormonal release or cardiovascular responses, which may then influence brain functioning indirectly. Additionally, more than one of these effects could act in concert. All of these factors must be taken into consideration in order to determine the functional significance of a pharmacological receptor or a neuroactive drug in a living animal. The purpose of the studies described in this dissertation was to investigate the influence of the serotonergic system on the electrophysiological activity of the HF, using both in vitro and in vivo preparations. The experiments conducted in this thesis are organized into three sections. The first two sections deal essentially with pharmacological studies, and investigate the effects of serotonin and serotonergic agents, in vitro and in vivo. The purpose of the experiments presented in these sections is to determine the modulatory effects of serotonergic compounds on evoked population responses, and thereby to make inferences about the roles of 5-HT receptor subtypes in the functioning of the HF. The first section compares the effects of serotonin and serotonergic ligands on electrophysiological responses in the DG and the CAl, using the in vitro hippocampal slice preparation. The second section examines the effects of serotonergic agents on electrophysiological responses in the DG in an anaesthetized, in vivo rat preparation. The third section examines the effects of stimulation of the MRN, the major source of serotonergic afferents to the HF, on synaptic plasticity in the DG. An introduction to this last topic will be presented separately, as the third section differs somewhat from the other two, both conceptually and methodologically. In all of these experiments, the dependent variable is the population response, recorded extracellularly in the HF. Responses were recorded in either the dentate gyrus or the CAl region, and evoked by either PP or Schaffer collateral stimulation, respectively. The recording of population responses has several inherent advantages over the recording of either intracellular potentials or patterns of unit activity. Intracellular recording from DGCs is difficult due to their small size; this is especially true in an in vivo preparation. The recording of unit activity, while desirable, would not allow for meaningful comparisons between in vivo and in vitro preparations, as spontaneous unit activity is observed less frequently in vitro (Misgeld et al., 1982). The recording of population responses offers the additional advantage of long term stability of recording both in vivo and in vitro. This facilitates the assessment of accurate time courses of pharmacological effects. In addition, the measurement of population responses, which provide a global indication of the state of a specific cell field, appears to be particularly appropriate when addressing the influence of a diffuse system such as the serotonergic projections to the HF. CHAPTER 2: IN VITRO PHARMACOLOGICAL EXPERIMENTS 2.1 INTRODUCTION The in vitro brain slice preparation, introduced by Yamamoto and Mcllwain (1966) is designed to facilitate the recording of neuronal activity, by maintaining an isolated portion of brain in an easily accessible artificial environment. The in vitro chamber is intended to simulate the conditions surrounding the tissue in the intact brain, by exposing it to warm, oxygenated artificial cerebrospinal fluid (ACSF). This preparation has been used with great success, as the viability of neural tissue may be maintained for more than eight hours at a time. Transverse slices of the HF are particularly amenable to in vitro preparation, as this allows for direct visualization of the cell fields, while essentially preserving the tri-synaptic circuitry of the region (Skrede and Westgaard, 1971; Andersen etal., 1971). 2.2 GENERAL METHODS A. PREPARATION OF HIPPOCAMPAL SLICES The in vitro experiments in this thesis were performed on tissue from male Wistar rats weighing 220-320 gm, which had been group housed, and maintained on a 12 hour light/dark cycle, with food and water available ad libitum. The animals were lightly anaesthetized with ether, and decapitated with a guillotine. The skin and muscle over the skull was cut, the surface of the skull scraped clear of tissue with a scalpel, and the dorsal surface of the cranium removed with rongeurs. The dura mater was cut carefully with scissors, and removed with fine forceps. Cold (4°C), oxygenated ACSF was poured immediately onto the exposed brain, to decrease metabolic rate, and so reduce the possibility of anoxic damage, and to firm the neural tissue, thereby reducing damage from handling. A transverse scalpel cut was made through the brain at the level of the coronal suture, and a scalpel handle was used to sever the cranial nerves and to remove the brain tissue caudal to the cut. The brain was placed on a filter paper, that had been placed on an overturned petri dish, and drenched with cold, oxygenated ACSF. Throughout the following procedures, until the cutting of the slices, cold, oxygenated ACSF was periodically poured onto the brain tissue. The cerebellum was removed with a transverse scalpel cut, and the two hemispheres were separated by a saggital incision through the cortex and brainstem. The right hemisphere was placed on its anterior cut surface, and a blunt dissecting instrument was used to separate the brainstem partially from the overlying cortex, and so expose the ventro-medial surface of the HF. Fine forceps were used to remove any remaining dura mater. The hemisphere was then placed on its lateral surface, and a blunt, non-metallic dissecting instrument was used to sever the fornix, and then to free the hippocampus from the neocortex. Any remaining cortical tissue attached to the hippocampus was carefully cut away. The HF was then washed onto a small piece of filter paper with cold, oxygenated ACSF, and was placed onto the stage of a Mcllwain tissue chopper. The stage was aligned so that the long axis of the HF was perpendicular to the chopper blade; this orientation was intended to preserve the intrinsic circuitry of the HF, and to disrupt only the longitudinal fibre tracts. Six slices, of approximately 400um thickness, were cut from the mid-region of the dorsal HF, along with a slice of filter paper underlying each slice. A fine brush was then used to lift each slice as it was cut, by contact with only the filter paper underneath, and place it into a petri dish filled with ACSF. This method reduced the amount of direct handling of the hippocampal slices. When all the slices were cut, they were transferred gently onto a nylon mesh net in the recording chamber, using either a fine brush or a pipette filled with ACSF. The above procedure, from decapitation to placement in the recording chamber, was usually accomplished in 5 - 8 minutes. B. T H E IN VITRO C H A M B E R The in vitro chamber is a device constructed out of clear plastic, and contains a recording well which is surrounded by a water jacket. Warmed, oxygenated ACSF is fed continuously by gravity, at a rate of about 3 ml/min, to the recording chamber, where it bathes the lower surfaces of the hippocampal slice. The water jacket is partially filled with warmed distilled water which serves to heat the incoming ACSF and the recording chamber, maintaining them at 34 _+ 0.5°C, and to heat and humidify a gas mixture of 95% C>2 and 5% CC*2 which is introduced into the atmosphere of the recording chamber. C. A R T I F I C I A L C E R E B R Q - S P I N A L F L U I D The composition of the ACSF was intended to simulate the ionic constituents of mammalian cerebro-spinal fluid (CSF). This solution contained, in millimolar concentration, 124 NaCl, 2 KC1, 1.25 KHP0 4, 1.5 CaCl 2, 1.5 MgS0 4, 24 NaHCOg, and 10 D-Glucose, and was equilibrated with a gas mixture of 95% O2 and 5% CO2 to a pH of approximately 7.3. Although this ACSF differs from mammalian CSF by not containing any organic molecules (e.g., amino acids or proteins), this modified Ringer's solution has been shown to maintain viability in brain tissue for 12 hours (Lee et al., 1981). In the in vitro experiments involving drug administration, the drugs were dissolved in this same ACSF. D. S T I M U L A T I O N T E C H N I Q U E S Bipolar stimulating electrodes were constructed from twisted, insulated, 62um diameter nichrome wire, with the insulation removed to expose the final 0.5 mm. These electrodes were mounted individually on Narashige micromanipulators, and lowered to the surface of the slice, under visual guidance with a binocular dissecting microscope (40X magnification). When potentials were recorded in the dentate gyrus, the tip of the stimulating electrode was placed in the outer third of the molecular layer of the DG, to activate fibres of the lateral perforant path; when recordings were made in the C A l region, the electrode tip was placed in the stratum radiatum of the C A l , to activate the Schaffer collaterals (Figure 2.1). Electrical stimulation was delivered by photon coupled, electrically isolated stimulation units (Medical Systems Corp., Model DS2), which were controlled by electronic timing and delay units (Medical Systems Corp. Digitimer, Models D4030 or D2030). Stimuli were square wave pulses of lOOwsec duration, presented at an intensity between 0.5 to 70 V. (approximately 5 - 200 uA.) During electrode placement, the intervals between successive stimuli were maintained at a minimum of 10 sec in the case of Schaffer collateral stimulation, and at a minimum of 20 sec for PP stimulation, to avoid any frequency dependent changes such as long-term potentiation (LTP) or habituation. Following electrode placement, stimuli were presented at the rate of one per minute in both regions. E. RECORDING TECHNIQUES Extracellular field potentials were recorded via glass microelectrodes (Fisher, Omega Dot, 1.5 mm outside diameter (od), 0.75 mm inside diameter (id)), which were drawn to a fine tip with a vertical electrode puller (Narashige model PE-2), and filled with 2M NaCl solution. The puller was adjusted to produce electrodes which, when filled, had a tip resistance of between 3 and 8 megohms; the resistance of each electrode was tested with an ohmmeter. The output of these electrodes was connected to an impedance matching amplifier (World Precision Instruments probe system, model KS 700), and reference was made to Ag/AgCl ground lead located in the recording well of the in vitro chamber. The signal was filtered (3KHz low pass), displayed on a Tektronix dual beam oscilloscope, and led to a PDP 11/23 computer system for storage and analysis. The recording electrodes were attached to Narashige fine micromanipulators, and positioned, either by hand, or with a Burleigh Inchworm, into the stratum pyramidale of the C A l region, or into the stratum granulosum of the DG, to an approximate depth of 75-150 um below the surface of the slice. Population responses in the C A l and DG were evoked by-electrical stimulation of the Schaffer collaterals or perforant path respectively. Only one pair of recording and stimulating electrodes were used per slice, and simultaneous recordings normally were taken from each of two slices in the recording chamber. Fine adjustments of the electrodes were used to maximize the amplitude of the evoked potentials. After equilibration, the stimulus intensity was adjusted to elicit a population spike of 40-60% of the maximal amplitude, with that amplitude measured as described in section 2.1.E below. With these stimulation parameters, the amplitude of the population spike is most labile, and, in the C A l , has been reported to be most sensitive to the effects of 5-HT (Beck and Goldfarb, 1985). F. QUANTIFICATION OF ELECTROPHYSIOLOGICAL RESPONSES The amplitude of the population spike was measured as the difference in voltage between the peak of the first positive wave (PI) and the peak of the first negative deflection (Nl) (Figure 1.2). This method of calculating the PSA produces qualitatively similar results to both the method of Steward et al. (1976) whereby PSA is calculated as the mean of the potential differences between PI and N l and between N l and the peak of the second positive wave (P2), and the method of McNaughton and Barnes, (1977) whereby PSA is calculated as the area under the tangent line connecting PI and P2. The method used here to measure population spike amplitude appears, theoretically, to be a better indication of the number of principal neurons activated synchronously by afferent fibre stimulation. The first positive wave reflects the source current for dendritic excitatory post-synaptic potentials (EPSPs), and is relatively unchanged if the action potential is blocked, for example by applied electric fields (Richardson et al., 1988); the population spike appears superimposed on this positive wave. The best measure of the number of action potentials evoked would be a calculation of the area of the population spike which is superimposed on the population EPSP. The true area of the population EPSP is not represented by the area under a line joining the PI and P2 waves, however, as the P2 wave is influenced greatly by ionic fluxes secondary to action potential generation, especially repolarizing K+ currents. For this reason, measures of population spike amplitude involving the P2 component may be less accurate. The population EPSP slope was measured as the maximum slope between baseline potential and the first peak positivity (the presence of a population spike precluded measurement of the population EPSP amplitude). G. PHARMACOLOGICAL EXPERIMENTS Population responses were evoked and recorded at the rate of one per minute. Drugs were administered after stable baseline responses (defined as no greater than 10% variability from the median population spike amplitude) were collected for 30 consecutive minutes. The confirmation of a stable baseline was necessary for two reasons. Firstly, a gradual increase in response amplitude was often observed for a period of time after electrode placement, presumably due to tissue equilibration. Secondly, the evoked responses were sometimes quite variable, and never met the criterion of stability, these slices were not used. Of the total of approximately 350 hippocampal slices recorded from, 260, or about 75% produced stable baseline recordings as defined by the above criteria. Drugs, dissolved in ACSF, were generally administered for a 10 minute period by means of a three-way stopcock. As some compounds, such as 8-OH-DPAT, are known to be sensitive to prolonged oxygenation, all of the drugs used were mixed into ACSF only 10 minutes prior to bath application, and oxygenated for only 3 min prior to, and during superfusion. In control experiments, drug-free ACSF, in a separate flask, was oxygenated and administered in the same manner as were drug solutions. Figure 2.1: Schematic diagram of the hippocampal slice preparation, illustrating electrode positions and representative evoked population responses. Responses recorded in the granular layer of the DG (Rl) were evoked by stimulation of the lateral perforant path fibres in the outer molecular layer of the DG (Si); responses recorded in the stratum pyramidale of the CAl (R2) were evoked by stimulation of the Schaffer collaterals in the stratum radiatum. (S2). 52 2.3 THE EFFECTS OF 5-HT, 8-OH-DPAT AND DOI ON EVOKED POTENTIALS  IN THE CAl AND DG IN VITRO A. INTRODUCTION As discussed in section I.5.C. above, a reduction in the PSA evoked in area C A l has usually been reported after application of 5-HT or 5-HT-^ agonists, suggesting an inhibitory role for the 5-HTj A receptor in the functioning of C A l hippocampal pyramidal cells (HPCs). To date, there have been no reports of the effects of serotonergic compounds on population responses in the DG, although studies of unit activity and intracellular potentials suggest similar inhibitory actions of 5-HT and 5-HT-j^ agonists on DGCs. Unlike the C A l region, the DG also contains moderate levels of 5-HT2 binding sites, which may also represent functional receptors. The purpose of the present in vitro experiments was to investigate the effects of serotonin, the 5-HT 1 A agonist 8-OH-DPAT, and the 5-HT2 agonist DOI on population responses recorded in the DG, and to compare these with the effects of these compounds on responses recorded in area C A l . The compound 8-OH-DPAT is the most selective agonist presently available for 5-HT 1 A receptor, and has an affinity for the hippocampal 5-HT-^ binding site in the low nanomolar range, which is several thousand times greater than its affinity for any other serotonergic binding site (Gozlan, et al., 1983; Middlemiss and Fozard, 1983; Hall, et al., 1985). Additionally, 8-OH-DPAT has been shown to be inactive at D 2, H^, a-^, GABA A, and benzodiazepine receptors (Johnson et al., 1987; Mynlieff and Dunwiddie, 1988). DOI is the most selective, and among the most potent of 5-HT2 agonists available, with an affinity at the 5-HT2 site about 100 times greater than at the 5-HT-L sites (Shannon et al., 1984; Glennon et al., 1987). Large discrepancies in dose-response curves have been reported for the effects of either 8-OH-DPAT or 5-HT on PSA in the C A l . For example, while Beck et al. (1985) report the effectiveness of 8-OH-DPAT to be between 30nM and 30uM, Peroutka et al. (1987) specify that the C A l population spike amplitude was unaffected at concentrations of less than 25uM, and that maximal responses were not obtained at lOOwM. Similarly, the concentrations of 5-HT reported to reduce C A l population spike amplitude by Olpe et al. (1984) are an order of magnitude greater than those found by Beck and Goldfarb (1985) to produce comparable responses. These discrepancies could be due to the various procedural differences between the studies, e.g.: methodology for preparing the hippocampal slices; composition, temperature and flow rate of the superfusate; partial versus complete submersion of the slices; stimulus frequency; or, in one study (Peroutka et al., 1987), the use of female rats. In addition, the concentration of drug at the neurons around the recording electrode tip may be dependent upon the depth of the electrode in the slice, a measure which is usually not reported. In order to provide an accurate comparison of the pharmacological effects between the C A l and DG, it is therefore necessary to conduct experiments on the C A l region utilizing the same techniques and procedures as used for the DG. In the present studies, therefore, experiments in area C A l and the DG were usually performed in parallel. Simultaneous recordings were usually made from the DG and the C A l of two slices in the same chamber, so that both slices were subject to identical conditions. This technique provides good control for the comparison of the two regions, especially when considering that simultaneous recordings were always made from slices prepared from the same rat. Dose-response curves for the inhibition of PSA in the C A l by serotonergic agonists have usually been derived by the repetitive administration of compounds, with the response to these compounds at each concentration then expressed as a percentage of the maximal response elicited in each individual preparation. This approach may be problematic in that some effects of serotonergic compounds clearly exceed the duration of application. For example, Makhov et al. (1982) have reported that a long latency (>40 min) increase in the unit activity of C A l pyramidal cells is often observed after the bath application of 5-HT. This excitatory effect appears not to have been a "rebound" from the initial inhibitory response, as the two effects were reported to occur independently. Similarly, the effects of 8-OH-DPAT on unit activity, intracellular potentials, or PSA in the C A l have been shown to last long after the application of this compound. In addition, 5-HT receptors are known to interact with complex second messenger systems which may mediate long-lasting biochemical effects. Finally, there is evidence that 5-HT^A agonists may produce a prolonged desensitization of 5-HT^A receptors (Kennett et al., 1987). Given the above factors, dose-response relationships were investigated in the present experiments with only one application of one concentration of drug per slice. This permitted accurate evaluation of the response to a given concentration of drug, while avoiding the possible influence of previous administrations. B. METHODS Hippocampal slices were prepared and maintained in vitro as described in the General Methods section for this chapter. Population responses in the C A l and the DG were evoked at the rate of 1/min, by electrical stimulation of the Schaffer collaterals or the lateral perforant path respectively. Stimulation intensity was adjusted to evoke population spikes of approximately 50% of maximal amplitude, as the responses have been reported to be most sensitive to 5-HT at this level of stimulation (Beck and Goldfarb, 1985). After stable baseline responses were collected, the chamber was perfused with ACSF containing drug, or with ACSF alone for 10 min. Population responses were evoked and recorded for three hours following drug application. The drug solutions contained either 1, 5, 10, 20, 50, or lOOuM 5-HT creatine sulphate (Sigma); 1, 5, 10, 20, or 50 uM 8-OH-DPAT HBr (Research Biochemicals Inc., Natick, Mass., USA); or 1, 5, 10, 20, or 50 uM DOI (Research Biochemicals Inc.). Each drug or control solution was applied to 6-10 hippocampal slices for each of the two recording sites. C. DATA ANALYSIS Population spike amplitude and population EPSP slope were calculated for each response, and these values were normalized relative to the mean of their respective baseline values. Data from each preparation were grouped in five minute bins for analysis, with each bin representing the mean of five responses from that preparation. Statistical analyses were performed on the mean value of the responses collected during the final 5-10 minutes of perfusion of 5-HT or DOI, and on the means of the responses collected between 10-15 mins following application of 8-OH-DPAT. The data from 5-HT, 8-OH-DPAT and DOI experiments (drug conditions) were analyzed separately. In each drug condition, PSAs and EPSP slopes were entered into a multivariate analysis of variance (MANOVA), with site (DG or CAl) and dose (including control group) as between subject factors. If MANOVA procedures indicated a significant overall effect (indicated by Wilk's lambda), these tests were followed by separate univariate analyses of variance (ANOVA) of PSA and EPSP, with dose and site as between subject factors. Following ANOVA procedures, PSA and EPSP values obtained at each dose were compared to control values using Dunnett's multiple comparison test, with critical t values adjusted to maintain familywise a levels at 0.05 or below (Glass and Hopkins, 1984). MANOVA and ANOVA procedures were run on the SPSS^ statistical package based on the University of British Colombia mainframe computer. D. RESULTS 1) CONTROLS Control preparations (N = 8 for each of DG and C A l recordings) exhibited stable population spike amplitudes and EPSP slopes recorded from both C A l or DG over three hours of recording; the variability of these responses, however, did increase with time. 2) 5-HT Superfusion of hippocampal slices with 5-HT produced a dose-related reduction in PSA and EPSP slope in both the C A l and DG, with a more pronounced effect on PSA in the C A l region (Figures 2.2a, 2.3, 2.4). The effects of 5-HT on evoked responses in both regions washed out rapidly in both regions, with responses recovering to baseline values usually within 10-15 min. MANOVA results revealed significant effects of dose (F(12,144) = 8.83, p<.001) and site (F(2,72) = 12.09, p<.001), as well as a significant site by dose interaction (F(12,144)= 1.89, p<.05). Univariate tests demonstrated a significant effect of dose on both PSA (F(6,73) = 20.54, p<.001) and EPSP slope ((F(6,73) = 2.49, p<.05), and a significant effect of site on PSA (F(l,73) = 24.52, p<.001), but no significant effect of site on EPSP. Univariate analysis did not confirm a site by dose interaction on either measure. Dunnett's comparisons revealed that population spike amplitude was reduced significantly (p < .05 ) by 20uM and greater concentrations of 5-HT in the DG, and by 5uM and greater concentrations in area C A l . In both regions, 50uM 5-HT produced a maximal effect. At this concentration, the population spike amplitude was reduced to 55 _+ 8% of baseline values in the C A l (£(11) = 3.72) and to 80 _+ 7% of baseline values in the DG (£(11) = 7.06), 10 minutes after superfusion. EPSP slopes were decreased significantly (p<,05) by 20uM and higher concentrations of 5-HT in the DG, and at lOuM and higher in the C A l . After superfusion with 50uM 5-HT, EPSP slopes were reduced to 83% +_ 6% of baseline values in the C A l (£(11) = 3.06), and 89% _+ 3% of baseline values in the DG (£(11) = 2.03). 3) 8-OH-DPAT Superfusion of the hippocampal slice with 8-OH-DPAT produced a dose-related reduction in the amplitude of evoked responses in both the C A l and DG, and, as with 5-HT, a more pronounced effect was observed on the amplitude of population spikes recorded in the C A l region (Figures 2.2b, 2.5, 2.6). Multivariate analysis showed significant effects of dose (F(10,136)= 11.95, p<.001) and site (F(2,68)= 10.34, p<.001), and a significant dose by site interaction F(10,136) = 2.28, p<.05). Univariate analysis of PSA revealed significant effects of dose (F(5,69) = 23.8, p<,01), site (F(l,69) = 20.91, p<.001), and dose by site interaction (F(5,69) = 4.42, p<.005). Univariate analysis of EPSP slopes revealed a significant effect of dose (F(5,69 = 8.17, p<.001), but not of site nor dose by site interaction. Dunnett's comparisons revealed significant (p<.05) effects of lOuM 8-OH-DPAT and above on PSA in both regions. In both area C A l and the DG, lOuM of 8-OH-DPAT produced a maximal effect on PSA. At this concentration, the population spike amplitude was reduced to 47 +_ 7% of baseline values in the C A l (f(ll) = 7.33) and to 79 +_ 5% of baseline values in the DG (f(13) = 3.22). Interestingly, in the C A l only, 50wM 8-OH-DPAT brought about an apparently smaller effect on population spike amplitude than lOuM; however, the planned format of the analyses prevented a legitimate statistical test of this difference. Population EPSP slopes were decreased significantly (p<.05) in both regions after administration of lOuM and higher concentrations 8-OH-DPAT, with values reaching 79 _+ 7% (t(ll) = 2.28) and 76 _+ 6% (((13) = 3.51) of baseline values in the C A l and DG, respectively, after superfusion with lOuM. The time course of these changes in evoked responses was prolonged. In both the C A l and DG, the maximum reduction in population spike amplitude was observed 20-30 min after the start of drug superfusion. The duration of these inhibitory effects varied with drug concentration, as population spike amplitudes returned to pre-drug baseline values within 90 minutes after perfusion with 5uM 8-OH-DPAT, whereas maximum inhibition was observed for the three hour duration of the experiment at concentrations of 10-20MM in C A l and 10-50uM in the DG. At 50uM, population spike amplitudes in the C A l returned to control values within two hours of superfusion. 4) DOI Superfusion of the hippocampal slice with DOI produced a dose-related reduction in the amplitude of evoked responses in the DG, but had no apparent effect on responses recorded in the C A l (Figures 2.2c, 2.7, 2.8). The lack of response in the C A l was indicated by preliminary data, and therefore experiments in C A l were confined to 10 and 50 uM concentrations of DOI. The different number of doses used in C A l and DG experiments required that two sets of analyses be run; one to allow comparison of C A l responses with DG responses at 10 and 50uM DOI, and a second to examine the entire range of doses used in the DG. The first MANOVA compared responses from both regions at 10 and 50uM, and revealed significant effects of dose (F(4,64) = 2.88, p<.05) and site (F(2,32) = 16.18, p<.001), and a significant dose by site interaction (F(4,64) = 3.64, p<.05). Univariate analyses revealed significant effect of dose (F(2,33) = 4.83, p<.05), site (F(l,33) = 23.13, p<.001) and dose by site interaction (F(2,33) = 3.65, P<.05), for PSA only, and no significant effects on EPSP slope. Dunnett's test disclosed significant (P<.05) effects of DOI on PSA in the DG, at both doses, with a reduction to 80 ± 4% (((12) = 3.917) of baseline values at lOuM. No significant effects of DOI were found on responses recorded in area C A l . The second series of analyses compared responses in the DG only, at all doses of DOI. MANOVA disclosed a significant effect of dose (F(5,32) = 2.36, p<.05). Univariate analysis, however, indicated no significant effects of dose on either PSA or EPSP slope,although the effects on PSA in the DG did approach significance (F(5,32) = 2.13, p = .087). Figure 2.2. Representative population responses recorded from the C A l and the DG of six different preparations, before and after drug superfusion. A. Responses recorded before, and 10 min after the start of 50uM 5-HT application. B. Responses recorded before, and 20 min after the start of lOuM 8-OH-DPAT application. C. Responses recorded before, and 10 min after the start of 10uM DOI application. 61 Figure 2.3 Dose-response to 5-HT of (A) population spike amplitudes and (B) population EPSP slopes in area C A l and DG, 5-10 min after drug application. All data points in Figs 2.3-2.8 represent the mean _+ s.e.m. of data from 6-10 hippocampal slices. X axis is log scale. Open circles: DG; closed circles: C A l . Figure 2.4 (A) Population spike amplitudes, (B) population EPSP slopes after administration of 50uM 5-HT Measurements are expressed as a percentage of mean pre-drug baseline responses. Open circles: DG; closed circles: C A l . 65 A. TIME (MINUTES) Figure 2.5 Dose-response to 8-OH-DPAT of (A) population spike amplitudes and (B) population EPSP slopes in area C A l and DG, 20-25 min after drug application. Open circles: DG; closed circles: C A l . Figure 2.6 (A) Population spike amplitudes, (B) population EPSP slopes after administration of lOuM 8-OH-DPAT. Open circles: DG; closed circles: CAl. Figure 2.7 Dose-response to DOI of (A) population spike amplitudes and (B) population EPSP slopes in area CAl and DG, 5-10 min after drug application. Open circles: DG; closed circles: CAl. 71 A. 120% -j 110% \ 10"6 10~5 10 ~ 4 CONCENTRATION OF DOI (M) B. 120%n 110% J CONCENTRATION OF DOI (M) Figure 2.8 (A) Population spike amplitudes, (B) population EPSP slopes after administration of lOuM DOI. Open circles: DG; closed circles: CAl. A. TIME (MINUTES) 2.4. BLOCKADE OF SEROTONERGIC EFFECTS BY METHYSERGIDE AND KETANSERIN A.INTRODUCTION All of the "classical" serotonin antagonists, e.g. spiperone, cyproheptadiene, methiothepin, are non-selective between the 5-HT2 and 5-HT^ receptor subtypes. Recently, selective antagonists for the 5-HT2 receptor have been developed; one of the more selective of these is ketanserin (Janssen, 1983). Selective antagonists for the 5-H T j A receptor are not currently available. Methysergide, like most other non-selective serotonergic antagonists, has a higher affinity for the 5-HT 2 than the 5-HTj binding site and does not differentiate between the 5 -HT 1 A and 5-HT^g receptors; its relative affinity for the 5-HTj sites, however, is greater than that of most other commonly used antagonists, and its use in the identification of 5-HT^ receptors is well documented (Bradley, et al., 1986). In order to examine further the role of 5-HT receptors, this experiment investigated whether ketanserin or methysergide could block the effects of 5-HT, 8-OH-DPAT or DOI on population responses in the DG and CAl. A. METHODS The procedures in Experiment 2 were identical to those described for Experiment 1 above, except for drug application. To assess the effects of the antagonists alone, slices were superfused with ACSF containing either luM methysergide or luM ketanserin (N = 6 slices for each drug, for each of DG and CAl recordings) for 15 min. In order to assess the combined effects of agonists and antagonists, ACSF containing antagonist alone was first superfused for 5 min, followed by ACSF containing a combination of that antagonist and an agonist. The following agonist-antagonist combinations were used: 20uM 5-HT and lwM methysergide, 20uM 5-HT and luM ketanserin, lOuM 8-OH-DPAT and luM methysergide, and lOuM DOI and luM ketanserin. Concentrations of agonists 75 were chosen w h i c h were shown in the previous experiment to produce significant responses w h e n applied alone. C A l and D G recordings were made f rom 6-8 slices each at every d r u g condition, w i t h the exception of the D O I and ketanser in combination, which was applied to slices w i t h D G recordings only. B. RESULTS Stat is t ica l analyses were conducted using the mean normal ized responses collected d u r i n g the final 5-10 minutes of drug application. The exceptions to this were the comparisons involv ing the combined 8 - O H - D P A T and methysergide condition, which, due to the prolonged time course of 8 - O H - D P A T , were conducted on data collected 10-15 minutes after d r u g application. Antagonist-alone superfusions were compared w i t h control preparations f r o m the previous experiment, while combined agonist-antagonist ) superfusions were compared with agonist-alone superfusions from the previous experiment. T h e results f rom this section are summarized i n F igure 2.9. Nei ther methysergide alone nor ketanser in alone produced significant effects on P S A or E P S P slope, in either the D G or area C A l . A l though the combination of 5-HT and methysergide produced less inhibition of the P S A and E P S P slope in both regions than did 5 - H T alone, this only reached significance for the effects on C A l E P S P . The inhibit ion of population responses produced by 5 - H T i n either region was not significantly different f rom that produced by the combination of ketanser in and 5-HT. T h e combination of methysergide and 8 - O H - D P A T produced significantly less inhibit ion of both P S A and E P S P slope in both the C A l and D G t h a n did 8 - O H - D P A T alonei Similar ly , - the combination of ketanserin and D O I produced significantly less inhibition of P S A in the D G , than did D O I alone. The following paragraphs give details of the stat ist ical analyses. The E P S P slopes and P S A s from the ketanserin-alone and methysergide-alone conditions were compared wi th the control (i.e. vehicle only) responses from the previous experiment in a M A N O V A , wi th drug condition and site ( D G or C A l ) as between subject factors. No significant effects of d r u g condition, site, or interact ion of the two were found. The responses from the 5 - H T plus ketanserin a n d 5 - H T plus methysergide conditions were compared w i t h the responses from the 2 0 u M 5 - H T condition from the previous experiment. The responses f r o m C A l and D G were entered into a M A N O V A which disclosed significant effects of d r u g condition (F(4,62) = 2,13, p < . 1 0 (Significant a values were set to .10 i n comparisons of agonist-only conditions w i t h combined agonist-antagonist conditions, as two-tailed tests were performed, although the comparisons were essentially one-tailed. The nul l hypothesis was that the presence of the antagonist would attenuate the effects of the agonist)). U n i v a r i a t e analysis revealed significant effects of drug condition on P S A only (F(2,34) = 2 .04, p < . 1 0 ) . F u r t h e r comparisons wi th Dunnett 's test determined that no significant differences existed between the combined 5 - H T plus ketanserin condition and the 5 - H T alone condition in either region. P S A s were inhibited to a significantly greater extent under the 5 - H T alone condition than under the combined 5 - H T plus methysergide condition in the C A l (f(12) = 2.41, p < . 0 5 , one-tailed), but i n the D G this effect only approached significance (r(12)=1.89, p<.10,one-tailed). The cr i t ical t values were corrected to m a i n t a i n f a m i l y w i s e a lpha at .05 . The responses from the combined 8 - O H - D P A T plus methysergide condition were compared with the responses f rom the l O u M D P A T condition f rom the previous experiment. Mult ivar iate analysis of D G a n d C A 1 responses indicated significant effects of drug condition (F(2,21) = 4.64, p < 0 5 ) , and univar iate analysis revealed significant differences between drug groups i n both P S A s ( F ( l , 2 2 ) = 7.30, p < . 0 5 ) and population E P S P slopes (F(l ,22) = 3.02, p<.10) . Responses were then compared us ing Student's t tests, which showed significant ( p < . 0 5 , one tai led, Dunn-Bonferroni corrected) differences between the two conditions i n both E P S P slope and P S A , i n both the D G and C A l ( C A l P S A : ( t ( l l )=10.6) ; D G P S A : (t(13) = 3.46); C A l E P S P : ( t ( l l ) = 6.17); D G E P S P : (t( 13) = 6.85). Figure 2.9 Antagonism of the effects of 5-HT, 8-OH-DPAT, and DOI by methysergide and ketanserin. Application of methysergide or ketanserin alone preceeded application of these compounds in combination with 5-HT, 8-OH-DPAT, or DOI by 5 mins. The effects of 5-HT and DOI were asessed during the final 5-10 min of application of these compounds; the effects of 8-OH-DPAT were assessed 10-15 min after application. A) CAl responses; B) DG responses Solid bars: population spike amplitudes; open bars: population EPSP slopes. 5HT: 20uM 5-HT 5HT + ME: 20uM 5-HT + luM methysergide 5HT + KE: 20uM 5-HT + luM ketanserin DP: lOuM 8-OH-DPAT DP + ME: lOuM 8-OH-DPAT + luM methysergide DO:10uMDOI DO + KE: lOuM DOI + luM ketanserin *: p<.05 vs agonist only condition. A Student's t-test was used to compare the D G P S A s recorded from the combined D O I plus ketanserin condition, and the l O u M D O I condition from the previous experiment. This comparison indicated a significant difference between the two conditions (t(10)— 1.98, p < . 0 5 , one-tailed). A s M A N O V A procedures in the previous experiment d id not indicate a significant effect of D O I , however, this apparent antagonism by ketanser in must be treated wi th caution. 2.5 DISCUSSION The present study found that a decrease i n P S A and E P S P was observed i n both area C A l and the D G following bath superfusion of 5 - H T or the 5 - H T j ^ agonist 8 - O H -D P A T . The effects of 5-HT and 8 - O H - D P A T were attenuated by the non-selective serotonergic antagonist methysergide, further indicating that the effects of these agonists are mediated by serotonergic receptors. Al though methysergide has previously been reported to hyperpolarize C A l H P C s in vitro (Andrade and N i c o l l , 1987), the present study found that superfusion with methysergide alone did not affect population responses. It is possible the iontophoretic route of drug application used b y Andrade and Nicol l (1987) m a y have resulted i n much higher local concentrations of the drug , w i t h resultant non-specific effects. The attenuating effects of 5-HT and 8 - O H - D P A T on population responses i n the C A l observed i n the present study are in agreement wi th ear l ier reports; however, unl ike the studies of Beck and Goldfarb (1985) and R o w a n and A n w y l (1985), no transient increase i n these measures was observed following superfusion w i t h 5 - H T . T h e inhibi tory effects of 5 - H T and 8 - O H - D P A T on population responses i n both regions are consistent w i t h previous studies using intracellular techniques, which have reported that application of either of these compounds produced membrane hyperpolar izat ion, increased g K , and inhibited unit activity in both D G C s and C A l H P C s . Interestingly, the present study indicated that i n the C A l , 5 0 u M 8 - O H - D P A T m a y be less effective at reducing P S A than l O u M . T h i s possible reduction in efficacy at 50uM may reflect a loss of specificity for the 5-HT 1 A site at higher concentrations of the drug. Alternatively, local circuits in the hippocampus may be affected differentially by changes in concentration, resulting in an altered balance of excitation and inhibition. The present results demonstrate a depression of cell layer population EPSPs coinciding with the inhibition of PSAs produced by 5-HT and 8-OH-DPAT in both regions. As the somatic layer EPSP reflects the synaptic current arising in the dendrites as a result of the action of neurotransmitter, a decrement in this measure indicates a reduction in net postsynaptic influx of current, which may underlie the concomitantly observed decrease in PSA. Two processes which could mediate the decrease in population EPSPs are decreased neurotransmitter release or shunting of ionic current by an increased membrane conductance; the latter mechanism appears to be more plausible in mediating the effects of the 5-HT-^ receptor, which appears to mediate an increased gK in HPCs and DGCs. The recording site was found to be a significant factor in the effects of both 5-HT and 8-OH-DPAT on PSA, with both of these compounds producing a greater effect on PSA in area CAl than in the DG. A significant "site by dose" interaction was found for the effects of 8-OH-DPAT on PSA, indicating non-parallel effects of this compound on the two regions; additionally, a site by dose interaction cannot be discounted for the effects of 5-HT. Several factors may underlie the differences between the two regions. Firstly, as no significant effect of site was found in the effects of 8-OH-DPAT or 5-HT on population EPSP slopes, a difference could exist between the DGCs and HPCs in the translation of EPSP slope to PSA. Secondly, the difference between the two regions may reflect a smaller conductance change produced by 5-HT ^ A receptors in the DG. This suggestion is supported by a report that iontophoretic application of serotonin hyperpolarizes CAl HPCs to a greater extent than DGCs, an effect which may be related to the more hyperpolarized resting potentials of the DGCs (Segal, 1981). Finally, the difference between the two regions could result from variations in receptor distribution. In the CAl, a dense concentration of 5 - H T ^ A binding sites is located i n the dendritic zones, while fewer sites are distributed in the cell regions (Kohler, 1984). I n the D G , a dense population of 5 - H T ^ A binding sites has been reported in the hi lar region, j u s t inferior to the D G C s ; this pattern is also reflected in the serotonergic innervation of this region. It has been suggested that 5 - H T 1 A binding sites i n the D G could be situated on G A B A e r g i c interneurons, as well as on dentate granule cells (Kohler, 1984, 1988). If 8 - O H - D P A T also exerts an hyperpolarizing action on these interneurons, it could serve to reduce their inhibitory tone onto the dentate granule cells, thereby counteracting the direct inhib i tory action of 8 - O H - D P A T at 5 - H T 1 A receptors on the granule cells themselves. The 5-HT2 agonist D O I had no effect on responses recorded i n area C A l . N o significant effects of D O I were found i n the D G , when responses were analyzed over the full range of doses; however, an inhibitory effect on P S A in the D G w a s indicated b y the analyses comparing 10 and 5 0 u M doses of D O I between sites. A l t h o u g h i t cannot legit imately be concluded from the present study that D O I has an action i n the D G , this possibil ity is supported by the time course and the apparent dose-dependence of the effects of this compound on P S A (Figure 2.6, 2.7). F u r t h e r examination of the effects o f this compound in the D G is therefore warranted. A s wi th methysergide, the selective 5-HT2 antagonist ketanser in did not affect population responses in either region when applied alone. K e t a n s e r i n w a s found to reduce the effects of D O I on P S A i n the D G , further suggesting the involvement of the 5-HT2 receptor i n this region. However , as the effects of D O I itself were not found to be statist ical ly significant, this finding must be regarded as provisional . F u r t h e r m o r e , the effects of 5 - H T were not attenuated by ketanserin suggesting either 5-HT a n d D O I m a y act on separate populations of receptors, or that the effects of 5 - H T w h i c h are mediated by 5-HT2 receptors are normal ly masked b y other processes, such as those mediated by 5 - H T j A receptors. To investigate this further, complete dose-response relat ionships should be derived for 5-HT and DOI in the presence of varying concentrations and combinations of antagonists. The suggestion of an inhibitory effect of DOI in the DG is of interest, given that the 5-HT2 receptor appears to mediate excitatory effects in other regions of the brain, such as the facial motor nucleus and the cortex, and that these effects are opposite to those mediated by the 5-HT^A receptor in those regions (Lakosi and Aghajanian, 1985; Davies et al., 1987; Aghajanian et al., 1987). A common inhibitory effect mediated by 5-HT^ A and 5-HT2 receptors would not be without precedent, however, as Maura et al. (1986) have reported that both 8-OH-DPAT and DOI inhibit the release of glutamate from cerebellar tissue. The effects of the bath perfusion of DOI and 5-HT on field potentials were found to be readily reversible after washout, with no noticeable long-term effects; the effects of 8-OH-DPAT were much longer lasting, however, consistent with previous studies (Beck and Goldfarb, 1985; Olpe et al., 1984; Beck et al., 1985). Similar relative time courses for these compounds have also been described using other electrophysiological measures. The hyperpolarization of DGCs and CAl HPCs observed following iontophoretic or pressure application of 8-OH-DPAT is prolonged relative to that produced by administration of 5-HT (Andrade and Nicoll, 1987, Baskys et al., 1986), while the hyperpolarization observed in other neurons (in the lateral septal nucleus) after bath perfusion of 8-OH-DPAT has been reported to last for at least 30 min after bath superfusion (Joels and Schinnick-Gallagher, 1987). In the present study, the duration of the inhibitory effects of 8-OH-DPAT, at the optimal dose of lOuM, exceeded the three hour duration of the experiment in both the CAl and DG. The reasons underlying the long duration of the effects of 8-OH-DPAT remain unknown, but may be related to the lipid solubility of this compound, for which, unfortunately, information is as yet unavailable. In summary, the data from this study suggest an inhibitory role for the 5-HT^A receptor in the DG and CAl , and the possibility of an inhibitory role for the 5-HT2 83 receptor in the DG. These findings are consistent with the results of binding studies, which indicate a high density of 5-HT^^ binding sites in both regions, and a significant concentration of 5-HT2 sites in the DG. CHAPTER 3: PHARMACOLOGICAL EXPERIMENTS IN VIVO 3.1 INTRODUCTION T h e experiments i n the previous section have examined the effects of 5 - H T and serotonergic agents on electrophysiological responses of the in vitro hippocampal slice preparat ion. The in vitro preparation allows the study of a discrete section of n e u r a l tissue i n isolation; this enables the direct examination of how a tissue responds to exper imental manipulations. W h e n neural tissue is part of a functioning b r a i n , however, it is subject to a wide variety of central and peripheral influences which m a y only be assessed i n an in vivo preparation. T h e experiments described i n the present section have utilized pharmacological tools to examine the influence of the serotonergic system on evoked field potentials i n the D G , i n the urethane anaesthetized rat . The first experiment investigated the effects of systemic administration of 5-hydroxytryptophan (5-HTP), the immediate metabolic precursor to 5-HT. This was followed by experiments examining the effects of compounds k n o w n to be active at 5 - H T ^ A and 5 - H T 2 receptors, both of which appear to be concentrated i n the D G . Responses to paired-pulse stimulation I n addition to recording population responses to individual s t i m u l i , as in the in vitro experiments in Chapter 2 above, m a n y of the experiments in this section assessed population responses evoked by pairs of electrical st imuli , which allows examinat ion of more complex characteristics of the system. In the D G , for example, a single s t i m u l a t i o n of the P P can modify the amplitude of a population spike evoked by a subsequent s t imulat ion of the same pathway. A t relat ively short (approx. < 60 msec.) intervals between the pulse pairs, the response to the second, or "test" s t imulat ion is attenuated relative to the response to the first, or "conditioning" st imulation. A t longer intervals (approx. 60-150 msec.) the test response is potentiated relative to the conditioning response; a n d at st i l l longer intervals (approx. 150-8000 msec.) the test response is again attenuated (Figure 3.1). T h e causes of this triphasic response appear to reside in both pre- and post-synaptic elements. In D G C s , the early period of inhibit ion is correlated wi th an inhibitory post-synaptic potential (IPSP), which appears to result f rom a G A B A ^ - r e c e p t o r mediated increase i n chloride conductance (gCl) (Misgeld et a l . , 1982, 1986). This I P S P reaches a m a x i m u m at about 10-15 msec after s t imulat ion , lasts for about 100 msec, and appears to be mediated largely by the release of G A B A f rom basket cells (Thalmann and A y a l a , 1982; A n d e r s e n et a l . , 1966a, L o m o , 1971). T h e late period of inhibition is correlated with a long last ing afterhyperpolarization ( A H P ) of the D G C s . This A H P is dependent on extracel lular K + and C a ^ + , and is a p p a r e n t l y mediated by a postsynaptic C a ^ + dependent g K (Thalmann and A y a l a , 1982; T h a l m a n n , 1984; N i c o l l and Alger , 1981; Rausche et a l . , 1988). The mechanisms u n d e r l y i n g this late inhibition are presently unclear, but m a y involve activation of G A B A g receptors (Hablitz and T h a l m a n n , 1987; N e w b e r r y and Nico l l , 1984). T h e potentiation seen at intervals of about 60 to 150 msec is l ikely to be due largely to presynaptic ca lc ium accumulat ion, and subsequently facilitated neurotransmitter release (Rauche et a l . , 1988; K o n n e r t h and H e i n e m a n n , 1983a,b), a phenomenon which has been shown i n severa l other synaptic systems (Dudel, 1983; K a t z and M i l e d y , 1970; Zucker and Stockbridge, 1983). F r o m a functional viewpoint, this sequence of inhibition-potentiation-inhibition may be looked at as a means by w h i c h the H F effectively filters incoming information. The processes of inhibition and potentiation serve to modulate information travel l ing through that gate, based upon frequency characterist ics; the paired-pulse paradigm provides a method to assess this modulat ion. Figure 3.1 DG responses to paired pulse stimulation of the perforant path from a single urethane anesthetized preparation. This figure illustrates the triphasic sequence of inhibition, potentiation, and inhibition of the population spike, at conditioning-test intervals of 20 msec (A), 80 msec (B) and 400 msec (0). Calibration bar: 10mV., 5 msec. r The recording of extracellular responses evoked by pairs of stimulation pulses at selected conditioning-test (C-T) intervals provides a convenient method with which toassess the individual inhibitory and facilitatory processes. This technique has been used effectively in the assessment of effects produced in the DG by pharmacological and electrophysiological manipulations (Tuff et al., 1983; de Jonge and Racine, 1987; Oliver and Miller, 1985). The magnitude of the early phase of paired-pulse inhibition has been shown to be dependent on the strength of stimulation, as have the processes thought to underlie the potentiation and late phase of inhibition (Oliver, 1986; Tuff et al., 1985; Rauche et al., 1988). In particular, greater early inhibition has been observed at increased stimulation intensities. This early period of inhibition appears to be mediated largely by recurrent activation of GABAergic interneurons, secondary to discharge of the DGCs. As stimulus intensity is increased, more DGCs are recruited to fire synchonously, ostensibly providing greater excitatory drive to these interneurons. These, in turn would then exert a greater inhibitory influence onto the DGCs. The potentiation phase of paired-pulse responses has also been shown to be dependent on stimulus strength, with a decrease in potentiation observed at higher stimulus intensities (Assaf and Miller, 1977; Robinson and Racine, 1985). This may be related to the continuing influence of increased recurrent GABAergic inhibition. Additionally, high stimulation intensities may already be inducing a near-maximal release of neurotransmitter, or, alternatively, evoking a near maximal post-synaptic response. The dependence of these processes on stimulus strength complicates the analysis of changes in paired-pulse responses produced by experimental manipulations. It is essential, therefore to determine if an effect on a paired-pulse measure is occurring independently of any effect on the initial, conditioning response. In order to control for this factor, the experiments in this thesis which utilized paired-pulse stimulation recorded responses at a range of stimulus intensities prior to performing experimental manipulations. 3.2 GENERAL METHODS: Male Wistar rats (350-500g.) were anaesthetized with an intraperitoneal (i.p.) injection of urethane (ethyl carbamate, 1.5 gm/kg dissolved in 0.9% NaCl at 0.23gm/ml). Although urethane anesthesia has been shown to inhibit the unit activity of DGCs to some extent (Buzsaki et al., 1986), it produces minimal effects on evoked potentials (Kamondi et al., 1988), and stands in contrast to barbiturate anaesthetics, which have been shown to produce a dramatic increase in inhibitory postsynaptic potentials in hippocampal neurons (Nicoll et al., 1975). Urethane is also advantageous in that it provides long-lasting anaesthesia, with supplemental doses not being required for periods of eight hours or more. The rats were mounted in a Kopf stereotaxic instrument. A longitudinal cut was made over the skull, and the surface of the skull scraped clear. The scalp was retracted with haemostats or wound clips to keep the skull exposed. Holes were trephined through the skull for the passage of electrodes, and the dura mater underneath slit carefully with the edge of a 26 gauge needle. The exposed cortex was kept moist by the application of warm mineral oil. A tungsten concentric bipolar stimulating electrode was lowered slowly (O.lmm/min) into the angular bundle to stimulate the mixed lateral and medial perforant path, at co-ordinates anterior-posterior (AP) -8.1 and lateral (L) 4.3 mm relative to bregma, and ventral (V) approximately 2.5 mm below the surface of the cortex. Glass recording electrodes were pulled from capillary tubes (Fisher "omega dot", 1.5mm od, 0.75 mm id), and filled with 2M NaCl. These electrodes were pulled in accordance with the method used for in vitro recording, except that the shank was drawn to a much more gradual taper, in order to minimize disruption of brain tissue. For some experiments, a single recording electrode was positioned in the stratum granulosum of the dentate gyrus ( A P -3.8, L 2.0, V 3.5 mm). Other experiments included an additional recording electrode i n the M L of the D G , wi th its tip directed towards a point 1.0 m m dorsal to the G L co-ordinates; this required each recording electrode to be incl ined at 2° to the vertical. A n A g / A g C l wire connected each recording electrode to a shielded cable, which led to a custom-built impedance-matching amplif ier. Reference was made to a ground lead connected to the edge of the wound w i t h a n al l igator c l ip. F i l t e r i n g , display, and storage of the signal were as described for the in vitro exper iments in Chapter 2 above. The f i n a l vert ical position of both electrodes w a s adjusted under electrophysiological control, i n order to maximize the evoked field responses. Af ter f inal electrode placement, the preparation was allowed to equilibrate for at least 30 minutes. Conditioning-test intervals of 20, 80, 400 and 2 0 0 0 msec were chosen for use i n the paired-pulse experiments in this section. The 20 msec i n t e r v a l is short enough to measure the effects of the ear ly period of inhibition, but is offset suff ic iently f rom the peak of that inhibition so that the population spike evoked by the test s t imulat ion is usual ly not completely inhibited. The 80 and 400 msec intervals were chosen to capture approximately the peak effects of the potentiation and late inhibi t ion, a n d the 2000 msec interval to fall within a period where the late inhibition might be assessed without the influence of potentiation or ear ly inhibition. Responses to paired-pulse s t imulat ion were not assessed in a l l experiments; additionally, due to the l i m i t a t i o n s of available equipment, some experiments assessed paired-pulse responses only at a C - T interva l of 20 msec. After equilibration, field responses were evoked and recorded by paired-pulse s t imul i at a range of st imulus intensities at all of the C-T intervals used i n a n y part icular experiment. Responses were evoked and recorded at a range of s t i m u l u s intensities, including those intensities where the conditioning s t imulus evoked a populat ion spike of approximately 25%, 5 0 % , 75%, and 100% of m a x i m a l ampli tude. A t each intensity, 4 paired s t imulus pulses were presented at each C-T interva l , and 40 sec were allowed between each st imulus pair to avoid the involvement of frequency dependent processes. (The recording of responses at a range of stimulus intensities provides a necessary control procedure to identify changes i n paired-pulse responses which m a y be related to changes i n P S A . I f a n exper imenta l manipulat ion produces changes in both P S A and paired-pulse measures, these controls m a y be used for comparison, to determine i f experimental effects on P S A and paired-pulse measures were distinct.) The s t imulus intensi ty was then adjusted to evoke a population spike of approximately one-half m a x i m a l amplitude. A t this intensity, the P S A appeared to be most labile, and so would be expected to be most easily affected by pharmacological manipulations; this has been s h o w n to be true in the response of the C A 1 population spike to 5 - H T in vitro (Beck a n d Goldfarb, 1985). Responses were evoked and recorded at the rate of 1/min. If paired-pulse s t i m u l i were used, pairs of pulses were presented at the rate of 1/min, cyc l ing between the C-T intervals used in that part icular experiment. After stable responses were recorded for 30 m i n , drug or vehicle injections were administered. I f a 30 minute period of stable baseline responses could not be recorded w i t h i n 2.5 hours, injections were not administered, and the experiment was terminated. Populat ion spike ampl i tudes and population E P S P s recorded f r o m the granular layer were analyzed as described i n Chapter 2 above. Populat ion E P S P s recorded from the molecular layer were quanti f ied as the m a x i m u m negative slope measured between baseline potential and a fixed latency (see Figure 1.2). Due to the var iabi l i ty i n the magnitude of responses between preparations, a l l measures were normal ized w i t h respect to the m e a n pre-drug baseline. In order to analyze effects on paired-pulse responses, P S A s evoked by test s t i m u l i were quantified as percentages of the P S A s evoked by their associated conditioning stimuli, where the parameter TQ was defined as (PSA evoked by test stimulation/PSA evoked by conditioning stimulation) X 100%. After drug injection, the changes in TQ from baseline values were calculated for every C-T interval used. These were. then compared statistically with changes in TQ observed in control animals after injections of saline vehicle. 3.3 THE EFFECTS OF 5-HYDROXYTRYPTOPHAN ON POPULATION  RESPONSES IN THE DG INTRODUCTION This experiment investigated the effects of presumably increased brain 5-HT content on population responses evoked in the DG. As 5-HT itself does not readily cross the blood-brain barrier, administration of 5-HTP, the immediate metabolic precursor to 5-HT, was utilized to increase the concentration of serotonin in the CNS. Conversion of 5-HTP to 5-HT is accomplished by the enzyme 1-aromatic acid decarboxylase. As this enzyme is present in excess in the brain, much of the 5-HTP reaching the CNS is converted to 5-HT. The systemic administration of 5-HTP requires the co-administration of a peripheral 1-aromatic acid decarboxylase inhibitor, to prevent the degradation of 5-HTP in the periphery (Bedard et al., 1971). METHODS Stimulating and recording electrodes were implanted in the PP and the granular layer of the DG, respectively, and the tissue was allowed to equilibrate, as outlined in the general methods section above. The stimulus intensity was adjusted to evoke a population spike of one-half maximum amplitude, and single responses were evoked at the rate of 1/min. After stable responses were recorded for 30 min, the animals received either i.p. injection of 20mg/kg of the peripheral 1-aromatic acid decarboxylase inhibitor DL-seryl-2-(2,3,4-trihydroxybenzyl)hydrazine hydrochloride (RO4-4602/1), followed 30 min later by i.p injection of 50 mg/kg 5-HTP (Sigma Corp.) (N = 6); RO4-4602/1 followed 30 min later by i.p. injection of 1.0 ml physiological saline (N = 2); or i.p. saline alone (N = 8). Both 5-HTP and R04-4602/1 were prepared in concentrations of 20 mg/ml in physiological saline solution. After injections, population responses were evoked and recorded at 1 min intervals for a further 5-7 hours. RESULTS Injection of saline vehicle or RO4-4602/1 alone had no observable effect on either PSA or EPSP slope, other than increasing variability with time. Injections of 5-HTP were followed consistently by an increase in PSA, which reached a mean of 167 _+ 22% of baseline values after 3.5 hours (Student's ((13) = 3.88, p<.05 vs saline injected controls) and usually remained elevated for the duration of the experiment (Figures 3.2a, 3.3). No change was observed in the slope of the population EPSP recorded in the granular layer. Only one animal was recorded from for more than 5 hours after 5-HTP injection; in this animal, a decrease in PSA to approximately baseline values was observed by about 7 hours (Figure 3.3a). DISCUSSION The time course of the increase in PSA observed after injection of 5-HTP and RO4-4602/1 is very similar to the increase in brain serotonin after administration of these compounds, as shown previously by electrochemical methods (Blaha, 1986; Lane et al., 1979). The presumably increased levels of brain 5-HT would be expected to activate all serotonergic receptor subtypes non-selectively, and therefore the mechanisms underlying the increase in PSA cannot be specified. Additionally, 1-aromatic acid decarboxylase is found in catecholaminergic as well as serotonergic neurons, and may effect conversion of 5-HTP in cells which do not normally express 5-HT, further complicating the issue. Figure 3.2 Representative DG responses evoked by PP stimulation A) prior to, and 3.5 hours after i.p. injection of 50 mg/kg 5-HT; B) prior to, and 0.5 hour after i.p. injection of 8-OH-DPAT. Scale: 5 mV, 5 msec. 95 A. 5 -HTP 1. C O N T R O L 2. 3.5 HR P O S T I N J E C T I O N B. 8 - O H - D P A T 1. C O N T R O L 2. 0.5 HR POST INJECTION Figure 3.3 The effects of i.p. injection of 50 mg/kg 5-HTP on DG population spike amplitude. A) Responses recorded from one animal. Note the return to baseline values after 7 hours. B) PSA (Mean +_ sem; N = 6) over time, expressed as percentage of baseline values. RO: i.p. injection of 20 mg/kg RO4-4602/1. A . CL O Q_ B . 5-> UJ o • z> •5 4-1 Q_ < UJ Q . to 3-1 -97 00 * o' o*_- oooo o o .-. -o oo o o o RO -1 TIME (HOURS) -i T——» r~—r——i 1 1 1 1—-i r r 1 2 3 4 ' 5 6 200% UJ § ' 180% UJ "« oo 160%-u_ O * 140% CO < Lo 120%-z •. o Sj 100% 80% / - i — r 1 2 3 TIME (HOURS) The remaining experiments in this section are an attempt to clarify the role of 5-HT in the modulation of evoked potentials in the DG, with the use of selective serotonergic agonists and antagonists. 3.4 THE EFFECTS OF 8-OH-DPAT ON POPULATION RESPONSES IN THE DG A. INTRAPERITONEAL ADMINISTRATION OF 8-OH-DPAT INTRODUCTION In order to investigate the role of the 5-HT^A receptor in the modulation of synaptic transmission in the DG in vivo, the present experiment examined the effects of i.p. administration of the selective 5-HT^A agonist 8-OH-DPAT. METHODS Stimulating and recording electrodes were implanted in the PP and the granular layer of the DG, respectively, and equilibration of tissue was allowed, as outlined in the general methods section above. The stimulus intensity was adjusted to evoke a population spike of one-half maximum amplitude, and single responses were evoked at the rate of 1/min. After stable baseline responses were recorded, the animals received i.p. injections of either 12.5, 25, 100, or 500 ug/kg 8-OH-DPAT HBr (N = 6 at each dose) in a volume of 1.0 ml physiological saline solution. Responses to single stimuli were evoked and recorded at 1 min intervals for 5-8 hours after injection. RESULTS A dose-dependent increase in PSA was observed following i.p. administration of 8-OH-DPAT (Figures 3.2b, 3.4). No change was observed in the slope of the population EPSP recorded in the GL after administration of any dose. Injection of 2 5 ug/kg 8-OH-DPAT produced an increase in PSA which attained a mean of 156 _+ 7% of baseline values after 30 min, and returned to baseline values within about 90 min. Comparable increases were observed following the administration of 10 Figure 3.4 The effects of i.p. injection of 8-OH-DPAT on DG population spike amplitude. Triangles: 12.5 ug/kg; squares: 25 ug/kg; diamonds: 100 ug/kg (n = 6 animals at each dose). 500 Mg/kg not shown, for clarity. 100 225%! TIME (HOURS) and 500 ug/kg 8-OH-DPAT, but the duration of the effect was extended. The lowest dose of 12.5 ug/kg produced no consistent effects on PSA. Statistical comparisons were made with responses recorded from the vehicle-injected animals in section 4.4.A above; for each animal, PSA was quantified as the mean of 5 responses, 28-32 minutes after injection. Following a one way analysis of variance (ANOVA) which confirmed a main effect of drug (F (4,25) = 9.03; p<.0002), Dunnett's test for multiple comparisons (Glass and Hopkins, 1984) revealed significant effects at doses of 25 ug/kg and higher (p<.05)-DISCUSSION This experiment demonstrated a potent effect of i.p. administration of 8-OH-DPAT on PSA recorded in the DG. The long time course of the effect resembles closely the reported pharmacokinetics of 8-OH-DPAT in vivo (Sethy and Francis, 1988), suggesting that the effects were not mediated by metabolites of this compound. To investigate further the processes mediated by 5-HT-j^ receptors in this region, the next experiment was designed to examine the effects of 8-OH-DPAT on dendritic field potentials and paired-pulse measures. An intravenous (i.v.) preparation was used in an attempt to decrease the variability and long time course of the effects produced by the i.p. administrations of this compound, and so to facilitate the analysis of dose-response relationships. B. INTRAVENOUS ADMINISTRATION OF 8-OH-DPAT METHODS Surgical preparation Male Wistar rats were anaesthetized with urethane, as described above, and an intravenous cannula was then implanted, as follows. With the rat in a supine position, an incision was made through the skin at the junction of the leg and abdomen. The underlying tissue was separated and the femoral vein was exposed, along with the attached artery and nerve. The vein was separated from the artery and nerve, and loops of suture thread were tied loosely around the proximal portion of the vein and tightly around the distal portion. A small slit was made in the vein with iris scissors, and a cannula of either polyethlyene (PE 20) or teflon tubing was inserted from the distal towards the proximal aspect of the vein, until the end of the tubing was past the loosely tied loop of suture thread. This loop was then tied around the vein, securing the cannula into place. Prior to insertion of the cannula, a syringe containing heparinized saline solution (approx. 10 i.u./ml) was attached, and following insertion, 0.1 ml of this solution was injected into the vein through the cannula in order to prevent clot formation. The cannula was then secured to the leg of the animal and the animal turned to a prone position and placed in a standard Kopf stereotaxic instrument. A stimulating electrode was then implanted in the angular bundle, and recording electrodes implanted in the ML and GL of the DG, following the procedures described in section 3.2 above. Stimulation, recording, and drug procedures Responses to paired-pulse stimuli at C-T intervals of 20, 80, 400, and 2000 msec were recorded at a range of stimulation intensities, as described in the section 3.2 above. Following this, the stimulus intensity was adjusted to evoke a population spike of approximately half-maximal amplitude, and paired-pulse responses were evoked and recorded once per minute. After stable potentials were recorded for a minimum of 30 minutes, either 3, 6, 12, 25, 50, 100, 200, 400, or 800 ug/kg of 8-OH-DPAT HBr in 0.1 ml of heparinized saline (N = 3 animals at each dose), or heparinized saline alone (N = 8), was injected i.v. at a rate of 0.05 ml/min. Population responses were evoked and recorded for 3-10 hours after injection. In some animals, multiple injections of 8-OH-DPAT were given; four of these received a series of injections in order of ascending dose, comprising the range of doses listed above. RESULTS A dose-related increase in PSA was observed after administration of 8-OH-DPAT. As repeated applications of 8-OH-DPAT appeared to produce a diminished effect, the responses recorded after initial administrations of 8-OH-DPAT were analyzed separately from the responses recorded during the series of injections. Initial drug administrations A dose-related increase in PSA was observed after administration of 8-OH-DPAT (Figures 3.5a, 3.6). A maximal increase was observed within 10 min after injection, reaching a mean of 162 _+ 12% of baseline values 5-10 min after injection of 25ug/kg 8-OH-DPAT. Doses of 200«g/kg and above, however, produced attenuated effects, and administrations of 800 ug/kg were followed by decreases in PSA. PSA, ML and GL EPSP slopes, and paired-pulse measures were entered into a MANOVA, which revealed a significant effect of dose at 5-10 min after injection (F(54,101) = 2.02, p<.005). Univariate analysis indicated a significant effect of dose on PSA (F(9,24) = 8.11, p<.001) and paired-pulse responses recorded at a 2000 msec C-T interval (F(9.24) = 2.68, p<.026); the effects on population EPSP parameters and on paired-pulse responses at other C-T intervals were not significant. Further analysis with the Dunnett method of multiple comparisons demonstrated that PSA was significantly different from control values at doses of 6-100 wg/kg (£(7) = 2.96 - 5.86, p<.05). After 8-OH-DPAT injection, paired-pulse responses at a C-T interval of 2000 msec exhibited a trend toward greater inhibition, i.e toward a decrease in TQ. Dunnett's test indicated that this change in TQ was significantly different from control preparations only at a dose of lOOug/kg (£(7) = 3.57, p<.05) (Figure 3.7a). Figure 3.7b illustrates 2000 msec C-T interval paired-pulse responses from each of the three animals in the lOOug/kg condition, recorded prior to and after drug injection. From this figure it is apparent that Figure 3.5 Mean Population spike amplitudes recorded 5-10 min after i.v. injection of 8-OH-DPAT. A) Closed circles: responses to initial administrations (N = 3 at each dose). Open circles: responses to a series of administrations of increasing doseage (N = 4 in total). Error bars represent 1 sem; Curves were fitted by eye. B) Open circle: Response to a series of administrations of 8-OH-DPAT only. This is the same plot as in Figure 3.5(A) above, at a different scale. Closed circles: Response to a series of administrations of 8-OH-DPAT, after pre treatment with i.v. administration of 1.5 mg/kg methysergide. A 220*1 200* J £ 180%.J 90*-I 1 . r , r — r - , ~i , , , , 3 6 12 25 50 100 200 400 800 16003200 DPAT (/ig/kg) Figure 3.6 A) Population spike amplitudes, B) granular layer population EPSP slopes and C) molecular layer population EPSP slopes recorded from one animal, demonstrating responses to a cumulative series of i.v. 8-OH-DPAT injections. This animal was not used in the analysis of responses to repeated administrations of 8-OH-DPAT, due to the long intervals between injections, but was chosen to illustrate the time course of a range of doses. Numbers above event markers indicate dose of 8-OH-DPAT in ug/kg. Note the obvious increase in PSA at each injection, and no discernable effect on GL EPSP slope. In this animal, an effect on ML EPSP slope was apparent; this was inconsistant between animals, however, and was not statistically significant. Note also the attenuated response of the PSA to the second administration of 6 ug/kg. EPSP SLOPE (mV./msec.) I I I at i ca O NJ A N>-T 2: x o c xn 01H CD • co-co -Figure 3.7 Modulation of late paired-pulse inhibition in the DG by 8-OH-DPAT. A) Changes from baseline T^ values 5-10 min after i.v. injection of saline vehicle (CON) or 3-800 wg/kg 8-OH-DPAT. TQ was calculated as (PSA evoked by conditioning stimulation)/(PSA evoked by test stimulation) X 100%. Increased paired-pulse inhibition at this interval was observed after injection of 8-OH-DPAT. *: p<.05 vs saline injected control. B) Paired-pulse responses at 2000 msec C-T interval from the three animals injected with 100«g/kg 8-OH-DPAT, plotted as a function of PSA evoked by conditioning stimulation. Open symbols: Responses evoked by a range of stimulus intensities, prior to injection. Closed symbols: Mean responses evoked 5-10 min after injection. Note that the paired-pulse responses from two of the animals are far out of the expected range. A. 109 1— 1 o 10% MSEC 0%-2000 -10%-<• o -i— -20%-2 CHANGE 30%-40% 4 CON 3 ^ r ~T~ 1 1 >— ' 1 . —, n 6 12 25 50 100 200 400 800 DOSE 8-OH-DPAT B. 150-r £ l 2 5 8 100 u. O fe5 75 CO < < to 0_ 50 {2 2 5 8 10 12 14 16 CONDITIONING PSA (mV.) the change in TQ in at least two out of the three animals could not be directly ascribed to changes in the size of the PSA evoked by the conditioning stimulus. Repeated administrations of 8-OH-DPAT Repeated administrations of 8-OH-DPAT produced smaller changes in PSA than initial administrations. For example, injections of 25wg/kg 8-OH-DPAT, when given alone, were followed by a mean increase in PSA to 162 _+ 12% of baseline values; when preceded by a series of lower doses, by a mean increase to only 131 _+ 7% of baseline values. In Figure 3.5a, which contrasts the changes in PSA after individual and repeated administrations of 8-OH-DPAT, it can be seen that this apparent desensitization occurs over a range of doses. Unfortunately, legitimate statistical comparisons are not possible between these conditions, as the values from the individual administrations are obtained between groups of animals, and the values from the repeated administrations from within groups. DISCUSSION Intravenous administration of 8-OH-DPAT was shown to increase PSA in the DG, with a much faster time course than i.p. injection. Additionally, a significant increase in the inhibitory processes assessed by paired-pulse stimulation at a C-T interval of 2000 msec was observed. Given the known selectivity of 8-OH-DPAT, the low doses (6wg/kg) of this compound which were found to be effective in increasing PSA indicate that this effect may be mediated by the 5-HT1A receptor. Blockade of the effects of 8-OH-DPAT on DG PSA by a serotonergic antagonist would provide support for this hypothesis. No selective antagonists are currently available for the 5-HT-^ receptor. Methysergide, although non-selective for serotonergic receptors, has been reported as being highly effective in blocking behavioural effects of 5-HT-^ agonists (Blackburn et al., 1987) and has also proven effective in blocking the effects of 8-OH-DPAT in the in vitro experiments in Chapter 2. The following experiment, therefore, investigated the ability of methysergide to attenuate the effects of 8-OH-DPAT on DG PSA. C. CO-ADMINISTRATION OF 8-OH-DPAT WITH METHYSERGIDE METHODS Surgical, stimulation, and recording procedures were all carried out as in section 4.4.C above, except that recordings were made only from the GL of the DG, and paired-pulse measurements were not made. After stable baseline potentials were recorded, each animal (N = 4) received an i.v. injection of 1.5 mg/kg methysergide followed at 15 min intervals by i.v. administrations of 6, 12, 25, 50, 100 ug/kg 8-OH-DPAT, 1.5 mg/kg methysergide, and 200, 400, 800, 1600 and 3200 ug/kg 8-OH-DPAT, in that order. Each dose of drug was administered in 0.1 ml physiological saline at a rate of 0.05 ml/min. Responses were evoked and recorded at the rate of 1/min until 30 min after the last injection. RESULTS The responses recorded from the animals in this experiment were compared with those obtained from the four animals from the previous experiment which had received a similar protocol of 8-OH-DPAT administration (Figure 3.5b). Univariate analysis of the PSAs at doses of 6-800ug/kg disclosed a significant difference between the two groups (F(l,6) = 7.04, p<.05), a significant effect of dose (F(7,42) = 4.82, p<.05), and a significant interaction between group and dose (F(7,42) = 7.71, p<.05) (the Greenhouse-Geisser correction was applied to the probabilities for the effects of dose and the interaction of dose and group). 112 DISCUSSION The administration of methysergide produced a significant change in the effects of 8-OH-DPAT on DG PSA. The dose-response curve appears to be shifted to the right, with no apparent difference in maximal response, suggesting a competitive antagonism of the effects of 8-OH-DPAT by methysergide (Figure 3.5b). Statistical analysis revealed a significant interaction of group and dose, which would not be expected from a simple rightward shift of the dose response curve. This may be attributed to the fact that statistical comparisons were made across a limited range of doses. D. INTRACEREBROVENTRICULAR ADMINISTRATION OF 8-OH-DPAT INTRODUCTION In order to investigate whether the increase in PSA observed following systemic administration of 8-OH-DPAT was mediated by actions on central or peripheral sites, the effects of intracerebroventricular (i.e.v.) injections of 8-OH-DPAT on evoked population responses were examined. METHODS Urethane anaesthetized animals were implanted with a stimulating electrode in the PP and a recording electrode in the GL of the DG. The injection needle of a lOul Hamilton microsyringe was implanted through an additional burr hole into the lateral ventricle ipsilateral to the recording electrode, at co-ordinates AP -0.7 mm, L 1.4mm relative to bregma, and D 4.4mm below the surface of the cortex. No recordings were made from the ML of the DG, as the presence of the microinjection cannula did not leave sufficient room for an additional electrode. Stimulation and recording procedures were carried out as in section 3.4.A above, except that paired-pulse measures were assessed only at a C-T interval of 20 msec. After recording stable baseline potentials for 30 minutes, either lOnmol (3.28ug) of 8-OH-DPAT in 1.0 ul of physiological saline (N = 6) or Figure 3.8 A) Mean population spike amplitudes and B) granular layer population EPSP slopes after microinjection of lOnmol 8-OH-DPAT (closed circles; N = 6) or saline vehicle (open circles N = 6)) into the lateral ventricle (LV). Injections of l.Oul were administered over 10 min; time 0 indicates start of injection. Injections of 8-OH-DPAT into the LV were followed by a significant increase in PSA, but not of GL population EPSP slope. 150% Ld 125% w 100% m , 75% Lu. o 50%-< CO 0- 25% 0% —I r-- . 5 H 1 1 1 1 H H h 0.5 1 TIME (HOURS) 1.5 B. 125%-. UJ 120%-z _J 11 1 115% -L U CO < 110%-CD U_ 105%-O 100%-CL 95%-CO CL LU 90%-85%--0.5 0 0.5 1 TIME (HOURS) the same volume of saline alone (N = 6) was injected into the lateral ventricle at a rate of 0.1 wl/min. Population responses were evoked and recorded every minute for a further 3 hours, after which the animals were perfused transcardially with 0.9% NaCl followed by 10% formalin, and the brains removed, sectioned, and stained with 0.9% thionin for histological verification of the injection site. In all animals, these histological procedures verified that the tip of the injection cannula had entered the lateral ventricle. RESULTS Statistical analyses were performed on data collected between 5 and 10 min after the end of the injection. The mean PSA, EPSP slope, and change in paired-pulse inhibition from each animal were entered into a MANOVA, which revealed a significant difference between the experimental and control groups (F(3,8) = 5.48, p<.05). Following this, ANOVA procedures disclosed a significant effect on PSA (F(l,10 = 7.95, p<.05), which reached a mean of 128 _+ 10% of baseline values in the experimental animals, versus 95 + 6% in the controls (Figure 3.8a). Univariate analysis did not demonstrate a significant effect on either EPSP slope or paired-pulse inhibition, although a trend toward an increase in EPSP slope was observed (Fig 3.8b). DISCUSSION -This experiment indicated that facilitation of DG PSA following administration of 8-OH-DPAT in vivo may be mediated by central receptors. The in vitro experiments in Chapter 2, however, indicate that application of 8-OH-DPAT directly to the HF has an inhibitory effect on population responses recorded in the DG. These experiments suggest that facilitatory effects of 8-OH-DPAT on PSA observed in vivo may be mediated by region of the brain other than the hippocampal formation. Serotonergic l A receptors have also been identified in the median raphe nucleus (MRN), where they may serve as inhibitory autoreceptors. This raises the possibility that the facilitatory effects of 8-OH-DPAT on DG population spikes may be mediated indirectly by a modulatory action on serotonergic neurons in the MRN. The next experiment, therefore, examined the effects of microinjection of 8-OH-DPAT directly into the MRN on population responses in the DG. E. INTRACRANIAL ADMINISTRATION OF 8-OH-DPAT METHODS Urethane anaesthetized rats were implanted with a stimulating electrode in the PP, and recording electrodes in the SM and SG of the DG, as described in section 3.2 above. In addition, the needle of a l.Oul Hamilton microsyringe was directed towards the MRN, at 0.8mm A to the intra-aural line, 2.2 mm L to bregma, and 6.9 mm below the surface of the cortex, at an angle of 20° from the vertical plane. Recording procedures were carried out as described above for i.e.v. administrations of 8-OH-DPAT, although the stimulation parameters were changed. The previous experiments involving either i.v. or i.c.v. administration of 8-OH-DPAT had all suggested an effect on both population EPSP slopes and PSA; however, analysis of the EPSP slopes did not yield statistically significant differences. The stimulus intensities which were used in those experiments were set at a level high enough to produce a population spike of half-maximal amplitude. At this level of stimulation, the EPSP slope is at about 90% of its maximum; as this is near saturation, the observation of any further drug-induced increase may be precluded. Therefore, changes in population EPSPs might be detected more readily at lower stimulation intensities. In order to investigate this, in this experiment stimuli were presented at two strengths: a high intensity, sufficient to produce a population spike of approximately one-half maximal amplitude (Avg. = 200uA), and a low intensity, below threshold for the generation of a population spike (Avg. = 30uA). At each stimulus strength, paired-pulse stimuli were presented at a C-T interval of 20 msec; stimulus pairs were presented every 30 sec and alternated between the high and low intensities. After stable baseline measures were recorded, either 5 nmol (1.64ug) of 8-OH-DPAT in 0.5 ul of physiological saline (N= 10) or the same volume of saline alone (N = 7) was injected into the MRN at a rate of 0.05 ul/min. Population responses were evoked and recorded for 3 hours after the MRN injection. HISTOLOGY On completion of data collection, the animals were perfused and their brains fixed, sectioned and stained for histological verification of the injection site, as in section 3.4.D above. In the brains from 12 out of a total of 17 animals, the cannula tip was localized to within 0.5mm of the MRN. Only the data from these animals were included in the analysis of evoked potentials. RESULTS Statistical analyses were performed on data collected between 5 and 10 minutes after the end of the injection. PSA and paired-pulse inhibition were assessed only at the high stimulation intensity condition, and GL and ML population EPSPs only at the low stimulation intensity condition. MANOVA analysis of these parameters identified a significant difference between the experimental and control groups (F(4,7) = 9.93, p<.01). Univariate analysis revealed significant effects on PSA (F(l,10) = 9.58, p<.05) and ML population EPSP slope (F(4,7) = 10.05, p<.05), but no significant effects on either GL population EPSP slope, or paired-pulse inhibition. In 8-OH-DPAT injected animals, the mean PSAs and ML EPSP slopes were 177 +_ 15% and 112 +_ 3% of baseline values, respectively, 5-10 minutes after injection (Figures 3.9, 3.10, 3.11). DISCUSSION This experiment demonstrated that an increase in PSA in the DG could be produced by injection of 8-OH-DPAT directly into the vicinity of the MRN. As a large population of 5-HT-^ receptors appear to be located on the somata and dendrites of serotonergic neurons in the raphe nuclei, it is possible that these neurons mediate the facilitatory effects of 8-OH-DPAT on PSA. Alternatively, the effects of 8-OH-DPAT could Figure 3.9 Representative responses evoked before (open arrow), and 20 min after (closed arrow) microinjection of 8-OH-DPAT into the median raphe nucleus. A) granular layer potential recorded at high intensity stimulation, B) molecular layer potential recorded at low intensity stimulation, and C) granular layer potential recorded at low intensity stimulation. Potentials in (B) and (C) were evoked by the same stimulus pulses. 119 5 msec. Figure 3.10 A) Population spike amplitudes and B) molecular layer population EPSP slopes recorded from one animal after microinjection of 8-OH-DPAT into the median raphe nucleus. 121 A. 14-r 12 10 8 6 4-2 % °oa o o o OB ° • 0 ° 0 "a % O oo o o o O ' —I— .5 -.5 1 —I— 1.5 2 TIME (HOURS) B. 5 j W E > 4--SLOPE 3--EPSP 2-POP. 1--0--o ° ©»- °o°> oo ' .'• * •'•** *•**•••»•••. \ 1 • • oo o o o oo 0 ° -.5 .5 —I— 1.5 TIME (HOURS) Figure 3.11 A) Mean population spike amplitudes, B) granular layer population EPSP slopes and C) molecular layer population EPSP slopes after microinjecion of 5nmol 8-OH-DPAT (N = 6) or saline vehicle (N = 6) into the median raphe nucleus. Injections of 0.5id were administered over 10 min. Microinjections of 8-OH-DPAT into the MRN were followed by a significant increase in PSA and ML "population EPSP slope, but not GL population EPSP slope. -0.5 0 0.5 1 1.5 2 TIME (HOURS) have been mediated by non-serotonergic neurons either within the MRN, or by diffusion of the compound outside of the raphe nuclei. . In order to investigate the necessity of serotonergic neurons in the expression of the facilitation of PSA by 8-OH-DPAT, the next experiment examined the effects of 8-OH-DPAT in animals treated with the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). When administered along with a catacholamine uptake blocker, such as desipramine, 5,7-DHT has been shown to produce a selective degeneration of serotonergic neurons, and a profound decrease of 5-HT in the brain (Bjorklund et al., 1974). F. ADMINISTRATION OF 8-OH-DPAT IN 5,7-DIHYDROXYTRYPTAMINE  TREATED ANIMALS METHODS Male Wistar rats were anaesthetized with an i.p. injection of sodium pentobarbital ("Somnotol", 60 mg/kg), and placed in a standard Kopf stereotaxic instrument. The skull was exposed, and a burr hole trephined. The needle of a lOul Hamilton microsyringe was lowered -into the brain, with the tip directed toward the right lateral ventricle, at co-ordinates AP -0.7 mm, L 1.4mm relative to bregma, and D 4.4mm BSC. After allowing 15 min for tissue equilibration, 30 mg/kg desipramine HCl, dissolved in 1.0 ml of physiological saline, was injected i.p. This was followed 30 min later by an i.e.v. injection either of lOOwg of 5,7-HT in 10 ul of physiological saline containing 1 mg/ml ascorbic acid (N = 6), or the ascorbate saline vehicle alone (N = 6). Solutions used for i.c.v. injections were delivered at a rate of 1.0 ul/min. After injection, the microsyringe needle was withdrawn slowly, the burr hole packed with bone wax, and the skin sutured. The rats were then returned to the animal colony, where they were housed in individual cages. Fourteen days after i.c.v. injections, the animals were anaesthetized with urethane, and implanted with a recording electrode in the GL of the DG, a stimulating electrode in the PP, and an intravenous cannula. Stimulation and recording parameters were carried out as in the General Methods section above, although paired-pulse responses were not assessed. After stable responses were recorded, each of these animals received i.v. administrations of 25 ug/kg 8-OH-DPAT, and responses were evoked and recorded for a further two hours. RESULTS An increase in PSA was observed in both groups following administration of 8-OH-DPAT. However, 10-15 minutes after injection, the PSA evoked in the 5,7-DHT treated animals reached 115 _+ 5% of baseline values, which was significantly less than the 150 _+ 5% of baseline values attained by the responses from the control animals (Student's £(10) = 2.23, p<.05). No changes were observed in the population EPSP slopes recorded from either group. DISCUSSION Pre-treatment of the animals by 5,7-DHT produced a significant attenuation of the effects of 8-OH-DPAT on PSA. Although analysis of the brains of these animals for 5-HT content was not performed, previous studies using similar 5,7-DHT pretreatment have demonstated a large decrease in brain 5-HT, and a toxic effect on brainstem serotonergic neurons (Bjorklund et al., 1974; Blackburn et al., 1980). This suggests strongly that the effects of 8-OH-DPAT on PSA are mediated by presynaptic receptors on serotonergic neurons. It is possible that the increase in PSA observed in some 5,7-DHT treated animals after injection of 8-OH-DPAT may have been mediated by serotonergic neurons which had survived the neurochemical lesion. 3.5 EXPERIMENTS WITH BUSPIRONE INTRODUCTION: Buspirone is an arylpiperazine derivative which displays high affinity for the 5-HT-^A site, and which appears to act as a partial agonist on 5-HT^A receptors on C A l 126 HPCs and on serotonergic neurons in the raphe nuclei (Bockaert et al., 1987; Mauk et al., 1988). The present study examined the effects of buspirone on population responses in the DG, in order to investigate the possibility of similar effects to 8-OH-DPAT. METHODS Animals were prepared with a stimulating electrode in the PP, recording electrodes in the GL and ML of the DG, and an intravenous cannula. Stimulation and recording procedures were carried out as in the General Methods section above, with responses to paired-pulse stimuli assessed at C-T intervals of 20, 80, 400 and 8000 msec. After stable baseline potentials were recorded, 100, 200 or 800ug/kg buspirone was injected i.v., in a volume of 0.1 ml physiological saline (N = 3 at each dose). RESULTS The most obvious effect of buspirone injection was a dose-dependent increase in PSA, which reached 146_+3% of baseline values, 5-10 min after administration of 800ug/kg (Figure 3.12) Repeated injections of buspirone were attempted, but abandoned after it was observed that the responses to multiple administrations were highly variable, and usually much smaller than to initial administrations (Figure 3.12b). Statistical analysis was performed on data collected 5-10 min after injection, and comparisons were made with the saline-i.v. control group from section 3.4.B. PSAs, ML and GL population EPSPs, and changes in paired-pulse responses were entered into a MANOVA. This analysis indicated that the effects of dose were not significant (F(21,21) = .069). Descriptively, however, dose dependent effects on PSA appeared evident. Univariate tests on PSA were therefore conducted, and these indicated a significant effect on PSA (F(3,13) = 28.38, p<.001). Given the MANOVA findings, however, the univariate analysis cannot strictly be justified. Figure 3.12 A) Mean PSA recorded 5-10 min after i.v. injection of buspirone. The mean effects were not statistically significant. B) Effects of buspirone on PSAs recorded from one animal, numbers over event markers indicate dose in ug/kg. Note attenuated responses to multiple injections. A. 128 LU 150%-, 125% UJ 100% -I CO < 75% 50%-| •< CO 0_ 25% 0% 100 200 , 400 800 DOSE BUSPIRONE (jug/kg) B. > CL < LJ CL CO CL O 0_ 7-, 6-5-4-3-2-1 oo% e 100 0 .5 400 o 200 T 1 1 1 1 1.5 2 2.5 TIME (HOURS) -r-3 —i 1 3.5 DISCUSSION Buspirone has been shown to be a ligand for the 5-HT-^ binding site, although it displays lower affinity and less selectivity for this site than does 8-OH-DPAT (Glaser and Traber, 1983; Riblet et al., 1984; Peroutka, et al., 1986) this compound has also been shown to act as a 5-HTj^ partial agonist, producing inhibitory effects on both C A l HPCs and serotonergic neurons (Peroutka et al., 1987; Mauk et al., 1988). In the present study, injection of buspirone produced no significant effects on population responses recorded in the DG. This failure to find a significant effect of buspirone may have resulted from\ the relatively conservative statistical procedures adopted. The MANOVA results approached statistical significance; when combined with the univariate results, these suggest that a facilitatory effect of buspirone on PSA cannot be discounted completely. These findings suggest that further investigation with this compound is required. 3.6 EXPERIMENTS WITH BMY-7378 INTRODUCTION BMY-7378 is a novel arylpiperazine derivative which displays high affinity and selectivity for the 5-HT 1 A site (Yocca et al., 1987). Administration of BMY-7378 has been reported to block the inhibitory effects of 5-HT and 8-OH-DPAT on the unit activity of C A l HPCs, and the inhibitory effects of 5-HT on DRN neurons (Chaput and DeMontigny, 1988). Additionally, low concentrations of BMY-7378 have been found to block the inhibition of adenylate cyclase produced by 8-OH-DPAT in hippocampal membranes (Yocca et al., 1987). These findings have prompted the consideration of BMY-7378 as a possible 5-HT^A receptor antagonist. However, recent reports that i.v. or iontophoretic administration of BMY-7378 inhibits the firing of DRN neurons indicate that this compound may have 5-HT^A agonist activity (Chaput and DeMontigny, 1988). 130 The present experiments investigated the effects of BMY-7378 on population responses in the DG. METHODS Animals were prepared with a stimulating electrode in the PP, recording electrodes in the GL and ML of the DG, and an intravenous cannula. Stimulation intensity was set to evoke an approximately one-half maximal PSA. Stimulation and recording procedures were carried out as in the General Methods section above, with paired-pulse stimuli presented at C-T intervals of 20, 80, 400 and 8000 msec. After recording stable baseline responses, either 50, 100, 200, 400 or 800 ug/kg BMY-7378 was administered i.v., in a volume of 0.1 ml of physiological saline (N = 3 at each dose). Responses were evoked and recorded for three hours following injection. RESULTS A dose-dependent increase in PSA was observed after injections of 200ug/kg or greater of BMY-7378, with a mean PSA of 135 _+ 14% of baseline 5-10 min after injection of 800ug/kg BMY-7378 (Figure 3.13). PSAs, EPSP slopes, and paired-pulse responses recorded at 5-10 min after injection were entered into a MANOVA along with the values obtained from the control group from section 3.4.B. Significant effects of BMY-7378 were not disclosed by MANOVA procedures (F(20,50) = 1.42, p = .158). As with buspirone, however, subsequent illegitimate univariate analysis indicated significant effects on PSA (F(5,18) = 7.02, p<.01), but not on any other measure. DISCUSSION BMY-7378 appeared to produce an increase in PSA, although to a lesser degree, and at much higher doses than 8-OH-DPAT, consistent with this drug acting as a 5-HT^ agonist with low activity. The results of the multivariate analysis indicate that these conclusions should be tentative, however, and only taken as a guide to further research. Figure 3.13 A) Mean PSA recorded 5-10 min after i.v. injection of BMY-7378. The mean effects were not statistically significant. 132 i 133 3.7 EFFECTS OF DOI AND KETANSERIN ON DG POPULATION RESPONSES INTRODUCTION As discussed in Chapter 1 above, the DG contains a moderate density of 5-HT2 binding sites. The in vitro experiments in Chapter 2 suggested that the 5-HT2 receptor might mediate inhibition of PSA in the DG. In order to examine the possible role of the 5-HT2 receptor in the modulation of electrophysiological activity of the DG in vivo, the present experiment examined the effects of systemic administration of the 5-HT2 agonist DOI (Glennon et al., 1986) and the 5-HT 2 antagonist ketanserin (Janssen, 1983). METHODS Animals were implanted with a stimulating electrode in the angular bundle, and recording electrodes in the ML and GL of the DG. Animals destined to receive DOI injections were also prepared with i.v. cannulas. Stimulation and recording procedures were carried out as in the general methods section, except that paired-pulse responses were assessed only at a C-T interval of 20 msec. Stimulation intensity was set to evoke a population spike of one-half maximal amplitude. After stable baseline responses were recorded, eight animals received i.v. administration of DOI in 0.1 ml physiological saline, -at concentrations ranging from 10 to 3200 ug/kg; four of these animals received repeated injections. Six other animals were injected i.p. with 2 mg/kg ketanserin HCl, in a volume of 2.0 ml physiological saline. RESULTS AND DISCUSSION Neither ketanserin, nor DOI at any dose, produced any discernable effects on PSA, GL or ML EPSP slope, or paired-pulse responses, within one hour after injection. Although the inhibitory effects of DOI on DG PSA observed in vitro in Chapter 3 did not reach statistical significance, the difference between this trend and the lack of effect of this compound in vivo is worthy of comment. One possible explanation for the apparent discrepancy between the two preparations is that the inhibitory effects observed in vitro are masked in the whole animal by influences from other brain regions. This might include the influence of populations of 5-HT2 receptors which could mediate excitatory influences in the DG. For example, 5-HT2 agonists are known to facilitate the activation of locus coeruleus neurons (Rasmussen et al., 1986; Rasmussen and Aghajanian, 1986), which, in turn, could enhance population responses. Such an influence could cancel direct inhibitory effects of DOI in the DG. 3.8 DISCUSSION OF THE IN VIVO PHARMACOLOGICAL EXPERIMENTS • A presynaptic hypothesis for the effects of 8-OH-DPAT The major finding of the experiments in this Chapter is that in vivo administration of the 5-HT2A agonist 8-OH-DPAT increases PSA in the DG, most probably by an action on presynaptic 5-HT-j^ receptors located on serotonergic neurons in the midbrain raphe nuclei. As these receptors are thought to be inhibitory (Aghajanian et al., 1987), this indicates that this serotonergic system normally functions to inhibit the flow of information in the HF. The involvement of the 5-HT-^ receptor in the facilitation of PSA in vivo is indicated by the known specificity of 8-OH-DPAT, by the effectiveness of the MRN injections, and the antagonistic effects of methysergide. Although the less selective 5-HT^A ligands buspirone and BMY-7378 did not produce statistically significant effects on PSA, this may have been related to the conservatism of the statistical analyses and the small sample sizes. Descriptively, dose-dependent increases in PSA were apparent after administration of both buspirone and BMY-7378, indicating that effect on PSA similar to that of 8-OH-DPAT cannot be discounted. Furthermore, both buspirone and BMY-7378 appear to be only partial agonists at the 5-HT^A receptor, and so would not be expected to exert as large an effect as 8-OH-DPAT. Several lines of evidence suggest that the facilitatory effects of 8-OH-DPAT on PSA are mediated by actions on serotonergic neurons in the brainstem. Firstly, DG PSA is increased by very low doses of 8-OH-DPAT which may be selective for presynaptic 5-H T 1 A receptors (Aghajanian et al., 1987). The facilitatory effects of 8-OH-DPAT on DG PSA are attenuated at higher doses, as would be expected if the higher doses induced post-synaptic effects. In the present study, the doses of 8-OH-DPAT found to be effective in increasing PSA, as well as the biphasic nature of the dose-response curve, are similar to those found for behavioural effects which appear to be mediated by the inhibition of raphe neurons by this compound. These include behaviours such as hyperphagia (Dourish et al., 1985; Hutson et al., 1986, 1987) and inhibition of male sexual behaviour in rats (Ahlenius and Larsson, 1987). The attenuation of the effects of 8-OH-DPAT by methysergide is compatible with the hypothesis of a presynaptic mechanism, as methysergide has been shown to block the inhibitory effects of 5-HT-^ agonists on presumably serotonergic DRN neurons (McMillen et al., 1987). The involvement of central serotonergic neurons in the MRN is supported by the increase in PSA which is observed following microinjection of 8-OH-DPAT either into the lateral ventricle or directly into the MRN, and by the attenuation of the effects of systemically administered 8-OH-DPAT on PSA following pretreatment with the serotonergic neurotoxin 5,7-DHT. It has been suggested that 5-HT^A receptors located on serotonergic neurons in the raphe nuclei function as inhibitory autoreceptors (Verge et al., 1985). Therefore, it is possible that the increase in PSA produced by 5-HT^A agonists in these experiments is secondary to the inhibition of serotonergic neurons in the raphe nuclei. This appears likely, as the tonic activity shown normally by serotonergic neurons in the raphe nuclei may be suppressed by iontophoretic or systemic administration of either 8-OH-DPAT or buspirone. 136 The increase in PSA following the presumed inhibition of serotonergic neurons may be most easily explained by a disinhibition of DGCs. The 5-HT which is presumably released by the tonic activity of raphe neurons might exert a direct inhibitory influence on the DGCs, and this inhibition may be removed by the effects of 5-HT-^ agonists on presynaptic autoreceptors. This would be consistent with the results of the in vitro experiments in Chapter 2 of this thesis, in which bath application of 5-HT was found to produce a decrease in PSA in the DG. A reduction of tonic release of 5-HT onto the DGCs is also indicated by the observation that systemic administration of 8-OH-DPAT was followed by significantly greater paired-pulse inhibition at a C-T interval of 2000 msec. The late period of inhibition assessed at this C-T interval correlates with an afterhyperpolarization of the DGCs which appears to be mediated by an increased Ca -dependent gK. Application of 5-HT, but not 8-OH-DPAT, has been shown to block this AHP in DGCs in vitro (Andrade and Nicoll, 1987; Colino and Halliwell, 1986) therefore, an increase in late inhibition in vivo is consistent with a decrease in the tonic release of 5-HT. It is also possible that inhibition of the raphe nuclei by 8-OH-DPAT facilitates transmission between the PP and DG by indirect means. This hypothesis is supported by recent studies investigating the effects of 5-HT-^A agonists on the substantia nigra and locus coeruleus. In vivo, systemic administration of 8-OH-DPAT or buspirone produces a large increase in the activity of nigral dopamine neurons (Fallon et al., 1983; Invernizzi et al., 1987; McMillen et al., 1987). The excitatory effects in the substantia nigra may also be produced by direct administration of 8-OH-DPAT into the DRN or MRN, and can be attenuated by neurochemical lesions of the serotonergic neurons by 5,7-DHT, indicating that the excitation is mediated by a direct action of 8-OH-DPAT on serotonergic neurons in the raphe nuclei. The doses of 8-OH-DPAT found by Invernizzi et al. (1987) to be effective in producing this facilitation of dopaminergic activity are very similar to those which facilitated PSA in the DG in the present study. Similarly, low doses of 5-HT 1 A agonists have been reported to produce a significant increase in the unit activity of locus coeruleus neurons (Wilkinson et al., 1987); whether this is mediated by pre- or post-synaptic receptors is not yet known. Excitation of either of these regions could underlie the increase in PSA observed after 8-OH-DPAT administration, in light of the previous studies showing that chemical or electrical stimulation of either the substantia nigra or the locus coeruleus result in an increased PSA in the DG (Assaf and Miller, 1978; Harley and Milway, 1986; Shin et al., 1987). Despite strong evidence for a central action of 8-OH-DPAT, the possibility exists that the enhancement of PSA is secondary to peripheral effects, such as changes in cardiovascular or thermoregulatory function. Serotonin has complex effects on blood pressure, generally producing a depressor response. Recent work (Mir and Fozard, 1988; McCall et al., 1987) has demonstrated that administration of 8-OH-DPAT also reduces blood pressure, most probably by activating 5-HT^A receptors at a brainstem site. The effective doses, and the time course of the depressor response parallel those which were found in the present study to increase DG PSA in vivo. This suggests that the decrease in blood pressure may be the indirect cause of the increase in PSA produced by 8-OH-DPAT or 5-HTP in vivo. Two lines of evidence indicate that this is unlikely. Firstly, in a recent study investigating the effects of ischaemia (Miller et al., 1987), population responses in the HF of the rat were recorded while mean arterial pressure (MAP) was gradually lowered to 40mm Hg by haemorrhage. In no case was an increase in PSA observed, either in the DG or area C A l , even though this decrease in MAP was up to 100mm Hg below the normal resting level. In actuality, a decrease in PSA was observed in both regions, after MAP had fallen to about 60mm of Hg. As the maximum decrease in blood pressure following 8-OH-DPAT administration has been reported to be about 40mm Hg below normal, the pressor effects of this compound probably do not account for its effects on PSA. Secondly, in the present study DOI was found to have no effect on population responses in vivo at doses which previously have been found to produce a significant increase in MAP (McCall et al., 1987); this indicates further a lack of sensitivity of the PSA to pressor effects. The systemic administration of 8-OH-DPAT has also been shown to produce hypothermia in rats, decreasing rectal temperature by up to 3°C (Gudelsky et al, 1986; Carlsson and Eriksson, 1987; Green and Goodwin, 1987; Blackburn et al., 1987). Changes in temperature have been shown to have profound effects on population responses recorded in the C A l region in vitro, with an approximately 50% increase in PSA reported after a 3° drop in temperature (Hooper et al., 1985). Although the temperature of the animals used in the present study was not monitored, hypothermia induced by 8-OH-DPAT was most probably not a primary cause of the increase in PSA produced by this compound. Although the time course of these two effects are similar, reaching a maximum at about 30 minutes after i.p. injection, a threshold hypothermic response is elicited by about 250ug/kg 8-OH-DPAT, and an asymptotic response at doses greater-than 2500ug/kg (Green and Goodwin, 1987). These doses are much higher than those found in the present experiments to increase PSA, and are consistent with a proposed postsynaptic mechanism of 8-OH-DPAT-induced hypothermia (Carlsson and Erikson, 1987). The effects of 5-HTP Presynaptic inhibition of serotonergic neurons may also underlie the increase in DG PSA observed after i.p. administration of 5-HTP. However, 1-amino acid decarboxylase is contained in excess in both neuronal and extraneuronal pools (Meltzer and Lowy, 1987). Therefore, even if the administration of 5-HTP did result in a decrease in the release of endogenous 5-HT, the global increase in 5-HT resulting from conversion of 5-HTP should result in an increased concentration of 5-HT at all physiological 5-HT receptors. This suggests that the increase in PSA observed after 5-HTP may be mediated by means other than presynaptic inhibition of serotonergic neurons. This could include actions on the 5-HT^g receptors which are located on presynaptic terminals of many transmitter systems; in the DG, this could increase the release of NE or ACh or decrease the release of GABA (Maura and Raiteri, 1986; Moldering et al., 1987), any of which could have an excitatory effect on DGCs. It is possible, however, that the reduced release of endogenous 5-HT more than compensates for the increase of 5-HT at the receptors involved. Desensitization of responses to 5-HTj^ agonists It was of interest that the repeated administrations of 8-OH-DPAT appeared to produce attenuated effects on PSA, as previous studies have reported a desensitization to 5-HT-j^ agonists on both physiological and behavioural measures. The reduction of electrophysiological responses to 5-HT-^ agonists after repeated administration has been investigated previously over a period of days, rather than minutes, and has involved chronic, rather than acute administration of these compounds (Blier and DeMontigny, 1987). Hyperphagia and decreased 5-HT metabolism have been observed following administration of 5-HT-^ agonists, and both of these effects appear to be mediated by presynaptic 5-HT-j^ receptors. Both of these responses can be attenuated by a single pre-treatment with 8-OH-DPAT, indicating that presynaptic 5-HT^A receptors may exhibit desensitization to an acute administration of this compound (Kennett et al., 1987). Whether the attenuated effects of repeated administration of 5-HTj^ agonists reflect down-regulation of 5-HT receptors is unknown. There is evidence that the number of 5-HT binding sites may be regulated rapidly by a number of systems. For example, DeKloet et al. (1986) have found that as soon as one hour after adrenalectomy, 5-HT^ binding was increased significantly in both the C A l and DG; binding could be reversed to normal levels by administration of either corticosterone or synthetic corticosteroids. The desensitization of presynaptic 5-HT-^ receptors may be of clinical importance. This phenomenon has been linked with electroconvulsive shock treatment and with chronic antidepressent drug treatment (DeMontigny and Blier, 1984). The desensitization of 5-HTj^ receptors may therefore be related to the possible antidepressant actions of 8-OH-DPAT (Cervo and Samanin, 1977) and buspirone (Goldberg and Finnerty, 1979; Schweizer et al., 1986). In summary, the present in vivo studies indicate that activation of 5-HT^ receptors in the midbrain raphe nuclei acts to increase PSA in the DG. As these receptors are thought to be inhibitory, the effects on PSA may be mediated by an attenuation of the tonic release of 5-HT. 141 CHAPTER 4: MODULATION OF SYNAPTIC PLASTICITY IN THE DENTATE  GYRUS BY STIMULATION OF THE MEDIAN RAPHE 4.1 INTRODUCTION Long-term potentiation (LTP) is characterized by a long lasting increase in synaptic efficacy following a brief, high frequency (tetanic) stimulation of afferent fibres. This phenomenon was first reported in the perforant path-dentate gyrus (PP-DG) synapse (Bliss and Lomo, 1973), and has since been found in many other pathways in the hippocampal formation. The long duration of LTP, combined with the well-known involvement of the hippocampus in memory formation, have prompted its consideration as a model of long term information storage in the brain (Teyler and Discenna, 1984). The induction of LTP at the PP-DG synapse by tetanic stimulation may be augmented by the concurrent, or near-concurrent stimulation of several locations outside of the HF, including the medial septum (Robinson and Racine, 1982) and mesencephalic reticular formation (Bloch and Laroche, 1985). Part of the attractiveness of such "co-operative" effects between brain regions is their ability to serve as models of associative learning. The present study investigated the modulation of PP-DG LTP by concurrent stimulation of the median raphe nucleus (MRN), which is the major source of serotonergic innervation of the DG. Several factors, including the high density of projections from the MRN to the DG and the high concentration of 5-HT receptors in the DG, indicate that the MRN may play a role in the modulation of LTP in the DG. Additionally, the connections between the MRN and the HF appear to be essential for some forms of behavioural conditioning (Solomon et al., 1980), while the serotonergic modulation of population responses in the DG is known to correlate with behavioural state, as would be expected for a system which might modulate a possible substrate of learning or memory (Winson 1980; 142 Srebro, et al., 1982). In the DG, stimulation of the medial septum alone will provide a brief period where the response to PP stimulation alone is facilitated (Robinson and Racine, 1982). These short-term effects of MS stimulation are augmented following the "cooperative" induction of LTP by the near-concurrent tetanic stimulation of the MS and PP (Moore and Racine, 1988). This has been viewed by these authors as an indication that the magnitude of LTP is greater when responses are evoked in the original "context" of the tetanic stimulation. Previous studies have shown that stimulation of the MRN alone will also provide a brief period where the amplitude of DG population spikes evoked by PP stimulation is increased (Assaf and Miller, 1978; Winson, 1980). The present study, therefore, also examined the influence of "context" on LTP, by investigating whether the short term modulatory effects of MRN stimulation may be influenced by the induction of LTP following tetanic stimulation of the MRN and PP. 4.2 METHODS A. GENERAL PROCEDURES Male Wistar rats (350-500 gm.) were anaesthetized with urethane and placed in a Kopf stereotaxic instrument. A stimulating electrode was implanted in the right angular bundle, to stimulate the mixed perforant path, and a glass recording microelectrode implanted in the GL of the right dentate gyrus, as in the General Methods section of Chapter 3 above. An additional stimulating electrode was implanted in the MRN, 0.7 mm anterior to the intra-aural line, 3.5 mm L and 7.5 mm BSC, at an angle of 20° to the vertical plane. The electrodes were lowered into position very slowly (0.2 mm/min) to minimize trauma to brain tissue. After placement of electrodes, 30 min were allowed for tissue equilibration. Population responses recorded in DG were evoked by electrical stimulation of the 143 perforant path. Stimulus intensity at the PP electrode was adjusted to evoke a population spike of approximately one-half maximal amplitude (Avg.: 150uA).. Stimulation intensity at the MRN electrode was set at 200uA. B. STIMULATION AND RECORDING PROCEDURES Paired-pulse 1: The short-term modulation of PP-DG responses by MRN stimulation was assessed by a paired-pulse procedure, whereby single "conditioning" stimuli of the MRN were followed by single "test" stimuli to the PP, and the population responses evoked by PP stimulation were recorded. After recording stable baseline responses to stimulation of the PP alone, paired-pulse responses were evoked at "conditioning-test" (C-T) intervals ranging between 0 and 100 msec. The stimulus pairs were presented in a fixed order of increasing C-T interval. Paired-pulse responses were evoked every 40 sec, and four responses were recorded at each C-T interval. Induction of LTP: A 100 Hz., 1 sec duration stimulus train ("tetanic stimulation") was then delivered either to the PP alone, concurrently to the PP and MRN, or to the MRN alone; previous experience had indicated that these stimulation parameters produced a minimal amount of LTP when applied to the PP alone. The concurrent stimuli to the PP and MRN were delivered 180° out of phase to each other, i.e. the interval between individual PP and MRN pulses during tetanus was 5 msec. After this tetanic stimulation, DG responses to PP stimulation were evoked and recorded once per minute for one hour. Paired-pulse 2: The stimulus intensity at the PP electrode was then adjusted to evoke a population spike of an amplitude equivalent to that observed before tetanization. The effects of tetanic stimulation on the short term effects of MRN stimulation were then examined, by repeating the paired-pulse procedures performed prior to tetanization. C. HISTOLOGY: Anodal current (lOuA, 10 sec) was passed through the stimulating electrodes, to mark the tip positions (Prussian Blue reaction). The animals were sacrificed with an overdose of urethane, and perfused transcardially with 0.9% saline followed by 5% potassium ferrocyanide in 10% neutral, buffered formalin. The brains were removed, sectioned in a cryostat at 200um thickness, stained with 0.9% thionin, and examined for electrode placement. Data analysis was performed only on the responses from animals which had a stimulating electrode placement within 0.5 mm of the MRN (PP tetanus alone: N = 11; combined PP and MRN tetanus: N = 9; MRN tetanus alone: N = 2). 4.3 RESULTS A. PAIRED PULSE Prior to tetanus, paired-pulse stimulation produced an increase in PSA at C-T intervals between 5 and 70 msec (Figure 4.3), with no discernable effect on population EPSP slope (not shown). B. TETANIC STIMULATION An increase was observed in mean PSA and population EPSP slopes after either PP-alone or combined PP and MRN tetanus, with an apparently greater effect on both measures following combined PP and MRN tetanus. The effects of the tetanic stimuli on PP-evoked responses were assessed at two time intervals, with analyses performed on the means of the responses collected at early (1-3 min) and late (55-60 min) periods after tetanus. Data from the animals which had received MRN tetanus alone were excluded from analysis, as this procedure produced no discernable effects on PP-evoked responses. PSAs and population EPSP slopes were first entered into a multivariate analysis of variance (MANOVA) with site of tetanic stimulation (PP or combined PP and MRN) as a between 145 group factor, and time period (early or late) as a within group factor; this revealed a significant overall effect (F(2,17) = 6;07, p = 0.0T). Univariate analysis of variance (ANOVA) identified significant effects of site of tetanic stimulation on both PSA (F(l,18)= 11.32, p<.005) and EPSP slope (F(l,18) = 4.83, p<.05), but no significant effects of time period or time period by site interaction. These results indicate that the PSA and EPSP slopes were significantly greater after combined MRN and PP tetanus than after PP tetanus alone, at both time periods (Figures 4.1, 4.2). The success of the two tetanic stimulation procedures in inducing LTP was then analyzed. Ninety-five percent confidence intervals were calculated about the means for EPSP slope and PSA at both early and late time periods, for each condtion. These were each then compared with the theoretical mean baseline value (100%), using the Dunn-Bonferroni correction to maintain the type I error rate below .05 for the eight comparisons (a =.0061) (Glass and Hopkins, 1984). After combined MRN and PP stimulation, PSA was significantly greater than baseline values at both early and late periods, and EPSP slope was significantly greater than baseline values only at the early period. Stimulation of the PP alone did not produce a significant increase in PSA or EPSP slope at either time period. C. TETANIC STIMULATION AND PAIRED-PULSE The effects of prior tetanic stimulation on paired-pulse facilitation were assessed by comparing the mean paired-pulse effect across all C-T intervals before tetanus with that observed in the same animals one hour after tetanus. Means were compared in t-tests, using the Dunn-Bonferroni correction to maintain the familywise alpha at 0.05. Using this measure, there was no significant change in the effects of paired-pulse stimulation after tetanus, in either the PP-tetanus alone or combined MRN-PP tetanus groups (Figure 4.3). 146 Figure 4.1: Representative responses evoked in the dentate gyrus by perforant path stimulation, before and one hour after tetanic stimulation. A) tetanic stimulation of PP alone; B) combined tetanus of PP and MRN. F i g u r e 4.2: Populat ion spike amplitudes (A) and population E P S P slopes (B) evoked after tetanic s t imulat ion of P P alone (open circles, n = 11) or P P and M R N combined (closed circles, n = 9 ) . B o t h P S A and E P S P slope showed significantly greater increases after combined P P and M R N st imulation. 149 TIME POST TETANUS (MINUTES) F i g u r e 4.3: Faci l i tat ion of PP-evoked population spike amplitude by prior st imulat ion of the M R N . " C - T i n t e r v a l " indicates the time between the conditioning st imulation of the M R N , and the subsequent test st imulation of the P P . Open circles: prior to tetanic s t imulat ion; closed circles: one hour after tetanic st imulation. N o change i n this short t e r m facil itation was observed after tetanic stimulation of P P alone (A) or combined tetanic s t imulat ion to the P P and M R N (B). A INTERVAL BETWEEN MR AND PP STIM (MSEC) 4.4 DISCUSSION The results from this study indicate that stimulation of the MRN increases LTP of both population spike amplitude and population EPSP slope, without affecting the paired-pulse modulation of DG responses by single stimuli to the MRN. Despite the apperent simplicity of the observed results, there are several possible underlying mechanisms. The most direct suggestion would be that the tetanus applied to the MRN increases the release of 5-HT onto the DGCs, and that this increased level of released 5-HT interacts with the processes normally underlying LTP to produce greater potentiation. Very little is known about the role of serotonin in the modulation of long term changes in synaptic efficacy in the HF. To date, only two studies have addressed the serotonergic modulation of LTP; both have examined the effects of serotonin depletion. Bliss et al. (1983) found that depletion of 5-HT by the neurotoxins 5,7-DHT or pCPA reduced the amount of LTP which could be induced in the DG of rats anaesthetized with pentobarbitone. Stanton and Sarvey (1985) reported that depletion of serotonin by 5,7-DHT had no effect on the amount of LTP which could be induced in the DG of in vitro hippocampal slices. These latter authors proposed that the serotonergic system may act to facilitate LTP indirectly, through oligosynaptic pathways, rather than via the direct serotonergic projections to the •DG. This appears possible, as the raphe nuclei project to many regions of the brain which are known to modulate activity in the DG, including the entorhinal cortex, septum, locus ceruleus, and amygdala. However, the results from the two depletion experiments also suggest that the release of 5-HT onto the DGCs may facilitate LTP directly, with this facilitation requiring intact serotonergic projections to the DG. Disruption of the serotonergic projections from the MRN during preparation of the hippocampal slices would eliminate any tonic serotonergic influence, and may account for the lack of difference in LTP 153 between slices prepared from 5-HT-depleted or control animals. Unfortunately, comparison of these two depletion studies is also made difficult by the difference in the methods of quantifying LTP, i.e. changes in population EPSP amplitude (Bliss et al., 1983) versus changes in population spike amplitude (Stanton and Sarvey, 1985). Although 5-HT has a hyperpolarizing effect on DGCs, which would be expected to block LTP (Malinow and Miller, 1986), it also decreases AHP, and the ability to accommodate to depolarizing stimuli (Baskys, et al., 1987, 1989). This latter effect may increase the responsiveness of the DGCs to the PP tetanus, thereby facilitating the induction of LTP. There is also indirect evidence to suggest an interaction of 5-HT with the processes normally underlying LTP. Induction of LTP in the DG is thought to be dependent upon activation of the N-methyl-d-aspartate (NMDA) -type of glutamate receptor. When paired with a concurrent depolarizing stimulus, such as tetanic stimulation, activation of this receptor is thought to lead to increased intracellular Ca , which may underlie the induction of LTP. Recent studies have demonstrated that 5-HT can act to potentiate the effects of NMDA on neocortical neurons (Reynolds et al., 1987; Baskys et al., 1988). If this form of modulation also holds true in the DG, then 5-HT released by tetanus to the MRN may potentiate LTP by an interaction with NMDA receptors on the DGCs. A facilitation of LTP by serotonin may be similar to that produced by norepinephrine (NE). Much evidence has accumulated for a LTP-like effect produced in the DGCs by NE, termed "NE-induced long lasting-potentiation" (NELLP), in both in vitro and in vivo preparations (Stanton and Sarvey, 1986; Neuman and Harley, 1983). The mechanism underlying NELLP is unclear, but may involve an increase in voltage-gated calcium conductances (Johnston et al., 1988). However, while application of either NE or 5-HT produce similar effects on membrane potential of DGCs, i.e. hyperpolarization, reduced accommodation, and reduced AHP, an NELLP like effect of 5-HT was not observed in any of the in vitro experiments in Chapter 2 of this thesis. This could be related to the positioning 154 of the stimulating electrode, as Sarvey et al. (1988) have recently reported that NELLP in the DG is specific to the medial perforant path, while in the experiments in Chpater 2 population responses in the DG were evoked by stimulation of the lateral perforant path. However, studies in our laboratory which are currently in progress indicate that bath application of 5-HT does not bring about an NELLP-like phenomenon in the DG even with medial perforant path stimulation. Stimulation of the MRN has been shown to elevate 5-HT concentrations in forebrain structures, although the stimulation parameters which have been used to achieve this are often extreme, consisting of continuous stimulation for many minutes (e.g. De Simone et al., 1987; Holman and Vogt, 1972). However, brief antidromic stimulation of serotonergic neurons in the DRN or MRN has been shown to produce a period of inhibition in the normal rhythmic activity of these cells, lasting up to 1000 msec (Wang and Aghajanian, 1977a,b,c). This "silent period" may be induced by the release of 5-HT onto inhibitory autoreceptors. It is possible that the tetanic stimulation of the MRN used in this study produces a similar period of inhibition of the MRN neurons, and therefore the potentiating effect of MRN stimulation on LTP may not be due to an increase, but to a decrease in the release of 5-HT. As 5-HT is known to hyperpolarize DGCs, the MRN tetanus could thus temporarily reduce a tonic hyperpolarizing influence on these cells during a crucial period of LTP induction. The paired-pulse data show that the short-term facilitation of the PSA by single stimuli to the MRN was not changed after either tetanization procedure. There is evidence, however, that the short-term effects of MRN stimulation are mediated by non-serotonergic fibres which either originate in, or pass through the MRN (Srebro et al., 1982). This indicates that the increase in LTP produced by MRN stimulation may result from activation of fibres which are unrelated to those producing paired-pulse facilitation. The existence of a non-serotonergic pathway from the MRN to the DG also raises the question of whether the enhancement of LTP by tetanic stimulation of the MRN is actually mediated by a serotonergic mechanism. Both the paired-pulse and LTP-modulating effects of MRN stimulation may be mediated by a common mechanism that is non-serotonergic; this would imply that the cooperative augmentation of LTP by this mechanism does not show "context dependency". Answers to these questions await experiments examining the effects of 5-HT depletion or pharmacological blockade of 5-HT receptors. 156 CHAPTER 5: GENERAL DISCUSSION This thesis has examined the serotonergic modulation of neuronal transmission in the hippocampal formation, and in particular, in the dentate gyrus. In the first two series of experiments, pharmacological agents were used in an attempt to identify the influence of serotonergic receptors on evoked potentials; the third series examined the influence of stimulation of the MRN, the major source of serotonergic projections to the HF, on stimulation induced plasticity in the DG. In all of the above experiments, the dependent variables were electrically evoked field responses. As these measures indicate the overall excitability of a population of neurons, they appear to be particularly appropriate in examining the diffuse and widespread influence of the serotonergic system on hippocampal activity. In the first series of experiments, either 5-HT, the 5-HT^A receptor agonist 8-OH-DPAT or the 5-HT2 receptor agonist DOI was applied to the in vitro hippocampal slice preparation, in order to investigate the effects mediated by serotonergic receptors. Previous studies of the effects of serotonergic compounds on evoked field potentials in the HF have been limited to the CAl region. In this section of the thesis, experiments were performed in parallel in both the CAl and the DG, explicitly to compare pharmacological effects in the two regions. Application of either 5-HT or 8-OH-DPAT inhibited population responses in both regions, with greater effect in the CAl. In addition, the effects of 8-OH-DPAT were attenuated by the serotonergic antagonist methysergide. These findings indicate an inhibitory role for the 5-HT-^ receptor in both area CAl and the DG. In vitro, the 5-HT2 receptor agonist DOI produced a decrease in population responses in the DG only, and this effect could be antagonized by the 5-HT2 receptor antagonist ketanserin. These experiments suggest the possibility of an inhibitory role for the 5-HT2 receptor in this region of the HF. The effects of DOI did not quite reach statistical significance, however, indicating that further investigations are required before 157 definate conclusions can be drawn about the role of the 5-HT2 receptor in this region of the brain. These results are consistent with previous studies which have examined the effects of serotonergic compounds on field potentials recorded in the C A l , and are the first demonstration of serotonergic effects on field potentials in the DG, in vitro. The inhibitory effects of 5-HT and 8-OH-DPAT on population responses indicate that the release of endogenous 5-HT may act to inhibit neural transmission in the HF at both the levels of the DG and the C A l cell fields, and so may act to restrict the processing of information through the hippocampal trisynaptic loop. The greater effects of 5-HT and 8-OH-DPAT observed on population spike amplitude in area C A l suggest that transmission in this area is more sensitive to serotonin than the DG; however, whether this relationship applies to the effects of endogenously released 5-HT remains unknown. The ionic and biochemical processes underlying the effects of these compounds on field potentials were not investigated; however given the effects of serotonergic compounds observed previously on intracellular potentials in both C A l HPCs and DGCs, it appears likely that the inhibitory effects on population responses described in this thesis reflect 5-HT^A receptor mediated hyperpolarizations of both of these cell types, probably resultant from an increased gK. The second part of the thesis investigated the effects of serotonergic compounds on population responses recorded in the DG in an in vivo preparation. In contrast to. the effects observed in vitro, intraperitoneal administration of 5-HTP or 8-OH-DPAT produced an increase in DG PSA, with no concomitant change in the GL population EPSP. Intravenous administration of either 8-OH-DPAT or the 5-HT^A ligands buspirone or BMY-7378 was also followed by an increase in DG PSA, although the effects observed following administration of the latter two compounds did not reach statistical significance. Descriptively, the effects of both BMY-7378 and buspirone on PSA were much smaller than those of 8-OH-DPAT. The smaller magnitude of these effects may be attributable to the lower efficacy of these compounds as agonists at the 5 - H T l A receptor than 8-OH-DPAT, especially considering that BMY-7378 has been proposed as an 5-HTj^ antagonist. Statistical significance of such smaller effects on PSA might have escaped the conservative statistical analyses used, when considering the small number of subjects injected with these compounds. Intravenous administration of the 5-HT2 agonist DOI appeared to produce no effects on population responses evoked in the DG in vivo. It is possible that the small and inconsistent inhibitory effects produced by this compound on DG population responses in vitro may have been masked by the larger amount of biological noise present in an in vivo preparation, or that they may have been suppressed by the use of anesthesia. The mechanisms underlying the effects of 8-OH-DPAT in vivo were investigated further, and several lines of evidence were developed which indicate that the facilitation of DG PSA by 8-OH-DPAT in vivo is mediated by 5 -HTj A receptors which are located on serotonergic neurons in the brainstem raphe nuclei. The involvement of 5-HT^A receptors is indicated by the known specificity of 8-OH-DPAT and by the attenuation of the effects of 8-OH-DPAT by the serotonergic antagonist methysergide. The involvement of brainstem serotonergic neurons is suggested by the increase in PSA which was observed after administration of 8-OH-DPAT either intracranioventricularly or directly into the vicinity of the median raphe nucleus, and by the attenuation of the effects of systemically administered 8-OH-DPAT on PSA following pre-treatment with the serotonergic neurotoxin 5,7-DHT. Such a similar pattern of effects has been observed for 8-OH-DPAT induced hyperphagia, which also appears to be mediated by effects of this compound on brainstem serotonergic neurons (Dourish et al., 1986a,b,c; Hutson et al., 1986, 1987). It appears likely that the effects of 8-OH-DPAT on the MRN are inhibitory, given the large number ,of electrophysiological studies which have shown inhibitory effects of 5 -HTj A agonists on raphe neurons. Further evidence for a presynaptic mechanism underlying the increase in PSA following i.v. administration of 8-OH-DPAT is provided by the biphasic dose-response relationship which was observed, with higher doses producing an attenuated facilitatory effect. This biphasic effect may have been due to actions of 8-OH-DPAT at both presynaptic and postsynaptic sites. As mentioned above, the facilitatory effect of 8-OH-DPAT on DG PSA appears to be mediated largely by presynaptic actions in the raphe nuclei. The attenuated facilitation of PSA observed at high doses in vivo may reflect the activation of inhibitory processes which are mediated by postsynaptic receptors in the DG, given the inhibition of population responses observed after application of 8-OH-DPAT in vitro. This biphasic response pattern may be explained by diferences in sensitivity between pre- and postsynaptic 5-HT-j^ receptors, as recent results suggest that serotonergic neurons in the raphe nuclei are more sensitive to 5-HTj.^ agonists than are hippocampal neurons (Aghajanian and Sprouse, 1987). However, even if raphe and hippocampal 5-HT-^ receptors were equally sensitive, such a biphasic response may still be explainable with reference to pre- versus postsynaptic effects. If the increase in PSA is mediated by a suppression of spontaneous activity of MRN neurons, then logically, this effect would reach a maximum at a dose of 8-OH-DPAT which would completely inhibit the unit activity of MRN neurons. If inhibitory effects of 8-OH-DPAT on evoked responses in the DG (such as are observed in vitro) are mediated by postsynaptic receptors on the DGCs, this inhibition may not reach an asymptotic effect at as low a dose, i.e. a dose of 8-OH-DPAT which may completely suppress unit activity of DGCs may not inhibit evoked responses to a maximal extent, as population responses in the DG may be evoked easily in the absence of unit activity. For example, at a low dose of 8-OH-DPAT, some DGCs may still be induced to fire by electrical stimulation, although all spontaneous activity of these neurons may be suppressed. At a higher dose of 8-OH-DPAT, the response to electrical stimulation may be inhibited further, even though no additional inhibition of DGC unit activity is possible. An alternative explanation of the biphasic response to 8-OH-DPAT may be found in the complex relationship of PSA to unit activity of DGCs. In vivo, increases in PSA may be brought about by mechanisms which have an inhibitory effect on DGCs. For example, stimulation of several brainstem regions, including the MRN, increases the amplitude of a population spike evoked subsequently in the DG, while concurrently inhibiting the spontaneous unit activity of DGCs (Assaf and Miller, 1978). A lower spontaneous firing frequency of a neuron may indicate that at any given time there will be a lower probability of that cell being in a refractory state. A reduction in unit activity, therefore, may increase the probability of activation by afferent fibre stimulation. Thus, a decrease in spontaneous activity of DGCs has been viewed as establishing a condition that could increase the "signal to noise ratio" in the PP-DG system, by allowing a greater response of DGCs to incoming signals (Assaf and Miller, 1978). From this line of speculation, it may be suggested that, at low doses, direct inhibitory effects of 8-OH-DPAT on DGCs may contribute to an increase in PSA, by decreasing spontaneous activity. Higher doses of 8-OH-DPAT may produce larger and more prolonged effects that could reduce PSA. The fact that spontaneous activity is diminished in the hippocampal slice preparation may explain why a similar facilitation of PSA at low doses was not seen in vitro. The increase in PSA produced by injections of 8-OH-DPAT into the brainstem is accompanied by an increase in the ML population EPSP but not the GL population EPSP, indicating that the facilitatory effects of 8-OH-DPAT may be mediated by actions in the dendritic region. However, a complex relationship exists between the responses of individual neurons and the extracellularly recorded population reponses in a cell field. Changes in the size of population EPSPs may reflect changes in several processes, including neurotransmitter release, membrane polarization, and membrane conductance. For example, an increase in population EPSP may be expected to reflect either an increase in the release of an excitatory neurotranmsitter, which would increase synaptic current; an hyperpolarization of the postsynaptic membrane, which would increase the driving force for EPSPs; or a decrease in postsynaptic gK, which would decrease the shunting of EPSP current. At present, whether the effects of 8-OH-DPAT on any of these processes underlies population EPSP changes remains unknown. Regardless of the mechanisms involved, the present results support the hypothesis that, in vivo, a decrease in serotonergic activity serves to increase the amplitude of evoked responses in the DG. If the PSA is taken as an index of the efficacy of transmission across the PP-DG synapse, this implies that under normal conditions, activity of the serotonergic neurons serves to inhibit the flow of information from the ER to the DG. As the DG appears to form a "gate" through which the bulk of the information entering via the perforant path must pass before reaching the hippocampus, this process of inhibition could be conceived of as regulating input to the entire HF, by restricting the flow through that gate. Additionally, the paired-pulse experiments in this thesis indicate that the serotonergic system may act to change the processing of information by the HF in a qualitative, as well as quantitative manner, by changing the frequency-dependent characteristics of the DGCs. Paired pulse measurements revealed that the i.v. administration of 8-OH-DPAT may act to decrease the late period of inhibiton observed after stimulation of DGCs'm vivo, with no effect on either the early period of inhibition, or of paired-pulse facilitation. Given that in the CAl HPCs, a reduction in afterhyperpolarization, which appears to underlie the late period of paired-pulse inhibition, has been found to correlate with behavioural conditioning (Coulter et al., 1989), it is possible that serotonergically modulated changes in the late period of paired-pulse inhibition in the DG may be of behavioural significance. The magnitude of the facilitation of the D G P S A by the presumed inhibition of serotonergic neurons which was reported i n this thesis indicates that the M R N m a y have a profound inhibitory influence on the functioning of the H F . This conclusion is supported by the results of the in vitro experiments, which have demonstrated a direct inhibitory effect of 5 - H T and serotonergic agonists in the H F . These findings emphasise the importance of non-local, b r a i n s t e m influences on the functioning of cortical structures. If the administration of 8 - O H - D P A T and other 5 - H T ^ A agoninsts in vivo produces effects i n the H F by inhibiting serotonergic neurons in the raphe nucle i , it is possible that the inhibition of raphe neurons also produces large effects in the other b r a i n structures which receive projections from these brainstem nuclei. The influence of the raphe nuclei on forebrain structures m a y be related closely to behavioral state, as the activity of serotonergic neurons is known to be correlated w i t h levels of behavioral arousal , w i t h the highest frequency of f ir ing during w a k i n g states, and the lowest d u r i n g slow-wave sleep (Trulson and Jacobs , 1979). T h i s hypothesis is supported by a series of experiments by W i n s o n a n d colleagues, who found that the amplitude of population spikes recorded i n the D G of chronical ly implanted rats was much larger during slow-wave sleep, than d u r i n g a s t i l l , a lert waking state. This difference i n cellular responsiveness appears to be mediated b y ascending serotonergic projections, as it is abolished by either mechanical lesions at the level of the midbrain or neurochemical lesions of serotonergic neurons w i t h 5 , 7 - D H T (Winson, 1980; Winson and Abzug, 1978a,b; Srebro et a l . , 1983, D a h l et a l . , 1983). Winsons 's experiments , therefore provide further support for an inhibitory role of 5-HT in the functioning of the D G . The experiments i n the third part of this thesis have examined the modulation of synaptic plasticity at the P P - D G synapse by s t i m u l a t i o n of the M R N . The finding that tetanic s t imulat ion of the M R N can facil itate the induction of L T P indicates that serotonergic processes may also modulate long-lasting changes in synaptic efficacy i n the HF. Given the aforementioned dependence of serotonergic activity on behavioural state, such a modulatory influence on LTP would be expected to vary with levels of arousal. Interestingly, Bramham and Srebro (1989) have reported recently that LTP in the PP-DG synapse of freely moving rats is most difficult to elicit during slow-wave sleep, when the activity of serotonergic neurons is lowest. As LTP has often been looked upon as a model of memory storage, these findings indicate a possible role for the endogenous serotonergic system in the formation and behavioural dependence of memory traces. Most comprehensive theories of hippocampal function acknowledge the importance of the ascending serotonergic projections, irrespective of whether their authors feel that the HF functions as a substrate for congnitive mapping (O'Keefe and Nadel, 1976), "behavioral inhibition" Gray, 1984), the integration of responses over discrete intervals of time (Rawlins, 1986), or the Freudian unconscious (Winson, 1984). All of these authors mention that the density of serotonergic projections indicate a profound influence on the functioning of the HF, especially at the level of the DG; the fact that none of them, however, make detailed theoretical speculation on the nature of this influence is most likely due to the paucity of previously available data. The concept of serotonergic influence on the HF as a behaviourally dependent, inhibitory gating system, at least at the level of the DG, could fit well with all of the above theories. Both the serotonergic system and the HF have long been implicated in anxiety (Gray, 1982; Soubrie, 1985). Recent interest in the possible serotonergic mechanisms underlying this disorder have arisen from the finding that several 5-HT^A agonists, including buspirone and 8-OH-DPAT, appear to be effective anxiolytic agents, both in animal models and clinical trials, without producing the unwanted side effects of the most commonly used anxiolytic compounds, the benzodiazepines (Goldberg, 1979; Eison, et al., 1985; Johnson et al., 1986; Carli and Samanin, 1988; Young et al., 1982, 1987). Given the inhibition of evoked responses which is produced by these compounds in the i n vitro hippocampal slice preparation, it has been suggested that the anxiolytic effects of 5-HT]^ agonsists may be mediated by a decrease in neuronal excitability in the HF (Beck and Goldfarb, 1985). The present studies indicate that in the whole animal, 5-HTj^ agonists may act to increase the efficacy of synaptic transmission between the ER and the DG. If the anxiolytic effects of these compounds are related to their effects in the HF, they may reflect increased, rather than decreased processing by this structure. Therefore, the current findings may have broad implications for the role of the HF and the serotonergic system in the expression of anxiety. Obviously, much more work remains to be done before a coherant picture can be formed of how the serotonergic system affects the HF. Much of the present thesis has rested upon the presumed selectivity of a few pharmacological compounds. For example, although 8-OH-DPAT is known to be highly selective for the 5-HTj^. receptor', the proper identification of the receptor involved in the effect with the 5 - H T ^ binding site requires assesment of the effects of a large number of compounds, and a comparison of their affinities for the 5 - H T ^ binding site. Similarly, experiments should be conducted for compounds active at the 5-HT2 receptor, particularly given the inconclusive effects of DOI in the present thesis. The role of the 5-HTjg receptor was not investigated in the present studies, largely because of the unavailability of selective agonists. While the 5-HT^g receptor appears not to be found in the human brain, it is abundant in the rat hippocampus, where it may play an important role by the presynaptic modulation of neurotransmitter release. In addition, conclusive identification of the effects of the 5-H T j A receptor awaits the development of selective antagonists for this subtype. Methysergide was used in the present experiments as it has been shown previously to block the effects of 5-HT^ agonists; like other available 5-HT^ antagonists, however, this compound is also effective at the 5-HT2 receptor. 165 The constraints inherent in the design of workable experiments may, of course, have introduced some bias. For example, only male rats were used as experimental animals; some data (Peroutka et al., 1987; Mauk et al., 1988) suggests that different results may have been obtained from female rats. That this was not investigated in the present thesis was due largely to the difficulty in controlling for hormonal factors in females. Additionally, preparations were used for experiments only if a robust signal could be evoked, and if it could be maintained at a stable baseline; this excluded approximately 30% of both the in vitro and in vivo preparations. In the present thesis, the experiments have focussed on the role of the MRN, which provides the bulk of the serotonergic innervation to the HF. The DRN cannot be excluded from playing a role in the phenomena reported here. Both the systemic injections of compounds and the treatment with 5,7-DHT would be expected to affect the DRN as well as the MRN; additionally, diffusion of_8-OH-DPAT from the MRN microinjections, and passive conduction of electical current from the MRN stimulation might both have impinged upon the DRN. As mentioned in Chapter 1, neurotoxins exist which are able to discriminate between the serotonergic neurons in the DRN and the MRN. Further studies with these compounds should be undertaken to investigate the individual roles of these nuclei. 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