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Studies on the effects of hemoglobin on synaptic transmission in the hippocampus Yip, Samuel 2000

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STUDIES ON THE EFFECTS OF HEMOGLOBIN ON SYNAPTIC TRANSMISSION IN THE HIPPOCAMPUS by SAMUEL YIP B.Sc. (Hon) (Pharmacology and Therapeutics), The University of British Columbia, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology and Therapeutics, Faculty of Medicine, The University of British Columbia) We accept this thesisasc^emoTming tojhejgo^ujrep^l^dar/: THE UNIVERSITY OF BRITISH COLUMBIA April 2000 © Samuel Yip, 2000 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 P^rnrYXTnlnnh £ jflfiiT? The University of British C o l u m b i a / Vancouver, Canada Date DE-6 (2/88) ABSTRACT During head injuries and hemorrhagic stroke, blood is released into the extravascular space. The pooled blood remains in the intracranial cavity for a prolonged period of time. During this time, it has been shown that the pooled erythrocytes get lysed and hemoglobin is released into the intracranial cavities. Since clearance of hemoglobin is slow, neurons may be exposed to hemoglobin and/or its breakdown products, hemin and iron, for long periods of time. It is, therefore, important to understand the effects of hemoglobin and its breakdown products on synaptic transmission. In this study, the electrophysiological actions of these agents on synaptic transmission in rat hippocampal CA1 pyramidal neurons were studied using extracellular field- and whole cell patch-recordings. It was found that commercially available hemoglobin samples produce inconsistent effects on synaptic transmission in hippocampal slices. The commercial hemoglobin which reversibly depressed synaptic transmission in CA1 neurons, was found to be contaminated with ammonium and bisulfate. These agents may be responsible for the observed synaptic depression. Since commercially available hemoglobin contains both methemoglobin and reduced-hemoglobin, the effects of these compounds were studied on synaptic transmission. Methemoglobin had no significant effect on synaptic transmission. Although reduced-hemoglobin, prepared with a method described by Martin et al. (1985), produced a significant reversible depression of synaptic - i i i -transients, the effects were actually due to the bisulfite that was introduced by the reducing procedure. Unlike hemoglobin, breakdown products of hemoglobin, ferrous chloride and hemin, produced a significant irreversible depression of field excitatory postsynaptic potentials. The importance of these effects of hemoglobin breakdown products in understanding neurological complications that follow head-injuries and hemorrhagic stroke awaits further investigation. Bhagavatula R. Sastry, Ph.D., Research Supervisor. Date18 t n April 2000 - iv -TABLE OF CONTENTS CHAPTER PAGE ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES xiii LIST OF TABLES xv LIST OF ABBREVIATIONS xvi LIST OF PHARMACOLOGICAL DRUG ACTIONS xviii ACKNOWLEDGEMENTS xix DEDICATION xx 1 INTRODUCTION 1 2 ANATOMY OF THE HIPPOCAMPUS 2 2.1 Definition of Hippocampus 2 2.2 Topography of the hippocampal formation 2 2.3 Stratification, Principle And Associated Cell Types Of The Hippocampal Formation 4 2.3.1 The dentate gyrus 4 2.3.2 The hippocampal proper 8 2.3.3 The subiculum 12 2.4 Connections 13 2.4.1 Intrahippocampal circuitry 13 2.4.2 Lamellar concept and longitudinal pathway 14 2.4.3 Extrinsic afferent connections 15 2.4.4 Extrinsic efferent connections 16 3 ELECTROPHYSIOLOGICAL PROPERTIES OF HIPPOCAMPAL PYRAMIDAL NEURON 17 3.1 Characteristic Of Hippocampal Pyramidal Neuron 17 3.2 Membrane Ionic Currents Of Hippocampal Neurons 18 3.2.1 Sodium current ( l N a) 18 3.2.2 Calcium current ( l C a ) 20 3.2.3 Potassium currents 23 4 EXCITATORY POSTSYNAPTIC POTENTIALS IN HIPPOCAMPAL CA1 PYRAMIDAL NEURONS 29 4.1 Excitatory Amino Acid Receptor In The Hippocampus 29 4.1.1 Electrophysiological properties of NMDA receptor 29 4.1.2 Electrophysiological properties of the Non-NMDA receptor 32 4.1.3 mGluR receptor 35 4.2 Distribution Of Glutamate Receptors In The Hippocampus 37 4.3 Excitatory Synaptic Transmission in Hippocampal CA1 pyramidal neurons 37 5 GABA RECEPTOR AND INHIBITION IN THE HIPPOCAMPUS. 40 5.1 GABA-A Receptor 40 5.2 GABA-B Receptor 42 5.2.1 Effector systems coupled to the GABA-B receptor 42 5.2.2 Pharmacology of the GABA-B receptor 45 5.3 Distribution of GABA-A and GABA-B Receptors 46 5.4 Inhibitory Circuit In The Hippocampus 46 5.4.1 Feed-forward Inhibition 47 5.4.2 Feedback (recurrent) Inhibition 47 - vi -5.5 Inhibitory Postsynaptic Potentials 48 5.5.1 GABA-A receptor mediated fast inhibitory postsynaptic potentials 48 5.5.2 GABA-B receptor mediated fast inhibitory postsynaptic potentials 49 5.5.3 Presynaptic GABA-B receptor 50 6 HEMOGLOBIN AND HEMORRHAGIC STROKE 52 6.1 Hemoglobin 52 6.1.1 Structure of hemoglobin 52 6.1.2 Anabolism of hemoglobin 54 6.1.3 Catabolism of hemoglobin 55 6.2 Hemorrhagic Strokes 56 6.2.1 Etiologies 57 6.2.2 Complications of hemorrhagic stroke 58 6.2.3 Medical management of hemorrhagic stroke 59 6.3 Pathophysiology of Hemorrhagic Stroke 60 6.3.1 Ischemic stroke 61 6.3.2 Clearance of erythrocytes after hemorrhagic stroke 64 6.3.3 Ferrous hemoglobin 65 6.3.4 Reactions of NO and hemoglobin 66 6.3.5 Iron and free-radicals production 68 6.3.6 Hemoglobin and cerebral vasospasm 68 6.3.7 Hemoglobin, epileptic seizures and neurotoxicity 70 7 Hypothesis 72 8 MATERIALS AND METHODS 74 8.1 Animal Source 74 8.2 Slice Preparation 75 - V l l -8.3 Slice Chamber 77 8.4 Superfusing Media 79 8.5 Pipette Solutions 83 8.6 Recording and Stimulating Equipment 84 8.6.1 Amplifiers 86 8.6.2 Stimulators and isolation units 86 8.6.3 Micromanipulators and hydraulic microdrive 87 8.6.4 Recording electrodes 87 8.6.5 Stimulating electrodes. 88 8.6.6 Recording systems 88 8.7 Electrophysiological Recordings 89 8.7.1 Extracellular Recordings 89 8.7.2 Conventional microelectrode recordings 89 8.7.3 Patch-Clamp recordings 91 8.8 Analysis of Extracellular and Intracellular Recordings 93 8.8.1 Extracellular recordings 93 8.8.2 Intracellular recordings 94 8.9 Studies Performed Using Mass Spectroscopy 95 8.10 Statistics 96 9 EXPERIMENTAL PROTOCOL 97 9.1 Electrophysiological Actions of Hemoglobin-1991 97 9.1.1 Electrophysiological actions of hemoglobin-1991 on hippocampal neurons 97 9.1.2 Electrophysiological actions of hemoglobin-1991 on synaptic transients, membrane potential and input resistance in hippocmapal CA1 neurons 97 - V l l l -9.1.3 Electrophysiological actions of hemoglobin-1991 on pharmacologically isolated synaptic transients in hippocampal CA1 neurons 98 9.1.4 Effects of Nco-Nitro-L-arginine on the actions of Hb-1991 on evoked potentials, membrane potential and input resistance 99 9.2 Electrophysiological Actions of Met-Hemoglobin and Reduced-Hemoglobin on Hippocampal CA1 Neuron 100 9.2.1 Effects of met-hemoglobin 100 9.2.2 Electrophysiological actions of reduced-hemoglobin on hippocampal CA1 neurons in the presence of picrotoxinin 100 9.2.3 Electrophysiological actions of reduced-hemoglobin on hippocampal CA1 neurons in the presence of DNQX and APV 101 9.2.4 Electrophysiological actions of reduced-hemoglobin on hippocampal CA1 neurons in the presence of picrotoxinin, DNQX and Mg 2 +-free medium... 102 9.2.5 Effects of reduced-hemoglobin on extracellular EPSPs and pre-synaptic volley on CA1 neurons 102 9.2.6 Effects of reduced-hemoglobin on depolarizing pulse-induced action potentials in CA1 neurons 103 9.2.7 Effects of reduced-hemoglobin on applied agonists in CA1 neurons 104 9.3 Effects of Reduced- Hemoglobin and L-cysteine 105 9.3.1 Effects of L-cysteine on CA1 neurons 105 9.3.2 Effects of L-cysteine and reduced-hemoglobin on field EPSPs in CA1 region 105 9.3.3 L-cysteine- and reduced-hemoglobin-induced depolarization 106 9.3.4 APV, DNQX and L-cysteine and reduced hemoglobin 106 9.3.5 Ca 2 +-free medium and L-cysteine- and reduced-hemoglobin-induced inward current 107 9.4 Studies on the Differences between Hemoglobin-1991 and Hemoglobin-1996 Samples 107 9.4.1 Effects of hemoglobin-1996 on field EPSPs 108 - ix -9.4.2 Effects of dialyzed hemoglobin-1991 on field EPSPs 108 9.4.3 Mass spectroscopic analysis of hemoglobin-1991 and hemoglobin-1996 109 9.4.4 Effect Of Bisulfate And Bisulfite On Whole Cell Patch Recorded EPSCs 109 9.4.5 Qualitative test for the presence of N H 4 + in Hb-1991 110 9.5 Possible Contamination of Reduced-Hemoglobin 110 9.5.1 Effects of dialyzed dithionite on field EPSPs 110 9.5.2 Effects of reduced-hemoglobin following prolong (18 hours) dialysis 111 9.5.3 Effects of coapplication of dialyzed dithionite and L-cysteine on field EPSPs 111 9.5.4 Mass spectroscopic analysis of reduced-hemoglobin 112 9.6 Effects Of The Components Of Hemoglobin On Field EPSPs 112 9.6.1 Effects of iron on field EPSPs 112 9.6.2 Effects of hemin on field EPSPs 113 10 RESULTS 114 10.1 Electrophysiological Actions Of Hb-1991 On Hippocampal CA1 Pyramidal Neurons 114 10.1.1 Effects of hemoglobin-1991 on field EPSPs recorded in hippocampal CA1 pyramidal neurons 115 10.1.2Effects of hemoglobin-1991 on the membrane potential in hippocampal CA1 pyramidal neurons: 115 10.1.3Effects of hemoglobin-1991 on the input resistance in hippocampal CA1 pyramidal neurons 118 10.1.4Effects of hemoglobin-1991 on the evoked synaptic responses in hippocampal CA1 pyramidal neurons 118 10.1.5Effects of hemoglobin-1991 on the evoked EPSPs and slow IPSPs in hippocampal CA1 pyramidal neurons in the presence of picrotoxinin 120 10.1.6Effects of hemoglobin-1991 on hippocampal CA1 pyramidal neurons in the presence of CNQX and APV 120 10.1.7Effects of hemoglobin-1991 on hippocampal CA1 pyramidal neurons in the presence of CNQX, APV and picrotoxinin 122 10.1.8Effects of Nco-nitro-L-arginine (0.1 and 0.5 mM) on the actions of hemoglobin 122 10.2 Effects of Reduced-Hemoglobin on Hippocampal CA1 Pyramidal Neurons 125 10.2.1 Effects of methemoglobin on hippocampal CA1 pyramidal neurons 125 10.2.2Effects of reduced-hemoglobin on evoked EPSCs in the presence of picrotoxinin 128 10.2.3Effects of reduced-hemoglobin on evoked IPSCs in the presence of DNQX and APV 128 10.2.4Effects of reduced-hemoglobin on evoked NMDA-EPSCs in the presence of DNQX, picrotoxinin, and Mg 2 +-free ACSF 131 10.2.5Effects of reduced-hemoglobin on extracellular field EPSPs, population spike, and presynaptic volley 131 10.2.6Effects of reduced-hemoglobin on depolarizing pluses-induced action potential in the presence of DNQX, APV, and picrotoxinin containing ACSF... 134 10.2.7Effects of reduced-hemoglobin on bath-applied AMPA-induced current in the presence of APV, picrotoxinin, TTX and Ca 2 +-free ACSF 134 10.2.8Effects of reduced-hemoglobin on bath-applied NMDA-induced current in the presence of DNQX, picrotoxinin, TTX and Mg 2 +-free ACSF 136 10.2.9Effects of reduced-hemoglobin on bath-applied THIP-induced current in the presence of DNQX, APV, TTX and Ca 2 +-free ACSF 136 10.2.10Effects of reduced-hemoglobin on bath-applied baclofen-induced current in the presence of DNQX, APV, picrotoxinin, TTX and Ca 2 +-free ACSF 139 10.3 Effects Of Co-Application Of L-Cysteine And Reduced-Hemoglobin On CA1 Pyramidal Neurons 141 10,3.1 Effects of reduced-hemoglobin and L-cysteine on field EPSPs 141 - xi -10.3.2Current-voltage relationship of coapplication of L-cysteine and reduced-hemoglobin 143 10.3.3Effects of APV, and DNQX on inward current produced by L-cysteine and reduced-hemoglobin 143 10.3.4Effects of co-application of reduced-hemoglobin and L-cysteine in slices perfused with EGTA and TTX containing Ca 2 +-free ACSF 147 10.4 Effects Of Hemoglobin-1991 May Be Due To Contaminants Present During The Preparation Of Hemoglobin 147 10.4.1 Effects of hemoglobin-1996 and hemoglobin-1991 on field EPSP s . 149 10.4.2Effects of dialysis on the hemoglobin-1991-induced depression on CA1 neurons 149 10.4.3Differences between hemoglobin-1991 and hemoglobin-1996 as compared by mass spectroscopy 151 10.4.4Effects of bisulfate and bisulfite on evoked EPSCs 155 10.4.5Presence of ammonium ion in the hemoglobin-1991 sample 155 10.5 Contamination By Dithionite During The Preparation Of Reduced-Hemoglobin 158 10.5.1 Effects of reduced-hemoglobin and dialyzed dithionite control on field EPSPs 160 10.5.2Differences between reduced-hemoglobin and non-reduced hemoglobin as studied by mass spectroscopy 160 10.6 Effects Of Breakdown Products Of Hemoglobin On Synaptic Transmission 163 10.6.1 Effects of ferrous chloride on field EPSPs 163 10.6.2Effects of hemin on field EPSPs 165 11 DISCUSSION 167 11.1 Differences Between Hb-1991 And Hb-1996 167 11.2 Contamination of Reduced-Hb with Dithionite 172 11.3 Implications Of These Findings 176 11.4 Iron and Hemin 177 - XII -11:4.1 Free Fe* + 177 11.4.2Hemin 179 11.4.3lron, hemin and ischemia 180 11.5 Future Directions 181 12 REFERENCES 183 - X l l l -LIST OF FIGURES Figure Page Fig. 2.1. Orientation of the hippocampal formation 3 Fig. 2.2. Organization of the hippocampal formation in transverse section 5 Fig. 2.3. Strata of the hippocampus, dentate granule cells and CA1 pyramidal neurons 6 Fig. 8.3. Recording chamber 78 Fig. 8.7.1 Placement of stimulating and recording electrodes in hippocampal slice. ..90 Fig. 10.1.1. Effects of hemoglobin-1991 on evoked field EPSPs recorded in the CA1 field 116 Fig. 10.1.2. Effects of hemoglobin-1991 on the membrane potential and input resistance in CA1 neurons of hippocampal slices recorded with nystatin perforated patch electrodes 117 Fig. 10.1.4. Effects of hemoglobin-1991 on synaptic responses in CA1 neurons 119 Fig. 10.1.5. Effects of hemoglobin-1991 on the evoked EPSPs and slow IPSPs 121 Fig. 10.1.6. Effects of hemoglobin-1991 on evoked fast IPSPs 123 Fig. 10.1.7. Effects of hemoglobin-1991 on evoked slow IPSPs 124 Fig. 10.2.1. Effects of met-hemoglobin on evoked EPSPs and fast IPSPs 127 Fig. 10.2.2. Effects of reduced-hemoglobin on evoked EPSCs 129 Fig. 10.2.3. Effects of reduced-hemoglobin on evoked fast IPSCs 130 Fig. 10.2.4. Effects of reduced-hemoglobin on evoked NMDA-EPSCs 132 Fig. 10.2.5. Effects of reduced-hemoglobin on field EPSPs and presynaptic volley 133 Fig. 10.2.6. Effects of reduced-hemoglobin on the ability of CA1 pyramidal neurons to generate action potentials in response to depolarizing current pulses 135 - xiv -Fig. 10.2.7. Effects of reduced-hemoglobin on bath-applied AMPA-induced current 137 Fig. 10.2.8. Effects of reduced-hemoglobin on bath-applied NMDA-induced current 138 Fig. 10.2.9. Effects of reduced-hemolobin on bath applied THIP-induced current 140 Fig. 10.2.10. Effects of reduced-hemoglobin on bath-applied baclofen-induced current 142 Fig. 10.3.1. Effects of coapplication of L-cysteine and reduced-hemoglobin on evoked field EPSPs .144 Fig. 10.3.2. I-V relationship of L-cysteine and reduced-hemoglobin induced current 145 Fig. 10.3.3. Effects of ionotropic glutamate receptor antagonists on the current induced by coapplication of L-cysteine and reduced-hemoglobin 146 Fig. 10.3.4. Effects of Ca 2 +-free an TTX containing solution on the L-cysteine and reduced-hemoglobin induced current 148 Fig. 10.4.1. Effects of hemoglobin-1996 and hemoglobin-1991 on evoked field EPSP s 150 Fig. 10.4.2. Effects of dialysis on the hemoglobin-1991-induced depression of evoked field EPSPs 142 Fig. 10.4.3. Mass spectroscopic analysis of hemoglobin-1996 and hemoglobin-1991 153-154 Fig. 10.4.4.1. Cumulative concentration-response relationship of bisulfate on evoked EPSCs 156 Fig. 10.4.4.2. Cumulative concentration-response relationship of bisulfate on evoked EPSCs 157 Fig. 10.5.1. Effects of reduced-hemoglobin and the control dithionite-dialyzed solution on evoked field EPSPs 161 Fig. 10.5.2. Mass spectroscopic analysis of contaminants in hemoglobin samples introduced by Martin's dialysis method 162 Fig. 10.6.1. Effects of ferrous chloride on evoked field EPSPs 164 Fig. 10.6.2. Effects of hemin on evoked field EPSPs 166 - XV -LIST OF TABLES Table Page Table 8.4. Compositions of perfusing medium 80 Table 8.5. Compositions of patch recording solutions 85 Table 10.1.7. Effects of Nco-nitro-L-arginine on the actions of Hb-1991 126 Table 10.4.5. Qualitative test for the presence of N H 4 + in Hb-1991 159 - xvi -LIST OF ABBREVIATIONS A C S F Artificial cerebrospinal fluid ALA delta-aminolevulinic acid AM PA alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid 4-AP 4-aminopyridine AHP Afterhyperpolarization AP3 2-amino-phosphonopropionic acid A P V 2-amino-phosphonovalerate ATP Adenosine triphosphate BAPTA K4-bis-(o-aminophenoxy)-A/,A/,A/'.A/'-tetraacetic acid CA Cornu ammonis cAMP Adenosine 3':5'-cyclic phosphate cGMP Guanosine 3':5'-cyclic phosphate C G P 35348 P-(3-aminopropyl)-P-diethoxymethyl-phosphinic acid CNS Central nervous system CNQX 6-cyano-7-nitroquinoxaline-2,3-dione CO Carbon monoxide DMSO Dimethylsulfoxide DNQX 6, 7-dinitroquinoxaline-2,3-dione DTX Dendrotoxin EGTA Ethylene Glycol-bis((3-aminoethyl Ether) E P SC Excitatory postsynaptic current E P S P Excitatory postsynaptic potential f EPSP Field EPSP GABA Gamma-aminobutyric acid GABA-T GABA transminase GAD Glutamic acid decarboxylase GIRK G-protein-mediated inward-rectifying potassium current GTP Guanosine triphosphate Hb Hemoglobin mGluR metabotropic glutamate receptor Hb-1991 Hemoglobin (Sigma; Cat# H2500 ; Lot# 101H9307) Hb-1996 Hemoglobin (Sigma; Cat# ; Lot#86H9310) HEPES A/-[2-hydroxyethyl]piperazine-N'-[2-ehtanesulfonic acid] HVA High-voltage activated ICP Intracranial pressure IPSP Inhibitory postsynaptic potential l(x) current of X iGluR ionotropic glutamate receptor L-AP4 L-2-amino-4-phosphonobutanoic acid LTP Long-term potentiation L-M Lacunosum-moleculare - X V I I -LVA Low-voltage activated mGluR metabotropic glutamate receptor MK-801 Dizolcipine NMDA /V-methyl-D-aspartate NO Nitric Oxide O/A Oriens/alveus PBG porphobilinogen Picro Picrotosinin PTX Pertussis toxin sensitive QX-314 Lidocaine N-ethyl bromide RBCs Red blood cells RMP Resting membrane potential SAH Subarachnoid hemorrhage S.E.M. Standard Error of the Mean SICH Spontaneous intracerebral hematoma STX Saxitoxin TEA Tetraethylammonium THIP 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride Trans-ACPD Trans-1-amino-1,3-cyclopentanedicarboxylic acid Tris 2-amino-2-(hyrdoxymethyl)-1,3-propanediol TTX Tetrodotoxin V Voltage - XV111 -LIST OF PHARMACOLOGICAL DRUG ACTIONS THIP GABA-A receptor agonist Picrotoxinin GABA-A receptor channel blocker Baclofen GABA-B receptor agonist C G P 35348 GABA-B receptor antagonist NMDA NMDA receptor agonist A P V NMDA receptor antagonist A M PA AMPA receptor agonist CNQX non-NMDA receptor antagonist DNQX non-NMDA receptor antagonist Nystatin Pore-forming antibiotic Dithionite Reducing agent Bisulfite Reducing agent - xix -ACKNOWLEDGEMENTS I would like to thank my supervisor Dr. Bhagavatula R. Sastry for his academic guidance and emotional support throughout this study. I am also grateful to my colleagues Dr. Zheng Xie, Dr. Wade Morishita and Dr. Joseph Ip, Mr. Trevor Shew, Dr. Mohammed Ouardouz, Mr. Newton Woo for their friendship, and hours of discussions over the work. I wish to thank all members of the Department of Pharmacology and Therapeutics for their academic guidance during my undergraduate and graduate years in this department. This project is funded by a grant to Dr. Sastry from the National Institute of Health. I would like to thank the Medical Research Council of Canada for their financial support in the last five years of my study. Finally, I would like to thank my parents and my sister for their unconditional support throughout these years. - XX -DEDICATION Dedicated to my mother and father 1 INTRODUCTION Head injuries, stroke and other cerebrovascular diseases are often associated with neurological complications such as dementia, paralysis and epilepsy (Gilman et al., 1992). To date most of the investigations related to stroke have been focused on the effect of ischemic/hypoxic conditions on central neurons; however, in hemorrhagic stroke or head injuries, extravascular pooling of blood in the surrounding brain tissue is known to occur (Barrows et al., 1955). In such cases, the intracranially pooled blood remains in the intracranial cavity for hours or days (Tourtelotte et al., 1964). Hemoglobin has been shown to be released from erythrocytes within hours after subarachnoid hemorrhage (Barrow et al., 1955; Tourtelotte et al., 1964). The hemoglobin released from hemolyzed blood remains in the intracranial cavity for a considerable period of time, and has been implicated in the prolonged cerebral vasospasm that is associated with subarachnoid hemorrhage (Miyaoka et al., 1976; Okwuasaba et al., 1981; Osaka et al., 1977, 1980). In addition, hematomas with high concentration of hemoglobin were reported to be present in patients with intraparenchymal hemorrhage (Kajikawa et al., 1979). As a result, in hemorrhagic stroke, neurons may be exposed to hemoglobin for prolonged periods of time. Moreover, hemoglobin is broken down to other components, which may have detrimental effects on neurons. Therefore, by examining the actions of hemoglobin and its breakdown products on central neurons, it is hoped that the role that hemoglobin plays in hemorrhagic stroke and traumatic head injury-induced neurological complications can be better understood. 2 ANATOMY OF THE HIPPOCAMPUS 2.1 Definition of Hippocampus Despite extensive studies, the terminology that is used to describe the various structures of the hippocampus remains confused and different definitions of the hippocampus exist. Some authors include the allocortical region (a term applied to cortical regions having fewer than six layers) or the three-layered regions as parts of the hippocampus. Therefore, in such usage, the hippocampal formation would be comprised of the dentate gyrus, the hippocampal proper and the subiculum. Others define the hippocampus as a region comprised of six cytoarchitectonically distinct regions, including the dentate gyrus, hippocampal proper, subiculum, presubiculum, parasubiculum and the entorhinal cortex. In this review, the former definition will be used and the structure of the dentate gyrus, hippocampal proper and the subiculum will be described. Moreover, as the hippocampus has been studied by many investigators, different names have been used to describe the same area. In this review, the terminology used by Ramon y Cajal, Blackstad and Lorente de No will be followed. 2.2 Topography of the hippocampal formation The rat hippocampi are situated bilaterally and symmetrically below the neocortex (Fig. 2 . 1 . ) . Each hippocampus lies along the wall of the descending horn of the lateral ventricle forming the medial margin of the cortical hemisphere. The hippocampus has a longitudinal axis running from the septal pole to the temporal pole known as the septo-temporal axis. The septo-temporal axis bends A TRANSVERSE SECTION SEPTAL Fig. 2.1. Orientation of the hippocampal formation. Line drawings showing the three-dimensional position of the hippocampal formation in the rat brain. In A, the hippocampus is shown in a transparent shell of the rat brain. Note that the hippocampus is C-shaped. In B, hippocampus without the transparent shell and the longitudinal axis, septal-temporal axis, of the hippocampus are indicated. Note that, the septal poles are rostrdorsally while the temporal ploes are caudoventrally. The transverse axis of the hippocampus is orentied perpendicular to the longitudinal axis. An example of the transverse section is shown in the inset. (Adapted from Amaral and Witter, 1995) from the septal nuclei rostro-dorsally to the incipent temporal lobe caudo-ventrally to give the hippocampus a C shaped appearance (Fig. 2.1.). Running perpendicular to the septo-temporal axis is the transverse axis. Transverse section of the hippocampus reveals 2 C-shaped fields interdigitating (Fig. 2.1. and 2.2). A number of terms used to describe the relative positions of the components in the transverse axis of a hippocampal field needs to be defined for the descriptions of the individual hippocampal formation components to be understood. The proximal and distal extremes of the hippocampal formation are defined as the dentate gyrus and the rhinal sulcus, respectively. In discussing the radial organization of a particular field, regions located closer to the pia or the hippocampal fissure are called superficial and the regions closer to the ventricle are called deep. 2.3 Stratification, Principle And Associated Cell Types Of The Hippocampal Formation In the following sections, the stratification, and the principal and associated cells found in the dentate gyrus, the hippocampal proper and the subiculum will be described (See. Fig. 2.2 and 2.3). 2.3.1 The dentate gyrus The dentate gyrus is divided into three layers: the molecular layer, the granule cell layer and the polymorphic layer. The molecular layer is a relatively cell-free layer closest to the hippocampal fissure (Fig. 2.2.). The granule cell -5 -Fig. 2.2. Organization of the hippocampal formation A three dimensional representation of a transverse section of the hippocampus is illustrated. Note that the major subfields and the intrinsic afferent pathways in a transverse hippocampal slices are shown, fim, fimbria; pp, perforant path; mf mossy fiber; sub, subiculum; Sch, Schaffer collaterals; com, commisural fibers; alv, alveus; DG, dentate gyrus; CA, comu ammonis; HF, hippocampal fissure. -6-alveus s. oriens s. pyramidale s. radiatum s. lacunosum/ moleculare s. polymorphe s. granulosum s. moleculare Fig. 2.3. Strata of the hippocampus, dentate granule cell and pyramidal neuron. In A, pyramidal neurons in the CA1 field is shown. Note that the various strata are illustrated. In B, a granule cells in the dentate gyrus is shown. Note the three strata are illustrated. layer lies deep in the molecular layer (Golgi, 1896). The granule cell and molecular layers form a V- or U-shaped structure. The granule cell layer that is adjacent to the Conus Ammonis 1 (CA1; see section 2.3.2. and Fig. 2.2. and 2.3.) is called, the suprapyramidal blade, and the portion that is opposite to the suprapyramidal blade is called the infrapyramidal blade (Blackstad et al., 1970; Ramon y Cajal, 1893). The region of the V or U that joins the two blades is called the crest (Teyler and DiSenna, 1984). The third layer is the polymorphic cell layer, synonymously known as the hilus. The granule cell is the principal cell type of the dentate gyrus and its cell bodies are densely packed in column stacks in the granule cell layer. The dentate granule cell has an elliptical cell body with a width of approximately 10 pm and a height of 18 pm (Golgi, 1896). The granule cell has a characteristic cone-shaped tree of spiny dendrites with all the branches directed towards the superficial portion of the molecular layer. Most of the distal tips of the dendritic tree end just at the hippocampal fissure or at the ventricular surface (Claiborne et al., 1990; Desmond and Levy, 1982). The axons of the granule cells, known as mossy fibers, extend through the polymorphic layer into the CA3 region of the hippocampal proper where they synapse with the CA3 pyramidal cells. In addition to the granule cells, many different types of interneurons that are immunoreactive for markers of GABA can be found in the dentate gyrus. These interneurons likely provide the inhibitory control of granule cell output. The pyramidally shaped GABA-ergic basket cells were found to be in the all three cell layers (Ramon y Cajal, 1911). In the molecular layer, at least two neuronal cell types are present. They include the multipolar or triangular cells (Hazlett and Farkas, 1978; Kosaka et al.; 1984, 1987; Ribak and Seress, 1983) and the "chandelier" or axo-axonic cell (Halasy and Somogyi; 1993; Kosaka, 1983; Somogyi et al., 1985; Soriano and Frotscher, 1989). The basket cells and the multipolar cells contribute to the basket plexus within the granule cell layer (Ramon y Cajal, 1911) while the chandelier cells terminate exclusively on the axon initial segments of the granule cells situated in the granule cell layer (Kosaka, 1983). In the polymorphic cell layer, the most common cell type is the mossy cell. The most distinctive feature of the mossy cell is that their proximal dendrites are covered by very large and complex spines called "thorny excrescencies" which are sites of termination of the mossy fiber axons from the granule cells (Frotscher et al., 1991; Ribak et al., 1985). Other cell types that are in the polymorphic layer are the fusiform-type cells, and the small, round or multipolar cells (Amaral, 1978). 2.3.2 The hippocampal proper The hippocampal proper can be clearly divided into two regions based on the differences in the size of the pyramidal cells, a large-celled proximal region and a small-celled distal region. The two regions have been named regio inferior and regio superior, respectively, by Ramon y Cajal (1911). Lorente de No (1934), however, has divided the hippocampal proper into three cornu ammonis (CA) fields, CA3, CA2 and CA1 (Fig. 2.2. and 2.3.). The CA3 and CA2 fields together are equivalent to the large-celled regio inferior and the CA1 is equivalent to the small-celled regio superior. This terminology is based not only on the size of the principal cells in the regions but also on clear-cut connectional difference. The terminology of Lorente de No has received more common usage; therefore, in this review, his CA3, CA2 and CA1 terminology will be used to describe the various fields of the hippocampal proper (Lorente de No, 1934). The lamellar organization is generally similar for all fields of the hippocampal proper (Fig. 2.2. and 2.3.). The CA3 field is divided into 6 strata: the alveus, the stratum oriens, the stratum pyramidale, the stratum lucidium, the stratum radiatum and the stratum lacunosum-moleculare (Kolliker, 1896; Lorente de No, 1934; Ramon y Cajal, 1911). The CA2 and the CA1 fields have essentially the same layers as those found in the CA3 field except for the s. lucidium which is missing; hence the CA2 and CA1 fields have only 5 layers (Fig. 2.2 and 2.3). In all three fields, the s. pyramidale is the principal cellular layer (Kolliker, 1896; Lorente de No, 1934; Ramon y Cajal, 1911). Lying deep to the s. pyramidale is a narrow, relatively cell-free layer called the s. oriens. The alveus is a fiber-containing layer (axon of the pyramidal neurons) that lies deep to the s. oriens. In the CA3 field, but not in the CA2 and CA1, the s. lucidium is a narrow acellular zone located superficial to the s. pyramidale where it is occupied by the mossy fiber axons originating from the dentate gyrus. Superficial to the s. lucidium in CA3 and lying superficially to the s. pyramidale in CA2 and CA1 is the s. radiatum. The s. radiatum is defined as the suprapyramidal region in which CA3 to CA3 associational connections and CA3 to CA1 Schaffer collateral connections are located (Fig. 2.2. and 2.3). The most superficial portion of the - 10-hippocampus is the s. lacunosum-moleculare where the perforant pathway fibers from the entorhinal cortex travel and terminate. The pyramidal cell, which makes up the vast majority of neurons in the pyramidal-cell layer, is the principal neuronal-cell type of the hippocampal proper. The cell bodies of these neurons have a diameter ranging from 25 to 40 u.m (Golgi, 1896). The cell soma is oriented so that the base of the triangular-shaped cell faces towards the s. oriens while the apical portion of the cell faces the s. lucidium (CA3) / s. radiatum (CA1) (See Fig. 2.2 and 2.3). The basal dendrites extend into the s. oriens while the apical dendrites extend to the hippocampal fissure. The length of the dendritic tree of pyramidal cells in the CA3 field is on the order of 16 mm whereas the length of the dendritic tree of pyramidal neurons in the CA1 field is on the order of 13 mm. In all the fields, a substantial portion of the dendritic tree is located in the s. oriens (Amaral et al., 1990). In addition to pyramidal neurons, different types of interneurons have been reported to exist in the various strata of the hippocampal proper. Basket cells are one of the most well-studied groups of interneurons in the hippocampal proper (Lorente de No, 1934; Ramon y Cajal, 1893; Seress and Ribak, 1984). The somata of the basket cells vary in shape from spherical to triangular and are on the average of 30 to 50 pirn in size. The basket cells lie very close to the pyramidal-cell layer. The basket cells predominately innervate the perisomatic region of the pyramidal cells. Another group of interneurons called the chandelier or axo-axonic cells can be found in the hippocampal proper. The cell bodies of the chandelier cells are situated within or immediately adjacent to the pyramidal-cell layer (Buhl et al., 1994). The main axonal branches of axo-axonic cells give rise to collaterals descending into the s. pyramidale, where they form characteristic bouton rows climbing on axon initial segments. The lacunosum-moleculare (L-M) interneurons are another group of neurons that were first described by Ramon y Cajal and Lorente de No (Kawaguchi and Hama, 1987; Kunkel et al., 1988; Lorente de No, 1934; Lacaille and Schwartzkroin, 1988a, 1988b; Ramon y Cajal, 1893, 1911). These neurons are located in the s. lacunosum-moleculare or at the border of the s. radiatum. The somata of the L-M interneurons are fusiform in shape and are 15 to 25 u.m in size. The axon originates from the soma or from the main proximal dendrites. Similar to the dendrite, the axon collaterals are also horizontally orientated. The axon collaterals arborize predominantly in s. lacunosum-moleculare or in the bordering region of s. radiatum. Other interneurons that are found in the hippocampal proper include the bistratified and horizontal trilaminar cells found within or near to the s. pyramidale or at the s. or/ens-alveus border (Buhl et al., 1994; Sik et al., 1995) and the O/A interneurons found localized at the s. oriens I alveus (O/A) border (Lacaille et al., 1987). Most of these interneurons are stained for glutamic acid decarboxylase (GAD) and are thought to be GABA-ergic neurons (Ribak, 1978). However, many of these neurons also expressed other neuropeptides, and it may help to further differentiate these interneurons by examining the peptide co-transmitter that they express. For instance, many of the O-LM neurons have somatostatin, - 12-but some are stain positive for neuropeptide Y. For more details of the interneurons of the hippocampus, please refer to an extensive review by Freund and Busaki (1996). 2.3.3 The subiculum The subiculum is considered to constitute the major output structure of the hippocampus. It is located distal to the CA1 field of the hippocampus and the CA1/subicular border is marked by an abrupt widening of the pyramidal-cell layer (Fig.2). Similar to the dentate and the hippocampal proper, the subiculum is considered to be an allocortex, a three-layered structure (Blackstad, 1956; Haug, 1976; Swanson et al., 1987). The s. radiatum and the s. oriens of the CA1 are not present in the subiculum. Instead, a wide molecular layer is present and can be subdivided into a deep portion that is continuous with the s. radiatum of CA1 and a superficial portion that is continuous with the molecular layer of the presubiculum and CA1. The principal cell layer in the subiculum is beneath the distal end of CA1 and continues in that position deep to Layer II of the presubiculum (Lorente de No, 1934; Ramon y Cajal, 1911). The cell layer is populated by pyramidal neurons that are relatively uniform in shape and size. Their apical and their basal dendrites extend into the molecular layer and the deeper portions of the pyramidal-cell layer, respectively. Many smaller neurons, presumably interneurons, also exist in the cell layer of the subiculum (See Swanson et al. (1987) for more details). - 13 -2.4 Connections In the following paragraphs, the intrahippocampal circuitry, the interhippocampal projections and the local inhibitory circuits will be described. 2.4.1 Intrahippocampal circuitry The circuitry of the hippocampus is very unique in that most of the connections are unidirectional. The major cortical projections are derived from the entorhinal cortex and enter the hippocampus via the perforant path (Blackstad, 1956; Lorente de No, 1933; Raisman et al., 1965; Ramon y Cajal, 1893). Cells of Layer II and III of the entorhinal cortex are considered to be the principal source of fibers for the perforant pathway. The perforant path terminates in all subdivisions of the hippocampal formation. The fibers, after leaving the entorhinal cortex, enter the underlying white matter and the angular bundle. They then transverse the pyramidal-cell layer of the subiculum and cross the hippocampal fissure to enter the dentate gyrus or be distributed to the molecular layer of the subiculum and the hippocampal proper (Blackstad, 1956; Hjorth-Dimonsen and Jeune, 1972). In the dentate gyrus, collateral of the perforant path from the entorhinal cortex predominately terminates on the dendrites of the granule cells. In turn, these granule cells give rise to a distinctive unmyelinated axon called the mossy fiber (Claiborne et al., 1986; Ramon y Cajal, 1911). The mossy fiber projects to the CA3 field of the hippocampus with few collateral. The presynaptic varicosities characteristic of the mossy fiber/CA3 cell contacts are distributed at 140 pm intervals along the course of the mossy fiber axons (Claiborne et al., - 14-1986). All mossy fibers extend throughout the full transverse extent of the CA3 field and terminate at the CA3/CA2 border. The mossy fibers travel both deeply and superficially to the pyramidal-cell layer, and its projections to the CA3 field of the hippocampus have a relatively lamellar trajectory (Gaarskjaer, 1978a, 1978b; Swanson et al., 1978). The CA3 hippocampal neurons give rise to highly collateralized axons called the Schaffer collaterals that project to the CA1 field. All portions of CA3 and CA2 project both septally and temporally to the CA1 field, but the distribution of terminations in CA1 depends on the transverse location of the CA3/CA2 cells of origin (Ishizuka et al., 1990). The CA1 continues this unidirectional synaptic projection by projecting axon collateral to the subiculum (Amaral et al., 1991; Finch and Babb, 1981; Finch et al., 1983; Tamamaki et al., 1988). Axons of the CA1 field descend into the s. oriens of the alveus and bend sharply toward the subiculum. The axon collaterals reenter the pyramidal-cell layer of the subiculum and ramify in the pyramidal-cell layer and in the deep portion of the molecular layer. This CA1 projection ends in a highly topographic columnar fashion in the subiculum. In addition, CA1 pyramidal cells also send their axons to neurons in the layer III of the entorhinal cortex. The subiculum projects to the pre- and para-subiculum as well as to the entorhinal cortex. The subiculum is the area where most of the efferent fibers leave the hippocampus. 2.4.2 Lamellar concept and longitudinal pathway The lamellar concept of hippocampal information flow that was developed in the early 1970s suggests that the major excitatory pathways of the - 15 -hippocampal formation were all oriented perpendicular to the long axis of the structure and had a restricted septo-temporal spread (Anderson et al., 1971). This view suggests that the hippocampal formation is comprised of a number of similarly organized but connectionally isolated slices stacked along the long axis. However, the lamellar concept of information flow in the hippocampus is not compatible with the information obtained from recent neuroanatomical data. As described earlier, many of the intrinsic hippocampal connections are extensively distributed in both the transverse axis and the septo-temporal axis (in CA3 to CA1 and in CA1 to the subiculum). The significance of the extensive distribution of the connections leads to a view that is more consistent with the neuroanatomical data, suggesting that the hippocampal formation can be treated as a series of three-dimensional networks of connections (For more details, please refer to a review by Amaral and Witter, 1989). 2.4.3 Extrinsic afferent connections In the hippocampus, extrinsic afferents can be divided into two groups, those that form between the cortical regions and those that form between the subcortical regions. While most of the cortical inputs originate from the entorhinal cortex via the perforant path, subcortical inputs are more prominent and are derived from a number of regions in the brain. For further details please refer to Lopes da Silva et al., (1990), Amaral and Witter (1989), and Amaral and Witter (1995). - 16-2.4.4 Extrinsic efferent connections The majority of the cortical efferents are projected from the subiculum (Amaral and Witter, 1995). As for the subcortical efferents, CA1 cells and subiculum forms the majority of the efferent pathways. The fiber systems that innervate subcortical and cortical regions from the hippocampus usually originate from the fornix-fimbria system formed by myelinated afferent and efferent fiber bundles originating from the ventricular surfaces, i.e., the "alveus", of the subiculum and the hippocampus. For more details of the efferent systems of the hippocampus, please refer to Amaral and Witter (1995) and Swanson et al., (1977 and 1987). - 17-3 ELECTROPHYSIOLOGICAL PROPERTIES OF HIPPOCAMPAL PYRAMIDAL NEURON 3.1 Characteristic Of Hippocampal Pyramidal Neuron The passive membrane properties of the hippocampal principal neurons (CA1, CA3 pyramidal neurons and the dentate granule cells) have been studied in great detail with the use of hippocampal slice preparation (Brown et al., 1981; Durand et al., 1983; Johnston, 1981; Spruston and Johnston, 1992; Spruston et al., 1994; Turner and Schwartzkroin, 1983). Prior to the application of whole cell patch clamp techniques, membrane properties of hippocampal neurons were primarily studied using microelectrodes. Using this recording technique, it was found that the resting membrane potentials of hippocampal pyramidal and granule cells range from -50 to -70 and -55 to -80 mV, respectively (Brown et al., 1981; Durand et al., 1983; Johnston, 1981; Turner and Schwartzkroin, 1983). The input resistances, calculated from the slope resistance within the linear range of the current-voltage curves, are 20 to 45 MQ and 40 to 60 MQ for pyramidal neurons and dentate granule cells, respectively. The membrane time constants are on average 12 to 16 ms for CA1 pyramidal neurons, 25 to 35 ms for CA3 pyramidal neurons and 9 to 11 ms for granule cells (Brown et al., 1981; Durand et al., 1983; Johnston, 1981; Turner and Schwartzkroin, 1983). With the application of patch clamp (whole cell and nystatin perforated) technique on slice preparation the resting membrane potential were observed to be similar to that recorded using conventional microelectrodes; however, the input resistance and the membrane time constant determined at resting membrane potential using patch clamp techniques are larger than those recorded using conventional microelectrodes (Edwards et al., 1989; Spruston and Johnston, 1992; Spruston et al., 1994). In nystatin perforated patch technique, the input resistances were found to be 104 MQ for CA1 pyramidal neurons, 135 MQ for CA3 pyramidal neurons and 446 MQ for dentate granule neurons (Spruston and Johnston, 1992). In addition, the membrane time constants were found to be 28 ms for CA1 neurons, 66 ms for CA3 neurons and 43 ms for dentate granule cells. The differences observed between perforated patch and microelectrodes can be accounted for by the reduction of a somatic leak in the patch clamp recording technique (Spruston and Johnston, 1992). 3.2 Membrane Ionic Currents Of Hippocampal Neurons The hippocampal pyramidal neurons have many different types of ionic currents. Much work has been done to investigate the biophysical and functional properties of these currents. It is beyond the scope of this thesis to review all the known currents in the hippocampal pyramidal neurons. The following paragraphs will briefly review the Na + , C a 2 + , and K + currents. For further details please refer to the reviews by Halliwell et al (1990) and Brown (1990). 3.2.1 Sodium current (lNa) Two types of sodium currents have been recorded from the hippocampal pyramidal neurons: the fast currents iNa(fast) and the slowly inactivating current lNa(slow)/(persistent). - 19-3.2.1.1 Fast sodium current (iNa(fast)) The iNa(fast) is observed in both the soma and the dendrites of the hippocampal neurons (Benardo et al., 1982; Huguenard, et al., 1989; Johnston et al., 1996; Sah et al., 1988a; Spruston et al., 1995b). In the soma, the iNa(fast) has an activation threshold of -60 mV and a time to peak of about 0.9 ms at 0 mV. Inactivation was complete throughout the activation range (Kaneda et al., 1988; Sah et al., 1988b). This iNa(fast) is responsible for the fast TTX-sensitive somatic action potentials (Benardo et al., 1982). In the dendrites, iNa(fast) has been shown to be important in the dendritic backpropagating action potentials (Spruston et al., 1995b). The dendritic Na + channel is believed to amplify EPSPs and may play a role in long-term potentiation (LTP) (Magee and Johnston, 1997). Both the somatic and dendritic iNa(fast) are sensitive to extracellular application of TTX, STX and intracellular QX-314 (Brown et al., 1990; Spruston et al., 1995b). 3.2.1.2 Persistent sodium current (lNa(P)) In addition to the iNa(fast), another Na + current with slow inactivation kinetics has been described in the hippocampal neurons (French and Gage, 1985). This current has been termed the slowly inactivating current, iNa(siow), or persistent current, lNa(P)- This current has an activation threshold of about 5 to 10 mV positive to the resting potential. The lN a(P) is much more resistant to inactivation than the iNa(fast), sustaining during a 400 ms depolarizing pulse, and it can be recorded at > 5 0 % of its normal amplitude when the transient current is completely inactivated (French and Gage, 1985). The pharmacology of lNa(P) is similar to that of the iNa(fast). It is blocked by extracellular TTX or intracellular QX-- 2 0 -314. l N A ( P ) can be found in both the soma and the dendrites of CA1 pyramidal neurons (Spruston et al., 1995b). Physiologically, it is believed that the IN 3(P) plays an important role in the repetitive firing of action potentials and may act as a 'pacemaker' current for the hippocampal activity (Brown, 1990; Crill, 1996). 3.2.2 Calcium current (lCa) Hippocampal neurons have been shown to exhibit two types of voltage gated C a 2 + currents: low-voltage activated (LVA) and high-voltage activated (HVA). The LVA calcium currents are mainly due to the activation of T-type calcium channels. On the other hand, the HVA calcium currents are mainly due to the activation of at least 5 types of C a 2 + channels, namely L, N, P, and Q. These C a 2 + channels are classified based on the differences in the biophysical properties of the currents they carry as well as by their sensitivity to various channel blockers (Tsien et al., 1988; Birnbaumeret al., 1994). In this section, the properties of the C a 2 + currents will be described. 3.2.2.1 Low-voltage activated Ca2+ currents The low threshold, transient, inward whole-cell current recorded in hippocampal neurons is called the T-type current. Other synonyms, including low-voltage activated current, low threshold-inactivating current or type I current, have also been used in the literature. The threshold membrane potential for activation of the T-type current is near membrane potential, -50 to -60 mV, and it reaches full activation at about -30 mV (Brown et al., 1990; Takahashi et al., 1991; Magee and Johnston, 1995; Huguenard, 1996). The inactivation is highly potential-dependent and this current requires extremely negative holding -21 -potentials to be completely reprimed (Kay and Wong, 1981; Mogul and Fox, 1991). The inactivation of T-type C a 2 + currents is normally complete at voltages (-100 to -40 mV) that are subthreshold for HVA activation. The time-constant of inactivation is approximately 20 ms to 50 ms at -40 mV (Ozawa et al., 1989; Takahashi et al., 1991; Magee and Johnston, 1995). Many investigators have used a low concentration of nickel to block T-type C a 2 + currents; however, the selectivity of nickel ions is relatively poor. The current is also weakly sensitive to dihydropyridine C a 2 + channel antagonists such as nicardipine; however, it is resistant to co-conotoxin and co-Aga-IVA (Randall and Tsien, 1997; Randall, 1998; Docherty and Brown, 1986b; Gahwiler and Brown, 1987a; Takahashi and Akaike, 1991). The T-type current plays an important role in many different cellular functions, such as synaptic integration, active properties of dendrites, spontaneous depolarization, generation of low-threshold spikes that can lead to burst firing and pacemaker (oscillatory) behavior (Tsakiridou etal., 1995; Huguenard, 1996). 3.2.2.2 High-voltage activated Ca2+currents Traditionally, high-voltage activated currents recorded in the hippocampus are divided into two categories: high-threshold, non-inactivating (L) and high-threshold inactivating (N). However, with the development of new selective antagonists, the high-voltage activated current can be divided into at least 4 different currents: L, N, P, and Q. -22-3.2.2.2.1 High voltage activated, non-inactivating (L) current The L-type C a 2 + current is a high-threshold, long-lasting whole-cell inward current. The current activates at potentials between -20 mV and -10 mV and inactivates at potentials between -20 mV and -60 mV (Kay and Wong, 1987; Ozawa et al., 1989; Mogul and Fox, 1991). This current inactivates very slowly with a time constant > 500 ms at 0 mV (Ozawa et al., 1989). The L-type current can be enhanced by B a 2 + and blocked by C a 2 + , C d 2 + and N i 2 + (Fisher et al., 1990; Mogul and Fox, 1991). The L-type current can be blocked by verapamil and by dihydropyridine C a 2 + antagonists, such as nimodipine and nifedipine, and can be enhanced by " C a 2 + agonist" Bay K 8644 (Brown et al., 1990; Docherty and Brown, 1986a; Segal and Barker, 1986; Gahwilerand Brown, 1987a). 3.2.2.2.2 High-voltage activated inactivating (N) current The N-type C a 2 + current is a high-threshold current that inactivates much faster than the L-type current. The N-type current has a threshold membrane potential for activation of -20 mV and inactivates between the potential of -30 mV to 110 mV (Ozawa et al., 1989; Takahashi et al., 1989). The time constant for inactivation of the N-type current is approximately 40 ms at 0 mV (Ozawa et al., 1989). The N-type C a 2 + current can be enhanced by B a 2 + and blocked by C d 2 + and N i 2 + (Brown et al, 1990). The current is also specifically blocked by co-conotoxin GVIA (Reynolds et al., 1986; Olivera et al., 1984; Cruz and Olivera, 1986). The N-type C a 2 + channel plays an important role in mediating synaptic transmission of the CA3-CA1 synapse in the hippocampal slice preparation (Wheeler etal., 1994, 1996; Ponceretal., 1997; Reidetal., 1997, 1998). -23 -3.2.2.2.3 Other high voltage activated C a 2 + currents The P-type C a 2 + current, originally found in Purkinje neurons, has also been reported to exist in the hippocampal CA1 neurons (Mintz et al., 1992a, 1992b). The P-type current has a high-activation voltage of -20 mV or more positive with little inactivation. This current is enhanced by B a 2 + and blocked by C d 2 + and Co 2 + . Another type of HVA C a 2 + current that is very similar to the P-type current is the Q-type C a 2 + current. Both the P- and the Q-type currents are sensitive to co-Aga IVA, but a higher concentration is required to block the Q-type current (Randall and Tsien, 1995; Wheeler et al., 1994). The Q-type C a 2 + current has been shown to play an important role in glutamatergic synaptic transmission in the hippocampus (Wheeler et al., 1994, 1995, 1996). 3.2.3 Potassium currents At least 7 types of potassium currents have been reported to exist in hippocampal CA1 neurons. Four of these potassium currents ( l A , ID, IK, and l M) are voltage-dependent and can be activated by depolarization. IK(IR> is activated by hyperpolarization. The other two are the Ca 2 +-dependent I A H P and lc. The biophysical properties of these potassium currents will be discussed in the following sections. 3.2.3.1 The fast transient hC current (lA) IA is a prominent current that can be recorded in hippocampal neurons (Segal and Barker, 1984a; Segal et al., 1984). IA is activated by depolarization beyond -60 mV, and it inactivates between -60 and -40 mV. IA activates and -24-inactivates with fast kinetics, and it also recovers quickly from inactivation with time constants in milliseconds range. The current is blocked by 4-amino-pyridine (4-AP) and dendrotoxin (DTX), but is resistant to tetraethylammonium (TEA) (Segal et al., 1984; Segal and Barker, 1984a; Numann et al., 1987; Storm, 1988a, 1990; Dolly et al., 1984; Halliwell et al., 1986; Gustafsson et al., 1982). IA plays an important role in delaying the onset of the discharge of the action potentials in response to depolarizing stimuli (for up to 100 ms) and hence permits slow repetitive firing (Gustafsson et al., 1982; Segal et al., 1984; Storm, 1984, 1988a). Moreover, l A has also been shown to be involved in early phase of the repolarization following action potentials (Storm, 1987a). 3.2.3.2 The delayed current (lD) l D is found to coexist with l A in the CA1 pyramidal neurons. ID differs from l A in its threshold for activation and inactivation, its inactivation kinetics, and its sensitivity to 4-AP (Storm, 1988a). I D is activated by depolarization beyond -70 mV, and it inactivates between -120 and -60 mV (Storm, 1988a). ID activates with fast kinetics having time constant typically of 20 ms; however, the kinetics of and the recovery from inactivation are very slow with time constants being over several seconds. ID is highly sensitive to 4-AP (30 u,M) and DTX but is resistant to TEA, B a 2 + and C s + (Buckle and Haas, 1982; Galvan et al, 1982; Rutecki et al., 1987; Benoit and Dubois, 1986). Due to its fast activation and slow inactivation kinetics, ID is believed to play an important role in providing the long delay in the onset of firing of action potentials in response to long-lasting depolarizing stimuli (Storm, 1990; Brown, 1990). In addition, due to its slow recovery from -25-inactivation, recruitment of depolarizing inputs over several seconds can take place; therefore, the response of the cell reflects the synaptic input it received in the preceeding seconds. 3.2.3.3 The delayed rectifier current (IK) I k is activated by depolarization beyond -40 mV at a relatively slow rate with time constant between 50 to 200 ms, at 0 mV (Numann et al., 1987; Segal and Barker, 1984a). The current inactivates over several seconds (time constants between 3 to 5 sec) and recovers from inactivation with a time constant of about 600 ms at -110 mV (Storm, 1987a). This current is blocked by high concentrations of external TEA, is slightly affected by 4-AP and is resistant to external C s + (Storm, 1990). Although this current activates slowly, it may contribute to the repolarization phase of action potentials (Storm, 1988a). In addition, it may cause brief AHP after a single spike but not following multiple spikes (Storm, 1989). 3.2.3.4 The M-current (lM) The M-current is a voltage dependent, non-inactivating current (Brown and Adams, 1980; Halliwell and Adams, 1982; Owen et al., 1990; Brown, 1988; Storm, 1989; Madison et al., 1987; Brown and Griffith, 1983). It is activated relatively slowly by depolarization beyond -70 mV (time constant of about 50 ms), and it does not inactivate (Adams et al, 1982). IM is sensitive to TEA and Ba 2 + , but it is resistant to C s + and 4-AP (Halliwell and Adams, 1982). IM underlies an earlier phase of spike frequency adaptation, and it seems to be involved in the medium after-hyperpolarization (mAHP) which follows a single spike or a spike -26-burst (Madison and Nicoll, 1984; Storm, 1989). IM can be modulated by a number of neurotransmitter substances. For instance, activation of M 2 receptor inhibits IM while somatostatin enhances IM (Dutar and Nicoll, 1988a; Moore et al., 1988; Watson and Pittman, 1988). 3.2.3.5 The fast inward rectifier hC current (IK(IR)) The l K(iR) is mainly activated by hyperpolarization (Owen, 1987). It is partially activated at resting potential (-60 mV) and peaks within 5 ms. It inactivates at potentials more negative than -100mV (time constant 35 ms at -115 mV). The current can be blocked by B a 2 + and C s + (Owen, 1987). A number of neurotransmitters, including GABA, 5-HT, and adenosine, have been shown to activate a G-protein-mediated inward-rectifying potassium current (GIRK) (Gahwiler and Brown, 1985; Newberry and Nicoll, 1985; Andrade et al., 1986; Luscher et al., 1997). The kinetics of this type of current have not been studied extensively; whether this channel is the same as K|R and whether these channels are activated in the absence of neurotransmitters require further investigation. 3.2.3.6 The fast Ca2'-dependent hC current (lc) l c is similar to other fast Ca 2 +-dependent K + currents recorded in various central neurons. It is formed by both C a 2 + and voltage-dependent non-inactivating K + currents (Marty, 1981; Barrett et al., 1982; Adams et al., 1982). Ic is activated by depolarization beyond -40 mV. It can be eliminated by Ca 2 + -free medium, by adding Ca 2 +-channel blockers into the perfusing medium or by injection of the fast C a 2 + chelator, BAPTA, into the cell (Storm, 1985, 1987a; Lancaster et al, 1991; Lancaster and Nicoll, 1987; Lancaster and Adams, 1986; -27-Madison et al, 1987; Storm, 1989). I C is also sensitive to K + channel blockers, external TEA and charybdotoxin (CTX) (Lancaster and Nicoll, 1987). Ic plays an important role in spike repolarization and is responsible for the fast and medium AHP (Storm, 1989, 1985; Lancaster and Nicoll, 1987). 3.2.3.7 The slow Ca2*-dependent hC current (IAHP) The IAHP in the hippocampal neurons are classified into two groups: I A H P and S IAHP - The S IAHP is not sensitive to apamin and has a slow activation and decay time lasting for seconds (Lancaster and Adams, 1986; Sah and Issacson, 1995; Sah, 1996; Stocker et al., 1999). The IAHP is sensitive to apamin and has a fast decay time (time constant in 90 ms) (Stocker et al., 1999). Both S IAHP and IAPH are Ca 2 +-dependent non-inactivating currents (Sah and Issacson, 1995; Sah 1996). In contrast to Ic, these two currents are not voltage-dependent and are not sensitive to TEA or 4-AP (Lancaster and Adams, 1986; Stocker et al., 1999). IAHP and S IAHP are clearly dependent on influx of C a 2 + ions as they are eliminated by Ca 2 + -free medium or by Ca 2 +-channel blockers (Lancaster and Adams, 1986; Stocker et al., 1999). The IAHP is thought to be responsible for the generation of the medium AHP which follows spike bursts, and is mainly responsible for the strong spike-frequency adaptation which is typical of pyramidal neurons (Madison and Nicoll, 1982, 1984; Lancaster and Nicoll, 1987; Stocker, 1999). The S IAHP is thought to generate the slow AHP which lasts for several seconds (Lancaster and Adams, 1986). - 2 8 -3.2.3.8 Leak currents Leak currents are passive currents that remain active around resting membrane potential when voltage and Ca 2 +-gated currents are suppressed. The current-voltage curve of leak currents negative to -50 mV is linear with a slope conductance of under 10 nS. One of the conductances that underlies this current is a voltage-insensitive K + conductance which is decreased by muscarinic agonists and somatostatin. These effects are probably mediated by a pertussis-toxin-insensitive GTP-binding protein. In addition to the K + conductance, several chloride conductances are also believed to be involved in the generation of leak currents (Brown, 1990). Franciolini and Petris (1988) described a background Cl" conductance with 62 pS in symmetrical 150 NaCl. This conductance is active at voltages between -80 and +80 mV, and its activity is independent of intracellular C a 2 + concentration. More studies have to be done to characterize the degree of involvement of these channels in the resting conductance of the cells. -29-4 EXCITATORY POSTSYNAPTIC POTENTIALS IN HIPPOCAMPAL CA1 PYRAMIDAL NEURONS In hippocampal region, low-frequency stimulation of the Schaffer Collateral pathway produces dual components: fast and slow EPSP/Cs in CA1 pyramidal neurons. The fast component is mediated by the activation of AMPA receptors while the activation of the NMDA receptor mediates the slow component of the evoked EPSP/Cs. In the following sections, the properties of the glutamate receptors will be reviewed, and their role in synaptic transmission will be discussed. 4.1 Excitatory Amino Acid Receptor In The Hippocampus Excitatory amino-acid receptors have been generally classified into two categories: 1) the ionotropic (iGluR) and 2) the metabotropic (mGluR) glutamate receptors. Based on in vitro differences in the electrophysiological as well as the pharmacological properties, the iGluRs are further divided into 3 subtypes: the NMDA, the AMPA and the kainate receptors (For review please refer to Mayer and Westbrook, 1987; Michaelis, 1998; Ozawa et al., 1998). On the other hand, based on the homology of the amino-acid sequence of the cloned mGluR 1-8, the mGluRs are subdivided into 3 groups: Group I, II and III (For review of this topic please refer to Conn and Pin, 1997). 4.1.1 Electrophysiological properties of NMDA receptor NMDA receptor is the best-studied glutamate receptor among all the subtypes. The receptor is a ligand-gated, voltage-dependent cationic channel. -30 -Activation of the NMDA receptor leads to the openings of channels that are permeable to N a \ K + and C a 2 + . Single-channel studies of hippocampal neurons have shown that the NMDA channels have a main-channel conductance between 40 to 50 pS (Ascher and Nowak, 1988; Jahr and Stevens, 1987; Nowak et al., 1984). The l-V relationship of the NMDA channel is highly non-linear (Nowak et al., 1984; Mayer et al., 1984). The maximum inward current through the NMDA receptor channel occurs at about -30 mV, and the current reverses at 0 mV. At membrane potential negative to -40 mV, little inward current flows through the channel. This voltage dependency is due to a M g 2 + block of the channel at membrane potential that is negative to -40 mV (Ascher and Nowak, 1988; Mayer et al., 1984; Mori et al., 1992; Nowak et al., 1984). The removal of M g 2 + from the perfusing medium produces l-V relationship that is linear throughout the membrane potential range of +30 to -100 mV. This voltage dependent M g 2 + block plays an important role as a coincident detector in the induction of LTP (Bliss and Collingridge, 1993). Another interesting characteristic of the NMDA receptor is that both glutamate and glycine are required to activate the receptor (Johnson and Ascher, 1987). Johnson and Ascher were first to show that the response of the NMDA receptor to glutamate is potentiated by glycine. Later, it became apparent that glycine is essential for the activation of the NMDA receptor and is, therefore, described as a co-agonist with glutamate (Klenkner and Dingledine, 1988). Through the use of concentration-response curves for glycine and glutamate, it was found that effective activation of the receptor requires the binding of at least -31 -two glutamate and two glycine molecules (Benveniste et al., 1990; Benveniste and Mayer, 1992; Clements and Westbrook, 1994; Patneau and Mayer, 1990). The binding of the co-agonist glutamate and glycine is likely to act at separate sites on the NMDA-receptor channel complex. However, it is generally believed that these sites are allosterically coupled with negative cooperativity. Desensitization of the NMDA receptor has been shown to exist in three different modes: 1) the Ca 2 +-dependent, 2) glycine-dependent, and 3) C a 2 + - and glycine- independent modes (Mayer et al., 1990). The Ca 2 +-dependent desensitization (or inactivation) requires the influx of C a 2 + , suggesting an intracellular site of action for this effect of C a 2 + (Legendre et al., 1993; Rosenmund and Westbrook, 1993 a and b). In addition, the time course of such inactivation is extremely slow and requires activation of the NMDA receptor over a period of several seconds (Legendre et al., 1993). On the other hand, the glycine-dependent desensitization is not truly desensitization but is rather due to the negative allosteric interaction of glutamate and glycine (Vylicky et al., 1990). When glutamate binds onto the NMDA receptors, it triggers the dissociation of glycine from its binding sites. This form of desensitization can be reversed by applying a sufficiently high concentration of glycine in the perfusing medium. The third kind of desensitization is independent of C a 2 + or glycine and is believed to be comparable to the desensitization seen in other receptor systems (Vylicky et al., 1990; Mayer etal., 1989). Glutamate is a potent agonist at the NMDA receptors with an EC 5 o of 2.3 u,M (Patneau and Mayer, 1990). Other agonists include L-aspartate and the -32-sulfur-containing amino acids analogues, such as cysteic acid, homocysteic acid and cysteine sulfinic acids (Collingridge and Lester, 1989; Watkins and Evans, 1981; Watkins et al., 1990). NMDA is a specific agonist at this receptor. Competitive antagonists commonly used in electrophysiological studies include A P V and C P P (Collingridge and Lester, 1989). In addition to the competitive antagonist at the glutamate binding sites, competitive antagonists at the glycine site, such as 5,7, dichlorokynurenic acid, HA-966, have also been developed. Non-competitive antagonists which act via a channel-blocking mechanism include MK-801, ketamine, and PCP (Ozawa, 1998). This class of antagonist provides a used-dependent blockade and has been shown to be effective as neuroprotective agents in vivo. The NMDA receptor function can be allosterically modulated by a number of endogenous substances such as proton, redox molecules, and polyamines. In addition, phosphorylation and dephosphorylation have been shown to modulate the NMDA receptor channel activity (Please refer to MacDonald et al., 1998). 4.1.2 Electrophysiological properties of the Non-NMDA receptor Traditionally, due to the lack of specific receptor antagonists, the AMPA and the kainate receptors have generally been termed as the non-NMDA receptors (Feldmeyer and Cull-Candy, 1994; Sommer and Seeberg, 1992). Two ways have been used to distinguish the AMPA and kainate receptors mediated responses from one another. One way is by comparing the size and the shape of the response to prolonged application of varying concentrations of AMPA and -33 -kainate. AMPA, being a potent agonist for the AMPA receptors, produces rapid and strong desensitization responses at low concentration via the activation of AMPA receptors. At higher concentration, it may produce a very small response via the activation of the kainate receptors (Sommer et al, 1990). On the other hand, kainate at low concentration activates kainate receptors to produce responses that show strong desensitization, while at higher concentration, kainate activates the AMPA receptors to produce a response with rapid but modest desensitization (Patneau et al, 1993). The second method that is used to distinguish the two currents is to the use of drugs that selectively reduce receptor desensitization and potentiate responses either at the AMPA or the kainate receptors. Pharmacological agent, such as cyclothiazide or aniracetam, selectively block AMPA-receptor desensitization whereas agents like concanavalin A (Con A) preferentially block the kainate-receptor desensitization (Partin etal., 1993). Activation of non-NMDA receptors results in an influx of Na + and K+. These receptors differ from that of NMDA receptors in their low permeability of C a 2 + (except recently it has been shown that a lack of G I U R B subunit infers C a 2 + permeability (Jonas and Burnashev, 1995; Ozawa and Lino, 1988)). The l-V relationship for the non-NMDA receptor-mediated current is linear and reverses at approximately 0 mV. The main single-channel conductance of the AMPA receptor channel is 8 to 10 pS while the kainate receptor channel has a single-channel conductance of approximately 4 pS (Jonas and Sakmann, 1992; Spruston et al., 1995). Due to their low single-conductances, detailed analysis of -34-their single channel properties has not been studied extensively. However, many macroscopic properties of non-NMDA receptors are well characterized. Recently, study of the activation kinetics of the AMPA receptor channels suggests that at least two glutamate molecules have to be bonded to the receptor in order to activate it efficiently (Clements et al., 1998). The affinity of glutamate for non-NMDA receptors is low compared to that of NMDA receptors, having a E C 5 0 of 0.5 mM (Patneau and Mayer, 1990; Trussell et al., 1988). The rate of deactivation and desensitization of the non-NMDA receptors is fast. For instance, in the hippocampal pyramidal neuron, the rate of deactivation and desensitization ranges from 2.3 to 3 ms and 9.3 to 11.3 ms respectively (Colquhoun et al., 1992; Jonas and Sakmann, 1992; Tang et al., 1991). AMPA is the most selective agonist for the AMPA receptors. Other potent agonists at the AMPA receptors include willardine and p-N-oxalyl-amino-L-alanine (BOAA), both of which are naturally occurring compounds, while, at low concentration, kainate selectively activates kainate receptors. Other agonists include acronelic acids A and B. Competitive antagonists at the non-NMDA receptors include the quinoxalinedione derivatives, DNQX, CNQX and NBQX (Collingridge and Lester, 1989). CNQX and DNQX are four times more potent than kainate in displacing AMPA whereas the NBQX has 30 times more specificity in displacing AMPA (Sheardown et al., 1990; Sheardown, 1993). Recently, specific non-competitive antagonists at the AMPA receptor have been developed. Structural analogues based on 2,3-benzodiazepines such as GYKI 52466 and GYKI 53655 were found to be highly selective noncompetitive -35 -antagonists at the AMPA receptors (Donevan and Rogawski, 1993; Ouardouz and Durand, 1991; Palmer and Lodge, 1993; Tarnawa et al., 1992; Zorumski et al., 1993). However, the mechanism by which these antagonists work is not well understood. As previously described, the AMPA and the kainate receptors can undergo desensitization. The degree of desensitization can be selectively blocked by using different pharmacological agents. At the AMPA receptors, desensitization can be blocked with agents such as cyclothiazide and aniracetam whereas lectin concanavalin A acts preferentially at the kainate receptors to block desensitization of the receptors. 4.1.3 mGluR receptor In addition to the ionotropic receptors, glutamate also activates another class of receptors that are coupled to effector systems through GTP-binding proteins, hence the name metabotropic glutamate receptors (mGluR). Since the cloning of the mGluRI in 1991 by two independent groups (Masu et al., 1991; Houamed et al., 1991), seven other genes have now been isolated (Conn and Pin, 1997; Michaelis, 1998; Ozawa et al., 1998). The mGluRs that are encoded by these genes are named mGluRI through mGluR8. In addition, splice variants have been found for mGluRI, mGluR4 and mGluR5 (Minakami, 1994; Pin et al., 1992; Tanabe et al., 1992). Based on their amino-acid-sequence identity, the eight mGluRs have been classified into three groups. Groups I includes mGluRI and mGluR5; group II includes mGluR2 and mGluR3; group III includes mGluR4, mGluR6, mGluR7, and mGluR8 (Conn and Pin, 1997). Interestingly, the -36-receptors that are categorized in the same group activate the same type of effector system. In every expression system examined, group I mGluRs, including the splice variants, stimulate phospholipase C and phosphoinositide hydrolysis. When expressed in mammalian cells, Group II mGluRs inhibit cAMP formation stimulated by either forskolin or a Gs-coupled receptor (Parmentier et al., 1996; Tanabe et al., 1992, 1993). This effect is inhibited by pertussis toxin, suggesting the involvement of Gj-type protein coupling. Similar to Group II mGluRs, group III mGluR also inhibit adenylyl cyclase activity via a pertussis toxin-sensitive G-protein; however, the inhibition is smaller than that observed with group II mGluRs (Duvoisin et al., 1995; Nakajima, 1993; Saugstad et al., 1994; Tanabe etal., 1993). The receptors within each group also share similar pharmacological properties. DHPG, and 3HPG are highly selective agonists for group I receptors (Ito et al., 1992); however, no selective antagonist for the group I receptors has been found. Selective agonists for the group II receptors are 2R- and 4R-APDC. In addition, MCCG has been proposed to be a specific antagonist of the group II mGluRs. Group III receptors are characterized by their sensitivity to L-AP4 as well as insensitivity to ACPD. MAP4 and A-methyl-L-AP4 have been proposed to be selective antagonists of the group III receptors (Gomeza, 1996; Johansen et al., 1995; Johansen and Robinson, 1995). -37-4.2 Distribution Of Glutamate Receptors In The Hippocampus In the hippocampus, NMDA binding is dense in the outer two-thirds of the molecular layer of the dentate gyrus and in the s. oriens and the s. radiatum of CA1 and CA3 fields (Monaghan and Cotman, 1985). In the subiculum, weak NMDA binding is also present (Monaghan and Cotman, 1985). For AMPA receptors, it was found that the AMPA binding is more prominent in the outer two thirds of the molecular layer of the dentate gyrus (Monaghan et al., 1984). In the s. oriens and the s. radiatum of both CA1 and CA3, AMPA binding is also dense. Subiculum has much lesser AMPA binding in comparison to the CA1 field. The distribution of kainate receptors was studied in kainate binding studies (Monaghan and Cotman, 1982). Kainate binding is most intense in the s. lucidium of CA3. In CA1 region kainate binding is virtually absent, while moderate binding exists in subiculum. 4.3 Excitatory Synaptic Transmission in Hippocampal CA1 pyramidal neurons Excitatory postsynaptic potentials/currents (EPSCs) can be recorded in CA1 pyramidal neurons following stimulation of the Schaffer Collateral-Commissural fibers in the stratum radiatum of the hippocampal slice. The EPSCs have a fast and a slow component (Hestrin et al., 1990). The amplitude of the fast component is voltage insensitive with a reversal potential of 0 mV. The rise time of the fast EPSCs is 1 to 4 ms, and the decay time ranges from 4 to -38-8 ms (Hestrin et al., 1990). Recently, through the use of AMPA receptor specific antagonist, it has been shown that the fast component of the EPSCs in hippocampal CA1 neurons is mediated by the activation of AMPA receptors. The slow component of the EPSCs is voltage sensitive with a region of negative slope resistance in the range of -70 to -30 mV (Hestrin et al., 1990). In the experiment, the rise time for the slow component was 7 to 20 ms, and the time constant for the decay ranged from 60 to150 ms (Hestrin et al, 1990; Lester et al., 1990). The slow component of the EPSC can be blocked with APV, CPP, or MK-801, indicating that this component is mediated by the activation of the NMDA receptor (Collingridge and Lester, 1989). In addition to the NMDA and AMPA receptors, kainate receptors are also present in high concentration in the hippocampus. Through the use of an AMPA receptor antagonist, GYIKI 53655, kainate-receptor-mediated EPSCs have been isolated. Castillo et al. (1997) and Vignes et al. (1997) have independently shown that repetitive-activation mossy fibers but not associational/commissural projections in the presence of AMPA- and NMDA-receptor antagonists produced EPSCs that are mediated by the activation of kainate receptors in CA3 neurons. However, similar stimulation protocol did not elicit EPSCs in the CA1 neurons (Castillo et al., 1997; Vignes et al., 1997). These data are consistent with the distribution of the kainate receptors in the hippocampus. The kainate-receptor-mediated EPSCs are dependent on the stimulation frequency and the amount of stimulation in the train. The synaptic current reverses at about 0 mV, and the l-V relationship shows slight outward rectification. The rise time as well as the decay -39-of the response, being approximately 7 and 103 ms respectively, are slower than the AMPA-mediated EPSCs. Further studies are required to understand the role that kainate receptors play in synaptic transmission in this pathway. The mGluRs have not been shown to play a part in the generation of EPSP/Cs in the hippocampal CA1 region; however, through the activation of a second messenger system, they may play an important role in modulating the excitability and synaptic transmission of the hippocampal neurons. For instance, the activation of mGluR receptors in the hippocampal pyramidal neurons has been shown to reduce l i e a k, IAHP, IM and ID, reductions which can result in an increase in the excitability of the neurons (Charpak et al., 1990; Desai and Conn, 1991; Gerber and Gahwiler, 1994; Guerineau et al., 1994; Luthi et al., 1996). In addition, transient activation of mGluR depresses evoked EPSC and IPSC as well as the frequency of mEPSC and mlPSC. It was suggested that mGluRs can act as autoreceptors at glutamatergic synapses to decrease the release of glutamate (Glaum and Miller, 1994). In hippocampal CA1 pyramidal neurons, group I and group III mGluR but not the group II mGluR were found to be responsible for such depression (Gereau and Conn, 1995; Manzoni and Bockaert, 1995). Further studies are required to understand the physiological significance of such modulation. -40-5 GABA RECEPTOR AND INHIBITION IN THE HIPPOCAMPUS GABA is a major inhibitory neurotransmitter in the mammalian CNS (For review, Kaila, 1994; Silvilotti and Nistri, 1991; Thompson, 1994). Immuno-histochemical staining studies show that GAD, an enzyme responsible for the production of GABA, is present in all hippocampal laminae (Ribak et al., 1978; Somogyi et al., 1983). In addition, the immunocytochemical staining for GABA (Storm-Mathisen et al., 1983) as well as to the GABA-transaminase (Nagai et al., 1983) have been shown in the hippocampus. It is generally believed that the interneurons in the hippocampus are inhibitory neurons that utilize GABA as neurotransmitter. When GABA is released from the inhibitory neurons, it activates the GABA-A and the GABA-B receptors on the postsynaptic cells and in most cases results in hyperpolarizing responses named the fast and slow inhibitory post-synaptic potentials/currents (IPSP/Cs), respectively. In the following sections, both the properties of the GABA-A and the GABA-B receptors as well as the properties of the inhibitory synaptic transmission in the hippocampus will be discussed. 5.1 GABA-A Receptor In most cases, activation of the GABA-A receptors results in a hyperpolarization of membrane potentials/ an outward current due to the opening of chloride channels (Review: Kaila, 1994; MacDonald and Olsen, 1994; MacDonald and Twyman, 1992; Silvilotte and Nistri, 1991). In steady-state single-channel studies of membrane patches from a variety of preparations, the -41 -GABA-A receptor channels were shown to have multiple conductance levels and bursts of flickering openings (MacDonald and Twyman, 1992). In cultured rat hippocampal neurons, the main conductance of the GABA-A receptor channels is in the region of 20 - 30 pS, and the duration of the burst of openings is around 20 to 30 ms at resting membrane potential (MacDonald et al., 1989; Segal and Barker, 1984a and b). The concentration-response curves of GABA on its native receptor channel have a sigmoidal shape and a Hill coefficient of two, suggesting that two molecules of GABA are required to fully activate a GABA-A receptor (Sakmann et al., 1983). Full agonists that possess activities similar to those of GABA at the GABA-A receptor include muscimol (Johnston et al., 1968), THIP (Krogsgaard-Larsen et al., 1983) and isoguvacine (Krogsgaard-Larsen et al., 1977). Antagonists, such as bicuculline and picrotoxin, have been used extensively in the study of GABA-A receptor-mediated inhibition (Johnston, 1996; Kerr and Ong, 1992). Bicuculline is a competitive antagonist which acts at the GABA recognition site on the GABA-A receptor (Curtis, et al., 1970a and b). On the other hand, picrotoxin is a noncompetitive antagonist. Because the blockade of the GABA-A receptor by picrotoxin requires GABA, it has been suggested that picrotoxin acts inside the channel to produce an open channel block (Yoon, 1993; Inoue and Akaike, 1988). The GABA-A receptors are also modulated by a number of compounds including barbiturates (MacDonald et al., 1989; Study and Barker, 1981), benzodiazepines (Study and Barker, 1981; Segal and Barker, 1984a) and -42-various protein kinases (Chen et al., 1990; Krishek et al., 1994; McDonald and Moss, 1994; Moss et al., 1992, 1995; Wang et al., 1995): The details of these modulations are reviewed in Thompson (1994), Johnston (1996), Kalia (1994) and Sivilotti and Nistri, (1991). 5.2 GABA-B Receptor The original interest in studying the effects of GABA-B receptors stemmed from the observations that baclofen, a GABA analogue, inhibited the release of noradrenaline in the peripheral nerve endings (Bowery and Hudson, 1979; Bowery et al., 1980; Bowery et al., 1981). These effects were mimicked by application of GABA, but they were neither mimicked by the specific GABA-A agonist, muscimol, nor blocked by the GABA-A antagonist bicuculline. These effects suggest that there may be another type of GABA receptor. In binding studies performed by Hill and Bowery (1981), the authors have shown that the GABA and baclofen bind to bicuculline-insensitive sites, and this form of receptor was termed the GABA-B receptor. Since then GABA-B receptors have been shown to play an important role in inhibitory synaptic transmission and modulation of transmitter release in different regions in the CNS. 5.2.1 Effector systems coupled to the GABA-B receptor Electrophysiological and biochemical studies have shown that the GABA-B receptors are coupled to a variety of effector systems via a pertussis-sensitive G-protein (Review in Bowery, 1993; Kerr and Ong, 1995; Mott and Lewis, 1994). Therefore, it is analogous to the metabotropic receptor in the glutamate-receptor -43-system. Activation of GABA-B receptors can cause the opening of inward-rectifying potassium channels, changing the inactivation kinetics of A-type K + channels, closure of L, T, P/Q and N voltage-gated calcium channels, inhibition/activation of the enzyme adenylyl cyclase, and increased/decreased phosphatidylinositol turnover. In the following section, the electrophysiological effects of GABA-B receptor activation will be described. 5.2.1.1 Potassium conductance: The activation of GABA-B receptors by baclofen causes a hyperpolarization, that is, an outward current and a reduction in input resistance in central neurons in the hippocampus, olfactory cortex, hypothalamus, neocortex, lateral septum, substantia nigra and locus ceruleus (Blaxter and Carlen, 1985; Deisz and Prince, 1989; Howe et al., 1987; Inoue et al., 1985a, b and c; Lacey et al., 1988; Misgeld et al., 1995; Osmanovic and Shefner, 1988; Scholfield, 1983; Soltesz et al., 1988;). The baclofen-induced hyperpolarization is believed to be mediated by the activation of K + conductances. In hippocampal CA3 neurons, it has been shown that the baclofen-induced K + conductance is inward rectifying (Gahwilerand Brown, 1985; Newberry and Nicoll, 1984 a and b, 1985; Sodickson and Bean, 1996). This potassium conductance is similar to those activated by other neurotransmitters, such as 5-HT, adenosine, somatostatin, and opiates (Andrade et al., 1986; Brown, 1990; North et al., 1987). In addition, the K + conductance is sensitive to pertussis toxin and GDP(3S, a G-protein inhibitor and, therefore, appears to be linked to a pertussis-toxin sensitive G-protein. Recently, a study using transgenic mice with a -44-knockout of G-protein inward rectifier K + 2 (GIRK2) subunits further suggests that the baclofen-induced outward current is due to an increase in an inward-rectifying potassium conductance. In these GIRK2-/- mice, baclofen failed to induce an outward current in CA3 pyramidal and dentate granule cells (Luscher etal., 1997). In addition to activating a K + conductance, a high concentration of baclofen has also been reported to alter the voltage dependence of the A-type K + current in cultured hippocampal neurons (Saint et al., 1990). The voltage-dependence inactivation of this current is shifted to a more positive potential; therefore, at the resting membrane potential, the activation of the A-type current is enhanced, and the duration of the action potential can be shortened. 5.2.1.2 Ca2+-conductance First described in the dorsal root ganglion and later reported in many central neurons, GABA-B receptor activation has been shown to inhibit multiple types of C a 2 + currents (Deisz and Lux, 1985; Dolphin and Scott, 1986, 1987, 1990; Dunlap, 1981). In hippocampal pyramidal neurons, baclofen depresses the N-type, the L-type, and the P-type C a 2 + currents whereas, in interneurons, baclofen inhibits the T-type C a 2 + currents (Fraser and MacVicar, 1991; Huston et al., 1990; Pfrieger et al., 1994; Scholz and Miller, 1991; Wojcik et al., 1990). The depression is blocked by GABA-B receptor antagonists, such as C G P 35348 and phaclofen, and is sensitive to pertussis-toxin. The physiological role of the inhibition of Ca 2 + currents on the postsynaptic cells is not well understood. -45 -5.2.2 Pharmacology of the GABA-B receptor Baclofen, a /?-/>cholrophenyl derivative of GABA, was introduced by Ciba Geigy in the late 1960s as an antispastic agent. However, the mechanism of action of baclofen was not well understood (Bein, 1972; Faigle and Keberle, 1972). While GABA binds to both GABA-A and GABA-B receptors with equal affinity, baclofen binds to GABA-B receptors with 1000-fold higher selectivity than to GABA-A receptors (Bowery, 1993). Baclofen is an optically active substance, and the (-) enantiomer is the active compound (Olpe et al., 1978; Haas et al., 1985; Hill and Bowery, 1981). Other agonists with higher binding affinity for the GABA-B receptors than baclofen are the 3-aminopropylphosphinic acid (3-APPA) and its methyl homologue (Lovinger et al., 1992; Ong et al., 1990; Seabrook et al., 1990). Selective antagonists for the GABA-B receptor have been developed based on the structure of baclofen. The first GABA-B receptor antagonist is a phosphinic analogue of baclofen called phaclofen (Chiefari et al., 1987; Kerr et al., 1987). Phaclofen is a weak antagonist requiring high concentrations (mM) to block the GABA-B receptor. Another GABA-B receptor antagonist, 2-OH saclofen has 10 times the potency than phaclofen and can effectively antagonize both pre- and post-synaptic GABA-B receptors (Curtis et al., 1988; Kerr et al., 1988). However, at higher concentrations (>400 pM), the antagonist has been reported to suppress GABA-A receptor-mediated responses. A more selective GABA-B antagonist, CGP 35348, has been shown to be selective for the GABA-B receptors and inactive at other receptor systems (Bittiger et al., 1993; Olpe et -46-al., 1990; Seabrook et al., 1990). Other more potent GABA-B antagonists based on this structure have been developed by Ciba-Geigy. Most of these drugs were screened by radioligand binding studies, and little is known of their effects on GABA-B receptor responses (Refer to Kerr and Ong, 1995 and Mott and Lewis, 1994, for SAR for GABA-B receptors). 5.3 Distribution of GABA-A and GABA-B Receptors Moderate levels of GABA-A receptors binding have been observed in all layers of CA1 to CA3 regions and dentate gyrus of the hippocampus using autoradiographic studies (Bowery et al., 1987). On the other hand, GABA-B receptors distributions are in a less homogenous manner (Bowery et al., 1987). In regions CA1 to CA3 and dentate gyrus, densities of GABA-B receptors are found to be higher in the principal cell layers, i.e. s. pyramidal and granular layer than in the dendritic layers of these regions (Bowery et al., 1987). 5.4 Inhibitory Circuit In The Hippocampus Inhibitory synaptic transmission in the hippocampal pyramidal neurons plays an important role in regulating the excitability of the principal pyramidal and granule cells. As already reviewed in section 2.3.3., many different morphological distinct interneurons have been identified in the hippocampus. In the hippocampus, these interneurons provide their inhibitory influences via the feed forward and/or the feedback recurrent circuit (Alger and Nicoll, 1982a; Buzsaki, 1984; Kandel et al., 1961). -47-5.4.1 Feed-forward Inhibition In the hippocampal proper CA1 region, feed-forward inhibitions are mediated by interneurons that are innervated by the Schaffer Collateral of the CA3 pyramidal cells (Alger and Nicoll, 1982a). The feed-forward inhibition consists of two components. First, the afferent fibers/Schaffer collaterals from the CA3 cells form an excitatory synapse onto inhibitory neurons. Inhibitory neurons, in turn, form GABA-ergic synapse with the pyramidal neurons. Orthodromic stimulation then can either directly activate the inhibitory neurons, or they can act via a polysynaptic pathway which initially activates the excitatory synapses and then onto the inhibitory synapses (Alger and Nicoll, 1982a; Davies et al., 1990). The basket cells, the O/A interneurons and L/M are believed to mediate the feed-forward inhibition (Alger and Nicoll, 1982a; Lacaille et al., 1987, 1988b; Traubs and Schwartzkroin, 1992). 5.4.2 Feedback (recurrent) Inhibition In addition to feed-forward inhibitions, recurrent inhibitions also play an important role in controlling the excitability of principal cells in the hippocampus (Anderson et al., 1964; Kandel et al., 1961). In the CA1 field, the axons of pyramidal cells in the alveus form excitatory synapses with local interneurons in s. oriens which then form inhibitory synapses onto nearby pyramidal neurons providing feedback inhibition. The basket cells and the O/A interneurons are believed to be involved in the recurrent inhibition in the hippocampal proper (Lacaille etal., 1987). -48-5.5 Inhibitory Postsynaptic Potentials Orthodromic stimulation of the s. radiatum or the s. oriens elicits a compound response consisting of both excitatory and inhibitory events. The hyperpolarizing inhibitory response has a fast and a slow component, termed the fast and slow IPSPs, respectively (Davies et al., 1990; Dutar and Nicoll, 1988a and c). Antidromic stimulation of the alveus also elicits an IPSP that has properties similar to those of the fast IPSPs. (Alger and Nicoll, 1982a and b; Newberry and Nicoll, 1984a and b; Solis and Nicoll, 1992). 5.5.1 GABA-A receptor mediated fast inhibitory postsynaptic potentials In the presence of CNQX, APV and C G P 35348, orthodromic stimulation of the s. radiatum or the s. oriens elicits an inhibitory response termed the fast IPSP/Cs (Davies et al., 1990). The rise time of the fast IPSC is rapid with half-rise times ranging from 0.2 ms to 0.6 ms (Davies et al., 1990). The decay of the IPSCs is best fitted with 2 exponentials with a fast-decay and a slow-decay time. The fast IPSP has a latency to peak of 30-50 ms and a duration of 200 - 300 ms. This fast IPSC have a reversal potential close to -70 mV and are carried by Cl" conductance (Davies et al., 1990). Picrotoxin and bicuculline block the fast IPSP/Cs, indicating that it is mediated by the activation of GABA-A receptors. Since the time course of the IPSC plays an important role in controlling synaptic integration, the mechanisms underlying the biphasic decay of the IPSC have been studied. Edwards et al. (1990) and Pearce (1993) have suggested that the two components are due to the activation of distinct receptor subtypes of GABA-A receptors with different kinetics and pharmacological properties (Banks -49-et al., 1998; Edwards et al., 1990; Pearce, 1993). While the GABA-A fast is blocked by furosamide, GABA-A slow is not. In addition, GABA-A fast is generated in the soma, and GABA-A slow is localized in the dendritic region. On the other hand, Jones and Westbrook reported that the biexponential decay of the fast IPSCs can be explained by the kinetics of the GABA-A receptors (Jones and Westbrook, 1995 and 1996). Using rapid application of GABA in outside-out patches from cultured hippocampal neurons, the ensemble average of the response mediated by fast application of GABA has kinetics that closely resemble those of fast IPSCs. It is estimated that the cleft concentration of GABA reaches at least 0.5 mM (Jones and Westbrook, 1995) which agrees with results generated in studies in other brain regions (Maconochie et al., 1994). The authors have shown that GABA-A receptor desensitization prolongs the deactivation of GABA-A receptors and is responsible for the slow-decay component of the fast IPSCs. However, one drawback is that the conclusions rely heavily on the kinetics model built by the authors and on the assumption that synaptic GABA-A receptors have kinetic properties that are similar to those in the outside-out patches. The role that desensitization plays in shaping the IPSCs may be better understood once drugs that specifically block desensitization of the GABA-A receptors are found. 5.5.2 GABA-B receptor mediated fast inhibitory postsynaptic potentials The orthodromically activated slow IPSPs have a latency to peak of 150-200ms and a duration of 400 - 1500 ms (Davies et al., 1990). They have a reversal potential of approximately -90 mV and are carried by K + conductance. - 5 0 -The IPSPs were blocked by phaclofen, 2-OH saclofen and C G P 35348, suggesting that the slow IPSPs are mediated by the activation of GABA-B receptors (Davies et al., 1990; Dutar and Nicoll, 1988 a and b; Misgeld et al., 1995; Solis and Nicoll, 1992). It is interesting that a higher orthodromic stimulation intensity, compared to the fast IPSPs, is required to elicit the slow IPSPs and that the antidromic stimulation only mediates fast IPSPs (Dutar and Nicoll, 1988a, 1988b). This suggests that the feed-forward inhibition activates the GABA-A and the GABA-B receptors whereas the feedback inhibition solely activates the GABA-A receptors (Samulack and Lacaille, 1993). Moreover, it has been suggested that the slow IPSPs are mediated by a subset of interneurons in the s. moleculare-lacunosum layer (Williams and Lacaille, 1992). Whether there are specific interneurons that mediate slow IPSPs requires further investigations. 5.5.3 Presynaptic GABA-B receptor In addition to the postsynaptic action of GABA-B receptors, activation of presynaptic GABA-B receptors has been shown to decrease the release of neurotransmitters in the CNS. In hippocampal pyramidal neurons, it has also been shown that application of baclofen depresses evoked EPSP, and IPSP at a concentration that did not produce any significant conductance in the postsynaptic cells (Dutar and Nicoll, 1988c). This GABA-B receptor mediated depression can be blocked by GABA-B receptor antagonists, 2-OH saclofen and C G P 35348, and is believed to be due to the activation of presynaptic GABA-B receptors (Dutar and Nicoll, 1988c; Solis and Nicoll, 1992; Thompson and -51 -Gahwiler, 1992). Moreover, bath application of baclofen has been shown to reduce the frequency of mEPSCs but not the mean amplitude of the mEPSCs further suggesting a presynaptic locus of action (Scanziani et al., 1992, Thompson eta l 1993; Thompson, 1994). The mechanism by which GABA-B receptors mediate the depression is not known. Presynaptic GABA-B receptors have been postulated to depress release by 1) activating a potassium conductance, 2) inhibiting of C a 2 + current and/or 3) acting via a mechanism that is independent of leak or voltage-dependent channels. Evidences for and against each mechanism have been reported in the literature and no conclusion has been made. (For review please refer to Thompson et al. (1993)). - 5 2 -6 HEMOGLOBIN AND HEMORRHAGIC STROKE In traumatic head injuries and cerebral vascular diseases such as hemorrhagic stroke, extravascular pooling of blood occurs. The pooled blood remains in the intracranial cavity for days to weeks. Hemoglobin has been shown to be released from erythrocytes and may play a role in various complications associated with hemorrhagic stroke. The following sections will describe the properties of hemoglobin and will review the current understanding of hemorrhagic stroke. 6.1 Hemoglobin Hemoglobin is a protein found only in the red blood cells and is responsible for the transportation of oxygen. It is the principal protein constituent of erythrocytes and its concentration inside the adult human erythrocytes is approximately 4.5 to 5.5 mM (Dickerson and Geis, 1982; Freedman, 1977). The structural properties as well as the metabolism (the biosynthesis and catabolism) of hemoglobin will be described in the following sections. 6.1.1 Structure of hemoglobin The hemoglobin molecule is a made up of four prosthetic groups and four globin chains. The prosthetic group is called heme. The heme molecule gives hemoglobin its characteristic red color. Each heme molecule consists of a ferrous iron and a tetrapyrrole ring compound called protoporphyrin IX (Dickerson and Geis, 1982; Freedman, 1977). The six unpaired electrons of the ferrous iron prefer to interact with six ligand sites in an octahedral coordination. In the heme -53-molecule, four of these sites are provided by the tetrapyrrole ring, each being a nitrogen atom on the porphyrin ring system. The fifth site is provided by a histidine residue (8 th) on the globin chain (F helix) of the protein portion of hemoglobin. The sixth sites is involved in the binding of O2 (in oxyhemoglobin) and H 2 0 (in deoxy- or met-hemoglobin) (Dickerson and Geis, 1982; Freedman, 1977). Each of the globin chains of hemoglobin is a protein. Three major classes of globin chains exist and are termed a , p and y. The human a-chain is comprised of 141 amino acid residues, whereas the p and y chains are both comprised of 146 amino acid residues. Each globin polypeptide chain adopts an a-helix structure such that the globin molecule is consist of eight helices (labeled A-H) that are linked by short polypeptide segments. As each subunit folds up on itself, the tertiary structure of hemoglobin is globular in shape. Each globin subunit contains a hydrophobic pocket, formed by helices E, F, G and the CD non-helical corner, which is capable of covalent attachment to the heme molecule (Dickerson and Geis, 1982; Freedman, 1977; Safer, 1978). The quaternary structure of hemoglobin is composed of 2 a and 2 non-a chains with the non-alpha chain changes during development. Most of the fetal hemoglobin is composed of 2 a and 2 y globins, while adult hemoglobin consists of 2 a and 2 p globin chains. The proportion of fetal hemoglobin declines with age and at the age of four years, more than 9 5 % of the hemoglobin is in the a-p form. Through predominantly hydrophobic interactions and hydrogen bonds, the a-p/a-y globulin chains assemble to form a heterodimer. Two a-p/a-y dimers in -54-turn associate to form a tetramer. The tertiary and the quaternary structures of hemoglobin provide an ideal environment where the iron can be kept in a reduced (ferrous) form so that it remains functional to load and unload 0 2 molecules at physiological partial pressures in blood (Dickerson and Geis, 1982; Freedman, 1977; Safer, 1978; Voet and Voet, 1990). 6.1.2 Anabolism of hemoglobin Hemoglobin is a protein synthesized in the marrow by the nucleated precursors of erythrocytes. The synthesis of hemoglobin requires the production of the heme prosthetic group and the expression of globin genes. 6.1.2.1 Heme synthesis The major sites of heme biosynthesis are erythroid cells and the liver. The heme produced by the erythroid cells accounts for approximately 8 5 % of the total body heme groups and is mainly used for the production of hemoglobin. The liver synthesizes most of the remaining heme groups which are mainly used as a prosthetic group of cytochrome P450 (Rimington, 1959; Voet and Voet, 1990). The synthesis of heme starts with the condensation of succinyl-CoA with glycine followed by decarboxylation (of carboxyl group from glycine) to form 8-aminolevulinic acid (ALA). This reaction is catalyzed by 5-aminolevulinate synthase. Two ALA molecules are then linked together to yield porphobilinogen (PBG) by the enzyme 8-aminolevulinic acid dehydratase. Four of the resulting PBG molecules were then condensed in a series of reaction catalyzed by uroporphyrinogen synthase and uroporphyrinogen III cosynthase to form uroporphyrinogen III. This molecule is then converted to protoporphyrin IX via - 5 5 -another series of reactions that involved uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and protoporphyrinogen oxidase. A ferrous iron is then inserted into the tetrapyrrole ring of the protoporphyrin IX by ferrochelatase to from heme (Freedman, 1977; Voet and Voet, 1990). 6.1.2.2 Globin synthesis The major site of globin biosynthesis is in the reticulocytes. The genes encoding the a and (5 (5 and y) globin chains are located in different gene clusters on separate chromosomes. The a-like gene cluster, found on chromosome 16, spans 28 kb and contains three functional genes: the embryonic gene and two slightly different a-genes, a1 and a2, which encode identical polypeptides. The P-like gene cluster, found on chromosome 11, spans more than 60kb and contains five functional genes: s, 2y, 8 and (3 genes. Cells that do not manufacture hemoglobin keep their globin genes completely silent and unexpressed. The rate of synthesis/expression of the globin is controlled by the level of heme. Heme stimulates protein synthesis in the reticulocytes and ensures that heme and globin are synthesized at a rate that is compatible with the rate of assembly of hemoglobin (Dickerson and Geis, 1982; Freedman, 1977; Safer, 1978). 6.1.3 Catabolism of hemoglobin The amount of oxyhemoglobin in circulating plasma is usually well controlled by a glycoprotein called haptoglobin. Haptoglobin binds to hemoglobin in a 1:1 ratio with the haptoglobin binding capacity at least a hundred-fold excess over normal circulating plasma hemoglobin. Any free oxyhemoglobin that is not -56-bound onto haptoglobin is first oxidized to methemoglobin so that the ferrous iron is converted to ferric form called the ferriheme. The ferriheme is then dissociated from methemoglobin. The resulting ferriheme is bound onto another glycoprotein called hemopexin in a 1:1 ratio. The remaining heme is subsequently catabolized by heme oxygenase to biliverdin through oxidative cleavage of the porphyrin rings. In this reaction, iron and CO is released (Freedman, 1977). The resulting biliverdin is further broken down to bilirubin. Bilirubin is lipophilic and is insoluble in aqueous solution and is therefore transported in blood in a complex with serum albumin. Bilirubin is transferred to the liver and is excreted into bile. Heme oxygenase is an important enzyme in this reaction. It has been shown that heme oxygenase expression is increased in the rat subarachnoid hemorrhage model as well as in the presence of heme and hemoglobin (Fukuda et al., 1996; Matz et al., 1996a and b; Turner et al., 1998). The iron that is released then binds onto transferrin and is transported to other cells. Once transported into a cell, the free iron then binds onto ferritin for intracellular storage. 6.2 Hemorrhagic Strokes Stroke is categorized into two major types: ischemic and hemorrhagic. Ischemic strokes are further subdivided into thrombotic and embolic. The thrombo-embolic ischemic stroke accounts for 85 to 9 0 % of all stroke cases. (Easton et al., 1998) Hemorrhagic stroke is subdivided into subarachnoid hemorrhage and intraparenchymal hemorrhage. These forms of stroke account - 5 7 -for the other 10% to 15% of all stroke cases (Easton et al., 1998). The effects of ischemia on central neurons have been studied extensively in the CNS; however, models of hemorrhagic stroke are not well characterized. In the following sections, a general overview of the current understanding of hemorrhagic stroke as well as the role that hemoglobin may play in the pathological complications following hemorrhagic stroke will be described. 6.2.1 Etiologies The major nontraumatic cause of subarachnoid hemorrhage is aneurysmal rupture. Aneurysm is balloon-like dilation of arterial wall that occurs at the bifurcation of major arteries. The precise mechanism of the formation of the aneurysm is unknown, but it is likely that both genetic and hemodynamic factors contribute to aneurysm formation. Other possible causes of subarachnoid hemorrhage include traumatic head injuries and infectious agents. Intraparenchymal hemorrhage can be caused by traumatic and non-traumatic events. Non-traumatic events account for 13 to 14 % of all stroke incidences and 14 to 20 % of deaths cause by stroke (Easton et al., 1998). The major non-traumatic cause of intraparenchymal hemorrhage/spontaneous intracerebral hematoma (SICH) is chronic arterial hypertension which accounts for 5 0 % of the cases in SICH (Easton et al., 1998). Other causes include cerebral amyloid angiopathy, post-stroke arterial infraction and venous occlusion, congenital vascular anomalies leading to aneurysms and drugs-induced causes such as anticoagulants, fibrinolytics and sympathomimetics. -58-6.2.2 Complications of hemorrhagic stroke 6.2.2.1 Subarachnoid hemorrhage In subarachnoid hemorrhage (SAH), patients usually suffer from three major complications: cerebral vasospasm, aneurysmal rebleeding and hydrocephalus. Cerebral vasospasm can occur acutely or chronically. Acute vasospasm occurs within hours of the SAH, whereas chronic vasospasm typically occurs three to four days after SAH, peaks on days 6 to 8, and resolves by day 14. Approximately 4 5 % of all SAH patients develop symptoms associated with cerebral vasospasm (Easton et al., 1998). Another 2 5 % of the patients has asymptomatic vasospasm that is evident angiographically (Easton et al., 1998). In a number of studies, vasospasm has been identified as a strong independent negative prognostic factor (Kassell et al., 1990a and b; Weir et al., 1975). The occurrence of rebleeding from unsecured aneurysms varies with time after the initial hemorrhage, being about 4 % on the first post-bleed day and approximately 1.5 % up to day 28 (Kassell and Torner, 1983). The mortality rate of rebleeding following the diagnosis of SAH exceeds 7 5 % (Nishioka et al., 1984). Hydrocephalus can occur in patients after SAH in an acute or chronic fashion. 19% of the patients present hydrocephalus upon admission and an additional 3 % of patients develop hydrocephalus one week after SAH (Hasan et al., 1989). Hydrocephalus does not appear to be a significant independent prognostic feature in aneurysmal SAH (Easton et al., 1998). - 5 9 -6.2.2.2 Intraparenchymal hemorrhage Increase in intracranial pressure (ICP) is the major complication associated with intraparenchymal hemorrhage. The increased ICP may reduce cerebral blood flow and promote ischemia. In addition, vasoactive substances may be released from the clot into the surrounding brain areas, causing exacerbation of ischemia. There is a direct correlation between a patient's neurological status and his or her ICP, increased ICP leads to poor outcome. 6.2.3 Medical management of hemorrhagic stroke 6.2.3.1 Subarachnoid hemorrhage Prior to 1970's, patients suffering from subarachnoid hemorrhage had relatively little treatment and were typically put to bed rest for 2 weeks, until the periods of maximal risk for rebleeding and vasospasm had passed. If the patients survived from the initial hemorrhage, then surgical treatments would be given. However, medical management for this condition has changed dramatically in the past 20 years. Currently emphasis is placed on the treatment of the complications of rebleeding, cerebral vasospasm and hydrocephalus (Bleck, 1997; Weaver and Fisher, 1994). Cerebral vasospasm is at present the major complication that physicians face in patients suffering from subarachnoid hemorrhage. Cerebral vasospasm is aggressively treated with one of or combination of 1) nimodipine, 2) Triple-H therapy (induce hypertension, hypervolemia and hemodilution), and 3) angioplasty (Dorsch, 1998). -60-ln treating rebleeding, the improvement in surgical instruments and techniques for obliteration of the aneurysm has tremendously reduced the risk of early (0-3 days post-bleed) surgical approaches such as intraoperative rupture. In addition, improved treatment for cerebral vasospasm has decreased the risk of early surgery. The treatment for acute hydrocephalus is ventriculostomy (ventricular drainage). However, a number of studies have found that this form of treatment is associated with a higher risk of rebleeding (Hasan et al., 1989; Pare et al., 1992). It is, therefore recommended that the aneurysm be secured surgically prior to ventricular drainage. 6.2.3.2 Intraparenchymal hemorrhage As discussed earlier, increased ICP is the major complications associated with intraparenchymal hemorrhage. Treatments are thus aimed to lower the intracranial pressure. A high ICP can be lowered by elevating the head and by hyperventilation. Mannitol is often used to reduce ICP as well (Allen and Ward, 1998; Qureshi et al., 1999). Drainage of cerebrospinal fluid using ventriculostomy catheter is also useful for the management of increased ICP. If the above treatment options are not effective in controlling the ICP, surgical removal of the hematoma will be considered. 6.3 Pathophysiology of Hemorrhagic Stroke After an episode of hemorrhagic stroke, blood pooling, as well as ischemia takes place where the bleeding occurs. To date, most investigations related to -61 -stroke have been focused on the effect of ischemic/ hypoxic condition on central neurons while the pathophysiology of hemorrhagic stroke is not well studied. Nonetheless, ischemia is thought to play an important role in brain damage due to hemorrhagic stroke. It is, therefore, worthwhile to review the pathophysiology of ischemic stroke. 6.3.1 Ischemic stroke Brain tissue almost exclusively utilizes oxidative phosphorylation for energy production, explaining its high consumption of glucose and oxygen. During a stroke, the delivery of these nutrients are compromised and the normal metabolic activities of neurons are affected. In addition, as aerobic metabolism of glucose ceases, lactic acid accumulates, leading to a dramatic decrease in pH in the ischemic tissue (Kristian and Siesjo, 1997; Marin et al., 1994). Energy dependent processes such as the maintenance of ionic gradients and uptakes of excitatory amino acids are impaired (Kristian and Siesjo, 1997; Martin et al., 1994). Under such circumstances, accumulation of glutamate and other amino acids take place in the extracellular space leading to over-activation of glutamate receptors (Choi and Rothman, 1990; Lee et al., 1999; Nicholls and Atwell, 1990). The over-activation of glutamate receptors and inhibition of ionic pumps will lead to efflux of K + and influx of Na + and Cl". The influx of Na + and Cl" creates an osmotic pull, leading to intracellular edema (Saito et al., 1990; Shinohara, 1990) which in turn increases intracranial pressure and may lead to vascular compression or even herniation. The efflux of K + will further depolarize the neurons. In addition, over activation of AMPA, NMDA and mGluR receptors will -62-lead to an increase in intracellular C a 2 + (Choir and Rothman, 1990; Lee et al., 1999). C a 2 + is an important second messenger (Putney, 1999) and can activate many different cellular processes such as proteolysis (Chen and Strickland, 1997; Furukawa et al., 1997), activation of protein kinases (Nestle and Greenland, 1999), phosphatases, phospholipase (Bonventre, 1997; Farooqui et al., 1997), cyclooxygenase (Bazan, 1998; ladecola and Ross, 1997), and NO synthase (Bolanos and Almeida, 1999), etc. These processes can lead to degradation of intracellular cytoskeletal proteins and extracellular matrix proteins. In addition, activation of phospholipase, cyclooxygenase and NO synthase can promote to free radical generation, lipid peroxidation and subsequent tissue damage. All the above events occur within minutes after the initial oxygen deprivation in the core region of the ischemic insult. Like other tissues in the body, damage to parenchymal cells often lead to inflammation. In the brain, inflammatory responses are activated following the initial ischemic damage from excitotoxicity (Stoll et al., 1998). Within four to six hours after ischemia, astrocytes become hypertrophic and microglia become active. Many proinflammatory mediators such as tumor necrosis factor a and interleukin i p (Rothwell and Hopkins, 1995) have been shown to be released following ischemic insults. Moreover, events that guide and assist in the migration of inflammatory cells to the target region takes place. Adhesions molecules such as P-selectins (Zhang et al., 1998), E-selectins (Haring et al., 1996) and ICAM-1 (Lindsberg et al., 1996) that are important in the migrations of inflammatory cells from the blood stream are expressed on the surface of the -63 -endothelial cells. Chemokines such as interleukin-8, growth-regulated oncogene and monocyte chemotactic protein are produced by the injured brain cells to help guide the migration of inflammatory cells (Ranosohoff and Tani, 1998). Five to seven days after ischemia, the predominant cells in the ischemic area are neutrophils, macrophages and monocytes (Stoll et al., 1998). These inflammatory responses are believed to potentiate the initial excitotoxic events. For further details please refer to Hopkins and Rothwell (1995), Ransohoff and Tani (1998), Rothwell and Hopkins, (1995) and Stoll et al. (1998). Following the initial excitotoxicity, cell death occurs. Two distinct patterns of cell death have been described following ischemic insults, necrosis and apoptosis (Lee et al., 1999; Lipton, 1999). Necrosis (infarction) predominates in acute, permanent vascular occlusion and is characterized by swelling of cell nuclei, organelles and plasma membrane with early loss of plasmalemmal integrity. In peripheral sites of the ischemic stroke where there is some partially preserved energy metabolism, penumbra, where destructive events take place at a slower rate. The other form of cell death, known as apoptosis, is typically unmasked in the penumbra. Apoptosis, also known as programmed cell death, has a distinct morphological appearance characterized by condensation and margination of large nuclear chromatin aggregates and as well as extrusion of membrane-bound cytoplasmic and nuclear material with progressive loss of cell volume (Wyllie et al., 1980). Notably, the integrity of plasma and mitochondrial membrane is initially maintained. Deprivations of growth factor, oxidative stress, and exposure to increase concentrations of inflammatory cytokines have been -64-implicated in triggering this distinct pattern of cell death. For further details of the role of apoptosis in brain ischemia, please refer to Lee and Choi (1999) and Lipton (1999). 6.3.2 Clearance of erythrocytes after hemorrhagic stroke After hemorrhagic stroke, the pooled blood can remain in the intracranial space or in the subarachnoid space for prolonged periods of time if surgical removal of the blood accumulated is not performed. The clearance of the blood cells is mediated mainly via two mechanisms: 1) hemolysis and or 2) phagocytosis by the macrophages. (Findlay et al., 1989) The extravasated erythrocytes undergo hemolysis not only because the CSF is slightly hypertonic compared to plasma, but also due to the loss of integrity of the erythrocytes membrane under hypoglycemic conditions (Findlay et al., 1989). Hemolysis releases the contents of red blood cells into the CSF, resulting in the clinically observed 'bloody CSF7CSF xanthochromia. Hemoglobin, one of the major constituents of red blood cells, is detected in the CSF two hours after subarachnoid hemorrhage. Typically, the concentration of hemoglobin in CSF peaks and reaches a plateau between day 2 to day 4. Clearance of the pigments has a highly variable time span ranging from 6 to 30 days. Slow clearing was associated with old age, diabetes, vascular diseases and greater size and severity of SAH (Tourtelotte et al., 1964). In patients with intraparenchymal hemorrhage, the concentration of hemoglobin in intracerebral hematomas can reach 0.6 to 22 mM (Kajikawa et al., 1979). Components and metabolic breakdown products of hemoglobin such as bilirubin and - 6 5 -methemoglobin have also been found in intracerebral hematoma and CSF of hemorrhagic stroke patients (Barrows et al., 1955; Kjellin and Soderstorm, 1974; Kjellin and Steiner, 1974; Wahlgren, 1988). These breakdown products also contribute to the color observed in the CSF. The concentration of bilirubin and the methemoglobin increases as the concentration of oxyhemoglobin decreases. In addition to hemoglobin, other components of erythrocytes are also released into the extravascular space, for instance, platelet, thrombin, fibrin, ATP, and fibrinogen (Findlay et al., 1989). However, the concentration at which these molecules are present in the CSF and hematoma is not clear. The presence of hematoma can often cause irritation in the meninges, resulting in the activation of macrophages. The activated macrophages then remove the red blood cells by phagocytosis producing free radicals in the process (Findlay et al., 1989). 6.3.3 Ferrous hemoglobin When hemoglobin is released from erythrocytes, three potential reactions can take place and could contribute to adverse effects: Rxn [1]: NO + Hb-Fe 2 + - *Hb-Fe 2 + -NO Rxn [2]: Hb-0 2 -» met-Hb + 0 2*~ Rxn [3]: Hb -Fe 2 + - 0 2 + H 2 0 2 Hb-Fe 4 + -0 2 " + 0 2 + H 2 0 In Rxn [1], ferrous hemoglobin binds to NO at the heme iron at an affinity that is 3000 times greater than that for CO and will thus scavenge any NO in its vicinity (Gibson and Roughton, 1957). In Rxn [2], the auto-oxidation of oxy-hemoglobin yield met hemoglobin and superoxide. The superoxide radical may - 6 6 -in turn react with hydrogen ions or NO, forming hydrogen peroxide and peroxynitrite, and hence causes lipid peroxidations. In Rxn [3], oxy-hemoglobin reacts with hydrogen peroxide, generating ferryl-hemoglobin (Giulivi and Davies, 1990; 1994). The biological activities of ferryl-hemoglobin are not well understood. Rxn [2] and Rxn [3] also take place at a very slow rate. In addition to the reactions described above, Hb also possessed pseudoenzymatic activities (Mieyal, 1978) such as cyclo-oxygenase activity (Zilletti, et al., 1994), peroxidative activity (Everse et al., 1994; Grishim and Everse, 1991), and catalytic activities in hydroxylation and dealkylation reactions (Mieyal and Starke, 1994). These enzymatic reactions may contribute to some of the physiological activities of hemoglobin. 6.3.4 Reactions of NO and hemoglobin Recently, NO has been shown to play an important role in the pathophysiology of ischemic stroke. It is possible that the extravascularly pooled hemoglobin may interact with NO. The following will review the physiological interactions of NO and hemoglobin. 6.3.4.1 Physiological importance of the interactions of NO and hemoglobin As described in Rxn [1], hemoglobin binds to NO with an affinity that is 3000 times higher than those with CO. Since CO increases the O2 affinity for other heme sites, it shifts the oxygen dissociation curve to the left. It was, therefore, initially believed that NO binding would also shift the oxygen dissociation curve to the left, decreasing the release of oxygen and hence increasing tissue hypoxia (Kon et al., 1977). However, in recent studies by -67-Kosaka and Seiyama (1997), it has been demonstrated that NO increases 0 2 release from hemoglobin and thus 0 2 supply to tissues. In addition to the promotion of 0 2 release, NO also has vasodilating effects and thus increases 0 2 supply. Stalmer and colleagues (Gow and Stalmer, 1998; Jia et al., 1996; Stamler et al., 1997) proposed that hemoglobin in the lungs is S-nitroslyated on the p-chain at the Cys93 residue. When the S-nitrosylated hemoglobin reaches peripheral circulation, NO dissociates and relaxes blood vessels, leading to an increase in blood flow. The physiological importance of such interactions between NO and hemoglobin needs further investigations. 6.3.4.2 NO and ischemic stroke NO is well established to be an important physiological messenger in the CNS (Bredt et al., 1994; Vincent, 1994). It is produced by NO synthase (Knowles and Moncada, 1994), of which a number of different types have been identified: endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible (iNOS). These NOS systems have different functions in ischemic stroke (Bolanos and Almeida, 1999). It is believed that NO produced by nNOS (Escot et al., 1998; Huang et al., 1994; Zhang et al., 1996) and iNOS (ladecola C et al., 1995 and 1997) have detrimental effects on neurons while NO generated by eNOS (Huang et al., 1996) may aid in neuronal survival. The fundamental differences between these enzymes are believed to be due to their distribution (Bolanos and Almeida, 1999; Chabrier, et al., 1999). NO produced by eNOS is important in vasodilatation, therefore, when released during ischemic stroke, it may help in the delivery of blood to the brain region with impaired blood flow. On the other - 6 8 -hand, NO generated by nNOS and iNOS may promote free radical generation by reacting with superoxide forming peroxynitrite, and leading to lipid peroxidation. 6.3.5 Iron and free-radicals production Iron is one of the major components of hemoglobin. Under prolonged incubations of hemoglobin or of methemoglobin, iron has been shown to completely dissociate from hemoglobin and becomes free Fe 2 + . In the presence of superoxide, free F e 2 + can be involved in a reaction known as the Fenton reaction (Rxn [4]) whereby it reacts with H 2 0 2 to produce highly reactive hydroxyl radical. Rxn [4]: F e 2 + + H 2 0 2 -» F e 3 + + 'OH + OH" The F e 3 + resulting from the reaction can then be recycled to F e 2 + by the reaction with another superoxide (Rxn [5]). Rxn [5]: F e 3 + + 0 2 '"-> F e 2 + + 0 2 With a source of superoxides, free radicals can be generated continuously through these reactions (Rxn [4] + Rxn [5]). Rxn [4] + Rxn [5]: 0 2 ' " + H 2 0 2 -» *OH + OH" + 0 2 Free radicals generated via the Fenton reaction can be detrimental to tissues. Moreover, lipid peroxidation can take place in the presence of iron. For further details please refer to Halliwell (1992). 6.3.6 Hemoglobin and cerebral vasospasm One of the major complications with SAH (may also occur in intraparenchymal hemorrhage) is cerebral vasospasm. The occurrence and -69-severity of cerebral vasospasm is often related to the size of the hemorrhage. Since vasospasm usually spreads to arteries that are proximal to, and downstream of, the hemorrhagic site it is often believed that spasmogen/s is/are released at the hemorrhagic site. The time course of the development of cerebral vasospasm and the increase in concentration of heme products in the CSF is closely associated; an observation which suggests that hemoglobin is the principle spasmogen involved in the mediation of cerebral vasospasm (MacDonald and Weir, 1991, 1994). In addition, it has been shown that oxyhemoglobin induces prolonged contraction in cerebral arteries both in vivo and in vitro, whereas, methemoglobin did not have such an effect. Oxyhemoglobin induced-vasospasm has been suggested to be mediated by the release of prostaglandins and endothelin (Machi et al., 1991; Masaoka et al., 1990; Sato et al, 1990), the production of heme, bilirubin (Duff et al., 1988; Miao and Lee, 1989), and lipid peroxides (Asano et al., 1980; Sasaki et al., 1979). Oxy-hemoglobin is also thought to induce vasospasm by inhibiting the actions of endothelium-derived relaxing factor (Toda et al., 1988; Tsuji et al., 1989) and damaging the perivascular nerves (Duff et al., 1987; Lee et al., 1984; Linnik and Lee, 1989). However, the involvement of oxyhemoglobin in cerebral vasospasm has been complicated by the findings that the hemoglobin used in those experiments may be contaminated with plasma membrane lipids, endotoxin and other stromal proteins; therefore, whether the cerebral vasospasm observed in SAH is due to hemoglobin is not known (Bolin et al., 1983; De Venuto et al., 1977; Feola et al., 1988, 1989; Rabiner, 1975). Recently, it was found that ATP -70 -has a high affinity for hemoglobin and it is thus possible that the hemoglobin used previously is also contaminated with ATP (MacDonald et al., 1998). When applied at a concentration that is present in hemoglobin samples, ATP has vasoconstricting effects. Therefore, it is possible that the observed vasospasm previously attributed to hemoglobin was in fact due to the ATP in the hemoglobin (MacDonald etal., 1998). In addition to hemoglobin, other compounds that are present in blood have also been implicated in the mechanism of cerebral vasospasm. Hemin (Letarte et al., 1993), bilirubin, biogenic amines, thrombin, thromboxane, and serotonin derived from platelet have also been implicated as the spasmogens involved in cerebral vasospasm. Based on these studies, it is possible that multiple mechanisms or agents may be involved in the cerebral vasospasm associated with subarachnoid hemorrhage. 6.3.7 Hemoglobin, epileptic seizures and neurotoxicity In patients with cerebral hematoma or subarachnoid hemorrhage, epileptic seizures are often observed (Caveness, 1963; Caveness and Liss, 1961; Jennett, 1975; Kaplan, 1961; Richardson and Dodge, 1954; Russel and Whitty, 1952, 1953; Ward, 1972). The extravasation of blood and the release of iron from the pooled blood during these cerebrovascular accidents have been implicated in the development of such or these post-traumatic epileptic seizures. Intracortical injection or pial iontophoretic application of whole blood, hemolyzed blood, hemoglobin, methemoglobin, hemin, ferritin, fibrinogen, ferrous as well as ferric chlorides in cats and guinea pig have been shown to induce epileptiform -71 -activities (Levitt et al., 1971; Hammond et al., 1980; Willmore et al., 1978 a, b, c). However, the mechanisms by which these substances cause epileptic seizures are not well understood. In addition to epileptic seizures, loss of neurons, iron deposition and glial proliferation has been shown to occur at the site of the intracortical hemorrhage (Hammond et al., 1980; Pollen and Trachtenberg, 1970; Rand and Courville, 1945). Hemoglobin and the free iron liberated from hemoglobin have been implicated in generating free radicals which may lead to peroxidation of neuronal membranes (Mori et al., 1990). In fact, Regan and Panter (1993) have shown that incubation in hemoglobin for >24 hours resulted in extensive cell death in neocortical cultured neurons. The hemoglobin-induced neuronal cell death is believed to be mediated by irons through the generation of free radicals, since both desferroxamine, an iron chelator, and Trolox, a lipid soluble antioxidants, were able to block the effects of hemoglobin (Scott and Panter, 1993). Further work is required to characterize the possible mechanisms by which hemoglobin induces epileptic seizures. -72-7 Hypothesis During hemorrhagic stroke and traumatic head injuries, extravascular pooling of blood occurs. Erythrocytes that are released into the extravascular space remain in the intracranial cavity for days to weeks. During such time the pooled erythrocytes are lysed and hemoglobin is released. Under these circumstances, neurons may be exposed to hemoglobin and/or its breakdown products. Hemoglobin has been shown to have neurotoxic effects on neocortical cultured neurons (Regan and Panter 1993). In addition, hemoglobin has been shown to promote free radicals production and lipid peroxidation (Everse and Hsin, 1997). Moreover, free radicals and lipid peroxidation has been shown to depress synaptic transmission in hippocampal CA1 pyramidal neurons (Pellmar and Neel, 1989). However, the effects of hemoglobin on synaptic transmission are not well understood. Therefore, the present experiments were designed to test the hypothesis that hemoglobin and its breakdown products, such as iron and hemin, have depressive effects on synaptic transmission. Objectives to address the hypothesis are outlined below. Effects of hemoglobin on synaptic transmission: 1. To investigate the effects of hemoglobin on excitatory synaptic transmission in hippocampal slices. 2. To investigate the effects of hemoglobin on inhibitory synaptic transmission in hippocampal slices. Differences between reduced-hemoglobin and met-hemoglobin: -73 -1. To investigate if there is a difference between the actions of reduced-hemoglobin and met-hemoglobin on synaptic transmission in hippocampal slice preparation. The mechanism underlying the effects of hemoglobin on synaptic transmission: 1. To investigate the effects of hemoglobin on action potential firing in hippocampal neurons. 2. To investigate the effects of hemoglobin on AMPA, NMDA, THIP or baclofen induced-responses in hippocampal neurons. Effects of Iron or hemin on synaptic transmission: 1. To investigate the effects of iron on synaptic transmission in hippocampal slice preparation. 2. To investigate the effects of hemin on synaptic transmission in hippocampal slice preparation. -74-8 MATERIALS AND METHODS In this section, the materials and methods that were used in the experiments performed will be discussed in details. 8.1 Animal Source Male Wistar Rats (100 to 150g) were used for intracellular microelectrode recordings and nystatin perforated-patch electrode studies. In studies in which the technique of whole-cell patch electrodes recordings was employed, male Wistar rats (2 to 3 week old, 25 to 40g) were used. These animals were provided by the Animal Care Centre of the University of British Columbia. After the animals reached the Department of Pharmacology & Therapeutics of the University of British Columbia, they were kept in wire cages (dimensions: 17" X 7" X 9.75") in the animal room. The 100 to 150g animals came in a shipment of six each. Since the 2 to 3 week old animals were not weaned, either a group of rats, usually containing 2 to 4 animals, was delivered on every working day, or a litter of rats, containing 10 to 12 animals with a mare, was delivered once every 4 to 5 days. Each shipment of animals was stored in a separate cage. The temperature of the animal room was controlled and kept between 20 to 22 °C. The lights of the room were kept on from 6 a.m. to 6 p.m. daily. Supplies of food and water were made available to the animals all the time and refilled daily from Monday to Friday. On every Friday, extra food and water were added to maintain the animals' diet through the weekend. The excretion trays of the wire cages were cleaned and replaced once every two days. - -75-8.2 Slice Preparation The animals were transferred from the animal room to the laboratory in a plastic cage (dimensions: 11.5" X 5" X 7.25"). The 100 to 150g rats were then placed on ice in a glass chamber in order to lower their metabolic rate. Inside the chamber, animals were anesthetized with 2 % halothane and carbogen (95% O 2 , 5% C 0 2 ) until surgical anesthesia was achieved (i.e. eyeblinking and the withdrawal reflexes were absent, and the respiration was thoracic in origin.). Due to difficulty in anesthetizing the young animals (25 to 40 g), they were sacrificed by decapitation. The head was then immediately submerged in a dish containing ice-cold physiological medium, artificial Cerebral Spinal Fluid (ACSF) bubbled with carbogen. Either after the surgical anesthesia was acquired or the decapitation was performed, a medial incision was made at the top to the base of the animal's head in order to expose the skull. A pair of nippers/spring scissors was used to create a small incision above the Bregma of the animal's skull. Through this incision, a pair of scissors was manipulated to cut along the sagittal suture line continuing up towards the tip of the head. Additional lateral cuts were made with the scissors on the tip as well as the base of the sagittal cut so that a flap-like structure was formed with the skull. A pair of ronguers/forceps was subsequently used to remove the bone and the dura. The brain was gently removed from the intracranial cavity by a spatula. Once the brain was removed from the intracranial cavity, it was immediately washed with cold (4°C) A C S F which was saturated with carbogen. The two cerebral hemispheres were divided into two by -76-cutting along the midline with a razor blade. Once the two hemispheres were separated, one of the hemispheres was oriented so that the ventral side was facing upwards. The remaining part of the midbrain was carefully removed, using a pair of spatula to expose the ventral side of the hippocampus. Two parallel cuts separated by 0.4 to 0.5 cm were made along the septo-temporal axis of the hippocampus. The hippocampus was then transferred to a filter paper to remove the excess ACSF. It was then glued onto an acrylic cutting platform with cyanoacrylate (Lepages Accu-Flo No. 8) having one of the transverse sides facing down. The mounted tissue was then placed in a bath chamber containing ice-cold physiological media oxygenated with carbogen. The bath containing the tissue was then mounted onto a Vibroslice machine (World Precision Instruments Inc.). The final orientation of the tissue with respect to the blade of the Vibroslice is such that the septo-temporal axis of the hippocampus was parallel to the blade of the slicing machine when mounted onto the tissue slicer. Transverse hippocampal slices with thickness of 400 to 425 pm were prepared and transferred into a petri dish containing cold oxygenated ACSF. This preparation procedure usually provided 3 to 4 healthy slices. The extra cortical tissue and the CA3 region were then removed with a pair of scissors. The CA3 region of the hippocampus slice was removed to minimize the epileptiform activity of CA1 neurons caused by spontaneous activity of CA3 neurons. The slices were stored in a holding chamber containing oxygenated physiological medium and gently placed on a partially submerged nylon net. The slices were allowed to equilibrate with the ACSF for at least one hour prior to being transferred to the recording -77-chamber. At the end of the equilibrium period, only slices with a discrete cell line were used for electrophysiological recordings. Slices stored in this manner were viable for eight hours post-surgery. Prior to transferring to the recording chamber, the slice was placed in a petri dish containing physiological solution oxygenated with carbogen. The slice was gently placed between two nylon meshes that were mounted onto two overlapping Plexiglas rings. The meshes were manipulated so that the slice was secured between the nets without physical damage. The secured slice was then transferred to a submerged-type recording chamber and superfused with oxygenated physiological medium at a flow rate of approximately 2.5 ml/min. 8.3 Slice Chamber The slice chamber was designed by Dr. B.R. Sastry and was made in the workshop at the Department of Pharmacology and Therapeutics (See Fig. 8.3). A detailed description of the slice chamber has previously been published (Pandanabonia and Sastry, 1984). Briefly, the slice chamber is made from a rectangular plexiglass block. A keyhole shaped superfusing chamber is drilled on the top surface of the plexiglass block. An inlet aperture is located at the circular end of the superfusing chamber while an outlet aperture is located at the narrow or rectangular end of the bath. Inside the keyhole chamber, a small ridge -78 Fig. 8.3. Recording Chamber. Diagram illustrating the slice chamber used to record electrophysiological potentials in the hippocampal slices. -79-is constructed around the sides of the chamber so that the nylon meshed ring that sandwiches the hippocampal slice is lifted slightly above the floor of the bath. This ridge enables the perfusing solution to reach both sides of the slice. The main inflow line is fed into the inlet aperture whereas a suction tube is fed through the outlet aperture and into the superfusing chamber, in order to maintain a constant level of superfusate in the recording chamber. The main inflow line is divided into seven different inlets by a manifold. Each inlet is connected to a 60 ml barrel. The barrels are adjusted to a height at which a flow rate of about 2.5 ml/min is obtained. 8.4 Superfusing Media Slices were superfused with oxygenated (95% 0 2 , 5% C0 2 ) A C S F containing (in mM) 120 NaCl, 3.1 KCI, 26 NaHC0 3 , 5 NaH 2 P0 4 , 2 MgCI 2, 2 CaCI 2, 10 dextrose (pH 7.4) at a flow rate of about 2.5 ml/min (See Table. 8.4.). In experiments in which the effects of ferrous chloride and hemin were studied, NaH2PC>4 was omitted, the 26 mM bicarbonate buffer was replaced with a 26 mM Tris-base/HCI buffer and the Tris-buffered solution was bubbled with 100% 0 2 in order to prevent precipitation of the drugs (See Table. 8.4.). In some experiments, a Ca 2 +-free medium was prepared by replacing 2 mM CaCI 2 with 2 mM MgCI 2 and 0.1 mM EGTA from the recipe (See Table. 8.4.) In some experiments, a Mg 2 +-free medium was prepared by omitting magnesium chloride and replacing it with 2 mM CaCI 2 (See Table. 8.4.). These solutions were made each morning prior to the beginning of the experiment. - 8 0 -Table. 8.4. Composition of Perfusing Medium Normal Ca^-free Mg2+-free Tris-Buffer ACSF ACSF ACSF NaCl (mM) 120 120 120 120 KCI (mM) 3.1 3.1 3.1 3.1 NaHC0 3 (mM) 26 26 26 0 MgCI 2(mM) 2 4 0 2 CaCI 2(mM) 2 0 4 2 Dextrose (mM) 10 10 10 10 N a H 2 P 0 4 (mM) 5 5 0 0 EGTA (mM) 0 0.1 0 0 Tris-base/Tris-HCI (mM) 0 0 0 26 Oxygenation Carbogen Carbogen Carbogen 100% 0 2 The drugs that were used in the present studies consisted of hemoglobin (Sigma), met-hemoglobin (Sigma); /V-co-nitro-L-arginine (Sigma), picrotoxinin (Picro; Sigma), tetrodotoxin (TTX, Sigma), 6,7-dinitroquinoxaline-2,3-dione (DNQX; Precision), D,L-2-amino-5-phosphonovaleric acid (APV; Precision), (RS)-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; Precision), A/-methyl-D-aspartate (NMDA; Precision), lidocaine A/-ethyl bromide (QX-314; Astra Pharmaceuticals), 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride (THIP; RBI), baclofen (Sigma), ferrous chloride (FeCI 2; Fisher), hemin (Sigma), bisulfate (Fisher) and bisulfite (Fisher). Bovine hemoglobin samples were purchased from Sigma with a catalogue number of H2500. In some experiments, the differences between two lots of hemoglobin (Lot #: 101H9307 and Lot #: 86H9310) were compared. For simplicity, Hb Lot 101H9307 will be referred to as Hb-1991 and Hb Lot #: 86H9310 will be referred to as Hb-1996. Reduced hemoglobin was prepared by a method described by Martin et al. (1985). Briefly, hemoglobin (1 mM) was reduced with a strong reducing agent, 10 mM sodium dithionite (Sigma). Upon application of the reducing agent, the hemoglobin solution changed from a brown to a bright red color. The excess reducing agent was dialyzed by placing the Hb solution in a dialyzing tube (Sigma). The solution was dialyzed with distilled water at least 100-fold the volume of the hemoglobin solution for 2 hours at 4°C. Control solutions in which only dithionite was dialyzed were prepared using similar methods. In some experiments, Hb was dialyzed for 18 hours with 3x 100-fold volume of distilled -82-water changing every 6 hours. The reduced-Hb was aliquoted into 5 ml bottles and was then stored in a -20°C freezer. The reduced-Hb solution was thawed when needed. The Hb solution was kept in the freezer for a maximum of 2 weeks. The spectrophotometric method was used to confirm the presence and concentrations of reduced-Hb. Concentrated stock solutions of APV (5 mM), Picro (5 mM), AMPA (1 mM), NMDA (1 mM), THIP (10 mM), baclofen (10 mM) and TTX (100 u.M) were prepared in distilled water and diluted with the physiological medium to the desired concentration. Stock solutions of CNQX (1 mM) and DNQX (1 mM) were made by first dissolving the drugs in dimethylsulfoxide (DMSO) and then diluting them with appropriate amounts of distilled water so that the final concentration of DMSO in the stock medium was 0.1%. Solid forms of bovine hemoglobin, Nco-nitro-L-arginine, FeCI 2, bisulfate, and bisulfite were directly dissolved in the superfusing medium to the desired concentration. In experiments in which the effects of ferrous chloride (FeCI2) and hemin were studied, the drugs were dissolved in a TRIS medium and oxygenated with 100% 0 2 . Hemin was dissolved by in 1 mM NaOH and was then dissolved in the TRIS medium with 1:10 ratio of albumin. The final concentration of albumin and NaOH in 0.5 mM hemin was 0.05 mM and 0.01 mM, respectively. The albumin was not treated to provide homogeneity or purity. The pH of all oxygenated superfusates, except FeCI 2, was about 7.4. FeCI 2 solution has a pH of approximately 7.34, therefore, control studies were performed with solutions, titrated with HCI, with a pH 7.34 to 7.32. -83-All superfusing media were oxygenated by carbogen before and during applications. The main barrel, which contained the principal superfusing medium, was continuously supplied with the medium from a reservoir located above the barrel. In order to minimize dead space in the superfusing tubing, air bubbles trapped in the lines were removed, before each experiment, by a suction device. All filled lines that were not in use were temporarily closed by butterfly clips. Whenever a particular filled line was needed, the butterfly clip of that line was removed while another butterfly clip was simultaneously placed on the previously running line. By doing this, the slice chamber received rapid exchange of different media with minimal flow disturbance. 8.5 Pipette Solutions Four different types of recordings were used in these studies: extracellular recordings, intracellular sharp electrode recordings, nystatin perforated-patch recordings and whole cell patch recordings. In each type of recording, the pipette electrode was filled with different types of recording solution. In extracellular field potential recordings, the recording electrodes were filled with the superfusing medium. In conventional pointed intracellular microelectrode recordings, , the recording electrodes were filled with 4M potassium acetate. The pH of the pipette solution was adjusted to 7.2 with acetic acid. In nystatin perforated-patch electrode recordings, the recording pipettes were filled with 30 mM potassium chloride, 130 mM potassium gluconate, 10 mM N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), and 160 pg/ml -84-nystatin in 0.16 % DMSO. The pH of the perforated patch electrode solution was adjusted to 7.2 with potassium hydroxide (see Table. 8.5.). In whole-cell patch electrode recordings, the recording pipettes were filled with ATP-regenerating solution (Forscher and Oxford, 1985) consisting of 135 mM potassium gluconate, 10 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), 10 mM potassium chloride, 1 mM K 4-bis-(o-aminophenoxy)-A/,A/,A/',/\/-tetraacetic acid (K4-BAPTA), 5 mM Mg-ATP, 0.1 mM CaCI2, 10 mM Na 2-phosphocreatine, 50 units/ml creatinine phosphokinase and 0.4 mM Na 2 -GTP (3 to 7 MQ) (see Table. 8.5.). In experiments in which the NMDA EPSCs were recorded, QX-314 (5 mM) was added into the pipette solution. The pH of the patch solution was adjusted to 7.2 with potassium hydroxide (See Table. 8.5.). Junction potentials for the patch pipette solutions were measured to be approximately between 6 to 10 mV. The membrane potential was adjusted to compensate for the potential difference. 8.6 Recording and Stimulating Equipment The recording set up comprised a superfusing recording chamber, a hydraulic microdrive (David Kopf) hooked up to an Opticon manual micromanipulator, and two horizontal carriers equipped with stands which support a headstage and two electrode carriers (one for the stimulating electrode and the other is for ground electrode). The equipment was mounted onto an aluminum base plate. The base plate was, in turn, placed on a vibration-free air isolated table. An aluminum wire-cage was used to shield the set up from -85-Table. 8.5. Composition of Patch Recording Solutions Whole-cell patch Whole cell patch for NMDA currents Nystatin-perforated patch Potassium 135 135 130 Gluconate (mM) HEPES (mM) 10 10 10 KCI (mM) 10 10 30 K 4 -BAPTA (mM) 1 1 0 Mg-ATP (mM) 5 5 0 CaCI 2 (mM) 0.1 0.1 0 Na 2 - 10 10 0 phosphocreatine (mM) Na 2 -GTP (mM) 0.4 0.4 0 Creatinine 50 50 0 phosphokinase (units/ml) QX-314 (mM) 0 5 0 Nystatin (pg/ml) 0 0 160 The pH of the patch solutions were adjusted to pH 7.2. The osmolarity of the patch solutions were 290 to 300 mOsmol. -86-electrical noises. Other recording and stimulating equipment included an amplifier (Axoclamp 2A), a two-channel stimulator (Grass 088), two isolation units, a data acquisition interface (Scientific Lab), and a 486-based computer with acquisition software pClamp 6. The equipment was mounted on two metal racks located next to the air-isolated table. The superfusing barrels were mounted on a separate freestanding stand next to the table. A Zeiss gross-dissecting microscope used to inspect the slice and direct the positioning of the recordings and stimulating electrodes were mounted on a movable stand. 8.6.1 Amplifiers Both extracellular and intracellular potentials were recorded with an Axoclamp 2A microelectrode clamp (Axon Instruments). The extracellular, intracellular microelectrodes and nystatin perforated-patch electrode recordings were made using the current clamp mode. Recordings obtained using the whole cell patch electrode were made under single voltage clamp recordings (sEVC). A precision resistor of 100 MQ in resistance, which was located in the headstage, set the headstage current gain (H) to be 0.1. At this value, the range of the bridge balance was 0 to 1000 MQ; the maximum DC current command was +/-10 nA; and the range of the capacitance neutralization was -1 to 4 pF. The signals from the Axoclamp were amplified ten times and filtered between 0.1 kHz and 1 kHz before they reached the recording systems. 8.6.2 Stimulators and isolation units A Grass S88 stimulator was used in all of the experiments. The stimulator has two outputs; they are connected to two Grass PSIU6 -87-photoelectric-stimulus isolation units with constant current output. One unit was connected to the Axoclamp 2A and was used either to feed squared-current pulses or to inject DC current into the recording microelectrode. The other unit was used to supply current to the stimulating electrode. 8.6.3 Micromanipulators and hydraulic microdrive An Optikon micromanipulator and a DKI 650 hydraulic microdrive (David Kopf Instrument) were used to manipulate the position of the recording electrodes. Briefly, recording electrodes were inserted into an electrode holder that was attached to the Optikon-DKI unit. In the extracellular studies, the recording electrodes were manipulated in the X, Y, and Z planes by an Optikon micropositioner.ln the intracellular studies, a DKI 650 hydraulic microdrive, which could lower the electrode by 2.5 p.m in 2.5 x 10-3 ms, was used in addition to the Optikon micropositioner. The stimulating electrode was installed on a DKI electrode carrier which also allowed the stimulating electrode to be moved in a three-dimensional manner. 8.6.4 Recording electrodes Both the intracellular and the extracellular recording electrodes were made from standard tin-walled borosilicate glass capillaries supplied by either Sutter Instrument or World Precision Instrument. The microelectrodes were pulled by a programmable Flaming/Brown P-87 model micropipette puller (Sutter Instrument). A 2 mm-wide horizontal trough filament (Sutter Instrument) was used. The null-bridge method was used to determine the resistance of the microelectrodes. -88-For extracellular recording electrodes and nystatin perforated- and whole cell- patch electrodes, borosilicate glass capillaries (WPI) with an inner and an outer wall diameter of the 1.17 and 1.5 mm, respectively, were used. A program that provides a 4-stage/cycle pull was used. The resulting electrodes had a tip diameter of approximately 4 pm. The resistance of these electrodes was about 3-5 MQ for the extracellular recording electrodes and 5-7 MQ for the nystatin perforated- and whole cell-patch electrodes. For intracellular pointed microelectrode recording, borosilicate glass capillaries (Sutter) with an inner and an outer wall diameter of 1.0 and 0.58 mm, respectively, were used. A program that provides a 1-stage/cycle pull was used. The resulting electrodes when filled with 3 M potassium acetate had a resistance ranging from 90 to 100 MQ. 8.6.5 Stimulating electrodes A bipolar concentric stimulating electrode (SNEX-100; Rhodes Medical Instruments) was used in all experiments. The stimulating electrode has a shaft length and a contact length of 50 mm and 0.75 mm, respectively. The tip/contact diameter of the electrode is 0.1 mm. The electrode had a resistance of approximately 1 MQ, and it was replaced when the electrode resistance exceeded 5 MQ. 8.6.6 Recording systems Both extracellularly and intracellular^ recorded potentials were monitored by a Tektronix type 511A dual beam oscilloscope. A Scientific Lab data acquisition interface (TL-1) was used to digitize the data at 10kHz. The data was -89-then acquired using either Clampex 6 or Fetchex 6 in a 485-based computer. The online waveform was monitored using the computer. All data was stored on computer and later transferred into either Zip disks or CDs. Hard copies of data acquired by Clampex 6 were printed with a HP 500 Desk Jet printer connected to the computer. 8.7 Electrophysiological Recordings Four different types of electrophysiological recordings were used in the study: extracellular field recordings, intracellular sharp microelectrode recordings, nystatin-perforated patch recordings and whole cell patch electrode recordings. The following sections will describe the recording techniques. 8.7.1 Extracellular Recordings In field EPSPs recordings, the recording electrode and the stimulating electrodes were placed in the s. radiatum of the CA1 region, separated by approximately 0.3-0.4 mm (See Fig.8.7.1). Field EPSPs were recorded in response to stimulations (0.2 ms; 10-150 nA) of the s. radiatum at 0.05 Hz. The stimulation strength was adjusted to produce field EPSPs of 0.5 to 1 mV in size. 8.7.2 Conventional microelectrode recordings The input resistance (MQ) of neurons was examined by measuring the change in membrane potential caused by rectangular hyperpolarizing current pulses (-0.05 to -0.2 nA, 200 ms) injected through the recording electrode. Changes in membrane potential at 150 ms of the rectangular hyperpolarizing current pulses, when the response flattened out, were measured. The excitability -90-STIM Fig. 8.7.1. Placement of stimulating and recording electrodes in hippocampal slice Intracellular responses in CA1 area were recorded by placing the recording electrode (REC) in the s. pyramidale layer of,the hippocampal slice. Stimulating electrode (STIM) was placed in the s. radiatum near the CA1-CA2 junction, (adopted from Ip, 1994) -91 -of the neurons was monitored by examining the number of spikes evoked by rectangular depolarizing current pulses (0.05 to 0.3 nA, 200 ms). Those neurons with stable membrane potentials of -55 to -65 mV and input resistances greater than 35 MQ were used in this study. The range of the membrane potential was chosen to allow recordings of EPSPs that were below threshold for action potentials and to allow recordings of IPSPs above their reversal potentials. Input resistances were chosen based on the fact that low resistance cells with leaky membranes are usually a sign of unhealthiness and synaptic responses as well as action potentials in these cells tend to be shunted as well. The stimulation strength was adjusted to produce EPSPs and IPSPs with amplitudes that were about 5 0 % of the maximum response. 8.7.3 Patch-Clamp recordings Perforated- and whole-cell patch clamp recordings, were made from CA1 hippocampal pyramidal neurons using the 'blind' patch-clamp technique in current clamp mode (Blanton et al., 1989; Korn and Horn, 1989). Briefly, the patch electrodes were inserted into the pipette holder which was connected to a 10 ml syringe via flexible teflon tubing. Through the syringe, a positive pressure was applied to the tip of the electrode before lowering it into the recording chamber. Once the electrodes were lowered into the superfusing solution, rectangular hyperpolarizing pulses with amplitude of 0.5 nA and 50 ms in duration were delivered through the recording pipette at a frequency of 50 Hz. The pipette was lowered into the s. pyramidale layer in the CA1 field using the hydraulic microdrive in steps of 25 pm/step. When the electrodes were -92-approaching or pressing onto a neuron, the voltage response to the rectangular pulses changed to an irregular sinusoidal waveform. The positive pressure was then released, and a negative pressure was applied. The voltage response then grew until it reached a resistance of > 1GQ. In whole cell recordings, once the seal resistance had reached > 1GQ, negative pressure was further applied until the membrane patch ruptured and whole cell access was gained. This was characterized by the membrane potential dropping to a potential close to the resting membrane of a neuron. In addition, the voltage responses dropped dramatically to give resistances that are close to those of neurons. In nystatin-perforated patch recordings, no further suction was given when the seal resistance has reached > 1GQ. The membrane potential of the neurons stabilized 5-15 minutes after the > IGQsea l was formed. In whole-cell patch recordings, only neurons with series resistance less than 30 MQ, usually between 25-15 MQ, were used. In nystatin perforated patch recordings, stable series resistances (between 60 to 45 MQ) were usually achieved in about 30 minutes after the formation of the GQ seal. In both types of patch recordings, the bridge balance was checked frequently to ensure the stability of the series resistance. If the series resistance changed by more than 15% of the control during the course of the experiments, the data from these experiments were discarded. The input resistances of the neurons were examined by measuring the change in membrane potential caused by rectangular hyperpolarizing current pulses (-0.01 to -0.1 nA, 200 ms in the nystatin perforated patch and whole cell - 9 3 -patch recordings) injected through the recording electrode. Those neurons with stable membrane potentials of -55 to -65 mV and input resistances greater than 85 MQ (for whole-cell and nystatin perforated patch electrode) were used in this study. Current-voltage curves were obtained by injecting 0.1 to -0.4 nA current of 400 ms duration. Intracellular evoked excitatory postsynaptic potentials (EPSP/Cs) and inhibitory postsynaptic potentials (IPSP/Cs) were recorded in CA1 neurons in response to 0.05 Hz stimulation of the s. radiatum with a bipolar stimulating electrode. The stimulation strength was adjusted to produce EPSP/Cs and IPSP/Cs of size that are 5 0 % of maximal response. Fifteen minutes of stable EPSP/Cs (+/- 15%) and IPSP/Cs (+/- 15%) were collected as control responses before drug applications. The membrane potential (+/- 1 mV), the input resistance (+/-10%), and the depolarization-induced discharge of action potentials (+/- 1 action potential with similar height, about 40-50 mV) were also ensured to be stable during this control period. 8.8 Analysis of Extracellular and Intracellular Recordings In the following sections, the method used to analyze the extracellular and the intracellular responses will be described. 8.8.1 Extracellular recordings The extracellular responses recorded have a characteristic waveform that consists of a baseline followed by a stimulus artifact then a presynaptic volley and then a field EPSP. The amplitude of an extracellularly recorded EPSP was determined manually by drawing a line extended from the baseline to the length -94-of the field EPSP. A perpendicular line extending from the baseline to the peak of the field EPSP response was drawn and the distance of that perpendicular line is the peak amplitude of the EPSP. The distance was then multiplied by a proper conversion factor to express the actual size of the response in mV. The conversion factor has a unit of mV/mm. The initial slope of the extracellularly recorded EPSPs was calculated manually using pClamp 6.0 by measuring the initial (10-90%) slope of the field EPSP and was expressed as mV/ms. The latencies (latency to peak) of the control field EPSPs (i.e., the time between the stimulus artifacts and the peak amplitude) were used as references for subsequent measurements. 8.8.2 Intracellular recordings The intracellular responses recorded using the pointed microelectrodes, nystatin-perforated patch electrodes as well as the whole cell patch electrodes have a characteristic waveform that consists of a baseline followed by a stimulus artifact and then the synaptic response (EPSP/Cs, NMDA-EPSCs, fast IPSP/Cs, and slow IPSPs). The synaptic responses were recorded at a 0.05 Hz. The peak amplitude of a synaptic response was determined by first drawing a line extending from the baseline to the length of the synaptic response. Then a perpendicular line extending from the baseline to the peak of the synaptic response was drawn and the distance of that perpendicular line is the peak amplitude of the synaptic response. This distance measured for the EPSP/Cs and the IPSP/Cs were then converted to mV/pA by multiplying the distances by a conversion factor. The conversion factor used, converts the distance to mV or pA -95-in units of mV/mm or pA/mm. The latencies (latency to peak) of the control synaptic responses (i.e., the time between the stimulus artifacts and the peak amplitude) were used as references for subsequent measurements. The initial (10 - 90%) slopes of the EPSP, IPSPs and slow IPSPs were calculated manually using pClamp 6.0 program and expressed as mV/ms. In some experiment, the kinetics of the control evoked current were compared to the currents evoked during drug application. The drug responses were scaled to the same amplitude as the control response and the kinetics of the response were compared by visual inspection. 8.9 Studies Performed Using Mass Spectroscopy Mass spectroscopic analyses of Hb-1991 and Hb-1996 were performedusing Micro Mass Quatro I (UBC, Faculty of Pharmaceutical Sciences, Mass Spect. Facility). Bisulfate, bisulfite and Hb were dissolved 5 0 % ACN/H 2 0 or H 2 0 and were injected and pumped into the mass spectrophotometer at a rate of 100 to 50 uJ/minutes. The mass spectrophotometer was operated under negative ionization with a cone voltage of 30 V. The source temperature was 150 °C and bath gas flowed at 450 l/hr. From the molecular weight and the Sigma preparation method, it is speculated that the substances with MW of 97 and 81 are bisulfate/sulfate and bisuIfite/suIfite, respectively. Standard curves for bisulfate and bisulfite were constructed using serial dilutions of bisulfite and bisulfate concentrations ranging from 4.9 p,M to 159 pM. Hb-1991 and Hb-1996 (0.1 mg/ml, each) at a concentration that is within the range used in the standard -96-curves were tested. Appropriate dilution factors were applied to find out the amount of bisulfate and bisulfite in 0.1 mM of Hb-1991 and Hb-1996 samples. 8.10 Statistics All control responses were pooled and the mean of these was taken as 100% and all controls and post-drug synaptic responses were then normalized (as a percent of the mean control response). All data were expressed as mean ± standard error of the mean (S.E.M.). Two tailed paired sample student t-test was used to determine the statistical significance of the effect of the drug on the input resistance, the change in membrane potential, and the depolarization-induced discharge of action potentials. The statistical significance of the drug on the synaptic responses at anytime was determined by analysis of variance (ANOVA) and the Duncan test, a post-hoc multiple comparison analysis. In both statistical tests, the level of significance (p) was selected to be 0.05. In other words, a probability of less than 0.05 was considered to be statistically significant. The paired sample t-test does not have the normality and equality of variance assumptions but assumes that the differences come from a normally distributed population of differences. On the other hand, the ANOVA and the Duncan test are parametric tests and have the underlying assumptions of population normality and homogeneity of variance. -97-9 EXPERIMENTAL PROTOCOL Previous studies (Ip, 1994) have shown that hemoglobin (0.01 to 1 mM) produced a depolarization, an increase in input resistance and a significant depression on synaptic transmission. Since it was found that the effects of 0.05 and 0.1 mM of hemoglobin produced significant effects, these concentrations were used in this present study. In the following sections the experimental protocol used in this study will be described. 9.1 Electrophysiological Actions of Hemoglobin-1991 In this section, the experimental protocol used to investigate the actions of Hb-1991 is described. 9.1.1 Electrophysiological actions of hemoglobin-1991 on hippocampal neurons Effects of Hb-1991 (0.1 mM) on synaptic transmission were examined using extracellular recordings. Stable field EPSPs were recorded for 15 minutes before oxygenated Hb-1991 was applied for 10 to 15 minutes. Following the Hb-1991 application, the slices were superfused with ACSF for 30 minutes. During the application of Hb-1991 and following the termination of Hb-application, synaptic transients were measured. 9.1.2 Electrophysiological actions of hemoglobin-1991 on synaptic potentials, membrane potential and input resistance in hippocampal CA1 neurons Effects of Hb-1991 (0.05 mM) on evoked EPSPs, IPSPs, membrane and -98-input resistance in hippocampal CA1 neurons were studied with nystatin perforated patch recordings. Stable membrane potential, input resistance and synaptic potentials were recorded for at least 15 minutes before oxygenated Hb-1991 was applied for 10 minutes. D.C. currents were injected into the neurons via the recording pipette to compensate for any changes in membrane potential during the application of Hb-1991. Following the Hb-1991 application, slices were superfused with ACSF for 30 minutes. During the application and washout of Hb-1991, synaptic potentials, input resistance, and membrane potential were measured. 9.1.3 Electrophysiological actions of hemoglobin-1991 on pharmacologically isolated synaptic transients in hippocampal CA1 neurons Intracellular^/ evoked synaptic potentials recorded in hippocampal CA1 neurons usually consist of multiple components. Each component is mediated by the activation of different glutamate and GABA receptors. In order to study the effects of Hb-1991 (0.05 mM) on the different components without the interference of other components, the synaptic transients were pharmacologically isolated using antagonists that block the other receptor-mediated synaptic currents. In studies in which EPSPs were recorded, picrotoxinin (0.02 mM) was used to block the GABA-A mediated response. In experiments in which the fast IPSPs were measured, APV (0.05 mM) and CNQX (0.02 mM) were used to block the non-NMDA- and the NMDA receptor- mediated EPSPs. In experiments -99-where the slow IPSPs were examined, CNQX (0.02 mM), A P V (0.05 mM), and picrotoxinin (0.02 mM) were used to block EPSPs and fast IPSPs. In these experiments, the antagonists were applied for at least 10 minutes prior to recording the control responses. Stable membrane potential, input resistance and synaptic potentials were recorded for at least 15 minutes before oxygenated Hb-1991 and antagonists were applied for 10 minutes. In order to study the effects of Hb-1991 on synaptic depression, independent of its action to depolarize the cells, D.C. currents were injected into the neurons via the recording pipette to compensate for any changes in membrane potential during drug application. Following the Hb-1991 application, slices were superfused with A C S F containing the corresponding antagonists for 30 minutes. During the application and washout of Hb-1991, synaptic transients, input resistance, and membrane potential were measured. 9.1.4 Effects of Nco-Nitro-L-arginine on the actions of Hb-1991 on evoked potentials, membrane potential and input resistance. Slices were incubated with Nco-nitro-L-arginine (0.1 mM) at least 1 hour before and throughout the experiment. In experiments where EPSPs and slow IPSPs were recorded, the slices were perfused with picrotoxinin (0.02 mM) and Nco-nitro-L-arginine containing ACSF throughout the experiments. In experiments where IPSPs were recorded, slices were perfused with CNQX (0.02 mM), A P V (0.05 mM) and Nco-nitro-L-arginine. Stable membrane potential, input resistance and synaptic transients were recorded for at least 15 minutes before oxygenated Hb-1991 (0.05 mM) and corresponding antagonists were applied for - 100-10 minutes. During depolarization, the EPSPs and IPSPs may be affected as the reversal potential of the EPSPs was approached and the reversal potential of the IPSPs were departed. Therefore, D.C. currents were injected into the neurons via the recording pipette to compensate for any changes in membrane potential during the application of Hb-1991. Following the Hb-1991 application, the slices were superfused with ACSF containing the corresponding antagonists for 30 minutes. During the application and washout of Hb-1991, synaptic transients, input resistance, and membrane potential were measured. 9.2 Electrophysiological Actions of Met-Hemoglobin and Reduced-Hemoglobin on Hippocampal CA1 Neurons 9.2.1 Effects of met-hemoglobin The effects of met-Hb (0.2 mM) were examined using nystatin-perforated patch electrodes. Stable membrane potential, input resistance and synaptic transients were recorded for 15 minutes before oxygenated met-Hb was applied for 10 to 15 minutes. Following the met-Hb application, the slices were superfused with A C S F for 30 minutes. During the application and washout of met-Hb, synaptic transients, input resistance, and membrane potential were measured. 9.2.2 Electrophysiological actions of reduced-hemoglobin on hippocampal CA1 neurons in the presence of picrotoxinin In order to study the effects of EPSCs without the interference of GABA-A - 101 -receptor-mediated fast IPSCs, GABA-A receptor-mediated fast IPSCs were blocked with picrotoxinin (0.05 mM), a GABA-A receptor channel blocker. Stable holding currents, series resistance, input resistance and synaptic transients were recorded for 15 minutes before oxygenated reduced-Hb (0.1 mM) and picrotoxinin were applied for 15 minutes. Following the reduced-Hb application, the slices were superfused with ACSF with picrotoxinin (0.05 mM) for 30 minutes. During the application and washout of reduced-Hb, synaptic transients, series and input resistances, and holding currents were measured. 9.2.3 Electrophysiological actions of reduced-hemoglobin on hippocampal CA1 neurons in the presence of DNQX and APV In order to study the effects of IPSCs without the interference of AMPA and NMDA receptor-mediated EPSCs, such EPSCs were blocked with DNQX (0.02 mM) and A P V (0.05 mM), which are non-NMDA and NMDA receptor antagonists, respectively. Stable holding currents, series resistance, input resistance and synaptic transients were recorded for 15 minutes before oxygenated reduced-Hb (0.1 mM), DNQX (0.05 mM) and A P V (0.02 mM) were applied. The drugs were applied for 15 minutes. Following the reduced-Hb application, the slices were reperfused with ACSF containing DNQX (0.02 mM) and A P V (0.05 mM) for 30 minutes. During the application and washout of reduced-Hb, synaptic transients, series and input resistance, and holding currents were measured. - 102 -9.2.4 Electrophysiological actions of reduced-hemoglobin on hippocampal CA1 neurons in the presence of picrotoxinin, DNQX and Mg2+-free medium The NMDA-mediated EPSCs were examined in the presence of DNQX (0.02 mM) and picrotoxinin (0.05 mM) in a Mg 2 +-free medium. Stable holding currents, series resistance, input resistance and synaptic transients were recorded for 15 minutes before oxygenated reduced-Hb (0.1 mM), DNQX (0.02 mM) and A P V (0.05 mM) were applied for 15 minutes. Following the application of reduced-Hb, the slices were reperfused with ACSF containing DNQX (0.02 mM) and APV (0.05 mM) for 30 minutes. During the application and washout of reduced-Hb, synaptic transients, series and input resistances, and holding currents were measured. 9.2.5 Effects of reduced-hemoglobin on extracellular EPSPs and pre-synaptic volley on CA1 neurons Since reduced-Hb depressed synaptic transmission, it was possible that its effect was produced via a depression of action potential firing in afferent fibers. Presynaptic volley is a field potential that reflects action potentials generated in axons. Hence, whether the effects of reduced-Hb on synaptic transmission were due to an effect on excitability of the afferent fibers, could be studied by examining actions on presynaptic volley. Studies examining the effects of reduced-Hb (0.1 mM and 0.2 mM) on field EPSPs and on the presynaptic volley recorded in hippocampal CA1 region were conducted. Stable evoked field EPSPs and presynaptic volleys were - 103 -recorded for at least 15 minutes before oxygenated reduced-Hb was applied for 15 minutes. Following the hemoglobin application, the slices were superfused with A C S F for 30 minutes. During the application and washout of reduced-Hb, the field EPSP and the presynaptic volley were measured. 9.2.6 Effects of reduced-hemoglobin on depolarizing pulses induced action potentials in CA1 neurons When rectangular depolarizing pulses are injected into the hippocampal neurons, those that are strong enough to depolarize the membrane potential to reach thresholds for action potential generation will naturally elicit action potentials. If reduced Hb-induced depression of synaptic transmission in CA1 neurons is via an effect on action potential generation mechanisms, it is argued that such an effect can be visualized by monitoring the depolarization-induced action potentials. Therefore, actions of the agent were examined on such action potentials. Rectangular depolarizing pulses (400 ms in duration) with three different intensities were used to examine the effects of reduced-Hb on the excitability of the neurons. The intensities of the pulses were selected such that the lowest stimulation strength give one spike, the medium strength produces 3 - 4 spikes and the highest strength produced 6 - 7 spikes. The same range of number of action potentials can be elicited consistently with each of these 3 intensities. By choosing three different stimulation intensities that are slightly above threshold for action potentials (rather than stimulations that are significantly larger than required for spike generation and induce a large number of spikes), it could be -104-possible to detect subtle changes threshold caused by the drug. The number of spikes and the amplitude of action potentials were, therefore, measured. Stable input resistance, membrane potential and depolarization pulse-mediated spiking patterns were recorded for 15 minutes before oxygenated reduced-Hb (0.1 mM) was applied for 15 minutes. Following the application of reduced-Hb, the slices were superfused with ACSF for 30 minutes. During the application and washout of reduced-Hb, input resistance, membrane potential and depolarization pulses-mediated spiking pattern were measured. 9.2.7 Effects of reduced-hemoglobin on applied agonists in CA1 neurons The effects of reduced-Hb on the postsynaptic neuronal response to bath-applied agonists were studied. Experimental protocols were similar in the AMPA, THIP, NMDA and baclofen studies. Two agonist-induced currents, similar in size, were used as control responses. These control currents were elicited by applying the agonist for 30 to 35 seconds in the superfusing medium. The agonist applications were separated by approximately 10 minutes each. Five minutes after the recovery of the second agonist-induced current, reduced-Hb (0.1 mM) was applied. At 10 minutes after the start of the application of reduced-Hb, the agonist was applied again in the presence of reduced-Hb containing ACSF for the same duration of time as in control. Following reduced-Hb application, the drugs were washed out with normal A C S F for 30 minutes. During the washout, the agonist was applied at 5 and at 30 minutes following the termination of reduced-Hb application. - 105 -9.3 Effects of Reduced- Hemoglobin and L-cysteine Since high concentrations of L-cysteine were shown to be released in ischemic conditions (Li et al., 1999; Slivka and Cohen, 1993; Landolt et al., 1992), the effects of a co-application of L-cysteine and reduced-Hb were studied. 9.3.1 Effects of L-cysteine on CA1 neurons Effects of L-cysteine (0.1 mM) on field EPSPs and the effects of L-cysteine (0.05 mM, 0.1 mM, 0.2 and 0.4 mM) on holding currents and input resistance of CA1 neurons were examined using extracellular field recordings and whole-cell patch recordings, respectively. Stable control responses were recorded for 15 minutes before oxygenated L-cysteine was applied for 15 minutes. Following the L-cysteine application, the slices were superfused with A C S F for 30 minutes. During the application and washout of L-cysteine, synaptic transients, input resistance, and membrane potential were measured. 9.3.2 Effects of L-cysteine and reduced-hemoglobin on field EPSPs in CA1 region The effects of the co-application of L-cysteine (0.1 mM) and reduced-Hb (0.1 mM) on field EPSPs in CA1 region were examined using extracellular field recordings. Stable field EPSP responses were recorded for 15 minutes before oxygenated L-cysteine and reduced-Hb were applied. The L-cysteine and reduced-Hb were applied simultaneously for 15 minutes. Following the coapplication of the agents, the slices were superfused with ACSF for 30 minutes. During the application and washout of the agents, synaptic transients, input resistance, and holding currents were measured. - 106-9.3.3 L-cysteine- and reduced-hemoglobin-induced depolarization In preliminary experiments, using whole cell patch clamp recordings, results have shown that the co-application of L-cysteine (0.1 mM) and reduced-Hb (0.1 mM) produced depolarizations that peaked at about 1 to 1.5 minutes following their applications. Based on this information, l-V curves for the inward current induced by L-cysteine and reduced-Hb were constructed. Two l-V control curves were constructed during the application of L-cysteine (0.1 mM). Following the washout of L-cysteine for 15 minutes, reduced-Hb and L-cysteine were applied simultaneously. During the peak of the inward current (1 to 1.5 minutes following the application), the same voltage step protocol was used to construct an IV curves. 9.3.4 APV, DNQX and L-cysteine and reduced hemoglobin Since L-cysteine-like molecules had been previously reported to activate glutamate receptors (Watkins and Evans, 1981), experiments were performed to investigate the effects of glutamate antagonists, namely, 1) A P V (0.05 mM), 2) DNQX (0.02 mM), and 3) APV (0.05 mM) and DNQX (0.02 mM), on the L-cysteine- (0.1 mM) and reduced-Hb- (0.1 mM) mediated inward current. The control response was induced by coapplying L-cysteine and reduced-Hb. In each experiment, two controls (separated by 10 minutes of A C S F washouts) were recorded prior to the drug treatments. Slices were then superfused with APV for at least 10 minutes, followed by coapplication of L-cysteine and reduced-Hb in the presence of APV. The drugs were then washed out with A C S F for 15 minutes and L-cysteine and reduced-Hb were applied again -107-to record a recovery of the response. DNQX was then applied for 10 minutes, followed immediately by the coapplication of L-cysteine and reduced-Hb in the presence of DNQX. Subsequently, both DNQX and APV were coapplied for at least 10 minutes. This was then followed by the coapplication of L-cysteine and reduced-Hb in the presence of APV and DNQX for at least 10 minutes. The experiment was then concluded by an ACSF washout (approximately 1 hour), and the subsequent recovery of L-cysteine and reduced-Hb induced current. 9.3.5 Ca2+-free medium and L-cysteine- and reduced-hemoglobin-induced inward current Experiments were conducted to investigate whether the effect of L-cysteine and reduced-Hb was due to a calcium-dependent and TTX-sensitive process. Therefore, experiments were conducted in the absence or the presence of a Ca 2 +-free superfusing medium containing TTX (0.1 uM). Two control responses induced by the coapplication L-cysteine (0.1 mM) and reduced-Hb (0.1 mM) with similar amplitude were recorded in the presence of ACSF. Slices were then superfused with the Ca 2 +-free medium containing TTX for at least 15 minutes prior to the co-application of L-cysteine and reduced hemoglobin. 9.4 Studies on the Differences between Hemoglobin-1991 and Hemoglobin-1996 Samples During a set of preliminary experiment in which the effects of Hb-1996 on field EPSPs were examined, it was found that unlike the Hb-1991, Hb-1996 did - 108-not have any significant effects of the field EPSPs. The differences of Hb-1991 and Hb-1996 were further studied. 9.4.1 Effects of hemoglobin-1996 on field EPSPs The effects of Hb-1996 (0.5 mM) on field EPSPs recorded in hippocampal CA1 region were studied. Stable evoked EPSPs were recorded for 15 minutes before oxygenated Hb-1996 was applied for 15 minutes. Following the Hb-1996 application, the slices were superfused with ACSF for 30 minutes. During the application and washout of the Hb-1996, the synaptic transients were measured. In some slices, Hb-1991 (0.5 mM) was applied following the 30 minutes of washout of Hb-1996. The Hb-1991 was applied for 15 minutes and the slices were superfused with ACSF for 30 minutes. During the application and washout of Hb-1991, the synaptic transients were measured. 9.4.2 Effects of dialyzed hemoglobin-1991 on field EPSPs In this study, Hb-1991 was dialyzed in dialyzing tubing at 4°C with 2 to 4 L of distilled water for 4 hours. The effects of dialyzed Hb-1991 (0.01 mM) on field EPSP s recorded in hippocampal CA1 region were then studied. Stable evoked EPSPs were recorded for 15 minutes before oxygenated dialyzed Hb-1991 was applied for 15 minutes. Following the Hb-1991 application, the slices were superfused with ACSF for 30 minutes. During the application and washout of the drug, the synaptic transients were measured. In some slices, Hb-1991 was applied following the washout of the dialyzed Hb-1991. The Hb-1991 was applied for 15 minutes and the slices were perfused with A C S F for 30 minutes. - 109-During the application and washout of Hb-1991, the synaptic transients were measured. 9.4.3 Mass spectroscopic analysis of hemoglobin-1991 and hemoglobin-1996 Mass spectroscopy was used to study the differences between the 1991 and 1996 hemoglobin samples. In preliminary experiments results suggested that there were differences in the amount of molecules with molecular weight of 97 and 83 in Hb-1991 and Hb-1996. After contacting Sigma, it was postulated that the possible contaminant in Hb-1991 may be bisulfate. Therefore, known concentrations of bisulfate and bisulfite solutions were prepared as standard, to estimate the concentration of bisulfate and bisulfite present in the Hb-1991 and Hb-1996 samples. 9.4.4 Effect Of Bisulfate And Bisulfite On Whole Cell Patch Recorded EPSCs Effects of bisulfate and bisulfite were examined using whole cell patch electrode recordings. Cumulative dose-response curve of the effects of bisulfate or bisulfite on evoked EPSCs were constructed by applying increasing concentrations of bisulfate (0.125, 0.25, 0.5, 1, 2 and 3 mM) or bisulfite (0.25, 0.5, and 1) to the hippocampal slices. EPSCs were recorded in the presence of picrotoxinin (0.05 mM), a GABA-A receptor channel blocker. Stable holding currents, series resistance, input resistance and synaptic transients were recorded for 15 minutes in picrotoxinin containing ACSF before oxygenated -110-bisulfate and picrotoxinin or bisulfite and picrotoxinin were applied. Each dose of the agents was applied for 10 minutes. Following the highest dose of bisulfate or bisulfite application, the slices were superfused with picrotoxinin-containing A C S F for 30 minutes. During application and washout of drugs, synaptic transients were measured. 9.4.5 Qualitative test forthe presence of NH4* in Hb-1991 A relatively simple qualitative test was used to test for the presence of N H 4 + in Hb-1991 sample. Concentrated NaOH solution (1N) was placed in N H 4 + (9 mM and 3 mM), Hb-1991 (0.3mM) and Hb-1996 (0.3 mM) solutions. Solutions were then heated up under a bunsen burner. A litmus paper was placed on the end of the test tubes and the gas produced in each samples were captured by the litmus paper. The color of the litmus paper and the intensity of the change in color were assessed with visual inspection. 9.5 Possible Contamination of Reduced-Hemoglobin After reviewing the literature, it was found that very few investigators who used the reduction method described by Martin et al. (1985), had done a control whereby dithionite alone was dialyzed using the same procedure. Therefore, experiments were conducted to study the effects of dialyzed dithionite on field EPSPs. 9.5.1 Effects of dialyzed dithionite on field EPSPs The effects of dialyzed dithionite on field EPSPs recorded in the hippocampal CA1 region were studied. Stable evoked field EPSPs were - I l l -recorded for 15 minutes before oxygenated dialyzed dithionite was applied for 15 minutes. Following the dithionite application, the slices were superfused with A C S F for 30 minutes. During the application and washout of dialyzed dithionite, the synaptic transients were measured. 9.5.2 Effects of reduced-hemoglobin following prolong (18 hours) dialysis Reduced-Hb (0.1 mM) was prepared using similar procedure as that described by Martin et al. (1985). However, the dialysis time was increased from 2 hours to 18 hours. The effects of reduced-Hb (18 hour) on field EPSPs recorded in hippocampal CA1 region were studied. Stable evoked field EPSPs were recorded for 15 minutes before oxygenated Hb (18 hour) was applied for 15 minutes. Following the application of the reduced-Hb (18 hour), the slices were superfused with A C S F for 30 minutes. During application and washout, the synaptic transients were measured. 9.5.3 Effects of coapplication of dialyzed dithionite and L-cysteine on field EPSPs The effects of dialyzed dithionite and L-cysteine on field EPSPs recorded in the hippocampal CA1 region were studied. Stable evoked field EPSPs were recorded for 15 minutes before oxygenated dialyzed dithionite and L-cysteine was coapplied for 15 minutes. Following dithionite application, the slices were superfused with ACSF for 30 minutes. During the application and washout of the agents, synaptic transients were measured. - 112-9.5.4 Mass spectroscopic analysis of reduced-hemoglobin Mass spectroscopy was used to analyze the composition of the reduced-Hb solution. In addition to Hb chains and hemin, considerable amount of molecules with molecular weight of 81 and 97 were found to be present in the reduced-Hb sample. From the reaction products of dithionite and water, it is postulated that the molecules corresponding with the molecular weights are bisulfate and bisulfite. Serial dilutions of bisufate or bisulfite were made and were subjected to mass spectroscopic analysis. The area under the curves for the corresponding signals and concentrations of bisulfate or bisulfite were plotted resulting in the generation of the standard curves. The concentrations of bisulfate and bisulfite left in the reduced-Hb were then estimated by using the standard curves and the area under the curves of signal produced by reduced-Hb sample. Standard curves were repeated for each experiment. 9.6 Effects Of The Components Of Hemoglobin On Field EPSPs Hemoglobin is composed of a number of molecules, i.e., iron, hemin and globin. The effects of ferrous chloride and hemin on field EPSPs were studied. 9.6.1 Effects of iron on field EPSPs The effects of ferrous chloride (0.05 mM and 0.1 mM) on field EPSP s recorded in the hippocampal CA1 region were studied. Stable evoked EPSPs were recorded for 15 minutes before oxygenated ferrous chloride was applied for 15 minutes. The slices were superfused with ACSF for 30 minutes. During application and washout of the agents, synaptic transients were measured. - 113 -Since ferrous chloride changes the pH of the solution from 7.4 to 7.35, control solution with pH adjusted to 7.35 were used to access the effects of pH on field EPSPs. During the application and washout of pH 7.35 control and ferrous chloride, the field EPSPs were recorded. 9.6.2 Effects of hemin on field EPSPs Since it is very difficult to dissolve hemin in ACSF, hemin was first dissolved in NaOH. The solution was then dispersed in an albumin-containing ACSF. The final solution gives hemin : albumin ratio of 10:1. The effects of the carriers, i.e., NaOH and albumin, on field EPSPs were studied. Stable evoked EPSPs were recorded for 15 minutes before the oxygenated carrier solution was applied for 15 minutes. Following the NaOH + albumin application, the slices were superfused with ACSF for 30 minutes. Hemin (0.5 mM) was then applied for another 15 minutes followed by a washout of 30 minutes. During the application and washout of carrier and hemin, the synaptic transients were measured. - 1 1 4 -10 RESULTS The results are separated into 5 sections. The first, second and third sections report the electrophysiological actions of Hb-1991, reduced-Hb and reduced-Hb and L-cysteine, respectively, on CA1 neurons. The fourth and fifth sections investigate the presence of contaminants in Hb-1991 and those that are introduced during the preparation of the reduced-Hb. 10.1 Electrophysiological Actions Of Hb-1991 On Hippocampal CA1 Pyramidal Neurons In preliminary experiments, the actions of rat hemoglobin (0.05 mM, Sigma) on hippocampal CA1 pyramidal neurons recorded using conventional sharp intracellular microelectrode recordings were compared with those of bovine hemoglobin (0.05 mM, Sigma). It was found that the agent from both sources produced a depolarization of the CA1 neurons associated with an increase in the input resistance. In addition, evoked EPSPs, fast IPSPs and slow IPSPs were suppressed by rat and bovine hemoglobin (Ip, 1994). Since bovine hemoglobin is much more economical, and there are no known differences between rat and bovine hemoglobins, bovine hemoglobin was used in subsequent experiments. Moreover, Ip (1994) has shown that these effects were dose-dependent (Hb concentrations of 0.01, 0.05, 0.1, 0.5 and 1.0 mM). In this study, concentrations of 0.1 and 0.05 mM were used to study the effects of hemoglobin on synaptic transmission. - 115 -The effects of Hb-1991 on hippocampal CA1 neurons were studied using extracellular field recordings, and intracellular recordings with nystatin perforated-patch electrodes. Studies using extracellular field recordings were carried out in a total of 20 slices. Nystatin perforated-patch electrode recordings were made in a total of 32 neurons with a resting membrane potential of -58.6 ± 0.4 mV and input resistance of 154 ± 9.1 MQ, respectively. 10.1.1 Effects of hemoglobin-1991 on field EPSPs recorded in hippocampal CA1 pyramidal neurons In extracellular field recordings, Hb-1991 (0.05, 0.1, 0.5 and 1.0 mM) was found to produce a dose-dependent depression of the slope of the field EPSPs (Ip, 1994). In the current studies, Hb-1991 (0.1 mM) produced a depression of the field EPSP (slope of field EPSP as a % of control: 75.3 ± 1.2; n = 3, Fig. 10.1.1) followed by a recovery (slope of field EPSP as a % of control: 94.6 + 5.3) at 30 minutes of washout. These results are consistent with those reported by Ip (1994). 10.1.2 Effects of hemoglobin-1991 on the membrane potential in hippocampal CA1 pyramidal neurons: In nystatin perforated-patch electrode recordings, 0.05 mM Hb-1991 induced a 2.7 ± 0.2 mV (n = 6; Fig. 10.1.2 A and Fig. 10.1.2 B) depolarization. The time courses of the onset of the depolarization as well as the recovery from the depolarization were similar to those measured with conventional pointed intracellular microelectrodes as reported by Ip (1994). In addition, in 1 out of 6 neurons, an after-hyperpolarization was observed following the depolarization. A - 116-Fig. 10.1.1. Effects of hemoglobin-1991 on evoked field EPSPs recorded in the CA1 field. Hemoglobin-1991 (Hb-1991; 0.1 mM) was applied for 15 min and the effects of the drug on the slope of the evoked EPSPs were plotted in the graph (n = 3). Data points were represented as mean ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) in this graph indicate that the values are significantly different frorrrtheir respective pre-drug controls. Note that Hb-1991 reversibly depressed the field EPSPs. - 1 1 7 -A Hb (0.05 mM) £ 10 m V B 5 min -63. mV-Control trntft -61 mV-Hb (0.05 mM) ttmrr Post-Hb -63mV.^. No current injection Current (nA) -0.3 -0.2 -0.1 0.0 0.1 -0.02 nA of current \5 mV 2 min i i i i i t i i i i ' i i 1 ; ' ' ' • Control • Hb(50uM) -40 CD ^ " 5 0 | CD -60 "§ CD -70 % < -80 ~ Fig. 10.1.2. Effects of Hb-1991 on the membrane potential and input resistance in CA1 neurons of hippocampal slices recorded with nystatin perforated patch electrodes. Hb-1991 (Hb; 0.05 mM) was applied for 10 min. The input resistance was monitored by injecting hyperpolarizing current pulses (0.05 nA for 200 ms duration) throughout the experiment. A illustrates the Hb-1991-induced depolarization while B shows the raw data of input resistance and membrane potential in control (Control), 5 min in Hb-1991 (Hb) and 15 min post- Hb-1991 (Post-Hb). C shows current-voltage curves of a neuron with resting membrane potential o f - 57 mV. In B (shown by the parenthesis below the trace) and C, the depolarization induced by Hb-1991 was compensated by injecting D.C. current into the CA1 neuron. The holding currents of-0.02 nA in C were subtracted from each test pulse for current-voltage curves. Note that there is no change in the slope resistance in C. - 118-high concentration of hemoglobin (0.5 mM) also depolarized the CA1 neurons, but the depolarization was much smaller (5 to 7 mV; n = 5) than that observed with pointed electrodes (Ip, 1994). 10.1.3 Effects of hemoglobin-1991 on the,input resistance in hippocampal CA1 pyramidal neurons With nystatin perforated-patch electrode recordings, 0.05 mM Hb-1991 did not have a significant effect on the input resistance (input resistance in hemoglobin as a % of control: 101.3 + 2.8, n = 6) of the CA1 neurons (Fig. 10.1.2B and 10.1.2C). In addition, a high concentration of Hb-1991 (0.5 mM) did not produce a consistent effect on the input resistance (n = 5). 10.1.4 Effects of hemoglobin-1991 on the evoked synaptic responses in hippocampal CA1 pyramidal neurons The drug effects on synaptic transients were examined and the records were quantified while current-clamping the neurons to pre-hemoglobin membrane potentials. In nystatin perforated-patch recordings, 0.05 mM Hb-1991 significantly suppressed the fast IPSP (response amplitude as a % of control: 54.9 ± 7.5, n = 6; Fig. 10.1.4A and 10.1.4B) and the fast IPSP recovered to control level when measured 30 minutes after the termination of the Hb-1991 application. However, the EPSP was not significantly affected by 0.05 mM Hb-1991 (EPSP slope and amplitude as a % of control: 104.7 ± 8.7 and 103.9 ± 8.7, n = 6, respectively; but see experiments carried out in the presence of picrotoxinin). - 119-A Control 10 m V 100 ms B 0 10 20 30 40 50 Time (min) Fig. 10.1.4. Effects of Hb-1991 on synaptic responses in CA1 neurons. Hb-1991 (Hb; 0.05 mM) was applied for 10 min. In A, records of synaptic responses shown were taken during control (Control), 10 min in Hb-1991 (Hb) and 30 min after termination of Hb-1991 application (Post-Hb) from a CA1 neuron. Records were taken during a current-clamp of the neuron at -60 mV which was the resting membrane potential. B illustrates the effect of Hb (0.05 mM) on the fast IPSP (n = 6). All points on the graph represent the means ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) indicate that the fast IPSPs are significantly different from the pre-drug control. Note that Hb-1991 suppressed the fast IPSPs without significantly changing the amplitudes of the EPSPs. - 120-10.1.5 Effects of hemoglobin-1991 on the evoked EPSPs and slow IPSPs in hippocampal CA1 pyramidal neurons in the presence of picrotoxinin In order to study the effects of Hb-1991 on the EPSP and the slow IPSP in the absence of the contamination of the GABA-A-mediated fast IPSP, nystatin perforated-patch electrode recordings of CA1 neurons were made in slices superfused with picrotoxinin (0.02 mM), a GABA-A antagonist, throughout the experiment. In these slices, 0.05 mM Hb-1991 significantly suppressed the E P S P (EPSP slope and amplitude as a % of control: 70.8 ± 4.4, and 57.3 ± 8.1, n = 6; Fig. 10.1.5A and 10.1.5B) and the slow IPSPs (response amplitude as a % of control: 32.9 + 4.8, n = 6; Fig. 10.1.5A and 10.1.5B). The EPSPs and the slow IPSPs recovered to control levels when measured 30 minutes after the termination of Hb application. 10.1.6 Effects of hemoglobin-1991 on hippocampal CA1 pyramidal neurons in the presence of CNQX and APV Hemoglobin-induced suppression of fast and slow IPSPs may be produced via the suppression of EPSP at glutamatergic synapses (either recurrent or feed forward inhibition); to minimize this possibility, glutamatergic transmission was blocked pharmacologically by superfusing the slices with CNQX (0.02 mM), a non-NMDA receptor antagonist, and APV (0.05 mM), a NMDA receptor antagonist. The blockade of the EPSP allowed the measurement of the slope of the fast IPSP. In the presence of CNQX and APV, fast IPSPs were, therefore, recorded with nystatin perforated-patch electrodes. It was found that 0.05 mM Hb-1991 significantly suppressed the fast IPSP (fast - 121 -A Control Hb Post-Hb |5mV 200 ms Control Hb Post-Hb 50 ms B • EPSP A sIPSP Picro Picro + Picro 0 10 20 30 40 50 Time (min) Fig. 10.1.5. Effects of Hb-1991 on evoked EPSPs and slow IPSPs. Hb-1991 (Hb; 0.05 mM) was applied for 10 min. The slices were superfused with picrotoxinin (0.02 mM) throughout the experiments to suppress the fast IPSPs. In A, records of synaptic responses shown were taken during control (Control), 10 min in Hb-1991 (Hb) and 30 min after termination of Hb-1991 application (Post-Hb) from a CA1 neuron. Records were taken during a current-clamp of the neuron at -60 mV which was the resting membrane potential. B illustrates the effect of Hb-1991 (0.05 mM) on the EPSP slope (•) and slow IPSPs amplitude (A ) (n = 6). All points on the graph represent the means ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) indicate that the responses are significantly different from the pre-drug control. Note that Hb-1991 suppressed the EPSPs and the slow IPSPs. - 122-IPSP slope and amplitude as a % of control: 35.7 ± 6.6 and 51.4 ± 4.6, n = 6; Fig. 10.1.6A and 10.1.6B). The fast IPSP recovered to control levels in about 30 minutes after the termination of the Hb application (Fig. 10.1.6A and 10.1.6B). The neurons were depolarized by 3.2 ± 0.4 mV (n = 6) by the agent. Effects of hemoglobin-1991 on hippocampal CA1 pyramidal neurons in the presence of CNQX, APV and picrotoxinin In some experiments, in addition to CNQX (0.02 mM) and A P V (0.05 mM), picrotoxinin (0.02 mM) was added to the superfusing medium in order to isolate the slow IPSP. In these slices, when nystatin perforated-patch recordings were made, 0.05 mM Hb-1991 significantly suppressed the slow IPSP (slope and amplitude as a % of control: 72.5 ± 7.0 and 57.6 ± 3.5, n = 6; Fig. 10.1.7A and 10.1.7B). The slow IPSP recovered to control level when measured 30 minutes after the termination of the Hb-1991 application (Fig. 10.1.7A and 10.1.7B). In the presence of CNQX, APV and picrotoxinin, 0.05 mM Hb-1991 continued to induce a 2.8 ± 0.3 mV (n = 6) depolarization. 10.1.7 Effects of Nco-nitro-L-arginine (0.1 and 0.5 mM) on the actions of hemoglobin In order to examine whether the actions of Hb-1991 were related to its NO scavenging property, both nystatin perforated-patch electrode- recordings were made in CA1 neurons in slices that were incubated with L-NOARG (0.1 mM) for at least 1 hour before hemoglobin was applied. In the presence of L-NOARG, the Hb-1991- (0.1 mM) induced depolarization, suppression of evoked synaptic 123 A Control Post - Hb 2 mV 200ms B 0 flPSP amplitude • flPSP slope c o O 100 CO CO CO OL CO Q_ CO CD CO c o Q . CO CD 50 0 i i i 10 20 30 40 Time (min) Fig. 10.1.6. Effects of Hb-1991 on evoked fast IPSPs. Hb-1991 (Hb; 0.05 mM) was applied for 10 min. The slices were superfused with 0.02 mM CNQX and 0.05 mM APV throughout the experiments to suppress the EPSPs so that the amplitude and the slope of the fast IPSPs can be measured. In A, records of synaptic responses shown were taken during control (Control), 10 min in Hb-1991 (Hb) and 30 min after termination of Hb-1991 application (Post-Hb) from a CA1 neuron. Records were taken during a current-clamp of the neuron at -57 mV which was the resting membrane potential. B illustrates the effect of Hb-1991 (0.05 mM) on the fast IPSP slope (A) and amplitude (•) (n = 6). All points on the graph represent the means ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) indicate that the responses are significantly different from the pre-drug control. Note that Hb-1991 suppressed the fast IPSPs slope and amplitude. - 124-C o O CD CO CO Q_ CO Q_ GO 100 75 co c o C L (/) Q) • s IPSP amplitude A s IPSP slope ^ 50 H 25 -J C N Q X + C N Q X + CNQX + A P V + Picro A P V + A P V + Picro Picro + Hb — i 1 1 1 r 0 10 20 30 40 50 Time (min) Fig. 10.1.7. Effects of Hb-1991 on evoked slow IPSPs. Hb-1991 (Hb; 0.05 mM) was applied for 10 min. The slices were superfused with 0.02 mM CNQX, 0.05 mM APV and 0.02 mM picrotoxinin (Picro) throughout the experiments to suppress the EPSPs and the fast IPSPs. In A, records of synaptic responses shown were taken during control (Control), 10 min in Hb-1991 (Hb) and 30 min after termination of Hb-1991 application (Post-Hb) from a CA1 neuron. Records were taken during a current-clamp of the neuron at -59 mV which was the resting membrane potential. B illustrates the effect of Hb-1991 (0.05 mM) on the slow IPSP slope (A) and amplitude (•) (n = 6). All points on the graph represent the means ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) indicate that the responses are significantly different from the pre-drug control. Note that Hb-1991 suppressed the slow IPSPs slope and amplitude. - 125 -transients on CA1 pyramidal neurons were not occluded (Table. 10.1.7 A and B.), indicating that these actions were unrelated to the NO scavenging property of hemoglobin. These findings were similar to those reported by Ip (1994). 10.2 Effects of Reduced-Hemoglobin on Hippocampal CA1 Pyramidal Neurons Since hemoglobin samples purchased from Sigma contained a mixture of oxyhemoglobin and methemoglobin (met-Hb), whether the effects of Hb-1991 are due to met-Hb or reduced-Hb is not known. Therefore the effects of met-Hb and reduced-Hb were studied. 10.2.1 Effects of methemoglobin on hippocampal CA1 pyramidal neurons Methemoglobin (0.2 mM) did not produce a significant effect on the membrane potential, input resistance, evoked EPSPs or IPSPs (Fig. 10.2.1 A and 10.2.1B, n = 5). Since met-Hb did not produce a significant depression of synaptic transmission on hippocampal CA1 pyramidal neurons, it was possible that the depression of Hb-1991 was due to reduced-Hb; therefore, the effects of reduced-Hb on CA1 neurons were studied. The effects of reduced bovine hemoglobin on hippocampal CA1 neurons were studied with whole cell-patch electrode recordings and extracellular field recordings in a total of 61 neurons and 12 slices, respectively. The resting membrane potentials and the input resistance of the neurons recorded with whole cell patch electrodes were 60 ± 3 mV and 153 ± 12.1 MQ, respectively. - 126-L-NOARG Hb-1991 fast IPSP Depolarization Input resistance (%) n (mM) (mM) (%) (mV) 0 0.05 54.9 ±7.5 2.7 ±0.2 101.3 ± 2.8 6 0.1 0.05 49.0 ±6.1 2.5 ±0.3 99.3 ±2.0 4 Table 10.1.7.A. Effects of Nco-nitro-L-arginine on the actions of Hb-1991 on evoked fast IPSPs, membrane potential and input resistance. Slices were incubated with 0.1 mM Nco-nitro-L-arginine (L-NOARG) throughout the experiment, starting at least 1 hr before the application of Hb-1991. Data from cells recorded with nystatin perforated patch electrodes are shown. Note that L-NOARG did not have an effect on Hb-1991-induced depression of fast IPSP, and depolarization. L-NOARG (mM) Hb-1991 (mM) EPSP ( %) slow IPSP (%) n 0 0.05 70.8 ±4.4 32.9 ±4.8 6 0.1 0.05 64.7 ±5.3 40.9 ±7.2 4 Table 10.1.7.B. Effects of Nco-nitro-L-arginine on the actions of Hb-1991 on evoked EPSPs and slow IPSPs. Slices were incubated with 0.1 mM Nco-nitro-L-arginine (L-NOARG) throughout the experiment, starting at least 1 hour before the application of Hb-1991. The EPSPs and slow IPSPs were recorded in the presence of picrotoxinin (0.02 mM). Note that L-NOARG did not have an effect on Hb-1991-induced depression of E P S P and slow IPSP. - 127-A Control met-Hb B 5 mV 200 ms c o O CO (Ii CO CD " O 3 140 120 100 80 £ 60 Q. < 40 CD </) c o CL Vi CD or 20 c o O Q_ CO D_ LU CO Fig. 10.2.1. Effects of met-hemoglobin on evoked EPSPs and fast IPSPs. Met-hemoglobin (met-Hb; 0.2 mM) was applied for 15 min and the effects of the drug on EPSPs and IPSPs examined. In A, records of synaptic transients were taken during control (Control) and 15 min in met-hemoglobin (0.2 mM; met-Hb). In B, the histograms show the effects of met-hemoglobin on the amplitude of EP SP s and fast IPSPs (n = 5). ANOVA was used to test for statistical differences. Data are represented as the mean ± S.EM. ANOVA was performed (p < 0.05). Note that met-hemoglobin did not have a significant effect on the synaptic transients. - 128-10.2.2 Effects of reduced-hemoglobin on evoked EPSCs in the presence of picrotoxinin In order to study the effects of reduced-Hb on EPSCs in the absence of the contamination of GABA-A receptor-mediated fast IPSC, whole cell-patch recordings were made in the presence of picrotoxinin (0.02 mM). Reduced-Hb (0.1 mM) significantly depressed the evoked EPSCs (response amplitude as a % of control: 62.3 ± 5.5, n = 5, Fig. 10.2.2). The EPSCs showed recovery following 30 minutes after the termination of reduced-Hb application. The kinetics of the EPSCs were not altered during the reduced-Hb-induced depression because when the amplitude of the depressed EPSC was scaled up to the peak amplitude of the control EPSC, (as illustrated in Fig. 10.2.2), there was little difference in the shape of the EPSC. 10.2.3 Effects of reduced-hemoglobin on evoked IPSCs in the presence of DNQXandAPV In order to study the effect of reduced-Hb on GABA-ergic transmission, IPSCs were pharmacologically isolated from the EPSCs with DNQX (0.02 mM) and A P V (0.05 mM). Reduced-Hb (0.1 mM) significantly depressed the evoked IPSCs (response amplitude as a % of control: 75.0 ± 3.5, n = 6, Fig. 10.2.3). The IPSCs recovered after 30 minutes after the termination of reduced-Hb application. The kinetics of the IPSC were not altered during the reduced-Hb-induced depression because when the amplitude of the depressed IPSC was scaled up to the peak amplitude of the controls (as illustrated in Fig. 10.2.3), there was little difference in the shape of the IPSC. - 129-A Control Hb Post-Hb Scaled 0 10 20 30 40 50 60 Time (min) Fig. 10.2.2. Effects of reduced-hemoglobin on evoked EPSCs. Reduced-Hb (Hb; 0.1 mM) was applied for 15 min. The slices were superfused with 0.05 mM picrotoxinin (Picro) throughout the experiments to suppress the IPSCs so that the amplitude of the EPSCs can be measured. In A, the records of synaptic responses shown were taken during control (Control), 10 min in reduced-Hb (Hb), 30 min after the termination of reduced-Hb application (Post-Hb) as well as scaled response of 10 min in reduced-Hb overlapping the control response (Scaled). B shows the effects of reduced-Hb (0.1 mM) on the amplitude of the EPSCs in graphs (n = 5). Data points were represented as mean ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) in this graph indicate that the values are significantly different from their respective pre-drug controls. Note that reduced-Hb depressed the amplitude of EPSCs. - 130-A Control Hb Post-Hb Scaled I 20 pA 50 ms B Hb + DNQX + APV DNQX + APV 10 20 30 40 50 60 Time (min) Fig. 10.2.3. Effects of reduced-hemoglobin on evoked fast IPSCs. Reduced-Hb (Hb; 0.1 mM) was applied for 15 min. The slices were superfused with 0.02 mM DNQX and 0.05 mM A P V throughout the experiments to suppress the EPSCs so that the amplitude of the fast IPSCs can be measured. In A, the records of synaptic responses shown were taken during control (Control), 10 min in reduced-Hb (Hb), 30 min after the termination of reduced-Hb application (Post-Hb) as well as scaled response of 10 min in reduced-Hb overlapping the control response (Scaled). B shows the effects of reduced-Hb (0.1 mM) on the amplitude of the fast IPSCs in graphs (n = 6). Data points were represented as mean ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) in this graph indicate that the values are significantly different from their respective pre-drug controls. Note that reduced-Hb depressed the amplitude of IPSCs. - 131 -10.2.4 Effects of reduced-hemoglobin on evoked NMDA-EPSCs in the presence of DNQX, picrotoxinin, and Mg2*-free ACSF Evoked NMDA responses were isolated by applying DNQX (0.02 mM) and picrotoxinin (0.05 mM) in Mg 2 +-free ACSF. The recording electrodes also contained 5 mM QX-314 in order to block both intracellular spikes and GABA-B receptor-mediated slow IPSCs (Nathan et al., 1990). Under these conditions, inward synaptic currents could be recorded when stimulations were applied to the s. radiatum. The inward current was sensitive to A P V (0.05 mM) as well as to 2 mM MgCI 2, suggesting that they were NMDA-EPSCs. Reduced-Hb (0.1 mM) significantly depressed the evoked NMDA-EPSC (response as a % of control: 65.5 ± 5.3; n = 6, Fig. 10.2.4). The evoked NMDA-EPSCs showed a recovery when measured at 30 min following the termination of Hb application. 10.2.5 Effects of reduced-hemoglobin on extracellular field EPSPs and presynaptic volley Since both evoked EPSCs and evoked IPSCs were depressed in the presence of reduced-Hb, the depression of synaptic transmission mediated by reduced-Hb may be due to interfering with the firing of action potentials. The presynaptic volley is a field potential generated by action potentials firing from afferent fibers; therefore, the relationships between the amplitude of field E P S P and the presynaptic volley may give some indications whether reduced-Hb interferes with action potentials. It was found that reduced-Hb (0.1 and 0.2 mM) depressed both field EPSP without depressing the presynaptic volley (n = 6, for each, Fig. 10.2.5). - 132-A Control B Hb Post-Hb 100 pA 400 ms Time (min) Fig. 10.2.4. Effects of reduced-hemoglobin on evoked NMDA-EPSCs. Reduced-hemoglobin (Hb; 0.1 mM) was applied for 15 min. The slices were superfused with 0.02 mM DNQX and 0.05 mM picrotoxinin in a Mg 2 +-free A C S F throughout the experiments to suppress the AMPA-mediated EPSCs and the fast IPSCs, so that the NMDA-mediated EPSCs can be measured. Recording pipettes contained solution with QX-314 to block action potentials and the slow IPSCs. In A, the records of synaptic responses shown were taken during control (Control), 10 min in reduced-Hb (Hb), and 30 min after the termination of reduced-Hb application (Post-Hb). B, shows effects of reduced-Hb (0.1 mM) on the amplitude of the NMDA-mediated EPSCs in graphs (n = 6). Data points were represented as mean ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) in this graph indicate that the values are significantly different from their respective pre-drug controls. Note that reduced-Hb depressed the amplitude of NMDA-EPSCs. In addition, a recovery was observed following 30 min of washout. - 133 -A Control Hb (0.2 mM) Post-Hb B1 B2 0.4 mV 10 ms c o O Ui ro to 0i in c o Q. CO CD 0) TJ "a. E < 140 120 100 80 60 40 20 0 -20 • field EPSP • presynaptic volley Hb(0.1 mM) T T 10 20 30 40 Time (min) 50 ~1 60 c o O CO CO co CO CD CO c o Q. CO o CD XI "5. E < 140 120 100 80 60 40 20 0 -20 field EPSP presynaptic volley Hb (0.2 mM) T T 10 20 30 40 Time (min) 50 60 Fig. 10.2.5. Effects of reduced-hemoglobin on field EPSPs and presynaptic volley Reduced-hemoglobin (Hb; 0.1 mM and 0.2 mM) was applied for 15 min. In A, the records of synaptic responses shown were taken during control (Control), 10 min in reduced-Hb (Hb, 0.2 mM), and 30 min after the termination of reduced-Hb application (Post-Hb). In B1, effects of reduced-Hb (0.1 mM) on the amplitude of the field EPSP (•) and the corresponding presynaptic volley (•) were shown in graphs (n = 6). B2 shows the effects of reduced-Hb (0.2 mM) on the amplitude of the field E P S P (•) and the corresponding presynaptic volley (•; n = 6). Data points were represented as mean ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) indicate that the values are significantly different from their respective pre-drug controls. Note that reduced-Hb depressed the amplitude of the field EPSPs but not of the presynaptic volley. - 134 -10.2.6 Effects of reduced-hemoglobin on depolarizing pulse-induced action potential in the presence of DNQX, APV, and picrotoxinin containing ACSF The effects of reduced-Hb on action potentials were further investigated using whole cell-patch recordings in the presence of DNQX (0.02 mM), A P V (0.05 mM) and picrotoxinin (0.05 mM). Three different D.C. depolarizing current pulses (0.5 to 1 nA; 400 ms) were injected alternatively at 0.05 Hz into the cell via the recording electrodes so that the pulses gave 1, 3 to 4 and 6 to 8 action potentials. After 10 minutes of stable controls, Hb was applied for 15 minutes. Reduced-Hb (0.1 mM) did not affect the number of action potentials generated by these depolarizing pulses (n = 6, Fig. 10.2.6). 10.2.7Effects of reduced-hemoglobin on bath-applied AMPA-induced current in the presence of APV, picrotoxinin, TTX and Ca2*-free ACSF Whether the effects of reduced-Hb induced-depression of the evoked EPSCs were due to a pre- and/or post-synaptic mechanism was unclear. Therefore, the effect of reduced-Hb on the postsynaptic AMPA receptor mediated current was studied. Bath application of AMPA-receptor agonist, AMPA (5 uM; for 30-35 sec; in the presence of APV, picrotoxinin, TTX and Ca 2 +-free medium) produced an inward current that could be blocked by DNQX (0.02 mM). During each experiment, at least two bath applied AMPA-induced current controls were completed prior to the application of Hb. Ten minutes after the start of the application of reduced-Hb (0.1 mM), the glutamate agonist was applied again in the presence of Hb. It was found that reduced-Hb did not - 135 -Control Hb +0.20 n A j L +0.26 nA J L Fig. 10.2.6. Effects of reduced-hemoglobin on the ability of CA1 pyramidal neurons to generate action potentials in response to depolarizing current pulses. Reduced-Hb (Hb; 0.1 mM) was applied for 15 min. The currents with intensity of 0.15, 0.2 and 0.26 each lasting for 400 ms were injected alternatively at 0.0 5 Hz into the cell via the recording electrodes so that the pulses gave 1, 3 to 4 and 6 to 8 action potentials, respectively. Single sweep records showing intracellularly recorded responses, in Control and in Hb, to 3 D.C. rectangular depolarizing pulses. Note that there is no difference in the number of action potentials generated by the depolarizing pulses in the presence of reduced-Hb. - 136-significantly affect the AMPA-induced current (response amplitude as a % of control: 100.4 ± 4.9, n = 6; Fig. 10.2.7). 10.2.8 Effects of reduced-hemoglobin on bath-applied NMDA-induced current in the presence of DNQX, picrotoxinin, TTX and Mg2*-free ACSF In the presence of reduced-Hb, the evoked NMDA-EPSC was depressed. This depression may have been mediated by a postsynaptic mechanism; therefore, the effects of reduced-Hb on postsynaptic NMDA receptor-mediated currents were examined. Bath-applied NMDA (10 pM for 40-45 sec in a Mg 2 + -free medium containing DNQX, picrotoxinin and TTX) produced an inward current which could be blocked by NMDA receptor antagonist, APV (0.05 mM). At least two stable controls of the NMDA-induced current were collected prior to the application of reduced-Hb. Ten minutes after the start of the reduced-Hb application, NMDA was applied in the presence of reduced-Hb. It was found that reduced-Hb significantly depressed the NMDA-induced responses amplitude as a % of control: 66.1 ± 3.6, n = 6; Fig. 10.2.8). The NMDA-induced current showed recovery 30 minutes after the termination of Hb. 10.2.9 Effects of reduced-hemoglobin on bath-applied THIP-induced current in the presence of DNQX, APV, TTX and Ca2+-free ACSF The locus of the action of reduced-Hb induced-depression of evoked IPSCs was studied by investigating the effects of hemoglobin on postsynaptic GABA-A receptor-mediated response. Bath-applied THIP (10 u.M for 1 minute in - 137-A Control A M P A B o 120 c o o ° 100 ro co co c <D \ i ZJ o •o cu o T3 O CD TJ Z3 "a. E < Hb Post-Hb V V "V 50 pA 100 sec Control Hb Post-Hb Fig. 10.2.7. Effects of reduced-hemoglobin on the bath-applied AMPA-induced current. Reduced-Hb (Hb; 0.1 mM) was applied for 15 min. The slices were superfused with 0.1 p.M TTX, 0.05 mM APV, 0.05 mM picrotoxinin dissolved in a Ca 2 + -free medium throughout the experiments. AMPA (5 u,M, applied for 30 sec, the horizontal line shown above the records) induces inward currents in control (Control), 10 min in reduced-Hb (Hb) and 30 min after the termination of reduced-Hb application (Post-Hb). In B, a histogram showing the effects of reduced-Hb (0.1 mM) on the amplitude of the AMPA-induced currents (n = 6). Data were represented as mean ± S.E.M. ANOVA was performed (p < 0.05). Note that reduced-Hb did.not produce a change in the amplitude of the AMPA-induced current. - 138-B Control NMDA . Hb Post-Hb 50 pA 100 sec c o o to to 3 o T3 (D O 3 T3 C i < Q o CD TJ 3 120 -, 100 - 80 CD 60 40 20 * T Control Hb Post-Hb Fig. 10.2.8. Effects of reduced-hemoglobin on the bath-applied NMDA-induced current. Reduced-Hb (Hb; 0.1 mM) was applied for 15 min. The slices were superfused with 0.1 uM TTX, 0.02 mM DNQX and 0.05 mM picrotoxinin dissolved in a Mg 2* free medium throughout the experiments. NMDA (0.01 mM, applied for 40 sec, the horizontal line shown above the records) induces inward currents in control (Control), 10 min in reduced-Hb (Hb) and 30 min after the termination of Hb application (Post-Hb). In B, a histogram showing the effects of Hb (0.1 mM) on the amplitude of the NMDA-induced currents (n = 6). Data were represented as mean ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) indicate that the values are significantly different from their respective pre-drug controls. Note that reduced-Hb depressed NMDA-induced current. - 139-a C a 2 + free medium containing DNQX, A P V and TTX) produced an outward current that could be blocked by picrotoxinin (0.02 mM). Two control THIP-mediated currents were obtained prior to the application of Hb. When THIP was applied in the presence of reduced-Hb (0.1 mM; 10 minutes after the start of the Hb application), the THIP-induced currents were not significantly different from those of the control (response amplitude as a % of control: 97.7 ± 5.2, n = 6; Fig. 10.2.9). It is interesting that GABA-ergic IPSCs are suppressed by reduced Hb while THIP effects are unaffected. These results suggest that the depressant effect is on GABA release or that if the effect is postsynaptic, the actions of THIP on non-synaptic receptors may be unaffected. 10.2.10 Effects of reduced-hemoglobin on bath-applied baclofen-induced current in the presence of DNQX, APV, picrotoxinin, TTX and Ca2*-free ACSF Whether reduced-Hb-induced depression of evoked slow IPSPs has a pre-or post-synaptic mechanism of action is unknown. The effects of reduced-Hb on the postsynaptic GABA-B receptor-mediated responses were studied using a bath-applied GABA-B receptor agonist, baclofen. Bath applied baclofen (0.1 mM, for 1 minute, in a Ca 2 +-free medium containing DNQX APV, picrotoxinin and TTX) produced an outward current that could be blocked with a GABA-B receptor antagonist, C G P 35348 (0.5 mM). At least two stable controls of the baclofen-induced current were obtained prior to the application of reduced-Hb. When applied again 10 minutes after the reduced-Hb (0.1 mM) application, the baclofen-induced current was decreased significantly (response amplitude as a - 140-A Control Hb Post-Hb T H I P 50 pA B 100 sec o 120 o o Ui ro c CD CJ T3 CD O 3 T3 C i Q_ X o CD T3 -*—« "a. E < 100 Control Hb Post-Hb Fig. 10.2.9. Effects of reduced-hemoglobin on the bath-applied THIP-induced current. Reduced-Hb (Hb; 0.1 mM) was applied for 15 min. The slices were superfused with 0.1 uM TTX, 0.02 mM DNQX and 0.05 mM APV dissolved in a Ca 2 + f ree medium throughout the experiments. THIP (0.01 mM, applied for 1 min, the horizontal line shown above the records) induces outward currents in control (Control), 10 min in reduced-Hb (Hb) and 30 min after the termination of reduced-Hb application (Post-Hb). In B, a histogram showing the effects of reduced-Hb (0.1 mM) on the amplitude of the THIP-induced currents (n = 6). Data were' represented as mean ± S.E.M. ANOVA was performed (p < 0.05). Note that reduced-Hb did not change the amplitude of the THIP-induced currents. - 141 -% of control: 68.6 ± 2.4, n = 6; Fig. 10.2.10). The baclofen-induced current showed recovery 30 minutes after the termination of Hb. The observations that baclofen-induced currents and evoked slow IPSCs were depressed by reduced-Hb, suggested that the postsynaptic GABA-B responses were sensitive to the drug. Given that the depression induced by reduced-Hb might be due to bisulfite or other breakdown products of dithionite, it was possible that these agents might interact with postsynaptic GABA-B receptors and/or the second messengers system that are coupled with GABA-B receptors. Moreover, it has been shown that sulphydryl groups are essential in the functions of G-proteins. Since dithionite and or bisulfite can interact with sulphydryl groups on proteins, it is possible that these agents may depress GABA-B receptor responses via an action on G-proteins coupled to such receptors. 10.3 Effects Of Co-Application Of L-Cysteine And Reduced-Hemoglobin On CA1 Pyramidal Neurons L-cysteine is one of the amino acids that is released into the brain during ischemic conditions (Li et al., 1999; Slivka and Cohen, 1993; Landolt et al., 1992). During hemorrhagic stroke or traumatic head injury, Hb and L-cysteine may interact with each other as both may be released. Therefore, the effects of reduced-Hb and L-cysteine on hippocampal CA1 neurons were studied. 10.3.1 Effects of reduced-hemoglobin and L-cysteine on field EPSPs L-cysteine (0.1 mM) alone was applied for 15 minutes prior to the - 142 -M Control Hb Post-Hb B a c 100 sec < Control Hb Post-Hb Fig. 10.2.10. Effects of reduced-hemoglobin on the bath-applied baclofen-induced current. Reduced-Hb (Hb; 0.1 mM) was applied for 15 min. The slices were superfused with 0.1 uM TTX, 0.02 mM DNQX, 0.05 mM APV, 0.05 mM picrotoxinin dissolved in a Ca 2 +-free medium throughout the experiments. Baclofen (0.1 mM, applied for 1 min, the horizontal line shown above the records) induces outward currents in control (Control), 10 min in reduced-Hb (Hb) and 30 min after the termination of reduced-Hb application (Post-Hb). In B, a histogram showing the effects of Hb (0.1 mM) on the amplitude of the baclofen-induced currents (n = 6). Data were represented as mean ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) indicate that the values are, significantly different from their respective pre-drug controls. Note that reduced-Hb significantly depressed the amplitude of the baclofen-induced currents. - 143 -coapplication of L-cysteine (0.1 mM) and reduced-Hb (0.1 mM). It was found that the combination of reduced-Hb and L-cysteine completely depressed the evoked field EPSP (response amplitude as a % of control: 0 ± 0, n = 6, Fig. 10.3.1). This depression was significantly larger than that produced by reduced-Hb (0.01 mM) when applied alone (See Fig. 10.2.5.A). Moreover, applications of L-cysteine (0.1 mM) for 30 minutes did not significantly affect the amplitude of the field E P S P (response amplitude as a % of control: 99.1 ±4.9, n = 6, Fig. 10.3.1). 10.3.2 Current-voltage relationship of coapplication of L-cysteine and reduced-hemoglobin When applied alone, L-cysteine (0.1 mM) did not produce a change in the holding current at membrane potential between -100 to +30 mV, as illustrated in the l-V. relationship, (n = 6, Fig. 10.3.2A) However, in 6 out of 8 neurons, co-application of reduced-Hb (0.1 mM) and L-cysteine (0.1 mM) produced an inward current associated with a decrease in input resistance. The inward current has a reversal potential of +20 to +30 mV (n = 6, Fig. 10.3.2A and B). 10.3.3 Effects of APV, and DNQX on inward current produced by L-cysteine and reduced-hemoglobin The inward current induced by coapplication of L-cysteine and reduced-Hb may be mediated via the activation of glutamate receptors. It was found that when L-cysteine and reduced-Hb were applied in the presence of A P V (0.05 mM) alone, the drugs-induced inward current was significantly depressed (response as a % of control: 23.1 ± 3.8, n = 5, Fig. 10.3.3). Fifteen minutes after the washout of APV, the L-cysteine (0.1 mM) and reduced-Hb (0.1 mM)- induced - 144-A B c o O CD in co D_ CO CL LU TJ CD CD T3 • "a. E < L-cysteine + L-cysteine Hb 20 40 Time (min) 60 c o O CO in CO CL W D_ H i XJ CU cu TJ 3 _-+^  "Q. E < 120 100 80 60 40 20 0 L-cysteine (0.1 mM) T T T -10 0 10 20 Time (min) 30 40 Fig. 10.3.1. Effects of coapplication of L-cysteine and reduced-hemoglobin on evoked field EPSPs. The slices were superfused with L-cysteine (0.1 mM) for 15 min prior to the coapplication of reduced-Hb (0.1 mM) and L-cysteine (0.1 mM) for an addition of 30 min. In A, graphs of the effects of coapplication of L-cysteine and reduced-Hb were shown (n = 6). In B, graphs the effects of L-cysteine (0.1 mM; applied for 30 min) on the amplitude of the field EPSP were illustrated (n = 6). Data points were represented as mean + S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) indicate that the responses are significantly different from their respective pre-drug controls. Note that coapplication of L-cysteine and reduced-Hb produced a significant depression of the amplitude of the field EPSP. - 145 -• Cys + Hb • Cys l n (nA) 1 I A - ^ A A A A A--120 -100 -80 -60 -40 i—i V h(mV) 40 B V h -60 mV +20 mV +40 mV 1 nA Fig. 10.3.2. l-V relationship of L-cysteine and reduced-hemoglobin induced current. The slices were superfused with L-cysteine (0.1 mM) and reduced-Hb (0.1 mM) for 1 min. At the peak of the current response, an i-V curve protocol was performed. In A, the l-V relationship was shown for L-cysteine ( A ; n = 6) and the coapplied L-cysteine and reduced-Hb (•; n = 6). In B, L-cysteine and reduced-Hb induced current recorded at -60 mV, +20mV and + 40 mV were shown. Note that coapplication of L-cysteine and reduced-Hb induced a current with reversal potential between +20 to +30 mV. - 146-A Control APV DNQX DNQX + A P V B 1 1 min Fig. 10.3.3. Effects of ionotropic glutamate receptor antagonists on the current induced by coapplication of L-cysteine and reduced-hemoglobin. In A, records of inward current induced by coapplication of L-cysteine (0.1 mM) and reduced-Hb (0.01 mM) for 1 min shown were taken during control, in the presence of A P V (APV), in the presence of DNQX (DNQX) and in the presence of DNQX and APV (DNQX + APV). In B, a histogram showing the effects of APV, DNQX and DNQX + APV on the amplitude of the L-cysteine and reduced Hb-induced current (n = 5). Data were represented as mean ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) indicate that the. responses are significantly different from their respective pre-drug controls. Note that APV, DNQX and DNQX + A P V depressed the inward currents induced by coapplication of L-cysteine and reduced-Hb. - 147-current showed recovery (See Fig. 10.3.3). DNQX (0.02 mM) was then applied for 10 minutes prior to the coapplication of L-cysteine (0.1 mM) and reduced-Hb (0.1 mM). It was found that the current-induced by L-cysteine and reduced-Hb was significantly depressed by DNQX (response amplitude as a % of control: 16.73 ± 3.27, n = 5, Fig. 10.3.3). In the presence of both DNQX (0.02 mM) and A P V (0.0 5 mM), the amplitude of the current induced by the drugs was further depressed (response as a % of control: 3.11 ± 2.09, n = 5, Fig. 10.3.3). 10.3.4 Effects of co-application of reduced-hemoglobin and L-cysteine in slices perfused with EGTA and TTX containing Ca2+-free ACSF Whether the L-cysteine and reduced-Hb induced depolarization was mediated via a C a 2 + and/or action potential-dependent release of glutamate was tested. In the presence of Ca 2 +-free and TTX-containing ACSF, coapplication of L-cysteine (0.1 mM) and reduced-Hb (0.1 mM) produced an inward current that was not significantly different from that of the control (amplitude of L-cysteine and reduced-Hb induced inward current as a % of control: 110.7 ± 16.7, n = 6; Fig. 10.3.4). This result suggests that L-cysteine and reduced-Hb induced inward current is independent of C a 2 + and action potential-induced release of glutamate. 10.4 Effects Of Hemoglobin-1991 May Be Due To Contaminants Present During The Preparation Of Hemoglobin Data presented in Section 10.1 showed that bovine Hb-1991 (0.05 and 0.1 mM) produced a significant depression on evoked synaptic transients on CA1 neurons. However, in a recent experiment, it was found that Hb-1996 did not - 148 -o i -»—1 c 8 120 -o Control Ca 2 +-free + TTX Fig. 10.3.4. Effects of Ca^-free and TTX containing medium on the L-cysteine and reduced-Hb induced current. L-cysteine (0.1 mM) and reduced-Hb (0.1 mM) were coapplied to the bath for one min in control and in Ca 2 +-free and TTX (0.1 ^M) containing medium. Histogram showing the effects of Ca 2 +-free and TTX containing medium on the amplitude of the L-cysteine and reduced-Hb induced current (n = 6). Data points are represented as the mean + S.E.M. Note that the Ca 2 +-free and TTX containing medium did not have a significant effect on the amplitude of th drugs-induced inward current. - 149-produce a significant effect on field EPSPs recorded in the hippocampal CA1 region. 10.4.1 Effects of hemoglobin-1996 and hemoglobin-1991 on field EPSPs Application of Hb-1996 (0.5 mM) did not produce a depression of the field EPSPs (slope of field EPSP as a % of control: 107.1 ± 5.0; n = 6, Fig. 10.4.1), while in the same slices, application of Hb-1991 (0.5 mM) depressed the field EPSPs (slope of field EPSP as a % of control: 11.8 ± 11.8; n = 2 (low sample size was due to limited amount of Hb-1991 sample but the data is similar to that reported by Ip (1994)). These results suggest that there are differences between the Hb-1991 and the Hb-1996 samples. It is possible that the effects of Hb-1991 on field EPSPs are due to substances other than hemoglobin. Dialysis was used to separate substances from Hb-1991 which may be causing the effects on field EPSPs. 10.4.2 Effects of dialysis on the hemoglobin-1991-induced depression on CA1 neurons Dialysis was used to determine whether the effects of Hb-1991 could be due to substances having a MW < 12000. Since Hb has a MW of 64500, and the dialyzing membrane has a cut-off of approximately 12000, it is possible to separate compounds with MW < 12000 from Hb. If the effects of Hb-1991 were due to substances having a MW < 12000, the active agents would be dialyzed out of the Hb solution and would render the Hb itself ineffective in producing a depression on the field EPSP. Consistent with this idea, dialyzed Hb-1991 (0.1 mM) did not produce a depression on the field EPSPs (slope of field EPSP as a - 150-Fig. 10.4.1. Effects of hemoglobin-1996 and hemoglobin-1991 on evoked field EPSPs. Hemoglobin-1996 (Hb-1996; 0.5 mM) and hemoglobin-1991 (Hb-1991; 0.5 mM) were applied for. 15 min. In A, records of field EPSP taken during control, 15 min in Hb-1996 and 15 min in Hb-1991, were shown. The graph in B shows the effects of Hb-1996 and Hb-1991 on field EPSPs with n = 6 for (•) and n = 2 (•). Data points in graphs represent mean ± S.E.M. Note that Hb-1991, but not the Hb-1996, produced a depression. - 151 -% of control: 109.0 ± 5.6; n = 5, Fig. 10.4.2); however, the non-dialyzed Hb (1991; 0.1 mM) depressed the field EPSPs (slope of field EPSP as a % of control: 73.9 ± 11.4; n = 2; low sample size was due to limited amount of Hb-1991, Fig. 10.4.2). The results suggest that the effects of Hb-1991 on field EPSP s are due to substances with MW < 12000. 10.4.3 Differences between hemoglobin-1991 and hemoglobin-1996 as compared by mass spectroscopy Further experiments were performed using mass spectroscopy to examine the differences between the Hb-1991 and Hb-1996. It was found that Hb-1991 and Hb-1996 did not show significant differences when scanned with positive ionization between molecular weight of 200 to 14000 (data not shown). However, when scanned with negative ionization, Hb-1991 was found to contain relatively large amounts of molecules with MW of 97 and of molecules with MW of 81 compared to those found in Hb-1996 (Fig. 10.4.3 C). From the molecular weight and the Sigma preparation method, it is speculated that the substances with MW of 97 and 81 could be bisulfate and bisulfite, respectively. Using known bisulfate and bisulfite concentrations, standard curves were constructed (Fig. 10.4.3 A1&2 and B1&2). Hb-1991 and Hb-1996 (0.1 mg/ml, each) at a concentration that is within the range used in the standard curves were used to estimate the amount of bisulfate and bisulfite in the Hb-1991 and Hb-1996 samples (Fig. 10.4.3 C1&2). The amounts of bisulfate and bisulfite for 0.1 mM of Hb-1991 were found to be in between 2 to 3.5 mM and 0.05 to 0.10 mM respectively (n = 3). However, in Hb-1996, it was found that bisulfate and - 152-A Dialyzed Non-dialyzed Control Hb Hb o -i , , , 1 . — 0 20 40 60 80 100 Time (min) Fig. 10.4.2. Effects of dialysis on hemoglobin-1991-induced depression of evoked field EPSPs. In A, records of field EPSP shown were taken during control (Control), 15 min in dialyzed Hb-1991 (0.1 mM; Dialyzed Hb), and 15 min in non-dialyzed Hb-1991 (0.1 mM; Non-dialyzed Hb). In B, the effects of dialyzed [n = 5, (•)] and non-dialyzed [n = 2, (O)] Hb-1991 (0.1 mM) on the field EPSP s were shown in graphs. Data points in graphs were represented as mean ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) in this graph indicate that the values are significantly different from their respective pre-drug controls.. Note that the dialyzed Hb-1991 did not depress the field E P S P while the non-dialyzed Hb-1991 did. - 154-C1 M.W. 97 Hb-1991 Hb-1996 385398 i • • • • i 43704 C2 M.W. 81 Hb-1991 Hb-1996 49318 - i — = » — r 1898 Fig. 10.4.3. Mass spectroscopic analysis of hemoglobin-1996 and hemoglobin-1991. Preliminary studies suggested that there are significantly higher concentrations of negatively charged molecules with molecular weight of 81 and 97 present in Hb-1991 when compared to Hb-1996. From the preparation method provided by Sigma [for Hb-1991 and Hb-1996], it is possible that the corresponding ions are bisulfate and bisulfite. Control standard curves were, therefore, constructed by using known concentrations of bisulfate and bisulfite. In A1, records of peaks of increasing concentrations of bisulfate were shown. In A2, the areas under the curve for the bisulfate peaks were plotted as a function of the concentrations. In B1, records of peaks of increasing concentration of bisulfite were shown. In B2, the areas under the curve for the bisulfite peaks were plotted as a function of the corresponding concentrations. In C1, records of bisulfate peaks detected in Hb-1996 and Hb-1991 were shown. In C2, records of bisulfite peaks detected in Hb-1996 and Hb-1991 were shown. Note that Hb-1991 has approximately 1.5 to 2 times as much of bisulfite and 15 times as much of bisulfate than the Hb-1996 sample. - 155 -bisulfite concentrations (for every 0.1 mM of Hb) were 0.2 to 0.4 mM and <0.01 mM, respectively (n = 3). 10.4.4 Effects of bisulfate and bisulfite on evoked EPSCs Consequently, cumulative dose-response relationships for bisulfate and bisulfite on evoked EPSCs were performed. Concentrations of 0.125 to 3 mM of bisulfate did not produce a significant depression of the evoked EPSCs (Fig. 10.4.4.1). In addition, bisulfite (0.5 to 1.0 mM) depressed the evoked EPSCs (amplitude of evoked EPSCs as a % of control: 80.6 ± 4.5 and 47.8 ± 7 . 1 , respectively; n = 5, Fig. 10.4.4.2), followed by a recovery in 10 minutes (amplitude of evoked EPSCs as a % of control at 10 minutes post-bisulfite: 92.4 ± 12.0; n = 5, Fig. 10.4.4.2). At a concentration of 0.25 mM, the agent did not show a significant depression of the evoked EPSCs. 10.4.5 Presence of ammonium ion in the hemoglobin-1991 sample Since the presence of bisulfate alone cannot fully explain the effects seen with hemoglobin, it was felt that other contaminants might be present. Since Sigma used ammonium sulfate to precipitate hemoglobin, it is possible that ammonium is present in the Hb-1991 sample. Since it was difficult to detect for the presence of ammonium using a standard test (which requires large amounts of the sample or one has to measure total nitrogen), a simple test was used to test for the presence of ammonium in the Hb-1991. The test makes use of the fact that N H 3 gas is basic and will turn litmus paper blue. Control solutions containing 9 and 3 mM of ammonium sulfate changed the litmus paper blue. A similar intensity of blueness was observed in the 0.1 mM Hb-1991 solution but - 156-o _l I Com 120 -4 — o 100 -CO 80 -as EPSCs 60 -40 -O itude 20 -AmpI 0 -o S— -1—' c E E E E E E o LO LO LO CM CO O 0.12 CM O o E o CD ZJ 1_ "O I to o CL [Bisulfate] Fig. 10.4.4.1. Cumulative concentration-response relationship of bisulfate on evoked EPSCs. Increasing concentrations of bisulfate (0.125 to 3.0 mM, 10 min each) was applied to hippocampal slice [in the presence of picrotoxinin (0.05 mM) throughout the experiment]. Histograms illustrating the effects of bisulfate (0.125 to 3.0 mM,) the amplitude of the EPSCs were shown (n = 5). Data were represented as mean ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) in this and subsequent graphs indicate that the values are significantly different from their respective pre-drug controls. Note bisulfate-did not produce a significant effect on the amplitude of evoked EPSCs. - 157-O 1 2 0 "c o O 1 0 0 CC (/) O CO CL LU o 0) TD 13 "5. 80 A 60 40 20 o L . +-< c E E E o LO LO O CN O O [Bisulfite] Fig. 10.4.5.2. Cumulative concentration-response relationship of bisulfite on evoked EPSCs. Increasing concentrations of bisulfite (0.25 to 1 mM, 10 min each) was applied to hippocampal slice [in the presence of picrotoxinin (0.05 mM) throughout the experiment]. Histograms illustrating the effects of bisulfite (0.25 to 1.0 mM) on the amplitude of the EPSCs were shown (n = 5). Data were represented a mean + S.E.M. ANOVA and Duncan test were performed (p< 0.05). The asterisks (*) in this histogram indicate that the values are significantly different from their respective pre-drug controls. Note that bisulfite reversibly depressed the amplitude of evoked EPSCs. - 158-not in 0.1 mM Hb-1996. A control solution containing no ammonium sulfate did not produce any response. The observation is summarized in the Table 10.4.5. 10.5 Contamination By Dithionite During The Preparation Of Reduced-Hemoglobin Prior to the realization that Hb-1991 was contaminated with sulfate and possibly ammonium, a series of experiments had been performed in order to investigate the effects of reduced-Hb on CA1 pyramidal neurons. These studies were done because it was found that met-hemoglobin did not produce a significant depression in evoked synaptic transients recorded in the hippocampal CA1 cells. Since the reduced-Hb produced depression on synaptic transients similar to those produced with Hb-1991, it was assumed that the effects of reduced-Hb are the same as those produced by Hb-1991. However, the findings that the effect of Hb-1991 may be due to contamination during the preparation of Hb-1991 make it difficult to explain why the reduced Hb-1996 produced a depression while non-reduced Hb-1996 did not. One can argue that the depressions of the synaptic transmission mediated by Hb are due to the application of reduced-Hb. On the other hand, it is also possible that the effects of hemoglobin are due to substances that were introduced during the reduction procedure of the Hb samples. It is interesting upon reviewing the literature from investigators who used the Martin et al.'s (1985) method to prepare reduced-Hb, no one reported the use of a dialyzed dithionite as a control. Therefore, the - 159-n N H 4 + (9 mM) N H 4 + (3 mM) Hb-1991 (0.3 mM) Hb-1996 (0.3 mM) Control without N H 4 + Intensity of blueness 3 +++ + + - -Table 10.4.5. Qualitative test for the presence of NH 4 + in Hb-1991. Concentrated NaOH solution (1N) was placed in the N H 4 + (9mM and 3 mM), Hb-1991 (0.3 mM) and Hb-1996 (0.3 mM) solutions. Solutions were then heated up. The ability of the resultant gas in turning the litmus paper from red to blue were assessed (n = 3). Note that Hb-1991 produced a blueness in litmus paper whereas Hb-1996 did not. - 160-effects of dialyzed dithionite control were studied in extracellular recordings in 18 slices. 10.5.1 Effects of reduced-hemoglobin and dialyzed dithionite control on field EPSPs Reduced-Hb (0.1 mM) depressed the field EPSPs (slope of field as a % of control: 71.7 ± 4.7; n = 6, Fig. 10.5.1 A). However, in parallel controls in which solution containing only dithionite was dialyzed, the dialyzed dithionite control also produced a depression in field EPSPs (slope of field EPSP as a % of control: 72.2 + 3.7; n = 6, Fig. 10.5.1B). Reduced-Hb following prolonged washing (18 h), however, did not depress the field EPSP (slope of field E P S P as a % of control: 93.5 + 3.7; n = 6, Fig. 10.5.1 C). The inability of reduced-Hb prepared with 18h of washing may reflect a washout of the dithionite. Moreover, when dialyzed dithionite control and L-cysteine were applied together, it was found that combination of these drugs completely blocked the field EPSPs (slope of field EPSP as a % of control: 0 ± 0%; n = 3). 10.5.2 Differences between reduced-hemoglobin and non-reduced hemoglobin as studied by mass spectroscopy Mass spectroscopic techniques were used to study the difference between the reduced- and non-reduced-Hb. Using negative ionization, it was found that the reduced-Hb (2 h, dialysis) and the dithionite control contain substances with MW of 81 and 97 (Fig. 10.5.2). From the reaction products produced by dithionite and the MW, it is possible that the agents with MW 81 and 97 are bisulfite and bisulfate, respectively. Using a standard curve, it was found that the - 161 -A 1 Control 120-, 2 h r dialyzed Hb Post 2 h r dialyzed Hb 0.4mV 0.4 ms 10 20 30 40 Time (min) B 1 Control 2 h r diayzed dithionite Post 2 h r dialyzed dithionite 20 30 40 Time (min) 60 120n 100T co Q- c W o - D O 80-Reduced Hb (18 hr wash) cu l u CL co o co 40-20 J 10 20 30 40 Time (min) 50 60 Fig. 10.5.1. Effects of reduced-hemoglobin and the control dithionite-dialyzed solution on evoked field EPSPs. A1&2 show effects of dialyzed reduced Hb (0.1 mM; 2hr dialyzed Hb; in A2 n = 6) while B1&2 show effects of dialyzed dithionite (in B2, n = 6). In C, the effects of reduced Hb (dialyzed for 18 hrs.) were shown (n = 6). Data points in the graphs were represented as the mean ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) in these graphs indicate that the values are significantly different from their respective pre-drug controls. Note that both 2hr dialyzed Hb or dithionite-control solution produced a significant reversible depression while the 18 hr dialyzed Hb did not. - 162-A B MW81 MW97 Control Control Commercial Commercial Hb-1996 Hb-1996 Reduced Hb Fig. 10.5.2. Mass spectroscopic analysis of contaminants in hemoglobin samples introduced by Martin's dialysis method. Preliminary studies suggested that there are significantly higher concentrations of negatively charged molecules with molecular weight of 81 and 97 present in reduced Hb-1996 when compared to Hb-1996. From the chemical reaction of dithionite with distilled water, it is possible that the products of such reactions are bisulfate and bisulfite, having molecular weight of 81 and 97, respectively. Control standard curves were, therefore, constructed by using known concentrations of bisulfate and bisulfite. These standards were repeated for each experiments and were used to estimate the amount of bisulfate and bisulfite in the Hb samples (As shown in Fig. 9.4.5.; n = 3). In A, records of bisulfite peaks detected in control commercial Hb-1996 and reduced Hb (using Martin et al's method) were shown. In B, records of bisulfate peaks detected in control commercial Hb-1996 and reduced-Hb were shown. Note that reduced-Hb prepared using Martin et al's method had 15 times as much bisulfate and, 50 times as much bisulfite than the commercial Hb-1996 sample. Reduced Hb - 163 -amount of bisulfite and bisulfate were 0.5 to 1.0 mM and 1 to 2 mM, respectively (n = 3, Fig. 10.4.3 A1&2 and 10.4.3 B1&2). From the data presented in section 10.4.4 (Fig. 10.4.4.1 and 10.4.4.2), application of bisulfate with concentration up to 3 mM did not significantly depress the evoked EPSCs. However, bisulfite with concentrations of 0.5 and 1 mM significantly depressed the evoked EPSCs, suggesting that the effects of reduced-Hb are due to bisulfite. 10.6 Effects Of Breakdown Products Of Hemoglobin On Synaptic Transmission During hemorrhagic stroke, the pooled erythrocytes are lysed and hemoglobin is released. Prolonged incubation of hemoglobin leads to release of heme and iron; therefore, neurons may be exposed to these compounds. The effects of these agents on synaptic transmission were investigated. 10.6.1 Effects of ferrous chloride on field EPSPs Ferrous chloride (0.05 and 0.1 mM) depressed the field EPSP (slope of field EPSPs as a % of control: 65.4 ± 6.6, n = 5 and 51.8 ± 6.7, n = 4, respectively, Fig. 10.6.1). The depressed field EPSP did not show a significant recovery 30 minutes following the washout of ferrous chloride (slope of field E P S P as a % of control: 46.0 ± 4.6, n = 5 and 41.4 ± 7.6, n = 4, respectively, Fig. 10.6.1). Since ferrous chloride (0.1 mM) slightly changes the pH of the solution from 7.4 to 7.35, control experiments were performed in which the effect of the medium with a pH 7.35 solution was tested. The pH 7.35 solution did not - 164-A Control Iron Post-Iron B CL ^ C O W -1 0.5 mV 10 msec 120 •_ _ C O p 100 C L ^ O 80 CD M _ M= O 60 40 20 Drug application 0 10 20 30 40 50 60 Time (min) Fig. 10.6.1. Effects of ferrous chloride on evoked field EPSPs. Ferrous chloride (0.1 and 0.05 mM) was applied for 15 min. In A, records of field EPSPs shown were taken during control (Control), 15 min in FeCI 2 (0.1 mM; Iron), and 30 min after the termination of FeCb (Post-Iron). In B, effects of FeCb [0.1 mM (•) and 0.05 mM (O); n = 5 and n = 4, respectively] and of pH control solution [n = 4, (•)] on the slope of field EPSPs, were shown in graphs. Data points in the graphs were represented as the mean ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) in these graphs indicate that the values are significantly different from their respective pre-drug; controls. Note that FeCb suppressed the field EPSP, and there was no recovery from this effect within the 30 min post-drug application. - 165 -produce a significant change in the field EPSP (slope of field EPSPs as a % of control: 102.5 ± 3.9; n = 4, Fig. 10.6.1). 10.6.2 Effects of hemin on field EPSPs Hemin (0.5 mM but not 0.1 mM) depressed the field EPSPs (slope of field E P S P as a % of control: 67.8 ± 5.3 and 95.0 ± 3.5; n = 6 and n = 4, Fig. 10.6.2). The depressed field EPSPs, as produced by hemin (0.5 mM), did not show much recovery 30 minutes following the washout of hemin (slope of field EPSP as a % of control: 63.8 ± 7.4, n=6, Fig. 10.6.2). Since the hemin was insoluble in solution, it was first dissolved in NaOH and was further dispersed in a Tris-buffered solution using albumin (1:10 ratio). Control experiments with media containing NaOH and albumin (0.05 mM) were performed on the same slices prior to the application of hemin (0.5 mM), and the carrier showed no significant effects on the field EPSPs (slope of field EPSP as a % of control: 98.3 ± 5.5, n = 6, Fig. 10.6.2). - 166-A B Control o Hemin — Post-Hemin 0.5 mV 10 ms 20 40 60 80 Time (min) 100 Fig. 10.6.2. Effects of hemin on evoked field EPSPs. Hemin (0.5 mM) was applied for 15 min. In A, records of field E P S P shown were taken during control (Control), 15 min in hemin (0.5 mM; Hemin) and 30 min after the termination of hemin (Post-Hemin). The graph in B shows the effects of hemin (0.5 mM in 0.05 mM albumin-containing medium) on the slope of field EP SP s (n = 5). Data points in the graphs were represented as the mean ± S.E.M. ANOVA and Duncan test were performed (p < 0.05). The asterisks (*) in this graph indicate that the values are significantly different from their respective pre-drug controls. Note that hemin (0.5 mM) suppressed the field EPSP which did not recover.within the 30 min post-drug application, and that albumin itself did not have a significant action. - 167-11 DISCUSSION During hemorrhagic stroke or traumatic head injuries, blood is released from the ruptured cerebrovasculature and is pooled in the surrounding brain tissue. (Barrows et. al., 1955) The extravascularly pooled blood can remain in the intracranial cavity for hours or days (Tourtelotte et al., 1964). Hemoglobin, a major component of erythrocytes, has been shown to be released from lysed blood cells within hours after subarachnoid hemorrhage (Barrows et al., 1955 and Tourtelotte et al., 1964). In addition, hematoma with high concentrations of hemoglobin can be found in patients suffered from intraparenchymal hemorrhage (Miyaoka et al., 1976; Osaka, 1977). The clearance of hemoglobin takes days to weeks; therefore, under these conditions, neurons may be exposed to hemoglobin for an extended period of time. This study is designed to test the hypothesis that hemoglobin and its breakdown products, iron and hemin, have effects on synaptic transmission. By examining the actions of hemoglobin on central neurons, it is hoped that the role that hemoglobin may play during hemorrhagic stroke and/or traumatic head injury can be better understood. 11.1 Differences Between Hb-1991 And Hb-1996 In this section of the discussion, the following results will be discussed: a. Hb-1991 depressed but Hb-1996 did not have an effect on field EPSPs in hippocampal slices. b. Effects of Hb-1991 on field EPSPs were significantly attenuated by the dialysis of Hb-1991 sample. - 168-c. Mass spectroscopic analysis of Hb-1991 and Hb-1996 found that Hb-1991 contains substances with MWof 81 and 97 in a much larger proportion than in Hb-1996. . d. Hb-1991, when heated, released a basic gas which may be ammonium. Hb-1991 produced a dose-dependent depression of synaptic transmission in hippocampal CA1 pyramidal neurons (Ip, 1994; Yip et al., 1996). However, 0.5 mM Hb-1996 had no effects on the field EPSP. At this concentration, Hb-1991 would have completely abolished the field EPSP (Yip et al., 1996). It, therefore, appeared that there might be differences between Hb-1991 and Hb-1996. It is possible that the Hb-1991-induced depressions are due to contaminants left behind during the preparation of hemoglobin. In order to address the question of whether Hb-1991 and Hb-1996 have different contaminants, the Hb-1991 sample was dialyzed. Hb has a molecular weight of 64,400 and the dialysis tubing had a molecular weight cut off of approximately 12,000. Therefore, it is possible to separate substances that have MW less than 12,000 from hemoglobin. If contaminant(s) were responsible for the depression of synaptic transmission mediated by the Hb-1991 sample and the contaminant(s) have molecular weights of less than 12,000, the dialysis procedure should have dialyzed the contaminant(s) from the Hb-1991 samples. Indeed, it was found that dialyzed Hb-1991 no longer depressed the field EPSP. This observation suggests that there are differences between Hb-1991 and -1996 and that the depressions mediated by Hb-1991 samples are due to substances other than hemoglobin. -169-Moreover, since the dialysis tubing has a MW cut off of approximately 12000, it is likely that the effects mediated by Hb-1991 sample were mediated by compound/s with molecular weights less than 12,000. One may argue that the effects of Hb-1991 may be due to the different components of hemoglobin. In our study, we found that iron and hemin did produce a significant depression on synaptic transmission; however, unlike Hb-1991, the iron- or hemin- induced depression of the field EPSP was irreversible. It may be argued that the concentrations of iron or hemin used in these studies are higher than those found in Hb-1991 and, therefore, produced an irreversible depression on the field EPSP. However, in studies where Hb-1991 (0.5 mM) was tested, the agent completely abolished the field EPSP, an effect that is clearly stronger than that observed with either iron or hemin, a recovery was still observed during the washout of Hb-1991. This suggests that the effects of Hb-1991 may be due to agents other than iron or hemin. In order to study the differences between Hb-1991 and -1996, the two samples were subjected to mass spectroscopic analysis. It was observed that in Hb-1991, the concentrations of negative ions with MW 97 and 81 were 30-fold and 3-fold, respectively, higher than those found in Hb-1996. Based on the molecular weights of these negative ions and the peaks that were detected using mass spectroscopy, it was likely that these compounds were bisulfates and bisulfites. It is possible that these compounds were responsible for the depression of synaptic transmission mediated by Hb-1991. Therefore, concentration response relationships of bisulfates and bisulfites on evoked - 170-EPSCs were performed. Bisulfates, when applied alone in the concentration ranges that were found in Hb-1991, did not produce a significant depression on the evoked EPSCs. Therefore, it is unlikely that the presence of bisulfates alone could explain the effect observed with Hb-1991. Although bisulfites produced a dose-dependent depression on the evoked EPSCs, the concentrations of bisulfites that were found in Hb-1991 were too low to account for the depression mediated by Hb-1991. The question of how such a high concentration of sulfate was present in the Hb-1991 sample remains unresolved. When examining the description of the preparation method printed on the bottles of the Hb-1996 samples, it was found that they were different from those found on the Hb-1991 samples. For Hb-1991 samples, it was reported that hemoglobin was purified by a '2X crystallized, dialyzed and lyophilized' procedure, while the Hb-1996 samples were prepared only by a 'lyophilized' procedure. This suggests that there may have been a change in the protocol for the preparation of Hb-1996, and may explain the differences observed between the two Hb samples in our studies. By contacting Sigma Chemical, it was found that the label used to describe the preparation of Hb-1991 was erroneous and the hemoglobin was not purified by crystallization; but, in fact, by a '2X precipitation, dialyzed and lyophilized' procedure. Therefore, Hb-1991 and Hb-1996 were prepared using the same procedure. I was told that Sigma used ammonium sulfate to precipitate the hemoglobin from solution. The sulfate and ammonium were then removed by using a dialysis procedure. The concentration of ammonium sulfate used as well as the dialysis - 171 -procedure were classified as confidential and were not disclosed to me. Apparently, the same procedure was used to prepare Hb-1996; therefore, the differences in the composition of the Hb samples cannot be explained by the differences in the method of preparation. However, the technical support personnel at Sigma admitted that they did not test for the presence of ammonium sulfate after the dialysis procedure. It is, therefore, possible that ammonium sulfate is present and its concentrations could be different in the two Hb samples. In this study, we found that a relatively high concentration of sulfate was present in the Hb-1991 sample compared to the Hb-1996. If sulfate is present in the hemoglobin sample, ammonium may also be present in the Hb-1991 sample. In fact, using a simple qualitative method that tests for ammonium, Hb-1991 was found to release ammonia, or at least a gas that is basic in pH, while Hb-1996 did not. Moreover, ammonium, at a concentration of 1-3 mM, has been shown to depress field EPSPs, intracellular EPSPs, IPSPs and depolarize hippocampal CA1 pyramidal neurons (Alger et al., 1983; Fan and Szerb, 1993; Theoret et al., 1985), effects that were similar to those observed in this study. A more quantitative determination of the ammonium concentration in the hemoglobin sample would be needed; however, the measurement of ammonium is limited by the test procedures and the amount of Hb required for these tests. Since we only have a limited, amount of Hb-1991 (less than 500 mg) and most of the commercially available tests either require a large amount of ammonium (limited by the source) to be sensitive, it became difficult to measure the ammonium under these conditions. - 172-11.2 Contamination of Reduced-Hb with Dithionite In this section of the discussion, the following results will be discussed: a. Met-Hb did not have an effect on synaptic transmission. b. Reduced-Hb as prepared using Martin et al.'s (1985) dialysis procedure, depressed the field EPSPs, EPSCs, NMDA-EPSCs and fast IPSCs. c. Coapplication of L-cysteine and reduced-Hb resulted in a depression of the field EPSPs, and generation of an inward current that is sensitive to DNQX and APV. d. Control dithionite solutions (in the absence of Hb), prepared with Martin et al.'s (1985) dialysis procedure, depressed field EPSPs. The magnitude of depression was similar to that found with reduced-Hb. e. Coappliction of L-cysteine and control dithionite solutions prepared with Martin et al.'s (1985) dialysis procedure, produced a depression of field EPSPs with similar magnitude as observed with L-cysteine and reduced-Hb. f. Reduced-Hb prepared using a modified Martin et al.'s (1985) dialysis procedure (i.e. increasing the dialysis time from 2 hours to 18 hours and changing the volume of dialysis from 100 fold volume to 3X 100 fold volume), did not produce significant effects on field EPSPs. - 173-g. Mass Spectroscopic analysis showed that reduced-Hb, prepared using Martin et al.'s (1985) method, contained bisulfate and bisulfite. h. Bisulfite, but not bisulfate, depressed evoked EPSCs. Hb exists in two forms: met- and reduced- Hb. It is possible that met-Hb and reduced-Hb have different effects on synaptic transmission; therefore, the effects of the drugs on excitatory and inhibitory synaptic transients were studied. When met-Hb was applied onto hippocampal slices, it did not produce a significant effect on evoked EPSPs and IPSPs. Since commercially available Hb contains a mixture of both met-Hb and reduced-Hb, in order to test the effects of reduced-Hb, the purchased Hb had to be reduced. Upon reviewing the literature, it was found that many laboratories studying the effects of hemoglobin reduced by the method of Martin et al. (1985). This procedure involves first reducing the Hb with dithionite, a strong reducing agent, followed by washing the dithionite with 100X volume of distilled water for 2 hours at 4°C with the Hb solution in a dialyzing tube. Reduced-Hb samples prepared using the above method were found to depress evoked EPSP, EPSC, IPSCs and NMDA-receptor mediated EPSCs. Control dialyzed samples containing dithionite but not Hb were prepared using the same methods as described by Martin et al. (1985). In these experiments, it was found that the control dialyzed samples also depressed the field EPSP to a magnitude similar to that by the reduced-Hb. This suggests that the observed synaptic depression is not due to hemoglobin but rather to dithionite or its breakdown product(s). Therefore, experiments were performed - 174 -with reduced-Hb that was dialyzed for 18 hours instead of 2 hours. Indeed, reduced-Hb that was prepared with 18 hours of dialysis did not produce a significant depression on the field EPSP. The inability of the reduced-Hb (18h) to produce an effect on the synaptic transmission following prolonged washout was not due to a lowered concentration of reduced-Hb because under UV spectrophotometry, the absorption readings in both the 2 hour- and the 18 hour-wash were similar. The above observations indicate that the actions of reduced-Hb samples on synaptic transmission are not due to reduced-Hb in the samples, but rather to contaminants present in the reduced-Hb samples. Furthermore, results obtained from mass spectroscopy indicated that the reduced-Hb and the dialyzed dithionite were contaminated with substances with MW of 81 and 97. Interestingly, bisulfite and bisulfate, reaction products of dithionite, had the same MW of 81 and 97, respectively. In addition, cumulative dose-response curves were performed to study the effects of bisulfite on synaptic transmission. The agent, at concentrations found in the reduced-Hb, produced a depression of the evoked EPSCs. Therefore, it is possible that bisulfite, a breakdown product of dithionite, is responsible for the depression caused by the reduced-Hb samples. During hypoxia/ischemia conditions, amino acids are released. L-cysteine can be one of the amino acids released in large quantities (Li et al., 1999; Slivka and Cohen, 1993; Landolt et al., 1992). Since L-cysteine has a sulphydryl group, it is possible that L-cysteine may interact with the sulphydryl group of hemoglobin and may prevent its oxidation. Using this logic, the effects of co-application of L--175 -cysteine and hemoglobin on hippocampal CA1 pyramidal neurons were studied. It was found that when L-cysteine and reduced-Hb were co-applied, the field EPSP s were greatly depressed. However, when L-cysteine and control dithionite solutions were co-applied, depressions of field EPSPs with the same magnitude were also observed. Therefore, the effects of co-application of L-cysteine and reduced-Hb on field EPSPs can be attributed to the interaction of L-cysteine with dithionite or its breakdown products rather than Hb itself. Moreover, since the depolarization induced by L-cysteine and reduced-Hb can be blocked by glutamate receptor antagonists, A P V and DNQX, it is likely that the effects are mediated via an action on the ionotropic glutamate receptors. Moreover, L-cysteine-S-sulfate has been shown to be neurotoxic and is believed to act through an action on the ionotropic receptors (Onley et al., 1974). It is possible that L-cysteine may react with bisulfate or bisulfite or yet other unidentified products of dithionite and result in the production of agents that can activate ionotropic receptors leading to strong depolarization of neurons. Further investigation is required in order to identify the active agents in producing the depolarization. Nevertheless it is interesting that a combination of L-cysteine and reducing agents have powerful effects on central neurons. Oxidative stress has been shown to be detrimental to the functions of a number of organ systems and is believed to be the cause of many diseases; therefore, antioxidants have been proposed as treatments for diseases that are believed to be caused by free radicals (Hogg, 1998). However, free radicals that are generated normally during cellular metabolism may have important biological - 176-functions (Kaln, 1998). Therefore, completely reducing a system may also have adverse effects to the function of such systems. In this study, we have shown that bisulfite and dithionite, which are strong reducing agents or antioxidants, have unwanted biological actions, namely powerful depression of synaptic transmission. Therefore, extreme oxidative or antioxidative stress is not ideal for the functioning of a system but rather a balance between oxidative and reducing states may be needed for optimal functioning of biological systems. Moreover, bisulfite is an agent that has been used as food preservatives. In this study, bisulfite is found to have strong biological activities. Whether these compounds when ingested exert any biological actions on humans requires further investigation. 11.3 Implications Of These Findings Since Hb is membrane impermeant, in a large number of studies reported in literature, it has been used as an extracellular NO scavenger in studying the role of NO in various biological system. In many of these studies, Hb was either purchased from Sigma (and other commercial sources) or prepared using Martin et al.'s (1985) method. In these reports, the investigators concluded that the effects they observed were due to Hb's NO scavenging property. However, in this study, we have identified two possible problems associated with the preparation of hemoglobin: first, the impurity of hemoglobin that is purchased from Sigma; second, the possible contamination of hemoglobin by strong reducing agents during the preparation of reduced-Hb using method described - 177-by Martin et al. (1985). Moreover, we have clearly demonstrated that contaminants in hemoglobin samples can have biological activities that are independent of hemoglobin. These findings may help explain some of the different experimental data collected among different laboratories. However, most important of all, many investigators have attributed the actions that they observed using hemoglobin samples from Sigma or hemoglobin prepared using Martin et al.'s (1985) dialysis method to reduced-Hb. Whether the effects reported were due to hemoglobin or the contaminants should be carefully determined before accepting conclusions in these papers. 11.4 Iron and Hemin From the present studies, it appears that hemoglobin itself may not have significant effects on hippocampal neurons. However, iron and hemin produced an irreversible depression of the field EPSPs in concentrations that can be achieved through the breakdown of hemoglobin released during head injuries and hemorrhagic stroke. Therefore, it would be worthwhile to further investigate the actions of these two agents and determine their possible involvement in neurological problems associated with head injuries and stroke. 11.4.1 Free Fe2* In hemorrhagic stroke or intracerebral hemorrhage, iron is released from the metabolized heme and under such circumstances, the iron handling of the neurons may be overwhelmed and intracellular accumulation of iron may occur. In fact, in post-traumatic head injuries, iron deposition is known to occur in - 178-cortical neurons and has been suggested to be related to stroke- or head injury-induced epilepsy (Rand and Courville, 1945; Kaplan, 1961). Moreover, pial application of ferrous and ferric chloride have been shown to cause epileptiform activities (Willmore et al., 1971a, 1971b). In our study iron produced an irreversible depression of synaptic transmission in CA1 neurons and whether this can translate to epileptiform activity requires further investigation. The intracellular accumulated free iron can catalyze two reactions: Reaction 1: 02" + H 2 0 2 -» 0 2 + OH ' + OH" Reaction 2: 2LH + 0 2 ^ LOH + H 2 0 + 2L/ Reaction 1 is called a Fenton reaction. In this reaction, iron catalyses the formation of hydroxyl radicals. The hydroxyl radicals are very reactive and can react with many organic compounds and has been suggested to be responsible for lipid peroxidations, DNA strand cleavage, DNA base destruction, enzyme inactivation, cell lysis, etc (Everse and Hsia, 1997). Reaction 2 describes the process of lipid peroxidation. In fact once lipid peroxidation started, it is a self-catalyzing process and unless the lipid radicals react with each other, otherwise the lipid peroxidation will occur until all lipids are oxidized. Both of these reactions can lead to oxidative injury. Regan and Panter (1993) had shown that prolonged applications of a low concentration of hemoglobin led to neuronal cell death in neocortical cultures (Regan, 1993). Since the neurotoxicity can be blocked with iron chelator, desferroxamine, and lipid soluble antioxidant, Trolox, it is likely that the effects of hemoglobin are due to the release of iron and the production of free radicals by the iron. Nevertheless, whether the effects of iron - 179 -on synaptic transmission as observed in this experiment are due to the production of free radicals ultimately leading to a neurotoxic effect is not clear. Moreover, iron can also act as an irreversible blocker of Ca 2 +-channels and if the channels are involved in exocytosis of transmitter release, synaptic transmission can be suppressed. 11.4.2 Hemin Hemin is a major breakdown product of hemoglobin. During extravascular accumulation of hemoglobin, auto-oxidation of hemoglobin to met-Hb takes place and results in the release of hemin. Hemin is a very hydrophobic molecule and therefore associates preferentially with membrane components (Beaven and Gratzer, 1978; Tipping et al., 1979). In such cases, hemin accumulates in membranes and causes rapid destruction of the integrity of the membrane. In the presence of free radicals such as superoxides, hemin has cytolytic activities towards erythrocytes as well as endothelial cell preparations (Chou and Fitch, 1981; Balla et al., 1991). In hippocampal neurons, it has been shown that superoxides are produced during synaptic activity (Bindokas et al., 1996). During low or high frequency stimulation of the Schaffer Collaterals, production of superoxides has been shown to take place. In addition, bath application of AMPA or NMDA has been shown to enhance the production of superoxides in hippocampal neurons (Bindokas et al., 1996). It is possible that these radicals react with hemin to produce neurotoxic effects and hence causing irreversible depression of field EPSPs. This is, however, a speculation and needs experimental support. Moreover, it is possible that the suppression of field -180-EPSPs caused by hemin may in fact be due to a release of iron from it; this requires further investigation. In addition, normally hemin is metabolized by heme-oxygenase, however, this enzyme can be overloaded during accumulation of hemoglobin (Maines, 1988). Hemin and hemoglobin are strong inducers of heme oxygenase in microglial and astrocytes. It has been shown that heme oxygenase expression was induced by injection of oxyhemoglobin, whole blood or lysed blood into the arachnoid as well as in rat subarachnoid hemorrhage model (Matz et al., 1996a and b; Turner et al., 1998). If heme-oxygenase expression is increased during extravascular pooling of blood, the clearance of hemin will also be increased. Since the breakdown products of hemin are CO, biliverdin and iron, an acceleration in the clearance may also increase the content of CO, iron and biliverdin in the surrounding tissue. Whether these released products have biological activities on synaptic transmission requires further investigation. 11.4.3 Iron, hemin and ischemia As discussed previously, during hemorrhagic stroke, not only is hemoglobin released into the extracellular space, ischemic insult is also taking place in the affected region. After the initial excitotoxic phase, inflammatory response can take place and dead tissues are remodeled by macrophages, microglial and astrocytes (Ransoholl and Tani, 1998; Stoll et al., 1998). These usually take place within days after the initial ischemic insults. Since inflammatory cells and microglial generate free radicals to 'clean up' the affected area, their presence may lead to local increases in the concentration of free - 181 -radicals. In hemorrhagic stroke, hemoglobin has been shown to be released within days of intracerebral hemorrhage ((Barrow et al., 1955; Tourtelotte et al., 1964; Kajikawa et al., 1979). Therefore, with the presence of hemoglobin, hemin and iron, free radical generation can be further enhanced and this may exacerbate the damage that can be caused by free radicals alone. Therefore, the effects of hemoglobin and iron on neuronal cells under ischemic conditions should be further studied. 11.5 Future Directions From the present studies, it appears that hemoglobin itself may not have significant acute effects on hippocampal neurons. However, long-term effects of hemoglobin on synaptic transmission remain to be investigated. Moreover, in hemorrhagic stroke, neurons are not only exposed to Hb but also to ischemic conditions, therefore, the effects of Hb and ischemic conditions on hippocampal neurons should be investigated. In addition, NO has been implicated in the pathogenesis of ischemic stroke. The effects of hemoglobin on NO generated by nNOS and iNOS are not well understood. If hemoglobin is available and acts as a sink for NO generated by these enzymes, it may help to prevent the toxicity mediated by NO. Moreover, if NO generated by eNOS is removed by hemoglobin, then, vasoconstriction may occur which in turn may slow down bleeding. However, if vasoconstriction is too extreme and lasts for a prolonged period of time, then it may have detrimental effects on the delivery of blood to the affected region. In - 1 8 2 -fact, scavenging of NO by hemoglobin has been suggested as a mechanism of cerebral vasospasm following hemorrhagic stroke (MacDonald and Weir, 1991, 1994). The interaction of hemoglobin and NO in hemorrhagic strokes need further studies. In this study, we have identified that iron and hemin produced an irreversible depression of field EPSPs in concentrations that can be achieved through the breakdown of hemoglobin released during head injuries and hemorrhagic stroke. 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