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Electrophysiological actions of hemoglobin on CA1 hippocampal neurons Ip, Joseph Ko Hung 1994

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Electrophysiological Actions of Hemoglobinon CAl Hippocampal NeuronsbyJOSEPH KO HUNG IPB.Sc. (Hon.), The University of British Columbia, 1992A THESIS SUBMflTED IN PARTIAL FULF[LLMENT OFTIlE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Pharmacology and Therapeutics,Faculty of Medicine)We accept this thesis as conformingto the required standard...THE UNiVERSITY OF BRITISH COLUMBIAJuly 1994© Joseph Ko Hung Ip, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of‘/W2t2C4YThe University of British ColumbiaVancouver, CanadaDate /1 14DE-6 (2/88)11ABSTRACTHemoglobin, the oxygen-carrying component of red blood cells, is known as a nitricoxide (NO) chelating agent. For this reason, hemoglobin has been used widely instudying the role of nitric oxide in long-term potentiation (LTP) and excitotoxicity.However, the direct electrophysiological actions of hemoglobin has not been examined.In this investigation, the actions of hemoglobin on rat hippocampal CAl neurons werestudied since hemoglobin may be present in hemorrhagic stroke and other head injuries.Superfusion of rat hippocampal slices with 0.1 mM of bovine hemoglobin for 15 minuteswas induced a significant depolarization associated with an increase in the inputresistance. In addition, hemoglobin suppressed the evoked synaptic responses andincreased the depolarization-induced discharge of action potentials, of rat hippocampalCAl neurons. These hemoglobin-mediated changes usually recovered partially 30minutes after the removal of hemoglobin.While the depolarizing action of hemoglobin was enhanced in a calcium-freemedium, it was not significantly changed by 2-amino-5-phosphonovalerate (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These observations suggest that thedepolarizing action of hemoglobin is independent of the presence of extracellular calciumand activations of the excitatory amino acid receptors. Because hemoglobin has beenobserved to suppress the depolarizing action of glutamate, it is possible that hemoglobinsuppresses the EPSP by interfering with the actions of glutamate. Although hemoglobinhas been suggested to suppress LTP and excitability by scavenging nitric oxide(Garthwaite et al., 1988; Haley et al., 1992; 0’ Dell et al., 1991; Schuman and Madison,1991), the reported actions of hemoglobin were not removed by pre-treatment with 100pM or 500 pM of No-nitro-L-arginine, a nitric oxide synthase inhibitor. Similar to the111scavenging property of hemoglobin, the iron content of hemoglobin probably did notcontribute to the actions of hemoglobin since 0.4 mM or 2.0 mM of ferric chloride did notsimulate the effects of hemoglobin.Because neurons can be exposed to hemoglobin in hemorrhagic stroke and headinjuries, the electrophysiological actions of hemoglobin on rat hippocampal CAl neuronsmay be relevant to the neurological complications associated with intracranial hemorrhageand head injuries. Further studies on mechanisms of the electrophysiological actions ofhemoglobin are necessary for understanding the role of hemoglobin in neuronal damagesassociated with hemorrhagic stroke and other head injuries.(Supervisor)ivTABLE OF CONTENTSChanter Title PafleA. ABSTRACT iiB. TABLE OF CONTENTS ivC. LIST OF FIGURES xiiD. ABBREVIATIONS xivE. ACKNOWLEDGEMENTS xvF. DEDICATION xvi1. INTRODUCTION 12. THE HIPPOCAMPAL FORMATION 32.1. Development of the hippocampal formation 32.2. Nomenclature and architectonics of therat hippocampus 32.2.1. The dentate gyrus 62.2.2. The hippocampus proper 72.2.3 The subicular complex 82.2.4. The hilus 83. LAMELLAR ORGANIZATION OF THE HIPPOCAMPUS 104. ORGANIZATION OF THE TRISYNAPTIC CIRCUIT 115. INTRINSIC CIRCUITRY OF HIPPOCAMPUS 125.1. Entorhinal cortex and the perforant path 125.1 .1. The perforant pathway 125.1.2. The lateral and medial componentsof the perforant pathway 13V5.2. Dentate gyrus and the mossy fibers5.3. Dentate gyrus and the associational projection5.4. Projections originating from CA35.5. Miscellaneous intrinsic connections6. EXTRINSIC CIRCUITRY OF THE HIPPOCAMPUS6.1. Extrinsic hippocampal afferents6.1 .1. Septal projections6.1.2. Isocortical projections6.2. Extrinsic hippocampal efferents6.2.1. Fornix-fimbria system6.2.2. Isocortical projections7. ELECTROPHYSIOLOGY OF HIPPOCAMPAL NEURONS8. IONIC CURRENTS IN HIPPOCAMPAL NEURONS8.1. Sodium currents8.2. Calcium currents8.2.1. High threshold sustained (L)calcium currentLow threshold transient (T)calcium currentHigh threshold inactivatingcalcium currentcurrentsDelayed rectifier current iK(DR)Transient (A) current IK(A)Slowly-inactivating “delay” current I K(D)141516171818181819202021232324242525262626278.2.2.8.2.3.8.3. Potassium8.3.1.8.3.2.8.3.3.vi8.3.4. M-current lK 278.3.5. Inwardly-rectifying K current IK(iR) 288.4. Chloride current 288.5. Current activated by hyperpolarization 288.6. Calcium activated currents 298.6.1. Calcium activated potassium currents IK(Ca) 298.6.2. Calcium activated chloride current ICI(Ca) 318.7. Miscellaneous membrane currents 318.7.1 Leak currents 318.7.2. Sodium activated current 318.7.3. ATP-gated potassium channel 329. FIELD POTENTIALS IN THE HIPPOCAMPUS 339.1. EPSPs and IPSPs 339.2. Population synchronization 3510. EXCITATORY AMINO ACID RECEPTORS IN THEHIPPOCAMPUS 3610.1. The NMDA receptor 3610.1.1. Transmitter recognition domains 3710.1.2. Allosteric modulation by glycine 3810.1.3. PCP channel binding site 3810.1.4. Modulation by polyamine 3910.1.5. Zn2 binding site 3910.2. Non-NMDA receptors 3910.2.1. The AMPA receptor 4010.2.2. The kainate receptor 41vii10.2.3. The L-AP4 receptor 4110.2.4. The GIUG receptor 4211. GABA-ERGIC SYNAPTIC TRANSMISSION 4411.1. Spontaneous IPSPs 4511.2. Characteristics of IPSPs evoked in CAl -CA3 regions 4611.3. Antidromic or feed-back IPSPs 4611.4. Orthodromic or feed-forward IPSPs 4711.5. IPSPs of granule cells 5011.6. Interneurons and inhibitory synaptic transmission 5111.6.1. Basketcells 5111.6.2. Interneurons at the border betweenthe oriens and the alveus 5211.6.3. Lacunosum-moleculare interneurons 5211.7. Presynaptic GABA Receptors 5312. HEMOGLOBIN 5612.1. Structure of hemoglobin 5712.2. Metabolism of hemoglobin 5812.2.1. Synthesis of hemoglobin 5812.2.2. Catabolism of heme 5912.3. Oxygenation of hemoglobin 6012.3.1. Oxygen binding and conformational changein hemoglobin 6012.3.2. 2,3-diphosphoglycerate (DPG) and oxygenaffinity of hemoglobin 61viii13. HEMOGLOBIN AND NITRIC OXIDE 6213.1. Nitrosylhemoglobin 6213.2. Nitric oxide and long term potentiation (LTP) 6214. HEMOGLOBIN AND CEREBRAL VASOSPASM 6515. HEMOGLOBIN AND EPILEPSY 6715.1. Nomenclature of epileptic seizures 6715.2. Pathogenesis of epileptic seizures 6715.3. Subarrachnoid hemorrhage-induced epileptic seizures 6815.4 Iron-induced epilepsy 6916. MATERIALS AND METHODS 7116.1. Animal source 7116.2. Slice preparation 7116.3. Slice chamber 7216.4. Superfusing media 7516.5. Recording and stimulating equipment 7616.5.1. Stimulators and isolation units 7616.5.2. Amplifiers 7616.5.3. Recording systems 7716.5.4. Electrodes 7816.5.4.1. Recording electrodes 7816.5.4.2. Stimulating electrodes 7816.5.4.3. Positioning of electrodes 78ix16.6. Electrophysiological recordings 7916.6.1. Extracellular recordings 7916.6.2. Intracellular recordings 7916.7. Analysis of extracellular and intracellular recordings 8016.7.1. Extracellular recordings 8016.7.2. Intracellular recordings 8016.8. Statistics 8117. EXPERIMENTAL PROTOCOL 8217.1. Electrophysiological effects of rat hemoglobin on rathippocampal CAl neurons 8217.1.1. Without compensation of the hemoglobin-induce change in membrane potential 8217.1.2. With compensation of the hemoglobininduced change in membrane potential 8217.2. Electrophysiological actions of bovine hemoglobin on rathippocampal CAl neurons 8317.2.1. Without compensation of the hemoglobin-induced change in membrane potential 8317.2.2. With compensation of the hemoglobin-induced change in membrane potential 8417.3. The current-voltage (IN) relationship for bovinehemoglobin 8417.3.1. Without compensation of the hemoglobininduced change in membrane potential 8417.3.2. With compensation of the hemoglobininduced change in membrane potential 8517.4. Calcium and the hemoglobin-induced depolarization 8517.5. Involvements of NMDA and Non-NMDA receptors inhemoglobin-induced depolarization 86x17.5.1. APV and the hemoglobin-induceddepolarization 8717.5.2. CNQX and the hemoglobin-induceddepolarization 8717.6. Glutamate-induced and hemoglobin-induceddepolarizations 8817.7. Nitric oxide synthase inhibitor and the actions ofhemoglobin 8917.8. Iron and the actions of hemoglobin 9017.9. Presynaptic volley and the suppression of synaptictransients 9118. RESULTS 9218.1. Effects of rat hemoglobin on rat hippocampalCAl neurons 9218.2. Effects of bovine hemoglobin on rat hippocampalCAl neurons 9418.2.1. The effects of hemoglobin on the evokedsynaptic responses, membrane potential,input resistance, and excitability of CAlneurons 9418.3. The effects of hemoglobin on the extracellularly recordedfield EPSPs 10218.4. Calcium and the hemoglobin-induced depolarization 10618.5. Involvements of NMDA and non-NMDA receptors inthe hemoglobin-induced depolarization 10918.5.1. APV and the hemoglobin-induceddepolarization 10918.5.2. CNQX and the hemoglobin-induceddepolarization 10918.6. Effects of hemoglobin on the glutamate-induceddepolarization 114xi18.7. Effects of Nco-nitro-L-arginine on the actions ofhemoglobin 11718.7.1. Low dose (100 pM) of Nw-nitro-L-arginine 11718.7.2. High dose (500 pM) of Nco-nitro-L-arginine 12418.8 Iron and the actions of hemoglobin 13118.8.1. Low dose (0.4 mM) of ferric chloride 13118.8.2. High dose (2.0 mM) of ferric chloride 13419. DISCUSSION 13919.1. Suppressions of the evoked synaptic responses 14019.2. Actions of hemoglobin on the membrane potential, theinput resistance, and the depolarization-induced dischargeof action potentials 14119.3. Possible involvement of metabotropic receptors in theactions of hemoglobin 14319.4. Implications of the actions of hemoglobin 14519.5. Nitric oxide (NO) scavenging property of hemoglobin 14619.6 The iron component of hemoglobin 14719.7. Future studies required for determining the role of hemoglobinin neurological deficits induced by cerebrovascular injuries 14820. CONCLUSION 14921. REFERENCES 151xiiLIST OF FIGURESFiure Pacie1. Orientation of the hippocampal formation 52. Slice chamber for electrophysiological recordings 743. Effects of hemoglobin on the membrane potential andthe input resistance of rat hippocampal CAl neurons 934. Effects of bovine hemoglobin on the input resistance and theexcitability of rat hippocampal CAl neurons 965. Effects of bovine hemoglobin on the evoked synaptic responses ofrat hippocampal CAl neurons 996. Time course of the effects of bovine hemoglobin on theevoked synaptic responses of rat hippocampal CAl neuronsafter current-clamping the membrane potential to the controllevel 1007. Effects of different concentrations of hemoglobin on the field EPSPand the presynaptic potential 1038. Calcium and the hemoglobin-induced depolarization 1079. Effects of APV on the depolarizing action of hemoglobin 11010. Effects of CNQX on the hemoglobin-induced depolarization 11211. Effects of hemoglobin on glutamate-induced depolarization 11512. Effects of 100 1iM Nc-nitro-L-arginine on the evoked synapticresponses of rat hippocampal CAl neurons 11913. Effects of hemoglobin on the evoked synaptic responses ofrat hippocampal CAl neurons in the presence of I OOjiMNo-nitro-L-arginine 12014. The effect of hemoglobin on the excitability of rat hippocampalCAl neurons in the presence of 100 iM Nw-nitro-L-arginine 12115. Actions of hemoglobin (0.1 mM) and Nco-nitro-L-arginine(100 jiM) on evoked synaptic responses 12216. Effects of No-nitro-L-arginine (500 jiM) on the evoked synapticresponses of rat hippocampal CAl neurons 126xlii17. Effects of hemoglobin on the evoked synaptic responses ofrat hippocampal CAl neurons in the presence of 500iMNco-nitro-L-arginine 12718. The effect of hemoglobin on the excitability of rat hippocampalCAl neurons in the presence of 500 IIM Nco-nitro-L-arginine 12819. Actions of hemoglobin (0.1 mM) and Nco-nitro-L-arginine(500 1iM) on evoked synaptic responses 12920. Effects of 0.4 mM ferric chloride on the evoked synapticresponses of rat hippocampal CAl neurons 13221. The effect of 0.4 mM ferric chloride on the excitability of rathippocampal CAl neurons 13322. Effects of 2.0 mM ferric chloride on the evoked synapticresponses of rat hippocampal CAl neurons 13523. The effect of 2.0 mM ferric chloride on the excitability of rathippocampal CAl neurons 13624. Time Course of the effects of ferric chloride on the evokedsynaptic responses of rat hippocampal CAl neurons 137xivABBREVIATIONSACSF Artificial cerebrospinal fluidAMPA c-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid4-AP 4-aminopyridineAHP AfterhyperpolarizationAP3 2-amino-phosphonopropionic acidAPV 2-amino-phosphonovalerateATP Adenosine triphosphateCA Cornu ammoniscAMP Adenosine 3’:5’-cyclic phosphatecGMP Guanosine 3’:5’-cyclic phosphateCNS Central nervous systemCNQX 6-cyano-7-nitroquinoxaline-23-dCO CarbonmonoxideDNQX 6,7-dinitroquinoxaline-2,3-dioneDTX DendrotoxinEPSP Excitatory postsynaptic potentialGABA y-aminobutyric acidGABA-T GABA transaminaseGAD Glutamic acid decarboxylaseGTP Guanosine triphosphateIPSP Inhibitory postsynaptic potentialL-AP4 L-2-amino-4-phosphonobutanoic acidLTP Long-term potentiationLEA Lateral entorhinal areaMEA Medial entorhinal areaMK8O1 DizolcipineNBQX 2,3-dihydroxy-6-nitro-7-sulphamoylbenzo(F)quinoxalineNMDA N-methyl-D-aspartateNO Nitric OxidePCP PhencyclidineRBCs Red blood cellsRMP Resting membrane potentialTEA Tetraethylammoniumtrans-ACPD trans-I -amino-I ,3-cyclopentanedicarboxylic acidTTX TetrodotoxinxvACKNOWLEDGEMENTSI would like to thank my supervisor Dr. Bhagavatula R. Sastry for hisencouragement and academic guidance. I am indebted to Dr. Zheng Xie and Mr. WadeMorishita for all the technical know-how. I also wish to thank Ms. Gitanjali Adlakha forproof reading my thesis, Mr. Trevor Shew, Mr. Samuel Yip and all members of theDepartment of Pharmacology and Therapeutics for their friendship and emotional support.I am grateful for financial assistance from the Medical Research Council of Canada andthe British Columbia Medical Services Foundation Summer Scholarship. Last but notleast, I want to say thank you to my parents, my sisters and Lorna for standing by me.DEDICATIONDedicated to my parentsxvi11) INTRODUCTION:Cerebrovascular diseases and head injuries are often associated with neurologicalcomplications. Dementia associated with loss of learning and memory is sometimesobserved as a consequence of cerebrovascular diseases and head injuries. Inhemorrhagic stroke and cerebrovascular injuries, extravascular pooling of blood in thesurrounding brain tissue occurs. Since the intracranially pooled blood takes hours to daysto disappear (Sornás et al., 1972), the brain tissue is exposed to an abnormalenvironment for prolonged periods of time. As a consequence, hemoglobin may leak outof erythrocytes and neurons may be exposed to this substance. The mechanismsinvolved in the above mentioned neurological complications are unclear. If, in fact,neurons are exposed to hemoglobin for prolonged periods of time, the possibility that thisagent is a contributing factor to neuronal damages associated with stroke and othercerebrovascular diseases, should be examined.NO has been suggested as a diffusible messenger which links activation ofpostsynaptic NMDA receptors to functional modifications in neighboring presynapticterminals and glial cells (Garthwaite et al, 1988). Since hemoglobin is known as a NOscavenger which is not membrane-permeant (Gibson and Roughton, 1957), this agenthas been used in studying the role of NO in excitotoxicity and synaptic plasticity.Hemoglobin has been found to suppress LTP (Haley, 1992; Musleh, 1993; O’Dell, 1991;Schuman and Madison, 1991) as well as the population EPSP (Garthwaite et al., 1988).However, the role of NO in LTP and in excitotoxicity is controversial (Izumi et al., 1992b;Pauwels and Leysen, 1992). Even though hemoglobin has been used widely as a NOchelator in studying the role of NO, the direct electrophysiological actions of hemoglobinon neurons have not been examined. It is possible that mechanisms besides chelation of2NO may be involved in suppression of the population EPSP, LTP, and excitotoxicity.Moreover, these unknown actions of hemoglobin may contribute to neuronal deficitsinduced by hemorrhagic stroke and cerebrovascular injuries. In this study theelectrophysiological effects of hemoglobin on rat hippocampal CAl neurons wereexamined. It is hoped that information from these studies will form a basis to determine ifthe agent is related to neurological complications associated with hemorrhagic stroke andother cerebrovascular injuries.32 THE HIPPOCAMPAL FORMATION:2.1) Development of the Hippocampal Formation:During ontogenic development, the cortical mantle is subdivided into theallocortex and the isocortex. While the isocortex separates completely from thecortical mantle to form the neocortex, the allocortex which contains the archicortexand the paleocortex remains attached to the cortical mantle (Filimonoff, 1947). Thearchicortex or hippocampus consists of the subicular complex, the hippocampusproper (Ammon’s horn), the dentate gyrus (fascia dentata), and both precommisuraland supracommissural hippocampi (Chronister and White; 1975; Schwerdtfeger,1984; Teyler and DiSenna, 1984). The paleocortex contains the olfactory bulb, theaccessory olfactory bulb, the anterior olfactory nucleus, the olfactory tubercle, theperiamygdala region, the septum, the diagonal region, and the periform region(Schwerdtfeger, 1984).Besides the archicortex and the paleocortex, an additional structure denoted asthe periallocortex exists between the allocortex and the isocortex. The periallocortexcomprises of the entorhinal, the peripalaeocortical, the clautral, the presubicular, theretrosplenial, and the periarchicortical cingulate cortices (Blackstad, 1956; Brodmann,1909; Chronister and White, 1975; Lorente De NO, 1934; Sandies, 1972; VazFerreira, 1951, White, 1959).2.2) Nomenclature and Architectonics of the Rat Hippocampus:The rat hippocampus, a bilaterally symmetrical structure which resembles acashew nut in shape, extends from the septal nuclei rostro-dorsally to the temporallobe caudo-ventrally (Green, 1964; Teyler and DiScenna, 1984). The hippocampus issituated below the neocortex and along the floor of the descending horn of the lateral4ventricle. The longitudinal or septo-temporal axis of the hippocampus runs from theseptal pole to the temporal pole while the transverse axis is perpendicular to thelongitudinal axis (Fig. 1).The hippocampus is composed of the Ammon’s horn, the dentate gyrus, andthe subiculum. The ventricular surface of the hippocampus is covered by a white fiberlayer, the alveus. These fibers, which are mainly constituted by axons from cells ofthe Ammon’s horn, converge to form the fimbria on the medial surface of thehippocampus (Teyler and DiScenna, 1984). The granular cell layer of the dentategyrus and the pyramidal cell layer of the Ammon’s horn are the two principal layers inthe rat hippocampus.Based on Golgi preparations, Ramón y Cajal (1911) subdivided Ammon’s horninto an area of large pyramidal cells (regio inferior) and a region of more denselypacked smaller pyramidal cells (regio superior). Unlike Ramón y Cajal, Lorente de No(1934) divided Ammon’s horn or hippocampus proper into four subfields, CAl to CA4.Although CA4 in Lorente de NO’s scheme corresponds to the polymorphic zone of thedentate gyrus described by Ramón y Cajal, it is unclear as to whether the CA4 regionbelongs to the dentate gyrus or the hippocampus proper. More recent studies tend tosupport that the polymorphic zone belongs to the dentate gyrus rather than to theAmmon’s horn (Blackstad, 1956; Amaral, 1978; Gaaraskjaer, 1981). While areas CA2and CA3 together correspond to the regio inferior, area CAl resembles the regiosuperior.5Subfiekls ofAmmon’s HornRhinalSubfields ofAmmon’s HornProximalEntorhinal CortexFigure 1. Orientation of the rat hippocampus. While the septotemporal axis runs fromthe septal pole to the temporal pole, the proximodistal axis runs from the hilus of thedentate gyrus toward the subiculum, Adapted from Witter (1989).Subfields ofAmmon’sDistalDentate Gyrus62.2.1) The Dentate Gyrus:In the dentate gyrus, granule cells, which are ovoid in shape and 15 to 25 urnin diameter, are the major cell type in the cell layer, stratum granulosurn (Golgi, 1886).The granule cell layer can be categorized, in relation to its location to the pyramidalcells of CA3, into a suprapyramidal (upper) and an infrapyrarnidal (lower) blade whichmerge at the crest of the dentate gyrus. While the blade which is adjacent to thehippocampal fissure is called the suprapyramidal blade, the opposite blade is theinfrapyramidal blade (Chronister and White, 1975; Swanson et al., 1978). As a resultof the sharp curvature of the dentate gyrus, the dentate gyrus contains the hilar areawhich is composed of several layers of polymorphic cells (Amaral, 1978; Lorente deNO, 1934).There are three strata, namely the stratum granulosum, the stratum moleculare,and the stratum polymorphe, in the dentate gyrus. While the stratum granulosum ispopulated by granule cells, the stratum moleculare is where the perforant pathprojections terminate. The stratum polymorphe contains various cell types such asbasket and modified pyramidal cells which give rise to at least two systems ofassociational connections that end within the dentate gyrus. The axons of granulecells, mossy fibers, collateralize in the polymorphic cell layer before entering the CA3field where they form en passant synapses on the proximal dendrites of the pyramidalcells (Blackstad et al., 1970; Claiborne et at., 1986; Gaarskjaer, 1978; Gaarskjaer,1986), whereas the dendrites project into the stratum moleculare layer.72.2.2 The Hippocampal Proper:Following the pyramidal cell layer of Ammon’s horn along its transverse axisfrom the dentate gyrus (proximal) to the subiculum (distal), the CA3 field mergesdistally with the CA2 field; the proximal part of CAl joins CA2; and the distal part ofCAl connects the subiculum. The CA2 subfield which is not innervated by mossyfibers is a transition zone between the CAl and CA3 subfields (Chronister and White,1975; Lorente de No, 1934). The CAl and CA3 subfields are further categorized intoCAIa, CAIb, CAIc, CA3a, CA3b, and CA3c (Lorente de NO, 1934). The principle celltype in Ammon’s horn is the pyramidal cell which is pear in shape and 25 to 40 tm indiameter (Golgi, 1886).In addition to the pyramidal cell layer or stratum pyramidale, the alveus whichlies next to the epithelium of the lateral ventricle, the stratum oriens which is situatedbetween the alveus and the stratum pyramidale, the stratum lucidum which is next tothe stratum pyramidale, the stratum radiatum, the stratum lacunosum, and stratummoleculare, can also be found in the Ammon’s horn (Kolliker, 1896; Cajal, 1911;Lorente de No, 1934). While the stratum alveus contains primary efferent axons ofpyramidal cells, the stratum oriens comprises of the basal dendrites of pyramidalcells, axons originating from the alveus, and non-pyramidal neurons. The stratumlucidum contains mossy fibers from the dentate gyrus. The straturr pyramidale isoccupied by the soma of pyramidal cells. The apical dendrites of pyramidal cells lie inthe stratum radiatum. In the stratum lacunosum-moleculare, both interneurons anddistal dendrites of the pyramidal cells can be found.In rat, the separation between the stratum radiatum and the stratum lacunosumis not well defined. Therefore, these two layers are collectively described as the8stratum radiatum (Lorente de No, 1934). For a similar reason, the stratum lucidumand the stratum pyramidale are considered as one layer (Lorente de NO, 1934). Theboundaries of the stratum radiatum and the stratum oriens mark the end of thehippocampus proper at the hilus. The abrupt termination of the stratum pyramidale inthe CAl field defines the boundary at the subicular end (Angevine, 1975; B)ackstad,1956; RamOn y Cajal, 1893; Lorente de NO, 1934).2.2.3) The Subicular Complex:The subiculum, which lies between the CAl subfield of the hippocampusproper and the presubicular region of the periallocortex, is replaced by the pre- andparasubiculum distally, the retrosplenial cortex dorsally, as well as the entorhinalcortex ventrally. The rat entorhinal cortex, which can be subdivided into LEA andMEA (Blackstad, 1956; Steward, 1976; Wyss, 1981; Krettek and Price, 1977), areconstituted by six cortical layers, namely the superficial (layers I-Ill) and the deep(layers lV-Vl) layers.2.2.4) The Hilus:Not only do the dentate granular cells send mossy fibers to CA3, but alsodistribute many axon collaterals within a relatively narrow lamella of the hilus of thedentate gyrus (Claiborne et al., 1986; Fricke and Cowan, 1978; Swanson et al., 1978).Most of the associational and commissural inputs to the dentate gyrus, whichterminate in the inner one-third of the molecular layer of the dentate gyrus, arise fromhilar cells. The hilar region receives extrinsic afferents predominantly from the brainstem and the septal area. These afferents include a prominent noradrenergic inputfrom the locus coeruleus, a serotonergic input from the median and dorsal raphe, acholinergic input from the medial septal nucleus, and a histaminergic input from the9supramammillary region. Even though these afferents are most dense in the hilus ofthe dentate gyrus, innervations of the other subfields of the hippocampus have alsobeen reported (Swanson et al., 1987). Because connections between the dendritictrees of hilar cells and the molecular layer of the dentate gyrus have been suggested(Amaral, 1978), these dendrites may be influenced directly by the major cortical inputsto the dentate gyrus. Therefore, the hilus is probably a functionally different unit ofthe hippocampus which may play a regulatory role in the dentate gyrus. It may eitherenhance or inhibit information flow in the dentate gyrus.103) LAMELLAR ORGANIZATION OF THE HIPPOCAMPUS:Electrophysiological data have suggested that the hippocampus is organized ina lamellar fashion (Andersen et al., 1971b). When the entorhinal area is stimulated,four pathways, namely the perforant pathway, the mossy fibers, the Schaffercollaterals, and the alvear fibers of CAl become activated successively (Andersen etal., 1971b). Each lamella is located perpendicularly to the longitudinal axis of thehippocampus. Connections from the dentate gyrus to the subiculum can be found ineach lamella. While the dentate gyrus, whose major cortical afferents originated fromthe entorhinal cortex, is the major input structure of the hippocampus, the subiculum isthe main source of the hippocampal efferents to subcortical and cortical areas,including the entorhinal cortex (Witter, 1986; Swanson et al., 1987).On the contrary, studies conducted by Hjorth-Simersen (1973), Laurberg(1979), and Swanson et al. (1978) suggest that the intrinsic circuitry of thehippocampus extends along the septo-temporal axis as much as in the transversedirection. By using an extended hippocampal preparation ( a procedure in which theseptal to temporal curvature of the hippocampus is corrected by flattening thehippocampus so that a more accurate representation of fibers being distributed in thesepto-temporal plane when transverse slices are cut) and Phaseolus vulgarusleucoagglutinin (PHA-L), the three-dimensional organization of the intrinsichippocampal circuitry, except mossy fibers, is not restricted to a lamellar array.114) ORGANIZATION OF THE TRISYNAPTIC CIRCUIT:Mossy fibers which arise from the dentate granule cells innervate the entiretransverse or proximodistal extent of CA3. These highly laminated fibers formsynaptic contacts with proximal dendrites of CA3 pyramidal cells. The proximalportions of CA3 preferentially interact with fibers that originate in the infrapyramidalblade, the crest, and the adjacent portion of the suprapyramidal blade (Claiborne etal., 1986). Neurons at the tip of the suprapyramidal blade are linked to more distalportions of the CA3 field. The mossy fibers which terminate on the basal dendrites ofmore proximally located CA3 pyramidal cells originate principally from theinfrapyramidal blade of the dentate gyrus.Schaffer collaterals, which arise from the pyramidal cells in field CA3, synapsein the stratum radiatum and stratum oriens with the dendrites of CAl pyramidal cells.The CAl pyramidal cells in turn give rise to projections to the subiculum whichterminate in the deep half of the molecular layer and in the pyramidal cell layer. Theconnections between CA3 and CAl and those from CAl to the subiculum areorganized perpendicularly to the cell layer in a columnar manner. Neurons in CA3and CAl constitute axonal columns in CAl and the subiculum, respectively. Whilefibers from proximal parts of CA3 and CAl interact with distal parts of CAl and thesubiculum, respectively, more distal parts of CA3 and CAl are linked to the moreproximal parts of CAl and the subiculum, respectively (Tamamaki et al., 1987;Ishizuka et al., 1988).125 INTRINSIC CIRCUITRY OF HIPPOCAMPUS:5.1) Entorhinal cortex and the Perforant Path:The topographical organization of the entorhinal-dentate projections and that ofthe afferents of the entorhinal cortex indicate that the lateral and caudal parts of theentorhinal cortex which mediate sensory information project densely to septal andintermediate parts of the dentate gyrus, in particular to the suprapyramidal blade.More medial parts of the entorhinal cortex project preferentially to more temporallevels of the dentate gyrus. These parts of the entorhinal cortex also show a slightpreference for the infrapyramidal blade. Because more medial parts of entorhinalcortex differ with respect to their afferents from the lateral part, different subsets ofdentate granular cells probably mediate different types of information (Witter et al.,1986, 1989). Projections to Ammon’s horn have also been reported by Steward(1976) and Wyss (1981). While projections to the dentate gyrus and CA3 arisemainly from neurons in layer II, projections to CAl originate from cells in layer Ill(Stewart and Scoville, 1976).5.1 .1) The Perforant Pathway:The entorhinal cortex gives rise to the perforant pathway which projectsstrongly to the dentate and the hippocampus (RamOn y Cajal, 1893; Lorente de NO,1933,1934; Blackstad, 1956, 1958; Raisman et aL, 1965). The fibers of the perforantpath travel dorsally and eventually along the transverse plane. They enter thedentate gyrus and the hippocampus after coursing through the pyramidal layer of thesubiculum along its long axis. Besides the pyramidal layer of the subiculum, perforantpath fibers also distribute within the molecular layer of the dentate gyrus, stratumlacunosum-moleculare of the hippocampus and the molecular layer of the subiculum13(Hjorth-Simonsen and Jeune, 1972; Nafstad, 1967; Steward, 1976; Witter et al.,1986). It has been reported that fibers of the perforant path cross the hippocampalfissure at the connection between the suprapyramidal and infrapyramidal blades ofthe dentate gyrus (i.e. the crest), bifurcate and extend branches that ramify into thesuprapyramidal and infrapyramidal blades of the molecular layer (Golgi, 1886).Although Blackstad (1956) and Hjorth-Dimonsen and Jeune (1972) suggestedthat the majority of the perforant pathway fibers traverse the molecular layers of thesubiculum and CAl, and subsequently cross the hippocampal fissure to reach themolecular layer of the dentate gyrus, Witter et al. (1988) has reported that fiberswhich follow the above pathway are not significant. Many of the entorhinal fiberstravel along the transverse axis of the molecular layer of the Ammon’s horn andsubsequently enter the suprapyramidal tip of the molecular layer of the dentate gyrus.These observations imply that entorhinal fibers can either reach the dentate gyrusdirectly or interact with cells in CA3 before reaching the dentate granule cells.5.1.2) The Lateral and Medial Components of the Perforant Pathway:The perforant pathway can be divided into two components which arise fromdifferent parts of the entorhinal cortex and differ with respect to their distribution in thehippocampus. The lateral perforant pathway is organized in a way which allows asmall part of LEA to interact with a large part of the hippocampus along its longitudinalaxis and one-third of the apical dendrites of the cells in the dentate gyrus and CA3.While the lateral perforant pathway has been reported to distribute preferentially tothe suprapyramidal blade of the dentate gyrus, the medial component either does notshow a preference or prefers the infrapyramidal blade of the dentate gyrus (Wyss,1981). Septal parts of the dentate gyrus are innervated by the lateral perforant14pathway which originates laterally in LEA and the medial perforant pathway whicharises from the most caudolateral part of MEA. The more intermediate parts of thedentate gyrus are influenced by the lateral and medial components originating atapproximately the same rostrocaudal level in LEA and MEA.In addition to the projections to the dentate gyrus, the lateral perforant pathwayprojects to the distal part of CAl, whereas the medial perlorant pathway projects tothe more proximal portion of CAl. Generally, fibers from LEA distribute moreextensively along the longitudinal axis of the hippocampus than do fibers thatoriginate in MEA (Witter et al., 1988).The difference in organization of the two components of the perforant pathwayimplies that these two components may exert different actions on the same targetneurons in the dentate gyrus and CA3. In the subiculum, they may affect the samecells in the same way even though both components differ with respect to theirneuroactive substances and electrophysiological characteristics (McNaughton, 1980;Fredens et al., 1984). In CAl, these two components may influence different cellpopulations.5.2) Dentate Gyrus and the Mossy Fibers:Mossy fibers are principally oriented transverse to the long axis of thehippocampal formation. Mossy fibers arising from the septotemporal level of thedentate gyrus minimally overlap with those originating from other septotemporallevels. After these fibers turn abruptly and caudally at the septal levels of thehippocampal formation as they approach the CAl field, they travel along the long axisof the hippocampus (RamOn y Cajal, 1893, Swanson et al., 1978). At mid andtemporal levels of the hippocampal formation, mossy fibers are not as temporally15directed (i.e. along the long axis) as the septal level. Mossy fibers which originatefrom the infrapyramidal blade travel in stratum oriens and terminate within subfieldsCA3b and CA3c, whereas those arising from the suprapyramidal blade travel in thestratum lucidum, course through CA3, and terminate at CA2 (Blackstad et al., 1970;Chronister and White, 1975; Lorente de NO, 1934). Throughout their course, mossyfibers form excitatory en passant synapses with the dendrites of pyramidal cells inCA3 (Andersen et al., 1966a) In addition to pyramidal cells, mossy fibers alsosynapse on basket cells and neurons in the hilus of the dentate gyrus. By using PHAL, the mossy fiber projection to the CA3 field has been reported to demonstrate asomewhat lamellar organization (Amaral and Witter, 1989).5.3) Dentate Gyrus and the Associational Projection:Unlike the mossy fiber projection to the CA3 field, the associational projectionto the dentate gyrus is not organized in a lamellar fashion. The associationalprojection to the dentate gyrus arises mainly from neurons in the polymorphic layer ofthe dentate gyrus, not from the pyramidal cells in CA3 (Hjorth-Simonsen andLaurberg, 1977; Laurberg, 1979; Laurberg and Sorensen, 1981). The inner one-thirdof the molecular layer of the dentate gyrus is innervated by fibers of ipsilateral origin,namely the cells of the polymorphic region or the CA3 pyramidal cells which are closeto the dentate gyrus (Swanson et al., 1978; Zimmer, 1971). This projection appears tobe divergent along the long axis of the dentate gyrus (Swanson et al., 1978). Theassociational projection is not organized to provide feedback to granule cells at thesame level in which polymorphic cells are located, but to levels as far as severalmillimeters away from the cells of origin (Ishizuka et al., 1990).16The polymorphic cells of the dentate gyrus do not project to the Ammon’s horn.The associational projections within CA3 and the Schaffer collateral system to CAloriginate exclusively in the CA3 and CA2 fields of the hippocampus (Hjorth-Simonsenand Laurberg, 1977; Laurberg, 1979; Laurberg and Sorensen, 1981).5.4) Projections Originating from CA3:The pyramidal cells of CA3 give rise to associational projections that terminatewithin CA3 and Schaffer collaterals which innervate CAl (Swanson et al., 1980;Swanson et al., 1981). The associational projections, which travel primarily parallel tothe long axis, link different levels of the hippocampus, whereas the Schaffercollaterals only link the CA3 and CAl fields of the same hippocampal level (Lorentede NO, 1934). The projections from CA3 to CA3 and from CA3 to CAl terminateextensively both in the stratum oriens and stratum radiatum, but not in the stratumlacunosum-moleculare (Hjorth-S imonsen, 1973).Schaffer collaterals course through CAl and make synaptic contacts withpyramidal cells on their basal dendrites in the stratum oriens and at the proximalthree-quarter of the apical dendrites in the stratum radiatum (Gottileb and Cowan,1973; Lorente de No, 1934). Fibers arising from CA3 cells which are close to thedentate gyrus project preferentially to the distal portion of CAl or where theyterminate in the superficial portion of the stratum radiatum. Fibers originating fromCA3 cells which are close to the CAl border distribute predominantly to parts of CAlthat are close to CA3 and to deeper portions of stratum radiatum. In general, CA3cells located close to the dentate gyrus project preferentially in a septal direction,whereas those which are located near the CAl border tend to project exclusively in atemporal direction.175.5) Miscellaneous Intrinsic Connections:Unlike the CA3 cells, CAl pyramidal cells do not give rise to projections toother levels of CAl. Fibers from CAl cells project to the subiculum in a columnarmanner (Finch and Babb, 1981; Finch et al., 1983; Tamamaki et al., 1987). Weakprojections from CAl to the deep layers of the entorhinal cortex have also beenobserved (Swanson et al., 1978).Other intrinsic connections such as projections from the subiculum to thepresubiculum, the parasubiculum, and the entorhinal cortex (Beckstead, 1978; Finchet al.; 1983, Köhler, 1985; KOhIer et al., 1978; Shipley, 1975), projections from thepresubiculum and parasubiculum to layers Ill and II of the entorhinal cortex,respectively (KOhler, 1985; Shipley, 1975), and projections in the entorhinal cortexwhich link various parts of the region and the deep layers with more superficial layers(Köhler 1986; Köhler, 1988; Witter et al., 1986), also exist in the hippocampus.1861 EXTRINSIC CIRCUITRY OF THE HIPPOCAMPUS:6.0 Extrinsic Hippocampal Afferents:6.1.1) Septal Projections:In addition to fibers of the perforant path, fibers which originate in the lateraland medial septal nuclei, as well as the nucleus of the diagonal band project to thedentate gyrus. These septo-hippocampal projections which are primarily cholinergicefferents (Kramis et al., 1975) are responsible for the rhythmic theta activity, whichare reflective of a behavioral state corresponding to intentional movement, in thehippocampus (Petsche et al., 1962; Vanderwolf et al., 1975). Because some neuronsin the medial septal nucleus and nucleus of the diagonal band are glutamic aciddecarboxylase (GAD) positive (Kohler et al., 1984; Panula et al., 1984), it is possiblethat GABAergic input to the dentate gyrus may exist.Besides the dentate gyrus, fibers from the medial septal nucleus also project toCA3. These projections which are mainly cholinergic (Frotscher et al., 1986; Houseret al., 1983) concentrate in stratum oriens (Nyakas et al., 1987). Projections from themedial septal nucleus to CAl are controversial since some investigators (Monmaurand Thompson, 1983; Sakanaka et al., 1980) observed the presence of this pathwaywhereas others (Powell, 1963; Rose et al., 1976; Swanson and Cowan, 1979) did not.6.1.2) Isocortical Projections:Projections from the isocortex to the hippocampus, which arise from theparietal, the temporal, and the perirhinal cortices, terminate exclusively in CAl,occasionally in CA3, and not in the dentate gyrus (Schwertfeger, 1979; Schwertfeger,1984). Because these projections link the hippocampus with different regions of thecortex, hippocampal function can be influenced by cortical sensory input.19In rats, the entorhinal cortex receives an input from the perirhinal cortex(KOhler, 1986). The perirhinal fibers which receive their input from widespreadsensory-related parts of the cortical surface (Deacon et al., 1983) predominantlyterminate in lateral portions of LEA and lateral and caudal parts of MEA (Witter et al.,1988). Although sensory convergence has been suggested to take place in thesuperficial layers of lateral parts of the entorhinal cortex (Vaysettes-Courchay andSessler, 1983), not much is known with respect to the afferents of more medial partsof the entorhinal cortex. The medial parts of the entorhinal cortex are thought toreceive information different from that received by the lateral parts (Witter et al., 1986,1989).6.2) Extrinsic Hippocampal Efferents:The hippocampus projects to various cortical and subcortical structures,including the entorhinal cortex, the septum, the nucleus accumbens, the amygdaloidcomplex, and the hypothalamus. Although these extrinsic projections originate mostlyfrom the subiculum, Ammons horn, especially CAl, also contributes to theseprojections (Swanson et al., 1987). While the projection to the nucleus accumbensoriginates in the cells that are located at the subicular-CAI border, the more distallylocated neurons in the dorsal subiculum projects to the retrosplenial and perirhinalcortices (Groenewegen et al., 1987). Although projections to the nucleus accumbens,and the retrosplenial and perirhinal cortices originated in differently located subicularcell populations, projections to the septum and entorhinal cortex arise from the sameneuronal population.206.2.1) Fornix-Fimbria System:The fornix-fimbria system is the most well studied extrinsic pathway in thehippocampus (O’Keefe and Nadel. 1978). Efferent fibers from the hippocampus andadjacent allocortical areas converge in the fimbria. These fibers then course throughthe septo-fimbrial nucleus and diverge into the post-commissural and precommissural fornices.Projections of the post-commisural fornix which arise from the presubiculum,the parasubiculum, and the subiculum, project to the thalamus, mammillary bodies,and the rostral brain stem (Chronister and DeFrance, 1979). The pre-commissuralfornix comprises of fibers from CAl and CA3 which distribute in the septal nucleus,the diagonal band of Broca, the bed nucleus of the anterior commissure, the lateralpreoptic region, and the lateral hypothalamus (Swanson and Cowan, 1977).6.2.2) Isocortical Projections:Efferent projections which serve to link the hippocampal formation with theisocortex have been reported between CAl and various parts of the frontal (Swanson,1981), the temporal (Schwerdfeger, 1979), the retrosplenial, and the perirhinalcortices (Swanson and Cowan, 1979).2171 ELECTROPHYSIOLOGY OF HIPPOCAMPAL NEURONS:The RMPs of pyramidal cells range from -50 to -70 mV, whereas that of granulecells range from -60 to -85 mV. Depolarization-evoked action potentials should rangefrom 50 to 110 mV for pyramidal cells and 70 to 140 mV for granule cells on average.By using the slope of a linear portion of an IN (Current vs Voltage) curve, the averageinput resistance of granule cells is calculated to be 40 - 45 Mf. While the averageinput resistance of CAl pyramidal cell is about 25- 30 Mc2, that of CA3 pyramidalcells is approximately 35 Mi These values are an order of magnitude higher thanthe input resistances recorded for spinal cord motorneurons (Barrett and Crill, 1974),but are comparable to those for neocortical neurons (Lux and Pollen, 1966; Connorset al., 1982). The membrane time constants, the time for membrane potential to reach1-l/e of its peak voltage in response to short current pulses (Spenser and Kandel,l96la), of the CA3 pyramidal, the CAl pyramidal, and the granule cells are 25, 15,and 11 ms, respectively. These time constants for hippocampal neurons are muchlonger than those for motorneurons (Barette and Crill, 1974). Based onelectrophysiological data derived from time constant measurements, the ratio of thecell’s dendritic and somatic conductances (p) has been reported to be approximately1.0- 1.5 (Brown et al., 1981; Johnson, 1981). The small p indicates that a large partof the cell is isopotential, including regions that are morphologically considered asdendritic. The electronic length (L) of hippocampal neurons is near I (Schwartzkroinand Mueller, 1987). This small L value indicates that current injected in the distalsynaptic region would have an appreciable effect on activity initiated at the somasince almost all of the injected current should reach the soma. Recent studies using22the multipolar cylinder model have found the L value to be 1/3 of the value mentionedpreviously (Glenn, 1988).2381 IONIC CURRENTS IN HIPPOCAMPAL NEURONS:8.1) Sodium Currents:A fast sodium (Na) current (INa(fast)) which exhibits the characteristics describedby Hodgkin and Huxley (1952) for squid giant axon, namely an activation threshold of-60 mV and a time to peak of about 0.9 ms at 0 mV, exists in hippocampal neurons(Kaneda et al., 1988; Sah et al., 1988a). Inactivation is complete throughout theactivation range. The permeability sequence for this current is Li> Na> hydrazine>formidine> guanidine > methylguanidine > monomethylamine.This fast Na current is primarily somatic in origin and is responsible for the fast,TTX-sensitive somatic action potentials. QX-312 sensitive Na-spikes which havesimilar rise times but different decay rates have been reported to exist on isolateddendrites of hippocampus (Benardo et al., 1982). Dendritic Na currents whichdemonstrate comparable time-course and current density to those in the soma wereonly observed in freshly dissociated neocortical neurons (Hugenard et al., 1989).Using the single microelectrode clamp technique, a slowly inactivating Nacurrent, which is sensitive to TTX and QX314 has also been recorded from rathippocampal CAl neurons (French and Gage, 1985). The activation threshold of thisslow Na current is about 5 to 10 mV positive to the resting membrane potential.Because equivalent rectification in isolated dendrites is insensitive to QX314 and isblocked by Mn2, the slow Na current may be confined to the soma (Bernado et al.,1982; French et al., 1990). This slow Na current may participate in hippocampalpacemaker activity (Brown et al., 1990) or repetitive firing of action potentials inresponse to prolonged depolarizations induced by intense synaptic activity (French etal., 1990).248.2) Calcium Currents:A high-threshold and sustained (L), a low-threshold (T), as well as a high-threshold and inactivating (N) calcium currents exist in hippocampal neurons (Fox etal., 1987a, b). The conductances of T-, L-, and N-type calcium channels are 7-8 pS,25 -27 pS, and 13 - 15 pS, respectively (Gray and Johnson, 1987).8.2.1) Hih Threshold Sustained (L) Calcium Current:The L-current is a slowly-activating and sustained inward current which isenhanced by 8a2 as well as blocked by Co2 and Cd2 (Johnson et al., 1980; Brownand Griffith, 1983a; Brown et aL, 1984; Docherty and Brown, 1986; Gâhwiler andBrown, 1987a). Using Ba2 as the charge carrier, this current demonstrated littleinactivation over several hundred of ms and appeared to be persistent between -50and -10 mV (Johnson et al., 1980; Brown and Griffith, 1983a). The L-current issensitive to verapamil (100 riM) (Brown and Griffith, 1983a), o-conotoxin (p.Mconcentrations) (Mogul and Fox, 1991), and dihydropyridine such as nimodipine (50nM-10 p.M), nifedipine (1 p.M), and PYIO8-068 (1 p.M). Bay K8644 (1-10 jiM), acalcium agonist, noradrenaline, and isoprenaline have been reported to enhance theL-current (Brown et al., 1984; Docherty and Brown, 1986; Segal and Barker, 1986;Gähwiler and Brown, I 987a; Gray and Johnson, 1987). Activation of the L-channelcan be described by the Boltzmann equation with a threshold of approximately -50mV, half-activation at -20 mV, a slope factor of 10 mV and a cooperativity factor of 2(Kay and Wong, 1987). The L-current has been suggested to be responsible forhippocampal burst-behavior (Johnson et al., 1980).258.2.2) Low Threshold Transient (T) Calcium Current:The low-threshold transient (T) current, which can be activated by adepolarization to about -60 mV from potentials as low as -100 mV and inactivated at -50 mV, has been observed in hippocampal CAl neurons (HalliweIl, 1983; Ozawa etal., 1989; Takahashi et al., 1989). While the T-current is blocked by Cd2 (100 pM),Ni2 (lC 0.2 - 0.4 mM), verapamil (100 pM), and phenytoin (Halliwell, 1983; Moguland Fox, 1991), the T-current is relatively resistant to dihydropyridine (Tsien et al.,1988). By using 10 mM of Ca2 as the charge carrier and a holding potential of -100mV, the threshold and the peak of the current were determined to be -60 and -30 mV,respectively. The T-current has been suggested to be involved in the initial phase ofburst potentials (Brown and Griffith, 1983a; Traub, 1982), the subthresholdprepotential which confers aCa2-dependent component of inward rectification (Brownand Griffith, I 983a) and pace-maker depolarizations (Llinás and Yarom, 1981).8.2.3) High Threshold Inactivating Calcium Current:The N-current which is activated by a depolarization to +10 mV decays over100 ms (Ozawa et al., 1989; Takahashi et al., 1989b). The N-current inactivates atpotentials around -70 mV (Ozawa et al., 1989). The N-type channel, whichdemonstrates a higher conductance for Ba2 than for Ca2 ions (Tsien et al., 1988), isblocked by Cd2, Ni2, and o-conotoxin, but is relatively insensitive to dihydropyridine.While activations of adenosine (Madison et al., 1987a) and muscarinic (Gãhwiler andBrown, 1987b; Toselli and Lux, 1989) receptors reduce the N-current, noradrenalineand isoprenaline have been reported to augment the current (Gray and Johnson,1987).268.3) Potassium Currents:Five different voltage-gated K currents, namely a delayed rectifier current IK(DR)a transient or A current IK(A), a delay current IK(D), a subthreshold and non-inactivatingcurrent (M current) IK(M), and an inwardly rectifying current IK(IR), are present inhippocampal neurons.8.3.1) Delayed Rectifier Current lK(DR):The IK(DR) has been recorded in depolarizing hippocampal neurons at -40 mV(Segal and Braker, 1984; Numann et al., 1987; Sah et al., 1988b). The activation ofthis current is slow. At 0 mV, the time-to-peak is approximately 50-200 ms. Atthreshold, the time constant is about 100 ms (Rogawski, 1986). The IC50 of TEA forthis current is about 10 mM (Segal and Barker, 1984). Even though this current issensitive to TEA, it is relatively insensitive to 4-AP.8.3.2) Transient (A) CurrentThis current has been recorded from hippocampal neurons in situ (Gustafssonet al., 1982; Zbicz and Weight, 1985), in culture (Segal et al., 1984; Segal and Barker,1984), and in acutely dissociated neurons (Numann et al., 1987; Alger and Doerner,1988; Sah et al., 1988b). The IK(A) activates (time constant = 5-10 ms) and inactivates(time constant = 20-30 ms) rapidly. This current is insensitive to TEA but is readilyblocked by 4-AP (>100 tM) and DTX (50-300 nM) (Halliwell et al., 1986).Noradrenaline and acetylcholine have also been reported to inhibit the IK(A) (Sah et al.,1985; Nakajima et al., 1986). This transient potassium current has been suggested tobe responsible for spike repolarization in hippocampal cells (Storm, 1987a & b).278.3.3’) S lowly-Inactivating “delay” currentRapid activation is also a characteristic of IK(D). IK(D), which showsmulticomponent inactivation over several seconds, has an activation threshold ofabout -75 mV. This slowly inactivating current is very sensitive to 4-AP (completeblock at 30-40 pM) (Storm, 1988) and DTX. In hippocampal cells, IK(D) is believed tointroduce a long delay in the firing induced by threshold depolarizations from RMP(Storm, 1988). In other words, firing can only commence when IK(D) is inactivated.Slow inactivation of IK(D) also provides recruitment during repetitive depolarizations.8.3.4) M-CurrentThis non-inactivating, subthreshold, and voltage-gated K current has beenreported in freshly dissected hippocampal slices (Madison et al., 1987b) and culturedneurons (Ghwiler and Brown, 1985a). The current is activated from about -70 mVupwards. At -45 mV and 30 °C, the time constant for activation is about 90 ms(Halliwell and Adams, 1982). Ba2, cholinergic receptor agonists, and serotonin havebeen reported to inhibit the M current (Colino and Halliwell, 1987; Halliwell andAdams, 1982; Gãhwiler and Brown, 1985a; Madison et al., 1987b), whereassomatostatin has been suggested to increase the current (Moore et al., 1988; Watsonand Pittman, 1988). The inhibition of IK(M) by acetylcholine is probably caused byinositol 1, 4, 5-triphosphate (Dutar and NicolI, 1 988a).Although IK(M) does not contribute much to the steady membrane current at thenormal RMP (-70 mV), it forms an appreciable fraction of the membrane conductancebetween -70 and -40 mV. Moreover, this current generates a component of postspikehyperpolarization and contributes to the accommodation which takes place duringtrains of action potentials (Madison and Nicoll, 1984).288.3.5) Inwardly-rectifying K currentIK(IR) is a rapidly activating (< 10 ms) K current (Owen, 1987). This currentpartially activates at resting membrane potential (i.e. -60 mV) and inactivates atpotentials which are more negative than -100 mV (Owen, 1987). GABA, 5-hydroxytryptamine, and adenosine have been reported to induce an inwardly-rectifying K current in hippocampal neurons (Gähwiler and Brown, I 985b; Newberryand Nicoll, 1985; Colino and Halliwell, 1987; Andrade and Nicoll, 1987). Theseeffects are probably mediated through a pertussis-toxin sensitive GTP-binding protein(Andrade et al., 1986; Zgombik et al., 1989).8.4) Chloride Current:A slow cr current which was activated by hyperpolarizing steps between -20and -100 mV has been observed in rat hippocampal slices (Madison et al., 1986).This chloride current is restricted to pyramidal cell processes. The reversal potentialfor the current was +9.5 mV when measured with microelectrodes filled with cr and -71 mV when recorded with microelectrodes filled with MeSO4 anion. Both Cd2(Selyanko, 1984) and phorbol dibutyrate (Madison et al., 1986) have been reported toblock I.85) Current Activated by Hyperpolarization:When hippocampal cells are hyperpolarized to potentials which are morenegative than -80 mV, a mixed cation (NaIK) current called the Q current (la) isobserved (Adams and Halliwell, 1982; Halliwell and Adams, 1982). Activation of thiscurrent is relatively slow. At 30 °C and -82 mV, the time constant of activation isabout 100 ms. This time constant increases with increasing hyperpolarization(Halliwell and Adams, 1982). Cs ions and tetrahydroaminoacridine block the Q29current (Brown et al., 1988). Activation of k serves to resist hyperpolarizingdeviations from the resting membrane potential. Deactivation of the currentcontributes to the rebound depolarization and excitation following a hyperpolarizingcurrent pulse. When action potentials (spikes) are initiated from membrane potentialswhich are negative to the normal resting potential (Storm, 1989), I may contribute tospike after-hyperpolarization.8.6) Calcium Activated Currents:Two potassium currents, a chloride current, and a cation current ofhippocampal cells are gated by calcium. These currents are activated by an increasein intracellular calcium produced by calcium entering through voltage-gated calciumchannels or through ligand-gated channels (Nicoll and Alger, 1981; Kudo and Ogura,1986), or by the release of intracellular calcium resulting from activation of muscarinicreceptors (Kudo et al., 1988) or metabotropic glutamate receptor (Furuya et al., 1989).8.6.1) Calcium Activated Potassium currents lK(Ca).In hippocampal neurons, two calcium activated potassium currents exist. Oneof these currents is a large, time- and voltage-dependent current (Ic) (Brown andGriffith, 1983b), while the other is a smaller and voltage-independent current (IAHP)(Lancaster and Adams, 1986).When a calcium charge flows through voltage-gated calcium channelsfollowing their activation by a depolarizing voltage-clamp pulse or during an actionpotential, I, which is a large current, is activated rapidly within I - 2 ms (Brown andGriffith, 1983b; Lancaster and Adams, 1986; Storm, 1987a; Lancaster et al., 1987).When the cell is repolarized, the current deactivates within 50- 150 ms (Brown andGriffith, 1983b). This high conductance channel (150- 270 pS) is blocked by I- 1030mM TEA. It requires a high concentration of calcium (> I tM) for activation(Franciolini, 1988). This strongly voltage-sensitive current (Franciolini, 1988)contributes to spike repolarization and generates the early phase of the spike afterhyperpolarization (Lancaster et al., 1987; Storm, 1987a, b).‘AHP, which is smaller than Ic in amplitude, rises slowly following calcium entryand declines slowly with a time constant of I - 1.6 s on repolarization (Lancaster andAdams, 1986). Although the decay rate is insensitive to voltage over the range -50 to-90 mV (Lancaster and Adams, 1986), it is determined by the rate of decline ofintracellular calcium (Knopfel et al., 1989). Activation of ‘AHP require a lowerintracellular calcium concentration than I (Knopfel et al., 1989). While ‘AHP isinsensitive to TEA and charybdotoxin (Lancaster and Adams, 1986; Storm, 1987a;Lancaster et al., 1987), it can be inhibited by acetylcholine (Cole and Nicoll, 1983;Madison et al., 1987b), noradrenaline (Madison and Nicoll, 1982; Haas and Konnerth,1983; Lancaster and Adams, 1986), histamine (Haas and Konnerth, 1983) and 5-hydroxytryptamine (Andrade et al., 1987; Colino and Halliwell, 1987). Acetylcholine,noradrenaline, histamine, and 5-hydroxytryptamine have been suggested to depress‘AHP by inhibiting the potassium current rather than by blocking the calcium transient(Knöpfel et al., 1989). IAHp which generates the long after-hyperpolarization followinghippocampal action potentials (Lancaster and Adams, 1986; Lancaster et al., 1987), isresponsible for the decline in firing frequency, and eventual cessation of firing, duringspike trains induced by prolonged depolarization (Madison and Nicoll, 1984). Thiscalcium activated potassium current does not seem to contribute to the restingmembrane current.318.6.2) Calcium Activated Chloride Currents IjA voltage-insensitive calcium activated chloride current (ICI(Ca)) which has aconductance of 20 pS has been reported in hippocampal cell membrane patches(Owen et al., 1988). This current probably contributes to the long calcium-dependenttail currents (Brown and Griffith, I 983b). In addition, it can generate depolarizingafter-potentials in sensory and spinal neurons (Owen et al., 1984; Meyer, 1985).However, whether ICI(Ca) contributes to after-potentials in hippocampal cells is not yetclear.Recent experiments with parallel Fura-2 recording show that a calciumactivated cation current probably contributes to an after-depolarization which replacesthe normal after-hyperpolarization after IAHP is suppressed with muscarinic agonists(Benardo and Prince, 1982; Gãhwiler, 1984; Knopfel et al., 1989).8.7) Miscellaneous Membrane Currents:8.7.1) Leak Currents:The currents which remain around the resting membrane potential whenvoltage- and calcium-gated currents are suppressed are referred as leak currents.One component of this residual conductance is a voltage-insensitive potassiumconductance which can be reduced by muscarinic agonists (Madison et aL, 1987b;Benson et al., 1988). Several types of chloride channels have also been suggestedas a component of this leak currents (Franciolini and Nonner, 1987; Franciolini andPetris, 1988; Owen et al., 1988).8.7.2) Sodium Activated Current:The prolonged after-hyperpolarization induced by tetanic stimulation of CA3neurons is probably caused by electrogenic sodium extrusion by the sodium pump32and a sodium activated potassium current (Gustafsson and Wigstrom, 1983). Thesignificance of this post-tetanic sodium activated potassium current to hippocampalcell behavior is unclear.8.7.3) ATP-Gated Potassium Channel:During anoxia, the potassium conductance of rat hippocampal CAl neurons isincreased (Hansen et al., 1982). Because hyperpolarization induced by the increasein potassium conductance during anoxia is blocked by sulphonylurea compounds, thispotassium conductance may be an ATP-blocked potassium channel. This ATPregulated potassium channel normally does not contribute to the resting membranepotential under normal situations since suiphonylurea does not alter the restingmembrane potential or conductance in the absence of anoxia (Krnjevic and Leblond,1988; Mourre et al., 1989).339) FIELD POTENTIALS IN THE HIPPOCAMPUS:Granule cells receive excitatory afferents from entorhinal cortex via theperforant pathway (Andersen et al., 1966a, b; Lomo, 1971). Perforant path fibers fromlateral entorhinal cortex produce EPSPs on the middle third of the dendritic tree(Blackstad, 1958; Hjorth-Simonsen and Juene, 1972; Steward, 1976; McNaughtonand Barnes, 1977). The rate of rise for more distal EPSPs is slower than that for themedial entorhinal EPSPs (Abraham, 1982). Like perlorant path fibers, commissuralafferents from contralateral pyramidal cells (Blackstad, 1956), associational fibersfrom CA3 and CA4 neurons (Zimmer, 1971), and septal fibers (Rose et al., 1976),which synapse on proximal dendrites, also produce excitatory effects (Steward et al.,1977; Fantie and Goddard, 1982).Mossy fibers from the granule cells in the dentate gyrus project a sufficientlylarge excitatory input to the proximal dendrites of CA3 pyramidal neurons, whereasCAl neurons receive their excitatory inputs from Schaffer collaterals. Besides mossyfibers, commissural, entorhinal, and septal inputs also contribute to excitatory inputsto CAl and CA3 cells (Andersen and Lomo, 1966; Andersen et al., 1966, 1971;Stanley et al., 1979).9.1) EPSPs and IPSPs:EPSPs produced by stimulation of the distal portions of the pyramidal andgranule cell dendrites have slower rise times than those generated at more proximallocations. The discrepancy between the rise times of the proximally and distallygenerated EPSPs has been attributed to electronic decay which occurs as the EPSPtravels from the distal dendrites to the soma. The different characteristics of the34inputs synapsing with the distal dendritic portions are also responsible for the variablerise times (Langmoen and Andersen, 1981; Andersen et al., 1980; Abraham, 1982).Excitatory afferents which synapse on pyramidal cell dendrites may beamplified by the intrinsic dendritic mechanisms such as dendritic spikes and dendriticcalcium depolarizations (Hamlyn, 1963; Andersen et al., 1966). The EPSP producedby the afferent volley is followed by a large IPSP which limits the excitatory responseto a single action potential (Kandel et al., 1961; Dunwiddie et al., 1980). Thisinhibition is probably mediated by inhibitory interneurons which demonstrate featuresof basket cells (Andersen et al., 1964a,b, 1969; Schwartzkroin and Mathers, 1978;Lee et al., 1980; Fox and Ranck, 1981). These basket cell-like interneurons arelocated basal to the pyramidal cell somata and are excited by axon collaterals ofpyramidal neurons (Knowles and Schwartkroin, 1981).Orthodromic stimulation of the stratum radiatum has been reported to producean early chloride-mediated IPSP and a later, slow hyperpolarization through activationof a feed-forward inhibitory system (Fujita, 1979; Thalmann and Ayala, 1982; Algerand Nicoll, 1982; Knowles et al., 1982; Alger, 1984). While the fast component ismediated by an increase in chloride conductance which has a reversal potential ofapproximately -70 mV (Spencer and Kandel, 1961a; Allen et al., 1977; Eccles et al.,1977; Dingledine and Langmoen, 1980; Alger and Nicoll, 1982), the slowercomponent is attributed to an increase in potassium conductance (Alger, 1984).Antidromic stimulation of alvear fibers has also been reported to produce IPSPs inpyramidal cells through activation of a recurrent inhibitory circuit (Andersen et al.,1964b; Kandel et al., 1961). While orthodromic stimulations of the stratum radiatumelicits both fast and slow IPSPs (Alger and Nicoll, 1982; Fujita, 1979), antidromic35stimulations of the alveus predominantly produce fast IPSPs (Alger and Nicoll, 1982;Andersen et al., 1964b; Dingledine and Langmoen, 1980). This observation suggeststhat the fast IPSP is generated near the pyramidal cell soma and dendrites and theslow IPSP is elicited at the dendrites.9.2) Population Synchronization:Both the afferent EPSP pathways and the local IPSP circuits may potentiallysynchronize in large populations of pyramidal cells. When many neurons aresynchronously activated, stimulation of an afferent input can generate the populationor field EPSP, which is characterized by a large negative wave when recording at thedendrites (sink) and a positive deflection when recording at the somata (source). Ifthe synaptic excitation exceeds the threshold for spike generation, the field EPSP willbe interrupted by a population spike which is a negative wave when recorded near thecell body layer and a positive wave when recorded near the dendrites. The number ofsynchronously discharging neurons can be estimated by the amplitude and width ofthe population spike (Andersen et al., 1971a). At a fixed stimulation strength, theamplitudes of the field EPSP and the population spike vary according to the distancebetween the source and the recording electrode.3610) EXCITATORY AMINO ACID RECEPTORS IN THE HIPPOCAMPUS:Excitatory amino acid receptors can be classified into five different types. Theyare the NMDA, the AMPA, the kainate, the L-AP4, and the metabotropic (GIuG)receptors. In this section, the properties of these receptors will be reviewed briefly.10.1) The NMDA Receptor:The NMDA receptor channel is voltage-dependent (MacDonald & Porietis,1982; MacDonald et al., 1982), permeable to K, Na, and Ca2 ions (MacDermott etal., 1986; Mayer and Westbrook, 1987b; Ascher and Nowak, 1988b), and blocked byMg2 in a voltage-dependent manner (Nowak et al., 1984; Mayer and Westbrook,I 987b; Jahr and Stevens, 1990). This receptor has a single channel conductancebetween 40 and 50 pS. Over a holding potential range of -100 to +30 mV, thecurrent-voltage (l-V) relationship recorded from neurons exposed to NMDA is biphasic(Mayer et al., 1984). This biphasic I-V relationship has been suggested to be due tothe voltage-dependent and uncompetitive blockade of the NMDA receptor channel byMg2 at negative potentials (Mayer et al., 1984; Mayer and Westbrook, 1987b; Nowaket al., 1984). While maximum inward current through the NMDA receptor channeloccurs at about -30 mV, the current reverses in direction at 0 mV. NMDA receptormediated synaptic currents demonstrate slow onset (8 - 20 ms) and long duration (60- 150 ms) (Collingridge et al., 1988; Forsythe and Westbrook, 1988; Hestrin et al.,1990). The long time course of the NMDA receptor evoked current has beensuggested to be the result of prolonged occupation of the receptor by glutamate(Lester et al., 1990).Compounds such as APV, PCP, ketamine, and MK 801 have been reported toblock NMDA receptor-mediated responses. While APV competitively blocks the37NMDA receptor (Davies et aL, 1981; Evans et al., 1982), PCP, ketamine, and MK 801block the channel in a highly voltage- and use-dependent manner (Hicks and Guedes,1981; MacDonald et al., 1987).NMDA binding is dense in the outer two-thirds of the molecular layer of thedentate gyrus, and in the stratum oriens and the stratum radiatum of CAl and CA3.While NMDA binding is absent in the stratum lucidum of CA3, NMDA binding is densein the stratum radiatum of CAl. In the subiculum, weak NMDA binding is alsopresent.Entry of Ca2 through the NMDA receptor channel has been shown to underlieLTP (Kelso et al., 1986; Malenka et al., 1988) and excitotoxic degeneration in culturedneurons (Garthwaite et al., 1986; Choi, 1987; Abele et al., 1990; Michaels andRothman, 1990).10.1.1) Transmitter Recognition Domains:Binding sites for agonists and antagonists on the NMDA receptor are notidentical (Watkins and Olverman, 1988; Fagg and Baud, 1988; Olverman andWatkins, 1989). While the 1-carboxyl and a-amino groups of agonists andantagonists interact with the same charged residues on the receptor surface, the oacidic terminals of agonists and antagonists bind to different sites on the receptor.3810.1.2) Allosteric Modulation by Glycine:By using patch clamp techniques in cultured neurons, submicromolarconcentrations of glycine have been reported to increase the frequency of NMDAinduced channel opening (Johnson and Ascher, 1987). Because this effect of glycineis insensitive to strychnine, it is not likely to be caused by the inhibitory actions ofglycine in the brainstem and spinal cord. For this reason, glycine may act as anagonist at an allosteric regulatory site on the NMDA receptor.Glycine has been reported to reduce desensitization of the NMDA current byincreasing the rate constant of recovery from desensitization (Mayer et al., 1989). Inaddition, glycine site agonists or partial agonists (glycine, D-serine, D-cycloserine)have been shown to enhance seizure activity (Larson and Beitz, 1988; Singh et al.,I 990a), to reverse the effects of PCP or MK 801 (Toth and Laijtha, 1986; Contreras,1990), and to enhance learning performance (Monahan et al., 1990).10.1.3) PCP Channel Bindinci Site:MK 801, a highly potent and selective NMDA receptor antagonist of the PCPtype (Wong et al., 1986), blocks NMDA receptor responses in an uncompetitive, usedependent, and voltage-dependent manner by binding inside the open channel andblocking transmembrane ion fluxes (Honey et al., 1985; Huettner and Bean, 1988;MacDonald et al., 1987; Martin and Lodge, 1985). Based on radioligand bindingstudies, PCP and related substances do not interact with the transmitter recognitionsite.3910.1.4) Modulation by Polyamine:Polyamines have been reported to increase the level of binding of glutamateand glycine. While spermidine has been shown to enhance NMDA-induced whole-cell currents in cultured neurons (Sprosen and Woodruff, 1990), arcaine has beenreported to block the NMDA-evoked release of[3H]noradrenaline from brain slices(Sacaan and Johnson, 1990). In vivo, spermidine has been observed to potentiateNMDA-induced seizures (Singh et al., 1990b). Ifenprodil which showsneuroprotective properties in vivo (Gotti et al., 1988) has been suggested to interactwith the polyamine site on the NMDA receptor (Carter et al., 1989; Schoemaker et al.,1990).10.1.5) Zn2 Binding Site:Low concentrations (pM) of Zn2 have been reported to block NMDA receptorresponses (Westbrook and Mayer, 1987; Peters et al., 1987). Unlike the Mg2blockade, Zn2 blockade is voltage-independent (Wesbrook and Mayer, 1987). Zn2has also been suggested to modulate the binding kinetics of[3H]MK 801 (Reynoldsand Miller, 1988) and to non-competitively inhibit the binding of[3H]glycine (Yeh et al.,1990). Because Zn2 is localized within and released from synaptic terminals duringexcitatory activity (Crawford and Connor, 1972; Assaf and Chung, 1984), it may play aregulatory role in NMDA receptor-mediated events in some regions of the CNS(Weiss et al., 1989).10.2) Non-NMDA Receptors:As a result of the voltage-dependent blockade by Mg2, the participation ofNMDA receptors in mediating the EPSP induced by a unitary stimulation is limited.While the NMDA receptor is responsible for the voltage-dependent component of the40EPSP, the voltage-independent portion of the EPSP is attributed to the kainate andAMPA receptors.10.2.1) The AMPA Receptor:AMPA receptors are distributed extensively in the cortex, hippocampus, lateralseptum, striatum, and the molecular layer of the cerebellum (Monaghan et al., 1984;Nielsen et al., 1988, 1990). Because the distribution of AMPA receptors correspondsclosely to that of NMDA receptors, these two receptor subtypes may act in concert toactivate postsynaptic neurons. The conductance of the AMPA receptor channel isintermediate in size between those of NMDA and kainate receptor channels (5 - 15pS) (Ascher and Nowak, 1988a; Cull-Candy et al., 1988). The reversal potential forAMPA receptor channels is 0 mV. These channels which show little voltage-dependence are permeable to Na and K (Mayer and Wesbrook, I 987a, b; Ascherand Nowak, 1988a).Although both AMPA and quisqualate activate the AMPA receptors, AMPAbinds the receptors with much higher selectivity than quisqualate (Krogsgaard-Larsenet al., 1980). Compounds such as CNQX, NBQX, and barbiturates have beenreported as antagonists of AMPA receptors (Honore et al., 1988; Sheardown et al.,1990). On the other hand, Zn2(Koh and Choi, 1988; Rassendren et al., 1990) andaniracetam agents (Ito et aL, 1990) have been suggested to potentiate AMPAreceptor responses.AMPA binding is more prominent in the outer two-thirds of the molecular layerof dentate gyrus. In the stratum oriens and the stratum radiatum of both CAl andCA3, AMPA binding is also dense. In comparison to CAl, AMPA binding in thesubiculum is less.4110.2.2 The Kainate Receptors:Neurophysiological actions of kainate have been suggested to be mediated viathe AMPA receptor (Watkins et al., 1990). In addition, cloning of the kainate bindingsite has implied that the kainate receptor is equivalent to the AMPA receptor (Gregoret al., 1989; Hollmann et al., 1989; Wada et al., 1989). However, neurotoxicity datasuggest that the pharmacology of kainate-induced excitotoxicity is not the same asthat of the AMPA receptor (Coyle et at., 1984). Electrophysiological studies haveidentified a group of C-fiber afferents to the spinal cord which are sensitive to kainatebut not to AMPA. In cultured neurons, kainate and AMPA activate channels whichdemonstrate different electrophysiological properties. Binding studies have indicateda high affinity[3H]kainate binding site which distributes differently from the AMPAreceptor (Monaghan and Cotman, 1982; Young and Fagg, 1990). In addition, theregional distribution of this high affinity[3H]kainate binding site is similar to thedistribution of kainate induced neurotoxicity (Pen et al., 1990; Teitelbaum et al.,1990).The KD of the kainate binding site is in the nM range and is blocked by kainateanalogues (domoate > kainate > quisqualate > glutamate), Ca2, CNQX, and DNQX.Kainate binding is most intense in the stratum Iucidum of CA3. A thin band of densebinding is present in the inner third of the molecular layer of dentate gyrus and inCA4. While kainate binding in CAl is virtually absent, moderate binding exists in thesubicul um.10.2.3) The L-AP4 Receptor:L-AP4 receptor was identified on the basis of the potent antagonistic propertiesof L-AP4 at excitatory synapses (Kd = 2.5 tM) (Koerner and Cotman, 1981; Collins,421982; Davies and Watkins, 1982; Lanthorn et at., 1984). While electrophysiologicalstudies have shown that L-AP4 blocks excitatory synaptic responses at lowmicromolar concentrations, L-AP4 does not block the depolarizing actions ofglutamate, NMDA, quisqualate, and kainate (Hon et aL, 1981; Ganong and Cotman,1982). L-AP4 receptor has been suggested to be a presynaptic autoreceptor whichinhibits transmitter release (Collingridge et al., 1984b; Anson and Collins, 1987).10.2.4) The Glu Receptor:In cultured mouse striatal neurons, quisqualate and L-glutamate were observedto stimulate phosphoinositol (P1) turnover via a pertussis toxin-sensitive G-protein withEC50’s of 0.16 and 4 riM, respectively (Sladeczek et al., 1985). Unlike quisqualateand L-glutamate, NMDA and kainate were less potent in stimulating the P1 turnover.Not only are these observations reported in mouse striatal neurons, but also incultured neurons from several different regions of the brain, cultured astrocytes,hippocampal slices, and synaptoneurosomes (Nicoletti et al., 1986b; Récasens et al.,1987; Schoepp and Johnson, 1988; Palmer et al., 1988; Ambrosini and Meldolesi,1989; Patel et aL, 1990; Pearce et al., 1990). The metabotropic receptor is mostabundant in the dentate gyrus and CA3. Binding in CAl is considerably less than inCA3 and the dentate gyrus.In comparison to quisqualate, ibotenate, and L-glutamate, trans-ACPD hasbeen shown to increase the P1 turnover in hippocampal slices with a higher selectivity(Palmer et al., 1989; Watson et al., 1990). Antagonists such as AP3 and AP4 havebeen reported to exhibit weak antagonistic property at the GIuG receptor (Schoeppand Johnson, 1988, 1989).43Glu0 receptors have been reported to be involved in synaptic plasticity(Sladeczek et al., 1988; Monaghan et al., 1989). The activity of the receptor declinesduring CNS maturation (Nicoletti et al., 1986a) and reappears followingdeafferentation, brain ischemia or kindling (ladorola et al., 1986; Nicoletti et al., 1987;Akiyama et al., 1989; Seren et al., 1989). Activation of the GIuG receptor also blocksthe slow afterhyperpolarization and accommodation of firing in CAl neurons followingdepolarizing current injections (Stratton et al., 1990).4411) GABA-ERGIC SYNAPTIC TRANSMISSION:GABA-ergic markers such as GAD which is the GABA synthetic enzyme (Ribaket al., 1978; Somogyi et al., 1983), GABA-T which is the GABA inactivating enzyme(Nagai et al., 1983), and GABA itself (Storm-Mathisen et al., 1983), are present in allhippocampal laminae. Around the somata of pyramidal cells in CAl -CA3 regions andaround granule cells in the fascia dentata, a dense plexus formed by GABA-ergicterminals and fibers has been reported in the literature (Ribak et al., 1978; StormMathisen et al., 1983; Somogyi et al., 1983; Misgeld and Frotscher, 1986; Woodson etal., 1989). Not only are GABA-ergic boutons found in the somata of pyramidal andgranule cells, but also on their axon hillock (Somogyi et al., 1983; Soriano andFrotscher, 1989), as well as the basal and apical dendrites (Somogyi et al., 1983;Woodson et al., 1989). While the density of GABA-ergic synapses is fairlyhomogenous in dendritic fields of CAl -CA3 area (Woodson et al., 1989), the densityof GABA-ergic synapses is highest in the outer third of the molecular layer of thefascia dentata (Woodson et al., 1989). In the dendritic layers of both CAl -CA3 areasand fascia dentata, GABAergic boutons are also present on the somata andprocesses of hippocampal GABA-ergic interneurons (Misgeld and Frotscher, 1986;Freund and Antal, 1988; Woodson et al., 1989).Using autoradiographic studies, moderate levels of GABAA receptors havebeen observed in all layers of CAI-CA4 regions and dentate gyrus (Bowery et aL,1987). Unlike GABAA receptors, GABAB receptors are distributed in a lesshomogenous manner. The densities of GABAB receptors in the CAl -CA4 pyramidallayer and the granular layer of the fascia dentata are lower than that in the dendriticlayers of these regions.45GABA-ergic cells, which constitute 11 % of the total population of hippocampalneurons (Woodson et al., 1989), are heterogenous in morphology. They includebasket, stellate, and horizontal cells which are short axon neurons (Ribak et al., 1978;Seress and Ribak, 1983; Nagai et al., 1983; Woodson et al., 1989). Somata of mostGABA-ergic neurons are located outside the pyramidal layers of CA areas and thegranule cell layer of fascia dentata, and predominantly in the CAI-CA3 dendriticregions.(Woodson et al., 1989).The hippocampal formation receives GABA-ergic afferents from the entorhinalcortex via the perforant path (Germroth et al., 1989), and from the septum via thefimbria-fornix (KOhier et al., 1984; Freund and Antal, 1988). Most GABA-ergichippocampal interneurons receive input from GABA-ergic septohippocampal fibers(Freund and Antal, 1988).11.1) Spontaneous IPSPs:Spontaneous IPSPs were observed in rat hippocampal slices by Alger andNicoll (1980a). By using microelectrodes filled with potassium acetate,hyperpolarizing spontaneous IPSPs were recorded from guinea pig CA3 pyramidalcells (Miles and Wong, 1984).Spontaneous IPSP5 are GABAergic in nature because they are sensitive tobicuculline (1-100 tM) (Alger and Nicoll, 1980a; Collingridge et al., 1984a) andpicrotoxinin (100 pM) (Miles and Wong, 1984). In addition to bicuculline andpicrotoxinin, spontaneous IPSPs are sensitive to pentobarbitone, flurazepam, dtubocurarine, and folic acid. Pentobarbitone (50-100 iiM) has been reported toenhance the duration and amplitude of the spontaneous IPSPs (Alger and Nicoll,1980a; Collingridge et al., 1984a). In mouse dentate granule cells, ionophoresis of46flurazepam was also reported to enhance the amplitude of spontaneous IPSPs(Biscoe and Duchen, 1985a). Both d-tubocurarine (10-50 1iM) and folic acid (0.1-1mM) have been observed to block spontaneous IPSPs in guinea pig CA3 neurons andrat CAl neurons, respectively, via a postsynaptic mechanism (Lebeda et al., 1982;Otis et al., 1985).11.2) Characteristics of IPSPs Evoked in CAI-CA3 Regions:Chloride-sensitive inhibitory synaptic potentials evoked by fornix stimulationwere reported in cat CA2-CA3 pyramidal cells by Kandel et al. (1961). Stimulation ofcommissural, septal, and fimbrial pathways can also elicit hyperpolarizing synapticpotentials (Andersen et al., 1963, 1964a,b). Activation of interneurons which synapseon the somata of pyramidal cells, such as basket cells, has been suggested tomediate hippocampal inhibition induced by stimulating the above pathways (Andersenet al., 1963, 1964a,b).11.3) Antidromic or Feed-back IPSPs:Stimulation of axons of hippocampal pyramidal cells such as the alveus and theSchaffer collaterals (i.e. antidromic stimulation) has been reported to evoke amonophasic fast hyperpolarizing potential in the cellular field of origin (Kandel et al.,1961; Dingledine and Langmoen, 1980; Biscoe and Duchen, 1985b). This fast IPSPwhich is associated with a decrease in the input resistance of the pyramidal cell(Dingledine and Langmoen, 1980) has a reversal potential of -65 to -75 mV whenmeasured with potassium acetate microelectrodes (Andersen et al., 1980; Alger andNicoll, 1982; Biscoe and Duchen, 1985b). Because the amplitude of the IPSPdecreases after extracellular chloride ions are replaced with ethionate (Alger andNicoll, 1982) and intracellular injections of cr reverse the antidromic IPSP (Kandel et47al., 1961). An increase in the membrane permeability to C1 has been suggested tounderlie the generation of the fast IPSP. In addition to its chloride sensitivity, the fastIPSP can be blocked by ionophoresis of bicuculline methiodide (Alger and Nicoll,1982) and by superfusion with picrotoxinin (1-100 riM) (Alger, 1984; Miles and Wong,1984). These observations suggest the involvement of GABAA receptors in theproduction of the fast IPSP.The antidromic IPSP is generated by a feed-back circuit which is formed byrecurrent collaterals of the efferent axons of pyramidal cells that synapse ontoinhibitory interneurons (MacVicar and Dudek, 1980; Knowles and Schwartzkroin,1981; Miles and Wong, 1984). Even though basket cells predominantly provideGABAergic synaptic contacts onto the somata of pyramidal cells, axo-axonicGABAergic cells which synapse onto the axon initial segment may also contribute tofeed-back or antidromic IPSPs since recurrent IPSPs have been reported in theabsence of axosomatic synapses (Somogyi et al., 1983).114) Orthodromic or Feed-forward IPSPs:Unlike antidromic stimulation, orthodromic stimulation elicits an EPSP and abiphasic IPSP (Alger and Nicoll, 1982; Newberry and Nicoll, 1984; Alger, 1984;Knowles et al., 1984; Biscoe and Duchen, 1985b). In addition to the synapticpotentials, an even slower hyperpolarization which is probably due to the activation ofa Ca2-dependent K current can be observed when the EPSP produces an actionpotential (Alger and Nicoll, 1980b; Hotson and Prince, 1980; Newberry and Nicoll,1984).Like the fast IPSP generated by antidromic stimulation, the orthodromicstimulation-induced fast IPSP is also associated with a decrease in the input48resistance which is caused by an increase in cr conductance (Knowles et al., 1984).GABAA receptors are also involved in the generation of the orthodromic stimulation-induced fast IPSP because superfusion with bicuculline (1-10 riM) or picrotoxinin (I-.10 i.tM) has been reported to remove the fast IPSP (Knowles et al., 1984). Thereversal potential of the fast IPSP is normally 10-20 mV more negative than theresting membrane potential and is identical to the reversal potential for GABA (BenAn et al., 1981; Knowles et al., 1984; Biscoe and Duchen, 1985b; Misgeld et al.,1986).The fast IPSP5 appear to be generated predominantly on the dendrites of thepyramidal cells since they are very sensitive to dendritic ionophoresis of bicucullinemethiodide (Alger and Nicoll, 1982). Depending on the proportion of pyramidal cellsthat reach the firing threshold at any given intensity of an orthodromic stimulation, theorthodromic fast IPSP may contain somatic components elicited by recurrent collateralfibers. In other words, the fast IPSP may be generated by inhibitory synapses whichare located on the pyramidal cell soma (Sivilotti and Nistri, 1991).In the presence of barbiturates, the hyperpolarizing fast IPSP can betransformed to a biphasic hyperpolarizing-depolarizing response. This depolarizingcomponent of the fast IPSP, which can be enhanced by GABA uptake inhibitors, isgenerated in the dendrites by GABAA receptor activation since it can be inhibited bydendritic ionophoresis of bicuculline methiodide or TTX (Alger and Nicoll, 1982).Normally, the depolarizing component is not detected because GABA uptake limitsthe diffusion of GABA from the synapses (Avoli and Perreault, 1987). The ionicmechanisms underlying the depolarizing component of the orthodromic fast IPSPshave been suggested to involve either the activation of a mixed Cl7cation49conductance or the activation of a cr conductance in parts of the dendritic tree whichhas an outward electrochemical gradient for cr (Silvilotti and Nistri, 1991).Unlike the fast IPSP, the slow IPSP appears at higher intensities of stimulation(Newberry and Nicoll, 1984), has a slower time course which lasts for hundreds ofmilliseconds (Newberry and Nicoll, 1984; Alger, 1984; Hablitz and Thalmann, 1987),and is associated with a modest increase in conductance (Knowles et al., 1984; Alger,1984). The reversal potential of the slow IPSP, which is sensitive to the extracellularK concentration has been estimated to be about -95 mV (Hablitz and Thalmann,1987). The slow IPSP is not sensitive to changes in intracellular and extracellular ciconcentrations (Knowles et al., 1984; Newberry and Nicoll, 1984; Biscoe and Duchen,I 985c) and is enhanced rather than being blocked by GABAA antagonists (Newberryand Nicoll, 1984). The enhancement of the slow IPSP by GABAA antagonists couldbe due to removal of GABAA-mediated inhibition of GABAergic interneurons(Newberry and Nicoll, 1984).Both phaclofen (0.2-0.5 mM) (Dutar and Nicoll, 1988b; Soltesz et al., 1988) and2-hydroxysaclofen (50-200 .tM) (Lambert et al., 1989) have been reported to block theslow IPSP. Pretreatment with pertussis toxin (Dutar and Nicoll, 1988c) or intracellularinjection of GTP-’y-S (Thalmann, 1988), which blocks the coupling of GABAB receptorsto K channels, can also abolish the slow IPSP.In general, the orthodromic IPSP is mediated by a direct activation of afferentfibers of GABAergic interneurons which synapse predominantly onto the dendrites ofpyramidal cells (i.e. feed-forward inhibition) (Alger and Nicoll, 1982). A somaticinhibitory component which is induced by feed-forward mechanisms or by thestimulation of feed back circuits brought about by the activation of the pyramidal cells50may also contribute to the mediation of the slow IPSP. In terms of recurrent inhibition,it is difficult to ascribe feed-forward IPSPs to the activity of a single set ofinterneurons.11.5) IPSPs of Granule cells:In hippocampal granule cells, inhibitory synaptic responses include a GABAAmediated fast IPSP and a GABAB mediated slow IPSP have been reported. The fastIPSPs which are Cr-dependent have been recorded following both orthodromic andantidromic stimulations (Thalmann and Ayala, 1982; Misgeld et al., 1986). Usingmicroelectrodes filled with potassium acetate and sulfate, the reversal potentials forthe GABAA mediated fast IPSP is -75 and -62 mV, respectively. Compounds such aspicrotoxinin (50 jiM) (Thalmann and Ayala, 1982), penicillin, bicuculline, andpentylenetetrazol (Fricke and Prince, 1984) have been observed to block the fastIPSP.Like the fast IPSP, the slow IPSP, which is sensitive to changes in extracellularlevels of K and picrotoxinin, has been observed in response to both antidromic andorthodromic granule cell stimulation. The reversal potential of this slow IPSP hasbeen estimated to range from -80 to -90 mV (Thalmann and Ayala, 1982; Biscoe andDuchen, I 985b).Even though dentate basket cells have been suggested to play a major role ingranule cell inhibition (Andersen et at., 1966), a GABAergic axo-axonic interneuronwhich may mediate the inhibition elicited by perforant path stimulation may also beinvolved (Soriano and Frotscher, 1989). While the dendrites of the axo-axonicinterneuron are mainly in the molecular layer where perforant path fibers terminate,their axons synapse on the axon of the initial segment of granule cells. In general,51both feed-forward and feed-back circuits contribute to granule cell inhibition (Buzsáki,1984).11.6) Interneurons and Inhibitory Synaptic Transmission:Hippocampal interneurons are large somata whose sizes range from 35 to 50urn on average. They are characterized by their aspinous dendrites and locallyarborizing axons (Ribak and Andersen, 1980). In this section, interneurons located instratum pyramidale, near the border between the oriens and the alveus, as well asnear the border between the stratum radiatum and lacunosum-molecular, which areinvolved in inhibitory synaptic transmission will be discussed.11.6.1) Basket Cells:Basket cells, whose axonal plexus resembles a basket around the targetsomata (Cajal, 1911; Lorente de NO, 1934), are present in both strata pyramidale andgranulosum. The somata of baskets cells have an average size of 45 pm. Theaspinous dendrites of baskets cells, which show periodic swellings, receive multiplesynaptic contacts. Basket cells have been reported to exert feedback (recurrent)inhibitions on pyramidal neurons (Kandel et al., 1961; Andersen et al., 1964). Eventhough basket cells are thought to be involved in the feed-back inhibition, basket cellsmay also take part in the feed-forward inhibition since stimulations of the Schaffercollateral/commissural afferents can activate basket cells (Alger and Nicoll, 1982;Ashwood et al., 1984; Buzsaki and Eidelberg, 1982).Because these interneurons are immunoreactive for GABA (Gamrani et aL,1986) and GAD (Ribak et al., 1978), GABA may be involved in the basket cellmediated inhibition. The membrane time constants of basket cells are approximately3 ms. Brief action potentials (0.8 ms) associated with large after hyperpolarizing52potentials (5-10 mV) and tonic depolarization-induced non-accommodating spikedischarges are both characteristics of basket cells.11.6.2) Interneurons at the border between the Oriens and the Alveus:The oriens/alveus interneurons, which are multipolar and 20 to 30im indiameter, have been reported to display both GABA-like (Gamrani et al., 1986) andsomatostatin-like immunoreactivity (Kohier and Chan-Palay, 1982; Morrison et al.,1986).. Aspinous dendrites with periodic swellings are also characteristics of theseinterneurons. While most dendrites of oriens/alveus interneurons are arrangedparallel to the alveus, some turn and project into the stratum oriens, the stratumpyramidale, the stratum radiatum, and the stratum lacunosum-moleculare. The axonsof oriens/alveus interneurons are distributed in the stratum oriens and the stratumpyramidale.This type of interneuron has a short membrane time constant of 6 ms. Likebasket cells, oriens/alveus interneurons also produce brief action potentialsassociated with large after-hyperpolarizations and tonic depolarization-induced non-accommodating spike discharge. These interneurons are involved in both feed-forward and feed-backward inhibitions since they can be activated either bystimulation of the Schaffer collateral/commissural afferents or by depolarizingpyramidal cells which are synaptically paired with the interneurons.11.6.3) Lacunosum-moleculare Interneurons:Lacunosum-moleculare interneurons, which are characterized by fusiform ormultipolar somata, are GABAergic inhibitory interneurons. These interneurons arelocated at the border between strata lacunosum-moleculare and radiatum (Kawaguchiand Hama, 1987; Lacaille and Schwhartzkroin, 1988). Dendrites of lacunosum53moleculare interneurons run parallel to stratum lacunosum-moleculare and project intothe stratum pyramidale and the stratum oriens of the hippocampus proper, as well asthe stratum moleculare of the dentate gyrus. Axons of these interneurons also projectin a similar fashion (Kunkel et al., 1988; Lacaille and Schwartzkroin, 1988). Becauselacunosum-moleculare interneurons are neither excited nor inhibited by pyramidalcells which are synaptically synapse with them, these interneurons have beensuggested to mediate feed-forward inhibition. (Lacaille and Schwartzkroin, 1988).Like basket and oriens/alveus interneurons, lacunosum-moleculareinterneurons also demonstrate spike after-hyperpolarizations and little spikeaccommodation to depolarizing current injections. The action potential duration andthe membrane time constant of the lacunosum-moleculare interneuron are 2 and 9ms, respectively.11.7) Presynaptic GABA Receptors:Baclofen, a GABA analogue which does not interact with muscimol- andbicuculline-sensitive GABA receptors, and GABA have been reported to inhibitrelease of noradrenaline (Bowery and Hudson, 1979; Bowery et al., 1981),acetyicholine (Brown and Higgins, 1979) from peripheral nerve endings, and that ofnoradrenaline (Bowery et al., 1980), dopamine (Bowery et al., 1980; Reimann et al.,1982), serotonin (Bowery et al., 1980; Schlicker et al., 1984; Gray and Green, 1987),and glutamate (Potashner, 1979) in the central nervous system. Similarly, the releaseof[3H]GABA elicited by 15 mM K from synaptosomes of the median eminence wasalso inhibited by baclofen (Andersen and Mitchell, 1985). Based on this observation,Andersen and Mitchell first implicated the existence of GABAB autoreceptors in thecontrol of GABA release in 1985.54In addition to the above observations, electrophysiological data also supportthe existence of the GABAB autoreceptor. Baclofen has been reported to attenuatepaired-pulse inhibition of hippocampal CAl pyramidal neurons induced by stimulationof Schaffer collaterals, which is mediated by postsynaptic GABAA receptors, atconcentrations which do not produce postsynaptic effects on pyramidal cells(Karisson and Olpe, 1989). For this reason, baclofen was suggested to reducepaired-pulse inhibition via reduction of GABA release from interneurons (Karisson andOlpe, 1989). Moreover, both phaclofen and CGP 35348, GABAB antagonists, havebeen reported to attenuate the effect of baclofen on paired-pulse inhibition (Pozza etal., 1989).Besides the reduction of paired-pulse inhibition, IPSPs elicited by orthodromicstimulations are also reduced by baclofen. Because this effect outlast thepostsynaptic effects of baclofen, the attenuation of IPSPs is probably mediated by areduction of GABA release (Deisz and Prince, 1989; Deisz and Zieglgansberger,1989).Although the existence of GABAB autoreceptors in synaptosomes of the ratmedian eminence, slices of rat hippocampus, and slices of the striatum has beenaffirmed (Andersen and Mitchell, 1985; Waldmeier et al., 1988), the existence of suchan autoreceptor is very controversial in the substantia nigra which contains highconcentrations of GABA and receives important GABAergic input from the striatumand the globus pallidus. While some investigators suggested that inhibition of GABArelease was not mediated by GABAA receptors (Arbilla et al., 1979; Floran et al.,1988), others thought the inhibition of GABA release was due to activation of GABAB55receptors (Giralt et al., 1989). For this reason, the role and existence of GABABautoreceptors in the substantia nigra remains to be determined.5612) HEMOGLOBIN:In the human body, approximately 7.5 x 1021 molecules of hemoglobin, anoxygen-transport protein, are present. The concentration of hemoglobin inerythrocytes of adult human is about 4.5 mM.In LTP, hemoglobin has been widely used as a NO scavenger. However, itseffect on LTP is controversial. While some investigators suggested that hemoglobinsuppressed LTP (Haley et al., 1992; Musleh et al., 1993; O’Dell et al., 1991; Schumanand Madison, 1991), others reported the opposite (lzumi et al., 1992a; Pauwels andLeysen, 1992).Slow hemolysis of erythrocytes with release of hemoglobin into the supernatantfluid has been reported to occur after 2 days of in-vitro incubation of blood (Asano etal., 1980; Barrows et al., 1955; Osaka, 1977; Miyaoka et al., 1976; Sonobe andSuzuki, 1978; Sasaki et al., 1979; Okwuasaba, 1981; Duff et al., 1987). Afterintracranial bleeding or hemorrhagic stroke, erythrocytes, which can remain in theintracranial cavity for days, are hemolysed in a similar fashion as in the in-vitroincubation (Barrows et al., 1955; Findlay et al., 1989). After 2 hours of subarachnoidhemorrhage, hemoglobin has been reported to be released from erythrocytes(Barrows et al., 1955). Because hemoglobin can remain in the cerebral spinal fluid forweeks (Barrows et al., 1955), neurons may be exposed to hemoglobin. In fact,hemoglobin has been suggested to induce cerebral vasospasm associated withsubarachnoid hemorrhage (Weir, 1987; Osaka et al., 1980). Moreover, its ironcontent may be related to stroke- or head injury-induced epilepsy (Hammond et al.,1980).57In this section, the biochemistry, the oxygen-binding, and the NO-scavengingproperties, of hemoglobin will be discussed.12.1) Structure of Hemoglobin:Normal hemoglobin is a tetramer which consists of two alpha globulin chainsand two beta globulin chains that face one another across a central cavity. Each achain comprises of 141 amino acid residues, whereas each f3 chain has 146 aminoacid residues. The a and 13 subunits assemble into tightly bound heterologous dimers(a-J3) which in turn associate into tetramers. Each globin chain (a or 13), whichresembles the structure of myoglobin, contains a hydrophobic pocket where a hememolecule or an iron [Fe (II)] protoporphyrin IX binds tightly and allows reversiblebinding of oxygen (Freedman, 1977; Hill, 1976). Both tertiary and quaternarystructures are ideally designed for keeping the heme iron in a ferrous state whichpermits the loading and unloading of the oxygen molecules at the physiological partialpressures present in the blood.The tertiary structure of each hemoglobin subunit is composed of eight a-helixes labeled A to H. The E, F, and C, helixes as well as the CD non-helicalsegment delineate a hydrophobic crevice where heme is tightly bound. While thenon-polar methyl and vinyl groups of heme extend into the hydrophobic interior ofeach subunit, the ionized carboxyl groups of heme are exposed to the aqueousenvironment. Six different ligands are required to hold the ferrous ion in place(Claude, 1992). Four of these are the nitrogen atoms of the porphyrin ring system.The fifth is the eighth residue in helix F which is a histidine. The sixth ligand is absentin deoxyhemoglobin, whereas oxygen in oxyhemoglobin functions as the sixth ligand.The non-polar side chains of the eleventh residue in helix E which is a valine and the58first residue in the CD non-helical segment which is a phenylalanine provide asterically hindered hydrophobic pocket for the heme group (Claude, 1992). Thehistidine F8 and the phenylalanine CDI are important for maintaining the iron atom ina ferrous state in the presence of oxygen and therefore reversible oxygenation(Rimington, 1959). Any change of these two residues can remove the functionalproperties of hemoglobin since reversible oxygenation cannot occur without sterichindrance in the hydrophobic pocket.Besides stabilizing heme, the heme pocket also favors oxygen binding over CObinding. The affinity of CO for free heme in aqueous solution is 25,000 times that ofoxygen (Claude, 1992; Rawn, 1989). If the affinity of CO to hemoglobin is as high asto free heme, oxygenation of hemoglobin would be impossible. For this reason, it isnecessary to lower the affinity of CO to hemoglobin. In free heme, the bond anglebetween carbon monoxide and iron is nearly at right angle to the plane of free heme(Claude, 1992; Rawn, 1989). In hemoglobin, steric hindrance caused by histidine E7and valine ElI, which are located at the distal part of the heme pocket, bend and,therefore, weaken the CO-iron bond (Claude, 1992; Rawn, 1989). On the contrary,steric hindrance does not influence the iron-oxygen bond because it is naturally bent.In addition to the steric hindrance, CO does not form a hydrogen bond with theimadazole nitrogen of the histidine E7 while 02 does. As a result of these structuralarrangements, hemoglobin can preferentially bind to 02.12.2) Metabolism of Hemoglobin:12.2.1) Synthesis of Hemocjlobin:The formation of aminolevulinic acid by the condensation of glycine andsuccinyl-coenzyme A in the mitochondria is a rate-determining step and the first step59in heme synthesis (Freedman, 1977; Safer, 1978). The final steps in heme synthesis,which are mediated by coproporphyrinogen oxidase and heme synthetase, also occurin the mitochondria. The heme synthesis is regulated by end-product inhibition ofheme synthetase and end-producer repression of heme synthesis (Freedman, 1977,Jacob et al., 1969; Rimington, 1959). A mutual interdependence of heme and globinsynthesis has been suggested (Bruns and London, 1965; Freedman, 1977; Hunt,1976; Jacob et al., 1969; Morris and Liang, 1968; Rabinovitz, 1974; Rimington, 1959).Low concentrations of heme have been reported to reduce globin synthesis (Brunsand London, 1975; Freedman, 1977; Gross and Rabinovitz, 1972a; Morris and Liang,1968). Moreover, heme has been shown to be necessary for globin synthesis in intactreticulocytes (Bruns and London, 1975; Freedman, 1977; Gross and Rabinovitz,1972a; Gross and Rabinovitz, 1972b).12.2.2) Catabolism of Heme:Heme is catabolized by the heme oxygenase through oxidative degradation(Brown and Grundy, 1977). The heme oxygenase, which is present in the microsomalmembranes of liver, kidney, brain, spleen, and bone marrow, is specific for heme, thec and f3 chains of hemoglobin, methemalbumin, and methemoglobin (Maines, 1977;Tenhunen et al., 1968). Heme groups of hemoglobin are eventually broken down intobilirubin in vivo (Barrows et al., 1955). Divalent cations such as cobalt, chromium,manganese, iron, copper, zinc, and lead have been shown to increase hemeoxygenase activity (Maines and Kappas, 1974; Maines and Kappas, 1975; Maines,1977).6012.3) Oxygenation of Hemoglobin:12.3.1 Oxygen Binding and Conformational Change in Hemoglobin:When hemoglobin changes from its deoxy state to the oxy state, one c-f3 dimerrotates by 150 relative to the other c-f3 dimer in hemoglobin (Dickerson and Geis,1983). Oxygenation of Hemoglobin is a cooperative process. Once an 02 molecule isbound, the binding of the succeeding 02 molecules is facilitated so that they can bindmore readily. The oxygen affinity of hemoglobin depends on the pH (German andWyman, 1937). The Bohr effect states that hemoglobin has lower affinity for 02 atlower pH values. The oxygen binding curve shifts to the right with decreasing pH sothat 02 can be unloaded from oxyhemoglobin more readily when muscle acidityindicates that more 02 is required for metabolic reactions.In the absence of oxygen, the four heme iron atoms in Hb A are in high-spinferrous state [Fe(Il)] with four unpaired electrons and one pair of electrons on eachiron atom. In the presence of oxygen, these heme iron atoms are converted to a low-spin, diamagnetic ferrous state. The change from the high to the low spin state is dueto unfavorable interaction of the electrons of 02 with the unpaired electrons in the highspin state. As a result of oxygenation, the bond between histidine F8 and the ferrousion becomes shorter and the interactions between the iron atom and the porphyrinnitrogen atoms become strengthened and more covalent in nature (Antonini andBrunori, 1971; Bunn and Forget, 1986; Dickerson and Geis, 1983; Edsall, 1972; Ho etal., 1982a, Perutz, 1989, 1990).In addition to the change in the spin state of the iron atom, oxygenation alsoproduces steric effects that alter the structure of hemoglobin. In deoxyhemoglobin,steric repulsion between the nitrogen atoms of the porphyrin and histidine F8 along61with the electronic repulsion between the it electrons of the porphyrin and the orbitalsof the ferrous ion causes the iron atom to be about 0.06 nm out of the plane of theporphyrin ring (Rawn, 1989). Upon oxygenation, the change in the electronicconfiguration of the ferrous ion causes the iron atom to move toward the plane of theporphyrin ring by about 0.039 nm (Rawn, 1989). Not only is the iron atom movedupon oxygenation, but also tyrosine HC2 and valine FG5. The movement of thesetwo residues disrupts the ion pairs which cross-link the chains of deoxyhemoglobinand therefore allows binding of oxygen to the heme group. When oxygen binds to theferrous ion of one subunit, the binding of the ferrous ions of other subunits will befacilitated because the other subunits are less constrained to undergo theconformational changes associated with oxygen binding.12.3.2) 2,3-Diphosphoglycerate (DPG) and Oxygen Affinity of Hemoglobin:The 2,3-DPG binds to the deoxygenated hemoglobin tetramer at the centralcavity between the two 13 chains in a molar ratio of one. DPG binds electrostatically toVal NAI, His NA2, and His H21 on both 13-chains as well as Lys EF6 on one of the f3-chains (Perutz and lmai, 1980). The valine residue at position NAI is important forDPG binding since valine allows the complete release of the initiator methionine andprevents the acetylation of the x-NH2 group by the erythrocytic N-x-acetyltransferasewhich would suppress two of the seven DPG binding sites per tetramer (Perutz andlmai, 1980). Because DPG stabilize the conformation of deoxyhemoglobin, theoxygen affinity of hemoglobin is lowered. The conformational changes involved inoxygenation disrupt the DPG binding site such that DPG cannot bind tooxyhemoglobin. In blood, DPG facilitates oxygen unloading by shifting the oxygenbinding curve to the right.6213) HEMOGLOBIN AND NITRIC OXIDE:13.1) NitrosyIhemoglobin:NO, which binds to the sixth coordination position of the heme inferrohemoglobin, possesses an extremely high affinity for the heme inferrohemoglobin. The affinity of NO to hemoglobin is approximately 3000 times that ofCO (Gibson and Roughton, 1957). NO derivatives of isolated X and f3 subunits ofhuman adult hemoglobin are dissimilar (Hille et al. 1977). In the presence of oxygen,nitrosylhemoglobin dissociates into methemoglobin and nitrates (Kon et al., 1977;Yoshida et al., 1980). Methemoglobin is then reduced to ferrous hemoglobin bymethemoglobin reductase in erythrocytes. While majority of nitrates produced by theconversion of nitrosyihemoglobin to oxyhemoglobin are excreted in urine, some ofthese nitrates can also be discharged into the oral cavity, where they are transformedto nitrites, through the salivary glands (Yoshida and Kasama, 1987). In general, mostof the metabolites of inhaled NO are excreted rapidly from the body within 48 hours(Yoshida et al., 1978). Because the regeneration of oxyhemoglobin is much fasterthan the dissociation of NO from nitrosylhemoglobin, this regenerative process plays aprotective role in NO intoxication.13.2) Nitric Oxide and Long Term Potentiation (LTP):NO synthase has been reported to be present in hippocampal interneuronslocated in the stratum oriens, the pyramidal cell layer, and the stratum radiatum (Leighet al., 1990; Mizukawa et al., 1989; Mufson et al., 1990; Seidel et al., 1991; Vincentand Hope, 1992; Vincent and Kimura, 1992). Majority of neurons in the medialseptum and the nucleus of the diagonal band of Broca, which project to the63hippocampus (Kinjo et al, 1989), also possess NO synthase (Kinjo et al., 1989;Mizukawa et al., 1989; Pasqualotto and Vincent, 1991; Schöber et al., 1989).NO has been suggested to be a possible retrograde messenger mediating LTP(Garthwaite et al., 1988). In addition, NO has also been thought to mediate NMDAaction in the hippocampal formation (Gaily et aI., 1990) because L-NAME, a nitricoxide synthase inhibitor, prevented the L-arginine-induced electroencephalogramdesynchronization and potentiation of the epileptogenic effects of NMDA (Mollace etal., 1991). In vivo, the increase in cGMP levels induced by local injections of NMDAinto the hippocampus were blocked by a NO synthase inhibitor (Wood et al., 1992).In hippocampal slices, cGMP production induced by the activation of the NMDAreceptor was also suppressed by NO synthase inhibitors (East and Garthwaite, 1991;Foster and Roberts, 1981). These observations support the speculation that NO isinvolved in mediating NMDA action.Studies on the role of NO in the induction of LTP have been conducted bymany groups (BOhme et al., 1991; Bon et al., 1992; Haley et al., 1992; Izumi et al.,1992b; Musleh et al., 1993; O’Dell et al., 1991; Schuman and Madison, 1991). NOsynthase inhibitors and hemoglobin which is known to be a membrane impermeantNO scavenger (Gibson and Roughton, 1957) have been commonly used in thesestudies. The role of NO in the induction of LTP is controversial. While someinvestigators have reported that LTP could be suppressed by NO synthase inhibitorsand hemoglobin (Böhme et al., 1991; Bon et al., 1992; Haley et al., 1992; Musleh etal., 1993; O’Dell et al., 1991; Schuman and Madison, 1991)., others concludedifferently (Izumi et al., 1992b; Kato and Zorumski, 1993; Williams et al., 1993).64This discrepancy can be attributed to the assumption that the inhibition of theinduction of LTP by oxyhemoglobin or NO synthase inhibitors means an involvementof NO or NO synthase, respectively. Oxyhemoglobin may have effects independent ofNO inhibition such as the inhibition of acetylcholinesterase (Linnik and Lee, 1986).On the other hand, NO synthase inhibitors, which are arginine analogs, may inhibitprotein and peptide synthesis, postranslational arginylation (Hallak et a!, 1991),arginase, arginosuccinase, Na/K-ATPase (Nagai et al., 1985), the formation ofthrombin, guanidinoacetate, and creatine, or arginine transport into cells (Bogle et al.,1992). Furthermore, the direct electrophysiological actions of NO synthase inhibitorsand hemoglobin on neurons have not been examined. It is possible that mechanismsother than chelation of NO and suppression of NO synthesis are involved in thesuppression of LTP. For these reason, the role of NO in LTP remains to be resolved.6514) HEMOGLOBIN AND CEREBRAL VASOSPASMS:Vasospasm has been reported to start and subside 3 days and 14 days aftersubarachnoid hemorrhage (SAH), respectively (Weir et al., 1978). Maximalvasospasm usually occurs 6 to 7 days after the hemorrhage (Weir et al., 1978). Whileintense polymorphonuclear cell infiltration of the meninges takes place within 24hours after SAH, breakdown of RBCs occurs by 16 to 32 hours after the hemorrhage(Bagley, 1928; Hammes, 1944; Alpers and Forster, 1945). Even though hemolysis ofRBCs peaks around day 7, intact RBCs can be present in the arachnoid space up to35 days after SAH (Bagley, 1928; Hammes, 1944; Alpers and Forster, 1945). Usingabsorption spectrophotometry to examine cerebrospinal fluid after SAH,oxyhemoglobin has been reported to appear 2 hours after SAH (Barrows et at., 1955).Although the amount of oxyhemoglobin continuously decreased while that of bilirubinincreased, the disappearance of both pigments did not occur until 2 to 3 weeks afterSAH (Barrows et al., 1955). These observations show that oxyhemoglobin is presentduring vasospasm and it may be the mediator of SAH-induced vasospasm(Simmonds, 1953; Dupontetal., 1961).Even though substances such as serotonin, biogenic amines, peptides, andeicosanoids may attribute to SAH-induced vasospasm, antagonists of thesevasoconstrictors including atropine, methysergide, cinanserin, ketanserin,phenoxybenzamine, phentolamine, mepyramine, chlorpheniramine, propranolol,salbutamol, angiotensin, sarcosine, alanine, theophylline, and quinine, are unable toreverse oxyhemoglobin-induced contractions of cerebral arteries in vitro and in vivo(Cook et al., 1979; Fujiwara et al., 1986; Fujiwara and Kuriyama, 1984; lwai et al.,1988; Kanamaru et al., 1987; Kajikawa et al., 1979; Nakagomi et al., 1988; Nakayama66et al., 1989; Ohmoto et al., 1979, Ohta et al., 1980a, b; Okamoto, 1982, 1984; Onoueet al., 1989; Ozaki and Mullan, 1979; Tanishima, 1980; Toda, 1980). However,agents such as papaverine, calcium channel antagonists, and some inhibitors ofeicosanoids synthesis have been reported to relax oxyhemoglobin-inducedvasospasm (Cook et al., 1979; Fujiwara et al., 1986; Fujiwara and Kuriyama, 1984;Kanamaru et al., 1987; Ohta et al., 1980b; Onoue et al., 1989; Tanishima, 1980).Oxyhemoglobin, which has been observed to increase the intracellular concentrationof inositol phosphates that are second messengers involved in smooth musclecontraction (Vollrath et al., 1990), can act via a variety of pathways over a prolongedperiod to produce arterial narrowing and ultrastructural damage to arteries after SAH.Vasospasm induced by oxyhemoglobin may involve direct effects on smooth muscle,release of vasoactive eicosanoids and endothelin (Machi et al., 1991; Masaoka et al.,1990; Sato et al., 1990; Shigeno et al., 1990) from the arterial wall, inhibition ofendothelium-dependent relaxation (Toda et al., 1988; Tsuji et al., 1975), production ofbilirubin (Duff et al., 1988; Miao and Lee, 1989) and lipid peroxides (Asano et al.,1980; Sasaki et al., 1979), and damage to perivascular nerves (Okada et al., 1980;Linnik and Lee, 1989; Lee et al., 1984). However, the relative contributions of thesemechanisms of action of oxyhemoglobin to SAH-induced vasospasm are yet to bedetermined.67151 Hemoglobin and Eoileøsv:15.1) Nomenclature of Epileptic Seizures:Epileptic seizures can be categorized into generalized and partial seizures.While generalized seizures often involve both cerebral hemisphere and alterconsciousness, partial seizures only affect a part of one cerebral hemisphere. Partialseizures can be further classified into simple and complex types. Simple partialseizures consist of focal neurologic events with intact consciousness, whereascomplex partial seizures involve focal events with impaired consciousness. Becauseseizure activity in the cerebral cortex can spread, simple partial seizures may developinto complex partial seizures. Both simple and complex partial seizures may alsoevolve into secondarily generalized seizures.In addition to the above classification, epileptic seizures are divided intoprimary and secondary types. While primary or idiopathic epileptic seizures areusually inherited, age-related, benign, and unassociated with identified structurallesions, secondary (symptomatic) epileptic seizures are caused by an identifiableunderlying disease or lesion.15.2) Pathogenesis of Epileptic Seizures:Epilepsy is characterized by recurrent epileptic seizures which result fromparoxysmal and abnormally synchronous discharges of cerebral cortical neuronscaused by an imbalance of excitatory and inhibitory synaptic processes (Engel, 1990).The imbalance between the two synaptic processes can occur as a result of artificialelectrical stimulation of brain tissue, metabolic disorders, hypoglycemia, and traumaticbrain injury. In secondary epileptic seizures, loss of neurons, synaptic reorganization,and the balance between levels of glutamate and GABA are the common causes.6815.3) Subarrahnoid Hemorrhage-Induced Epileptic Seizures:Posttraumatic epilepsy is often observed in patients with cerebral hematoma,cortical laceration, or subarachnoid hemorrhage (Caveness, 1963; Caveness andLiss, 1961; Jennett, 1975; Kaplan, 1961; Richardson and Dodge, 1954; Russell andWhitty, 1952, 1953; Ward, 1972). Under normal physiological conditions, biologicaliron, which is normally bound in transferrin and hemoglobin, is released into braintissues by vascular endothelium (Harrison, 1971; Kristensson and Bornstein, 1974;Rigby, 1971; Strassmann, 1945). In head injury or hemorrhagic cortical infarction,extravasation of blood and deposition of iron within the neutrophil can occur (Kaplan,1961). Because topical cortical application of hemolyzed red blood cells (Levitt et al.,1971) or intracortical injection of ferrous orferric chloride (Willmore et al., 1978a, b, c)have been reported to generate seizures, blood or its metabolites may play a role ingeneration of epileptic seizures induced by intracranial hemorrhage. Indeed, focalepileptiform paroxysmal discharges were reported after intracortical injection of wholeblood, hemolyzed erythrocytes, methemoglobin, ferritin, ferrous chloride, ferricchloride, fibrinogen, hemin, and cottonoid in cats and guinea pigs (Hammond et al.,1980). Besides epileptic seizures, iron deposition, loss of neurons, and glialproliferation have also been observed at the site of brain lesions (Hammond et al.,1980; Rand and Courville, 1945; Pollen and Trachtenberg, 1970). Becausehemoglobin and iron liberated from hemoglobin have been implicated in generatingoxygen free radicals, peroxidation of neuronal membranes may occur as a result ofthe generation of oxygen free radicals (Mon et al., 1990).6915.4j Iron-Induced Epilepsy:Injection or ionophoretic deposition of iron salts into rodent has been shown tocause acute epileptiform discharges, formation of focal cerebral edema, cavitarynecrosis, and the occurrence of chronic or recurrent seizures (Wilimore et aI., 1978a,b, c). Lipid peroxidation of neuronal membranes which may produce epileptic foci hasbeen attributed to the generation of active oxygen-free radicals (02, •OH, •OOH)induced by iron (Hiramatsu et al., 1983, 1984). Besides the above phenomenon, thelevels of aspartate and GABA have been reported to decrease while the levels ofalanine and glycine increase in iron-induced epilepsy (Shiota et Ia., 1989). Thedecrease in aspartate and GABA levels has been attributed to the accelerated releasein the acute epileptic focus (Shiota et al., 1989). Alterations of excitatory andinhibitory amino acid levels may relate to the decrease in the activity of cerebralNa/K-ATPase which can cause brain edema accompanied by convulsion (Shiota etal., 1989).Using eletrocorticographic characteristics, three stages have been identified inseizures induced by injection of iron into rat cerebral cortex (Moriwaki et al., 1992).The first stage includes an increase in the frequency of isolated spikes, which areoften present on one side of the cortex, in the first 30 days after the injection. Thesecond stage occurs when an increase in the number of spike and wave complexes,which exhibit a bilateral appearance, is noted between 30 days and 6 months after theinjection. In the third stage, the proportion of the brain showing spike and wavecomplexes remains almost constant. In addition, alterations in the cerebral cortexsuch as changes in the adenosine- or norepinephrine-sensitive cAMP generatingsystems of the cortices probably occur in this stage. Similar to Moriwaki et al.,70Mizukawa et al. (1991) also observed 3 stages of responses induced by ironinjections by using c-fos-immunohistochemistry. At 3 hours after the ironadministration, c-fos-immunopositive neurons were observed in the dentate gyrus,CAl, CA2, the vicinity of the iron injected cerebral cortex, and amygdala. Whileseveral heavily stained cells and nerve fibers appeared around the injection site threedays after the administration, large cavities and gliosis were present at three weeksafter the injection.7116) MATERIALS AND METHODS:16.1) Animal Source:Male Wistar Rats (100-1 50g) provided by the Animal Care Center of the Universityof British Columbia were used in all studies. After the animals reached the Department ofPharmacology & Therapeutics of the University of British Columbia, they were kept in awire cage (dimensions: 17” X 7” X 9.75”) in the animal room. Animals came in ashipment of six animals. Each shipment of animals was stored in separate cages. Thetemperature of the animal room was carefully controlled and kept between 20 to 22°C.Lights of the room were kept on from 6 a.m. to 6 p.m. daily. Chow contained in acompartment attached to the wire cage and water contained in an inverted 250 ml bottlewere available to the animals. Food and water supplies were refilled daily from Monday toFriday. On every Friday, extra food and water were supplemented to maintain theanimals’ diet through the weekend. The excretion trays of the wire cages were cleanedand replaced once every two days.16.2) Slice Preparation:Rats were transferred from the animal room to the laboratory in a plastic cage(dimensions: 11.5” X 5” X 7.25”). The animals were then placed on ice in a glasschamber in order to lower their metabolic rate. Inside the chamber, animals wereanesthetized with 2% halothane and carbogen (95% 02, 5% C02) until surgicalanesthesia was achieved (i.e. the eyeblinking and the withdrawal reflexes are absent, andthe respiration is thoracic in origin.).After surgical anesthesia was acquired, a medial incision was made at the top ofthe animal’s head in order to expose the skull. A pair of nippers was used to create asmall incision at the base of the animal’s skull. Through this incision, a pair of scissors72was manipulated to cut along the sagital suture line. A pair of ronguers was subsequentlyused to remove the bone and the dura. The brain was gently removed from theintracranial cavity by a spatula. Once the brain was removed from the intracranial cavity,it was immediately washed with cold (4°C) ACSF which was saturated with carbogen.The hippocampi were exposed by removing the two cerebral hemispheres laterally. Oneof the hippocampi was peeled away from the remaining portion of the brain. Thehippocampus was then transferred to the cutting platform of a Mcllwain tissue chopperand positioned such that the septo-temporal axis of the hippocampus was perpendicularto the blade of the chopper. The hippocampus was cut transversely into slices of 400 jimin thickness. The slices were then separated by a pair of spatulas in a petri dishcontaining cold oxygenated ACSF and stored in a holding chamber which wascontinuously oxygenated by carbogen.The CA3 region of all slices was removed. Slices were allowed to equilibrate withthe superfusate for at least one hour before any electrophysiological recordings weremade. Slices stored in this manner were viable for eight hours post-surgery. At the end ofthe equilibrium period, only slices with a discrete cell line were used forelectrophysiological recordings. In order to avoid the slice from moving in the recordingchamber, the slice was placed in between two nylon meshes which are mounted on twoseparate overlapping plastic rings. The meshes were manipulated such that the slice wassecure between the nets without any physical damages.16.3) Slice Chamber:The slice chamber comprised of a rectangular plexi glass block, the superfusingchamber, the inlet, the suction, and the oxygen apertures (Fig. 2). The superfusingchamber resembles the shape of a key hole. While the circular section of the superfusing73chamber is located close to the inlet aperture, the narrow or rectangular section is closerto the suction aperture. The main inflow line was fed into the inlet aperture, whereas asuction tube for maintaining a constant circulation of superfusate was fed through thesuction aperture and into the superfusing chamber. Before the main inflow line reachedthe inlet aperture, it coursed through an aluminum heating block which was locatedunderneath the slice chamber. The temperature of the superfusate was carefullycontrolled and maintained at a constant temperature by a feed-back temperature sensingdevice in the control panel of the heater.A ground wire, which has a silver pellet at its end, was secured on the plexi glassblock by plasticine. The silver pellet of the ground wire was placed inside the rectangularsection of the superfusing chamber.The main inflow line is divided into seven different inlets by a manifold. Each inletwas connected to a 60 ml barrel. The barrels were adjusted to a height where a flow rateof about 1.6 mI/mm was obtained.I I ICD C.0 0Cl) g 0 CD0 0.= CD = CD ==7516.4) Superfusing Media:Slices were superfused with oxygenated (95% 02, 5% C02) artificial cerebrospinalfluid containing (in mM) 120 NaCI, 3.1 KCI, 26 NaHCO3,2 MgCI2, 2 CaCI2, 10 dextrose(pH 7.4) at a flow rate of about 1.6 mI/mm. In some experiments, CNQX (TocrisNeuramin), APV (Sigma), TTX (Sigma), picrotoxinin (Sigma), rat or bovine hemoglobin(Sigma), ferric chloride (Fed3: Nichols), tn-sodium-citrate (B.D.H.), or No-nitro-L-arginine(Sigma) was added to the superfusing medium. Concentrated stock solutions of CNQX,APV, and TTX were prepared in distilled water and diluted with ACSF to the desiredconcentration. Rat or bovine hemoglobin, ferric chloride, tn-sodium-citrate, and N-nitroL-arginine, which were in powder form, were directly dissolved in the superfusing mediumto acquire the desired concentration. In cases where the total concentration of divalentcations was significantly altered, an appropriate amount of calcium ions were removedfrom the recipe of the superlusing medium to compensate the change in the totalconcentration of divalent cations. The pH of all oxygenated superfusates was about 7.4.In some experiments, a Ca2-free medium was prepared by omitting calciumchloride from the recipe. Even though contamination from other chemicals was possible,the amount of Ca2 contributed by other chemicals was likely to be insignificant.All superfusing media were oxygenated by carbogen before and duringapplications. The main barrel which contained the principal supenfusing medium wascontinuously supplied with the medium from a reservoir located above the barrel. In orderto minimize dead space in the superfusing tubings, air bubbles trapped in the lines wereremoved, before each experiment, by a suction device. All filled lines which were not inuse were temporarily closed by butterfly clips. Whenever a particular filled line wasneeded, the butterfly clip of that line was removed while another butterfly clip was76simultaneously placed on the previously running line. By doing this, the slice chamberreceived rapid exchange of different media with minimal flow disturbance.16.5) Recording and Stimulating Equipment:The superfusing chamber, as well as the stimulating and recording electrodes wereall mounted on an aluminum plate. The base plate was in turn placed on a vibration-freetable. An aluminum wire-cage was used to shield the set up from electrical noises. Allrecording and stimulating equipment were mounted on two racks which are located to theright of the vibration-free table. The superfusing barrels were mounted on a stand whichwas positioned to the left of the table. A Zeiss gross dissecting microscope mounted on amovable stand was also situated on the same side as the superfusing barrels.16.5.1) Stimulators and Isolation Units:A Grass S88 stimulator which were connected to two Grass PSIU6 photoelectricstimulus isolation units with constant current output was used in all experiments. Whileone unit fed square wave current pulses or DC current through the Axoclamp into therecording microelectrode, the other unit supplied current to the stimulating electrode.16.5.2J Amplifiers:Both extracellular and intracellular potentials were recorded with an Axoclamp 2Amicroelectrode clamp (Axon Instruments) in the current clamp (CC) mode. A precisionresistor of 100 M2 in resistance, which were located in the headstage, set the headstagecurrent gain (H) to be 0.1. At this value, the range of the bridge balance was 0 to 1000M2; the maximum DC current command was ± 10 nA; and the range of the capacitanceneutralization was -1 to 4 pF. The signals from Axoclamp were amplified ten times andfiltered between 0.1 kHz and 1 kHz before they reached the recording systems.77The null-bridge method was used to determine the resistance of themicroelectrodes. When the microelectrode was placed in the superfusing medium, thevoltage response elicited by a 0.2 nA and 200 ms square pulse stimulation was removedby adjusting the bridge balance of the Axoclamp. The value registered on the bridgebalance was the resistance of the microelectrode. After the microelectrode impaled aneuron, the voltage deflection caused by the same square pulse was once againeliminated by further adjustment on the bridge balance. The value of the bridge balanceafter the second adjustment was the sum of the resistance of the microelectrode and thatof the cell membrane. At the end of each experiment, the above processes wereperformed in reverse order to ensure that the resistance of the recording microelectrodewas not altered in the experiment.16.5.3) RecordinQ Systems:lntracellularly recorded potentials were monitored by a Tetronix type 564 storageoscilloscope and analyzed by a DATA 6000 waveform analyzer. Each recording from theDATA 6000 waveform analyzer represented an average of four consecutive records.While the recordings from the Data 6000 waveform analyzer were recorded by a HP7470-A graphic plotter (Hewlett Packard), the responses from the oscilloscope were recordedby a Grass 79D polygraph.Extracellularly recorded potentials were recorded in a similar manner. However,each recording from the DATA 6000 waveform analyzer only represented an average oftwo consecutive responses.7816.5.4) Electrodes:16.5.4.1) Recording Electrodes:Both Intracellular and extracellular recording electrodes were made from standardwall borosilicate glass capillaries supplied by Sutter Instrument. The microelectrodeswere pulled by a programmable Flaming/Brown P-87 model micropipette puller (SutterInstrument). A 2 mm wide horizontal trough filament (Sutter Instrument) was used.The inner and outer wall diameters of the extracellular electrodes were 1.17 and1.5 mm, respectively. The program setting used to pull the extracellular electrodes was:Heat = 390 units; Pull = 0 units; Velocity = 86 units; Time = 255 units. The electrodeswere filled with ACSF. The tip resistance of these electrodes was about 5 MQ.The inner and outer wall diameters of the intracellular electrodes were 1.0 and 0.58mm, respectively. These electrodes were pulled with the following setting: Heat = 390units; Pull = 250 units; Velocity = 80 units; Time = 160 units. A 3M potassium acetateelectrolyte was used in most intracellular recordings. The tip resistance of the intracellularmicroelectrodes ranged from 90 to 110 ME.16.5.4.2) Stimulating ElectrodesjA bipolar concentric stimulating electrode (SNEX-100; DKI) was used in allexperiments. The shaft length and the contact length of the electrode was 50 mm and0.75 mm, respectively. The contact diameter of the electrode was 0.1 mm. Theresistance of the electrode was around I Mfl.16.5.4.3) Positioning of Electrodes:In extracellular studies, the recording electrode was manipulated in the X, Y, and Zplanes by an Optikon micropositioner. In intracellular studies, a DKI 650 hydraulic79micropositioner, which could lower the electrode by 2.5 tim in 2.5 x i0 ms, was used inaddition to the Optikon micropositioner. The stimulating electrode was installed on a DKIelectrode carrier which also allowed the stimulating electrode to be moved in a threedimensional manner.16.6) Electrophysiological Recordings:16.6.1) Extracellular Recordings:Field EPSPs evoked by stimulation (0.05 Hz; 0.2 ms; 10-150 1iA) of the stratumradiatum with a bipolar electrode (SNEX 100; Rhodes Electronics) were recorded next tothe CAl cell body region. The stimulation strength was adjusted to produce field EPSPsof 0.5 to I mV in size. Fifteen minutes of stable responses were obtained as controlvalues before any drug applications.16.6.2) Intracellular Recordings:Intracellular responses evoked by stimulation (0.5 Hz; 0.2 ms; 10-l5OpA) of thestratum radiatum with a bipolar electrode (SNEX 100, Rhodes Electronics) were recordedin CAl neurons of the hippocampus. The input resistance of neurons was examined bymeasuring the change in membrane potential caused by rectangular hyperpolarizingcurrent pulses (-0.05 to -0.2 nA, 200 ms) injected through the recording electrode.Excitability of the neurons was monitored by examining the number of spikes evoked byrectangular depolarizing current pulses (0.05 to 0.3 nA, 200 ms). The stimulation strengthwas adjusted to produce EPSPs of 5 to 10 mV in size and IPSP5 of 2 to 5 mV inamplitude. Fifteen minutes of stable EPSPs and IPSPs were collected as controlresponses before any drug applications. The membrane potential, the input resistance,and the depolarization-induced discharge of action potentials were also ensured to bestable during this control period.80In some experiments, neurons were current-clamped to compensate for the drug-induced change in membrane potential (i.e. bringing the membrane potential back tocontrol level) when records of the EPSPs, the IPSPs, the input resistance, and thedepolarization-induced discharge of action potentials were taken during drug application.These records were then compared to the control responses at the same membranepotential.16.7) Analysis of Extracellular and Intracellular Recordings:16.7.1) Extracellular Recordings:The height of the extracellularly recorded EPSPs was determined by the distancemeasured from the peak of the response to the baseline. The distance was thenmultiplied by a proper conversion factor to express the actual size of the response inmillivolts. The initial slope of the extracellularly recorded EPSPs was also computed andexpressed in mV/ms by the DATA 6000 waveform analyzer16.7.2) Intracellular Recordings:The amplitude of the EPSPs and the IPSPs was measured by the distancebetween the baseline and the peak of the EPSPs, and those between the peaks of theIPSPs and the baseline, respectively. The actual size of the EPSPs and the IPSPs inmillivolts were obtained by multiplying the distances by a conversion factor. The latenciesof the control synaptic responses (i.e. the durations measured from the stimulation artifactto the peak of the EPSP, the fast IPSP, and the slow IPSP) were used as references forsubsequent recordings. The initial slope of the EPSP was also computed and expressedin mV/ms by the DATA 6000 waveform analyzer.8116.8) Statistics:All control and post-drug synaptic responses were normalized to the mean of thecontrol responses. All data were expressed as mean ± S.E.M.. Two tailed paired samplet-test was used to determine the statistical significance of the effect of the drug on theinput resistance, the change in membrane potential, and the depolarization-induceddischarge of action potentials. The statistical significance of the drug on the synapticresponses at any time was determined by ANOVA and the Duncan test, a post-hocmultiple comparison analysis. In both statistical tests, the level of significance (p) wasarbitrarily selected to be 0.05. In other words, a probability of less than 0.05 wasconsidered to be statistically significant.8217) EXPERIMENTAL PROTOCOL:17.0 Electrophysiological Effects of Rat Hemoglobin on Rat Hippocampal CAlNeurons:17.1.1) Without Compensation of the Hemoglobin-Induced Change in MembranePotential:Rat hemoglobin (0.05 mM) was prepared by dissolving the appropriate amount ofhemoglobin powder (Sigma) in ACSF. After the EPSP, the input resistance, themembrane potential, and the depolarization-induced discharge of action potentials wereobserved to be stable for 15 minutes, oxygenated rat hemoglobin (0.05 mM) was appliedfor 15 minutes. After the hemoglobin application, the slice was reperfused with ACSF for30 minutes. During drug application and reperfusion, the EPSP, the input resistance, themembrane potential, and the depolarization-induced discharge of action potentials weremeasured quantitatively.17.1.2) With Compensation of the Hemoglobin-Induced Change in Membrane Potential:During depolarization, while the input resistance of neurons may increase as aresult of closures of voltage-dependent ion channels, the EPSP may be suppressed asthe reversal potential of the EPSP is approached. Since the EPSP and the inputresistance could be changed by the hemoglobin-induced change in membrane potential,the membrane potential was current clamped to the pre-hemoglobin level when the EPSPand the input resistance were measured during drug application. In cases where themembrane potential during reperfusion was not the same as that of the control period,compensation of the change in membrane potential was also done to allow comparison ofthe responses measured during reperfusion to the control responses.8317.2) Electrophysiological Actions of Bovine Hemoglobin on RatHippocampal CAl Neurons:The effects of rat hemoglobin (0.05 mM; Sigma) were compared to those of bovinehemoglobin (0.05 mM; Sigma). There was no significant difference between the actionsof rat and bovine hemoglobins on the membrane potential, the input resistance, and theEPSP. Because bovine hemoglobin is much more economical, and there are no knowndifferences between rat and bovine hemoglobins, bovine hemoglobin was used insubsequent experiments.17.2.1) Without Compensation of the Hemoglobin-Induced ChanQe in MembranePotential:Different concentrations of bovine hemoglobin (0.01 to 1 mM) were used todetermine the appropriate concentration to induce significant effects consistently in CAlneurons. Bovine hemoglobin was prepared by dissolving the appropriate amount ofhemoglobin powder (Sigma) in ACSF to reach the desired concentration. After thesynaptic transients, the input resistance, the membrane potential, and the depolarizationinduced discharge of action potentials were observed for 15 minutes for stability,oxygenated hemoglobin was applied for 15 minutes. Following the hemoglobinapplication, the slice was reperfused by ACSF for 30 mm.. During drug application andreperfusion, the synaptic transients, the input resistance, the membrane potential, and thedepolarization-induced discharge of action potentials were measured quantitatively.8417.2.2) With Compensation of the Hemoglobin-Induced Change in Membrane Potential:If the membrane potential was changed during hemoglobin application orreperfusion, the membrane potential was current clamped to the control value when theelectrophysiological responses were measured. In some experiments, 50 pM ofpicrotoxinin was included in the superfusing medium in order to examine the effects ofhemoglobin on GABAB-mediated slow IPSPs without contamination by the fast IPSPs.17.3) The Current-Voltage (IN)Relationship for Bovine Hemoglobins:Like the synaptic transients, the input resistance can also be changed by a changein membrane potential. In order to assure that the hemoglobin-induced change in inputresistance was not due to the change in membrane potential caused by hemoglobin, theIN relationship for hemoglobin was studied with and without compensation to thehemoglobin-induced change in membrane potential in the same neuron.17.3.1) Without Compensation of the Hemoglobin-Induced Change in MembranePotential:Voltage deflections elicited by depolarizing and hyperpolarizing rectangular currentpulses (0.1 nA to -0.2 nA) injected through the recording electrode were measured duringcontrol and during hemoglobin application. For hyperpolarizing pulses, the membranepotentials at the peaks of the current pulse-induced voltage responses were calculated bysubtracting the absolute values of voltage deflections elicited by the current pulses fromthe membrane potential recorded when the current pulses were injected. For depolarizingpulses, the membrane potentials at the peaks of the current pulse-induced voltageresponses were calculated by adding the absolute values of the voltage deflectionselicited by the current pulses to the membrane potential recorded when the current pulseswere injected. The membrane potentials at the peaks of the current pulse-induced85voltage deflections and the corresponding current strengths measured during control andhemoglobin application (0.1 mM; 15 mm) were then plotted on a voltage vs current curve.The input resistance of the neuron in control medium and in hemoglobin was representedby the slope of the respective graph.17.3.2) With Compensation of the Hemoglobin-Induced Change in Membrane Potential:During hemoglobin application (0.1 mM; 15 mm.) and reperfusion, the membranepotential was current clamped to the control value when the depolarizing andhyperpolarizing current pulses were injected through the recording electrode. While themembrane potentials at the peaks of the depolarizing current pulse-induced voltagedeflections were calculated by adding the absolute values of the current pulse-inducedvoltage deflections to the control membrane potential, those at the peaks ofhyperpolarizing current-induced voltage deflections were computed by subtracting theabsolute values of the current pulse-induced voltage deflections from the controlmembrane potential. The absolute membrane potentials and the respective currentstrengths were then plotted on a voltage vs current graph.17.4) Calcium and the Hemoglobin-induced Depolarization:In order to determine if the depolarizing action of hemoglobin wasCa2-dependent,a Ca2-free medium containing i07’ M of TTX, 3.5 mM Mg2, and 0.5 mM Mn2 was usedfor superfusion The total concentration of divalent cations in the Ca2-free medium wasmaintained at 4 mM by increasing the concentration of Mg2 from 2 to 3.5 mM and byincluding 0.5 mM of Mn2 in the medium.86In normal ACSF containing i0’ M TTX, a depolarization induced by superfusionwith 0.1 mM of hemoglobin for 15 minutes was obtained as a control response. At the endof the application of hemoglobin, the slice was reperfused with the normal medium for 30minutes. At the end of the reperfusion period, the normal superfusate was replaced bythe Ca2-free medium. After superfusing the slice with the Ca2-free medium for 15minutes, 0.1 mM hemoglobin prepared with the Ca2-free medium was applied for 15minutes. A reperfusion with the normal medium (30 mm) followed the second hemoglobinapplication. At the end of the second reperfusion, 0.1 mM hemoglobin prepared in thenormal medium was applied for 15 minutes. After the third application of hemoglobin, theslice was reperfused with the normal medium for 30 minutes. All three hemoglobinapplications were done at the same membrane potential so that the responses recordedin the normal medium (i.e. the control response and the response from the thirdapplication of hemoglobin) could be compared with that recorded in the Ca2-free mediumat the same membrane potential.17.5) Involvements of NMDA and Non-NMDA Receptors in the HemoglobinInduced Depolarization:Besides calcium, the depolarizing action of hemoglobin may be due to theactivation of glutamate receptors. In order to determine whether activation of excitatoryamino-acid receptors was necessary for the depolarizing action of hemoglobin,hemoglobin was applied in the presence of APV or CNQX.8717.5.1) APV and the Hemoglobin-Induced Depolarization:The stock solution of APV was diluted with a Mg2-free medium containing I 0 MTTX to reach a final concentration of 50 pM. The concentration of the divalent cations inthe Mg2-free medium was maintained at 4 mM by increasing the Ca2 concentration from2 to 4 mM. A control hemoglobin-induced depolarizing response was obtained bysuperfusing the slice for 15 minutes with 0.1 mM hemoglobin, which was prepared withACSF containing 10’7M TTX. At the end of the hemoglobin application, the slice wasreperfused with the normal medium for 30 minutes. Following the reperfusion period, thenormal medium was replaced with the Mg2-free medium. After superfusing the slice withthe Mg2-free medium for 5 minutes, 0.1 mM hemoglobin prepared in the Mg2-freemedium was applied for 15 minutes. A 30 minutes long reperfusion with the normalmedium followed the second application of hemoglobin. At the end of the secondreperfusion, 0.1 mM hemoglobin prepared with the normal medium was applied for 15minutes again. After the third application of hemoglobin, the slice was also reperfusedwith the normal medium for 30 minutes. All hemoglobin-induced depolarizing responseswere elicited at the same membrane potential to allow comparison between the responsesrecorded in the normal medium (i.e. the control response and the response from the thirdapplication) and that obtained in the Mg2-medium.17.5.2) CNQX and the Hemoglobin-Induced Depolarization:The stock solution of CNQX was diluted with a normal medium containing i0’ MTTX to reach a final concentration of 20 pM. A control hemoglobin-induced depolarizingresponse was obtained by superfusing the slice with 0.1 mM hemoglobin, which was88prepared with ACSF containing I o’ M TTX, for 15 minutes. At the end of the hemoglobinapplication, the slice was reperfused with the normal medium for 30 minutes. Followingreperfusion, the normal medium was replaced with the CNQX containing medium. Aftersuperfusing the slice with the CNQX containing medium for 10 minutes, 0.1 mMhemoglobin prepared with the same medium was applied for 15 minutes. A 30 minuteslong reperfusion with the normal medium followed the second application of hemoglobin.At the end of the second reperfusion, 0.1 mM hemoglobin prepared with the normalmedium was again applied for 15 minutes. After the third application of hemoglobin, theslice was also reperfused with the normal medium for 30 minutes. All depolarizingresponses were elicited at the same membrane potential so that the responses recordedin the normal medium (i.e. the control response and the response from the thirdapplication) could be compared to that acquired in the CNQX containing medium.176) Glutamate-Induced and Hemoglobin-Induced Depolarizations:A stock solution of concentrated glutamate was diluted with ACSF containing 1 0qM TTX to reach a final concentration of 5 mM. Glutamate (5 mM) was applied for Iminute to elicit a depolarization. Two glutamate-induced depolarizations which weresimilar in size were used as control responses. During the 15 minutes application of 0.1mM hemoglobin, 5 mM glutamate was applied for 1 minute at the peak of the hemoglobininduced depolarization. Glutamate was also applied for 1 minute at 5 and 30 minutespost-hemoglobin.When glutamate was applied during hemoglobin application, the membranepotential was current-clamped to the control value. In general, all glutamate-induced89responses were recorded at the same membrane potential so that responses recordedbefore, during, and after hemoglobin-application could be compared.17.7) Nitric Oxide Synthase Inhibitor and the Actions of Hemoglobin:In order to determine if the actions of hemoglobin were caused by the NOscavenging property of hemoglobin, the actions of hemoglobin were examined in thepresence of a nitric oxide synthase inhibitor, Nco-nitro-L-arginine. A low (100 riM) and ahigh (500 i.tM) doses of Nco-nitro-L-arginine were used in this investigation.No-nitro-L-arginine was prepared by dissolving the appropriate amount of theagent powder in 20 ml of ACSF whose pH was lowered to about 1.5 by HCI. The aliquotwas then diluted to the desired concentration (100 iM or 500 jiM) with ACSF. The pH ofthe final solution with oxygenation was about 7.4. Hemoglobin (0.1 mM) was preparedwith ACSF containing either 100 or 500 jiM of Nco-nitro-L-arginine.In the presence of either 100 or 500 jiM of No-nitro-L-arginine, the synaptictransients, the input resistance, the membrane potential, and the depolarization-induceddischarge of action potentials were determined to be stable for 15 minutes beforehemoglobin was applied. At the end of the hemoglobin application, the slice wasreperfused with ACSF containing either the low or high dose of No-nitro-L-arginine. Themembrane potential was current-clamped to the control value when the synaptictransients, the input resistance, and the depolarization-induced discharge of actionpotentials were recorded.9017.8) Iron and the Actions of Hemoglobin:As a component of hemoglobin, iron could contribute to the effects of hemoglobin.In this study, the effects of iron were compared to those of hemoglobin.A low (0.4 mM) and a high (2 mM) dose of iron which corresponded to the ironcontent in 0.1 and 0.5 mM of hemoglobin, respectively, were used in this investigation.The 0.4 mM iron solution was prepared by dissolving the appropriate amount of ferricchloride powder with a phosphate-free medium containing 0.4 mM of tn-sodium citrate.The concentration of NaCI was reduced from 120 to 119.4 mM to compensate for theinclusion of 0.4 mM ferric chloride and 0.4 mM tn-sodium citrate, as well as the exclusionof 1.8 mM sodium dihydrogen phosphate in the iron containing medium. Like the ironsolution, the control superfusate also contained 0.4 mM tn-sodium citrate.The 2 mM iron solution was prepared as descried for the 0.4 mM counterpart. Theconcentration of NaCI was reduced from 120 to 109.8 mM to compensate for the inclusionof 2 mM ferric ions and 2 mM tn-sodium citrate, as well as the exclusion of 1.8 mM sodiumdihydrogen phosphate in the iron superfusate. For the control superfusate, theconcentration of NaCI was only reduced from 120 to 115.8 mM since only tn-sodiumcitrate (2 mM) but not ferric ions were present in this medium.After the synaptic transients, the input resistance, the membrane potential, and thedepolarization-induced discharge of action potentials were determined to be stable for 15minutes, iron solution of 0.4 or 2 mM was applied for 15 minutes. After removal of either0.4 or 2 mM iron solution, the slice was reperfused with the corresponding controlsuperfusate for 30 minutes. The membrane potential was current-clamped to the control91value when the synaptic transients, the input resistance, and the depolarization-induceddischarge of action potentials were measured.179) Presynaptic Volley and the Suppression of Synaptic Transients:Three different concentrations of bovine hemoglobins (0.1, 0.5, or 1 mM; Sigma),were applied for 15 minutes after the extracellularly recorded field EPSPs weredetermined to be stable for 15 minutes. After each hemoglobin application, the slice wasreperfused with ACSF for 30 minutes.. The amplitude of the field EPSP was plottedagainst the presynaptic volley on a graph (i.e. amplitude of the field EPSP vs amplitude ofthe presynatic volley graph).9218) RESULTS:18.1) Effects of Rat Hemoglobin on hippocampal CAl neurons:Superfusion of rat hippocampal slices with 0.05 mM of rat hemoglobin for 15minutes changed the membrane potential of the neurons from -68.1 ± 1.0 mV to -59.2 ±1.8 mV (n=21; p < 0.05) (Fig. 3A). The membrane potential usually returned to the controllevel shortly after the removal of hemoglobin. In 7 out of 21 neurons, an afterhyperpolarization was observed. This hyperpolarization lasted for 5 to 10 minutes. Rathemoglobin also increased the input resistance and suppressed the intracellularlyrecorded EPSPs (% of the control EPSP slope: 37.3 ± 8.5%; n = 7; p < 0.05). Partialrecoveries of the EPSPs and the input resistance were observed during the first thirtyminutes after the removal of hemoglobin. In these 7 neurons, the membrane potentialduring the application was not current clamped to the pre-drug level when records ofsynaptic transients were taken.Since it is possible that the suppression of the EPSP was due to the depolarizationinduced by hemoglobin, in some neurons the membrane potential was current clamped.In these cells, EPSPs were suppressed and the input resistance was enhanced (EPSPslope in hemoglobin as a % of control: 36.1 ±8.9; n = 7; p <0.05).93A2OmVB2OmVFigure 3. Effects of hemoglobin on the membrane potential and the input resistance of rathippocampal CAl neurons. Hemoglobin was applied for 15 minutes. The inputresistance was monitored by injecting hyperpolarizing current pulses throughout theexperiment. A) Rat hemoglobin (0.05 mM) caused a depolarization and increased theinput resistance. The resting membrane potential of this neuron was -58 mV. B) Bovinehemoglobin (0.1 mM) showed similar actions on the membrane potential and the inputresistance in CAl neurons of rat hippocampal slices as rat hemoglobin. 10q M TT)( wasadded in the superfusate in order to avoid firing of action potentials during thedepolarization caused by hemoglobin. The resting membrane potential of this neuronHb5 mm.Hb5 minwas -69 mV.9418.2) Effects of Bovine Hemoglobin on hippocampal CAl neurons:18.2.1) The effects of hemoclobin on the evoked synaptic responses, the membranepotential. the input resistance, and the excitability of CAl neurons.The effects of bovine hemoglobin on hippocampal CAl neurons were studied in atotal of 53 neurons with an average resting membrane potential of -65.6 ± 5.2 mV.Different concentrations of bovine hemoglobin (0.01- 1 mM) were used todetermine the appropriate concentration for inducing significant effects consistently inCAl neurons (n = 27). A concentration of 0.1 mM (applied for 15 minutes) was used toinduce a 5.9 ± 0.9 mV (n = 26; p < 0.05) depolarization of the neurons (Fig. 3B). As in thecase of rat hemoglobin, the effect of bovine hemoglobin on the membrane potentialusually disappeared 2 - 5 minutes after terminating its application. Although themembrane potential usually returned to the control value upon removal of hemoglobin, anafter-hyperpolarization that lasted for approximately 15 minutes was observed in 10 out of26 neurons. Bovine hemoglobin also increased the input resistance by 51.61 ± 5.94 % (n= 26; p <0.05). The hemoglobin-induced increase in the input resistance is clearly shownby the increase in the slope of the current-voltage curve recorded in the presence of 0.1mM hemoglobin (Fig. 4A). This increase in the input resistance caused by hemoglobinwas found to be independent of the hemoglobin-induced depolarization (Fig. 4B).Superfusion of hippocampal slices with 0.1 mM hemoglobin for 15 minutes also produceda significant suppression of the evoked synaptic responses of CAl neurons duringapplication (n = 6). While a depolarization of 41.2 ± 3.6 mV and an increase of 63.4 ±11.4 % in the input resistance were observed when 0.5 mM hemoglobin was applied for15 minutes (n = 9; p <0.05), a depolarization of 53.6 ± 6.2 mV and an increase of 103.8 ±28.7 % in the input resistance were seen with a 15 minutes application of 1.0 mM95hemoglobin (n = 5; p < 0.05). Both 0.5 mM (n = 9) and 1.0 mM (n = 5) hemoglobincompletely abolished the evoked synaptic responses during application. Because 0.1 mMproduced less extreme effects on the membrane potential, the input resistance, and theevoked synaptic responses than the higher doses, this concentration of hemoglobin wasapplied for 15 minutes in further experiments.Upon application of 0.1 mM hemoglobin, the number of spikes evoked byrectangular depolarizing pulses increased (Fig. 4C). This increase in the depolarizationinduced discharge of action potentials either partially or completely recovered 30 minutespost-drug application when recordings were terminated.96Figure 4. Effects of bovine hemoglobin on the input resistance and the excitability of rathippocampal CAl neurons. A) Current-voltage curves recorded during control and in thepresence of hemoglobin (0.1 mM; 15 mm). Note the increase in the slope of the curve forhemoglobin. B) Current-voltage curves for control and bovine hemoglobin recorded aftercurrent-clamping the membrane potential to the control value. The hemoglobin-induceddepolarization was compensated by a hyperpolarizing DC current. The slope of the curvefor hemoglobin is steeper than that for control. Both A and B were recorded in the sameneuron. The resting membrane potential of this neuron was -68 mV. C) The effect ofbovine hemoglobin on the excitability of rat hippocampal CAl neurons. The cellexcitability was monitored by the number of spikes induced by injecting a 0.3 nAdepolarizing rectangular pulse (200 ms). Depolarizing pulses were injected into theneurons during control (a), immediately before the end of the 15 minutes application ofhemoglobin (b), and 30 minutes after the application of hemoglobin (c). The restingmembrane potential of this neuron was -64 mV.97Figure 4—0.25 —0.15<>Bbo Control—0.25 —0.15o Ebo ControlCurrent (nA)00200 ms200 ms—0.05 0.05 0.15088000000o0o0Current (nA)—0.05 0.05ABC0E0.15—558lOmV00000/b98Without adjusting the membrane potential to the pre-drug level, the EPSP wasdepressed by 0.1 mM hemoglobin (EPSP slope in hemoglobin as a % of control: 26.95 ±5.16, n = 6; p < 0.05). In these neurons, the fast and slow IPSPs were suppressed,enhanced or not changed. These varied results may be due to the depolarization causedby the drug.When neurons were current-clamped to pre-hemoglobin membrane potentials, theagent depressed the EPSP (slope of EPSP in hemoglobin as a % of control: 27.23 ±3.24, n = 8; p < 0.05), the fast IPSP (response in hemoglobin as a % of control: 44.56 ±3.66, n = 10; p <0.05), and the slow IPSP (response in hemoglobin as a % of control:20.24 ± 6.92, n = 4; p <0.05) (Fig. 5A & B; 6D, E, & F). The EPSP (% of control EPSPslope: 52.65 ± 10.66, n = 8; p <0.05) and the slow IPSP (% of control response: 41.99 ±6.31, n = 4; p < 0.05) showed only partial recoveries when measured 30 minutes after theremoval of hemoglobin (Fig. 6D & F). Unlike the EPSP and the slow IPSP, the fast IPSP(% of control response: 102.48 ± 15.17, n = 10; p > 0.05) completely recovered from thehemoglobin-induced depression (Fig. 6E).99J 520 msB5 mV200 msFigure 5. Effects of bovine hemoglobin on the evoked synaptic responses of rathippocampal CAl neurons. Bovine hemoglobin (Hb, 0.1 mM) was applied for 15 minutes.The synaptic responses were recorded in response to 0.05 Hz stimulation of the stratumradiatum. A) The effects of hemoglobin on the EPSP. The EPSP traces were takenduring control (a), immediately before the end of the application of hemoglobin (b), and at30 minutes after removal of hemoglobin (c). B) The effects of hemoglobin on both fastand slow IPSPs. The synaptic responses were recorded during control (a), immediatelybefore the end of the application of hemoglobin (b), and at 30 minutes after removal ofhemoglobin (c). The resting membrane potential of this neuron was -69 mV.100Figure 6. Time course of the effects of bovine hemoglobin on the evoked synapticresponses of rat hippocampal CAl neurons after current-clamping the membranepotential to the control level. The synaptic responses were recorded in response to 0.05Hz stimulation of the stratum radiatum. The depolarization caused by hemoglobin wascompensated by a hyperpolarizing DC current so that synaptic responses could berecorded at the control membrane potential. A) Time course of the effects of normalACSF on the EPSP. The curve was plotted with the average data of five differentneurons. B) Time course of the effects of normal ACSF on the fast IPSP. The curve wasplotted with the average data of five neurons. C) Time course of the effects of normalACSF on the slow IPSP. The curve was plotted with the average data of five neurons. D)Time course of the suppression of intracellularly recorded EPSP by bovine hemoglobin(0.1 mM; 15 mm). The curve was plotted with the average data of eight different neurons.E) Time course of the suppression of the fast IPSP by bovine hemoglobin (0.1 mM; 15mm). The curve was plotted with the average response often different neurons. F) Timecourse of the suppression of the slow IPSP by bovine hemoglobin (0.1 mM; 15 mm). Thecurve was plotted with the average response of four different neurons. Slices weresuperfused with medium containing picrotoxinin (50 mM) throughout the experiment inorder to remove the fast IPSP.0Figure60120120120100f.Talsoaa8080606060C——CE40404080fl=580_____________20Switch20Switch—20Switch00iII0i010203040506070010203040506070010203040506070Time(mm.)Time(mm.)Tune(mm.)ABC120120120100a10001.80808060f80*60*C**EIS40*C*804o***‘S020***80n=1080*4n82020SmRb___Hb0II0•III0•10203040506070010203040506070010203040506070Time(mm)Time(mi)Time(mm)CDE10218.3) The effects of hemoglobin on the extracellularly recorded Field EPSPs:Superfusion of slices with 0.1 (n = 3), 0.5 (n = 3), and 1.0 mM (n = 3) bovinehemoglobin for 15 minutes induced paroxysmal depolarization shifts as well as dose-dependently suppressed the extracellularly recorded field EPSPs and the presynaticvolley. At 0.1 and 0.5 mM hemoglobin, the field EPSPs started to recover at 40 and 50minutes post-drug application., respectively. At 1.0 mM bovine hemoglobin, the fieldEPSPs did not start to recover until 60 minutes post-drug application. While thepresynaptic volley was significantly suppressed by 0.5 mM hemoglobin, minimal changeon the presynaptic volley was observed with 0.1 mM hemoglobin (Fig. 7A & B).103Figure 7. Effects of different concentrations of hemoglobin on the field EPSPs and thepresynaptic volley. A) 0.1 mM (n = 3). B) 0.5 mM (n = 3). The size of the field EPSP(mV) was plotted against the size of the presynaptic volley (mV). Three differentstimulation strengths were used in all six experiments. Records were taken during control,immediately before the end of drug application, and at 60 minutes after the removal ofhemoglobin.>Eci(I)0LJ0ci)-oDE>E0C,)0..LI0ci)0:30.E>E0C/)0LI0ci)-D:30Figure 7A104o ControlL]HbRecovery0.0.2 0.3 0.4Amplitude of Presynoptic Volley (my)0.52.82.42.01.6o ControlDUbRecovery1.20.40.0 0.2 0.4Amplitude of Presynaptic Volley (mV)2.72.31.91.5o Control[]IThRecovery1.10.7—0.10.0 0.1 0.2 0.3 0.4Amplitude of Presynciptic Volley (mV)0.5105>E0(I)00V-o0EFigure7Bo ControlDHbA Recovery0.1—0.10.0 0.2 0.4 0.6 08 1.0Amplitude of Presynoptic Volley (my)o ControlDRA Recovery02.72.31.9ft.1.50Vo 1.10.70.3—0.10.01.31.1>0.9e 0.700.5-o0.3E0.1—0.10.00.4 0.8 1.2 1.6 2.0 2.4Amplitude of Presynoptic Volley (my)o ControlDUbA Recovery0.2 0.4 0.6 0.8Amplitude of Presynaptic Volley (mV)1.010618.4) Calcium and Hemoglobin-Induced Depolarization:In a Ca2-free medium, the depolarization induced by hemoglobin (0.1 mM; 15 mm)was enhanced significantly (% of control response: 223.50 ± 28.31; n = 10; p <0.05) (Fig.8A & B). By 30 minutes after the replacement of the Ca2-free medium with the controlmedium, the enhancement in the hemoglobin-induced depolarization was reversed (% ofcontrol response: 102.54±2.56; n = 10; p > 0.05) (Fig 8A & B).107Figure 8. Calcium and the hemoglobin-induced depolarization. Depolarizations inducedby 0.1 mM hemoglobin prepared in a normal medium containing i0’ M TTX werecompared to that prepared in a calcium-free medium which also contained I 0’ M TTX. A)A histogram was constructed with the average response of ten different neurons. B) Rawtraces recorded in a neuron whose resting membrane potential was -59 mV.0Figure8A____250n=1O200-150-100-ControlCa-freeRecoveryBj2OrnVHbNb+Ca2-freemediumab10mm.10918.5) Involvements of NMDA and Non-NMDA Receptors in the HemoglobinInduced Depolarization:18.5.1) APV and Hemoglobin-Induced Depolarization:In the presence of 50 jiM APV, the depolarization induced by 0.1 mM hemoglobin(15 mm) was decreased (% of control response: 78.25 ± 12.45; n = 10) (Fig. 9A & B).However, this APV-mediated suppression was statistically insignificant (p > 0.05). Whenhemoglobin was applied at 30 minutes after the removal of APV, the response recovered(% of the control response 89.54 ± 6.75; n = 10; p > 0.05) (Fig 9A & B).18.5.2’) CNQX and Hemoglobin-Induced Depolarization:Superfusion of slices with a medium containing 20 jiM CNQX enhanced thedepolarization evoked by 0.1 mM hemoglobin (15 mm) (% of control response: 120.14± 17.01; n = 6) (Fig. 1OA & B). Similar to the effect of APV, the enhancement in thehemoglobin-induced depolarization caused by CNQX was statistically insignificant (p> 0.05). The effect of CNQX was reversed by 30 minutes after the removal of CNQX(% of control response: 104.01 ± 11.27; n = 6; p > 0.05) (Fig. I OA & B).110Figure 9. Effects of APV on the depolarizing action of hemoglobin. Depolarizationsinduced by 0.1 mM hemoglobin (15 mm) were evoked in a normal medium containingio’ M TTX as well as a Mg2-free medium containing i0’ M TTX and 50 1iM APV. A)A histogram was constructed with the average response of 10 different neurons. B)Raw traces recorded in a neuron whose resting membrane potential was -69 mV.Figure9A120n=1O180.-600a)40,02000—____——ControlAPVRecoveryHbRbRb+APVabcT20naV10nun.112Figure 10. Effects of CNQX on the hemoglobin-induced depolarization.Depolarizations induced by 0.1 mM hemoglobin (15 mm) were evoked in a normalmedium containing I 0 M TTX as well as a medium containing I 0 M TTX and 20 jiMCNQX. A) A histogram was constructed with the average response of 6 differentneurons. B) Raw traces recorded in a neuron whose resting membrane potential was-72 mV.()AFigurelO160-j140n=61?010040.a200-—_____—_____ControlCNQXRecoveryJ{T+CNQXHbT.j2OmVabc10mm.11418.6) Effects of Hemoglobin on the Glutamate-Induced Depolarization:When 5 mM glutamate was applied at the peak of the depolarization inducedby hemoglobin (0.1 mM; 15 mm), the glutamate response was suppressed (% of thecontrol response: 53.62 ± 9.51; n = 8; p < 0.05) (Fig. hA & B). When 5 mMglutamate was applied at 5, 30, and 60 minutes after the removal of hemoglobin, theglutamate-mediated response was 83.81 ± 13.41 %, 82.22 ± 21.46 %, and 95.01 ±25.56% of the control response, respectively (n = 8; p > 0.05).115Figure 11. Effects of hemoglobin on the glutamate-induced depolarization.Depolarizations induced by 5 mM glutamate were evoked in normal ACSF and at thepeak of the hemoglobin-mediated depolarization. A) A histogram was constructedwith the average response of 8 different neurons. B) Raw traces recorded in aneuron whose resting membrane potential was -61 mV.Figure11140-An=8120-080-I*20-0-————————controlHb5post30’post60’postB2OmV-—5niin.GluGluGluGluGluControlHb5’post30’post60’ post117IBJ) Effects of Na-nitro-L-arginine on the Actions of Hemoglobin:18.7.1) Low Dose (100 1iM) of No-nitro-L-arginine:Superfusion of slices with 100 i.iM Nco-nitro-L-arginine for one hour caused ahyperpolarization of 3.20 ± 1.07 mV (n = 5; p <0.05). The NO synthase inhibitor alsocaused a statistically insignificant increase in the input resistance (change in the inputresistance as a % of control: 4.48 ± 8.91 % (n = 5; p > 0.05). Superfusion of hippocampalslices with 100 iM of No-nitro-L-arginine for 1 hour produced a suppression of the EPSP(% of the control slope: 56.67 ± 9.17; n = 5; p <0.05), the fast IPSP (% of the controlresponse: 76.79 ± 7.51; n = 5; p > 0.05), and the slow IPSP (% of the control response:62.11 ± 5.66; n = 5; p < 0.05) of CAl neurons at the end of the application (Fig. 12A & B;15 A, B, & C). The number of spikes evoked by rectangular depolarizing pulses wasfound to be increased at the end of the one hour application of No-nitro-L-arginine (Fig.14A & B).When 0.1 mM hemoglobin (15 mm) was applied after an hour of superfusion with100 iM No-nitro-L-arginine, the EPSP (slope of EPSP in hemoglobin as a % of control:35.94 ± 9.10, n = 5; p <0.05), the fast IPSP (response in hemoglobin as a % of control:41.22 ± 15.40, n = 5; p < 0.05), and the slow IPSP (response in hemoglobin as a % ofcontrol: 34.07 ± 9.69, n = 5; p < 0.05) were depressed at the end of the hemoglobinapplication (Fig. 13A & B; 15D, F, & F). The EPSP (% of control EPSP slope: 65.91 ±15.08, n=5; p > 0.05), the fast IPSP (% of control response: 99.83 ± 17.16, n = 5; p>0.05), and the slow IPSP (% of control response: 88.03 ± 16.95, n = 5; p > 0.05)recovered from the hemoglobin-induced suppression at 30 minutes after the removal ofhemoglobin (Fig. I 3A & B; 1 5D, E, & F).118In addition to the suppression of the evoked synaptic responses, hemoglobin alsocaused a depolarization of 7.40 ± 1.57 mV (p < 0.05) and a 91.51 ± 18.59 % (p < 0.05)increase in the input resistance. An increase in the number of depolarization-induceddischarge of action potentials was observed during Nco-nitro-L-arginine and hemoglobinapplications (Fig. 14A, B, & C). The increase in the number of depolarization evokedaction potentials was still present at 30 minutes after the removal of hemoglobin (Fig.14D).119AmV50 insBniV200 insFigure 12. Effects of Nonitro-L-arginine on the evoked synaptic responses of rathippocampal CAl neurons. Nco-nitro-L-arginine (100 tM) was applied for I hour. Thesynaptic responses were recorded in response to 0.05 Hz stimulation of the stratumradiatum. A) The effects of Nw-nitro-L-arginine on the EPSP. The EPSP traces weretaken during control (a), and immediately before the end of the application of Nco-nitro-Larginine (b). B) The effects of Nco-nitro-L-arginine on both fast and slow IPSPs. Thesynaptic responses were recorded during control (a), immediately before the end of theapplication of Nco-nitro-L-arginine (b). The resting membrane potential of this neuron was -69 mV.ab5mV50ms120BFigure13.EffectsofhemoglobinontheevokedsynapticresponsesofrathippocampalCAlneuronsinthepresenceof100j.tMNo-nitro-L-arginine.Hemoglobin(0.1mM)wasappliedfor15minutes.Thesynapticresponseswererecordedinresponseto0.05Hzstimulationofthestratumradiatum.A)TheeffectsofhemoglobinontheEPSP.TheEPSPtracesweretakenduringcontrol(a),immediatelybeforetheendoftheapplicationofhemoglobin(b),andat30minutesaftertheremovalofhemoglobin(c).B)TheeffectsofhemoglobinonbothfastandslowIPSPs.Thesynapticresponseswererecordedduringcontrol(a), immediatelybeforetheendoftheapplicationof hemoglobin(b),andat30minutesaftertheremovalofhemoglobin(c).Therestingmembranepotentialofthisneuronwas-69mV.abC5mV200ms/a b121d10 mV200msFigure 14. The effect of hemoglobin on the excitability of rat hippocampal CAl neuronsin the presence of 100 pM No-nitro-L-arginine. The cell excitability was monitored by thenumber of spikes induced by injecting a 0.3 nA depolarizing rectangular pulse (200 ms).A depolarizing pulse was injected into the neurons during control (a), immediately beforethe end of the 1 hour application of hemoglobin (b), immediately before the end of the 15minutes application of hemoglobin (c), and 30 minutes after the application of hemoglobin(d). The resting membrane potential of this neuron was -64 mV.122Figure 15. Actions of hemoglobin (0.1 mM) and No-nitro-L-arginine (100 tM) on evokedsynaptic responses. A) Time course of the No-nitro-L-arginine-induced suppression ofthe EPSP. B) Time course of the NCD-nitro-L-arginine-induced suppression of the fastIPSP. C) Time course of the Nco-nitro-L-arginine-induced suppression of the slow IPSP.D) Time course of the suppression of intracellularly recorded EPSP by hemoglobin (0.1mM; 15 mm) in the presence of 100 iM No-nitro-L-arginine. E) Time course of thesuppression of the fast IPSP by hemoglobin in the presence of 100 i.tM Na-nitro-Larginine. F) Time course of the suppression of the slow IPSP by hemoglobin in thepresence of 100 jiM No-nitro-L-arginine. The synaptic responses were recorded inresponse to 0.05 Hz stimulation of the stratum radiatum. The depolarization caused byhemoglobin was compensated by a hyperpolarizing DC current so that synapticresponses could be recorded at the control membrane potential. The curves wereconstructed with the average response of five different neurons.cy,CNFigure15120100f100100180*8060*f60600040E4Q040*20o520n520no50I0-III.10i010203040506070800102030405060708001020304050607080Time(miii.)Time(miii.)Time(miii.)ABC120120120100100a1fff80Jj6060fff600IC04Qc3°*f*40f*___**20fl=520______n520RbRbn5Rb0III0III0010.203040506070010203040506070010203040506070Time(miii.)Time(miii.)Time(miii.)DEF12418.7.2) Hih Dose (500 uM) of Nco-nitro-L-ariinine:Superfusion of slices with 500 M Nco-nitro-L-arginine for one hour caused a6.33 ± 1.02 mV (n = 6; p < 0.05) hyperpolarization. The NO synthase inhibitor alsoincreased the input resistance by 36.73 ± 13.02 % (n = 6; p < 0.05). Superfusion ofhippocampal slices with 500 tM Nco-nitro-L-arginine for 1 hour produced a suppression ofthe EPSP (% of the control slope: 77.02 ± 10.03; n = 6), and an enhancement of the fastIPSP (% of the control response: 173.18 ± 33.18; n = 6) as well as the slow IPSP (% ofthe control response: 126.70 ± 14.90; n = 6) of CAl neurons at the end of the application(Fig. 16A & B; 19 A, B, & C). However, the No-nitro-L-arginine-induced changes in theEPSP, the fast IPSP, and the slow IPSP were statistically insignificant (p > 0.05). Thenumber of spikes evoked by rectangular depolarizing pulses was increased at the end ofthe one hour application of No-nitro-L-arginine (Fig. 1 8A & B).When 0.1 mM hemoglobin (15 mm) was applied after an hour of superfusion with500 jiM No-nitro-L-arginine, the EPSP (slope of EPSP in hemoglobin as a % of control:27.46 ± 10.06, n = 6; p < 0.05), the fast IPSP (response in hemoglobin as a % of control:53.60 ± 16.54, n = 6; p < 0.05), and the slow IPSP (response in hemoglobin as a % ofcontrol: 49.05 ± 13.00, n = 6; p <0.05) were depressed (Fig. 17A & B; 19D, E, & F). TheEPSP (% of control EPSP slope: 70.38 ± 1.05, n =6; p > 0.05), the fast IPSP (% of controlresponse: 94.81 ± 12.66, n = 6; p > 0.05), and the slow IPSP (% of control response:110.54±5.32, n = 6; p > 0.05) recovered from the hemoglobin-induced suppression at 30minutes after the removal of hemoglobin (Fig. 17A & B; I 9D, E, & F).Besides the changes in the evoked synaptic responses, hemoglobin also caused adepolarization of 11.00 ± 3.42 mV (p < 0.05) and a 72.13 ± 6.20 % (p <0.05) increase inthe input resistance. An increase in the number of depolarization-induced discharge of125action potentials was observed during and at 30 minutes after hemoglobin application(Fig. 18C & D).ABa b50 ins1265 mVa5 mV200 insFigure 16. Effects of No-nitroL-arginine (500 1iM) on the evoked synaptic responses ofrat hippocampal CAl neurons. No-nitro-L-arginine (500 1iM) was applied for 1 hour. Thesynaptic responses were recorded in response to 0.05 Hz stimulation of the stratumradiatum. A) The effects of N-nitro-L-arginine on the EPSP. The EPSP traces weretaken during control (a), and immediately before the end of the application of N—nitro-Larginine (b). B) The effects of Nco-nitro-L-arginine on both fast and slow IPSPs. Thesynaptic responses were recorded during control (a), immediately before the end of theapplication of No-nitro-L-arginine (b). The resting membrane potential of this neuron was -58 mV.b1275niV50msBabc200maFigure17.EffectsofhemoglobinontheevokedsynapticresponsesofrathippocampalCAlneuronsinthepresenceof500i.tMNo-nitro-L-arginine.Hemoglobin(0.1mM)wasappliedfor15minutes.Thesynapticresponseswererecordedinresponseto0.05Hzstimulationofthestratumradiatum.A)Theeffectsof hemoglobinontheEPSP.TheEPSPtracesweretakenduringcontrol(a),immediatelybeforetheendoftheapplicationofhemoglobin(b),andat30minutesafter theremovalofhemoglobin(C).B)TheeffectsofhemoglobinonbothfastandslowIPSPs.Thesynapticresponseswererecordedduringcontrol(a),immediatelybeforetheendoftheapplicationofhemoglobin(b),andat30minutesaftertheremovalofhemoglobin(c).Therestingmembranepotentialofthisneuronwas-58mV.128lOmV200 msFigure 18. The effect of hemoglobin on the excitability of rat hippocampal CAl neuronsin the presence of 500 1iM No-nitro-L-arginine. The cell excitability was monitored by thenumber of spikes induced by injecting a 0.1 nA depolarizing rectangular pulse (200 ms).A depolarizing pulse was injected into the neurons during control (a), immediately beforethe end of the 1 hour application of hemoglobin (b), immediately before the end of the 15minutes application of hemoglobin (c), and 30 minutes after the application of hemoglobin(d). The resting membrane potential of this neuron was -60 mV.a bC d129Figure 19. Actions of hemoglobin (0.1 mM) and Nw-nitro-L-arginine (500 .iM). A) Timecourse of the No-nitro-L-arginine-induced suppression of the EPSP. B) Time course ofthe Na-nitro-L-arginine-induced suppression of the fast IPSP. C) Time course of the Nconitro-L-arginine-induced suppression of the slow IPSP. D) Time course of thesuppression of intracellularly recorded EPSP by hemoglobin (0.1 mM; 15 mm) in thepresence of 500 iiM N-nitro-L-arginine. E) Time course of the suppression of the fastIPSP by hemoglobin in the presence of 500 iiM No-nitro-L-arginine. F) Time course ofthe suppression of the slow IPSP by hemoglobin in the presence of 500 jiM No-nitro-Larginine. The synaptic responses were recorded in response to 0.05 Hz stimulation of thestratum radiatum. The depolarization caused by hemoglobin was compensated by ahyperpolarizing DC current so that synaptic responses could be recorded at the controlmembrane potential. The curves were constructed with the average response of sixdifferent neurons.CFigure19160120,160I100fH‘JI140120f f{fffH}ffiltit80f120I6080e060404040n=620n=6n=6200IIII0-III0-II010203040506070800102030405060708001020304050607080Thne(mm.)Time(nun.)Time(mOn.)ABC120100.IQ.‘a80-ff fTITI_80TI0.I•60480606040T40_______*120n=620_______20___________n=6Hbn6HbHb0i-iI0•III-0II010203040506070010203040506070010203040606070Time(mOn.)Time(mm.)Time(mlii.)DEF13118.8) Iron and the Actions of Hemoglobin:18.8.1) Low Dose (0.4 mM) of Ferric Chloride:Superfusion of slices with 0.4 mM ferric chloride for 15 minutes produced asuppression of the EPSP (% of the control slope: 80.94 ± 4.62; n = 8), the fast IPSP(% of the control response: 90.16 ± 6.84; n = 8), and the slow IPSP (% of the controlresponse: 77.90 ± 6.01; n = 7) when measured at the end of the application (Fig. 20A& B; 24A, B, & C). However, the iron-induced changes in the evoked synapticresponses were statistically insignificant (p > 0.05). At 30 minutes after the removal ofiron, the EPSP (% of the control slope: 83.05 ± 10.76; n = 8), the fast IPSP (% of thecontrol response: 80.30 ± 10.24; n = 8), and the slow IPSP (% of the control response:78.77 ± 8.39; n = 7) were not significantly different (p > 0.05) from those recorded atthe end of the iron application (Fig. 20A & B; 24A, B, & C). The membrane potential,the input resistance, and the number of depolarization-induced discharge of actionpotentials, of the neurons were not changed during iron application (Fig. 21).5mV132Figure20.Effectsof0.4mMferricchlorideontheevokedsynapticresponsesofrathippocampalCAlneurons.Ferricchloride(0.4mM)wasappliedfor15minutes.Thesynapticresponseswererecordedinresponseto0.05Hzstimulationofthestratumradiatum.A)TheeffectsofferricchlorideontheEPSP.TheEPSPtracesweretakenduringcontrol(a),immediatelybeforetheendoftheapplicationofferricchloride(b),andat 30minutesaftertheremovalof ferricchloride(c).B)TheeffectsofferricchlorideonbothfastandslowIPSPs.Thesynapticresponseswererecordedduringcontrol(a), immediatelybeforetheendoftheapplicationofferricchloride(b),andat30minutesaftertheremovalofferricchloride(c).Therestingmembranepotentialofthisneuronwas-63mV.abBC5mV50msabC200ms133lOniV200insFigure21.Theeffectof0.4mMferricóhloñdeontheexcitabilityofrathippocampalCAlneurons.Thecellexcitabilitywasmonitoredbythenumberofspikesinducedbyinjectinga0.2nAdepolarizingrectangularpulse (200ms).Adepolarizingpulsewasinjectedintotheneuronsduringcontrol (a),immediatelybeforetheendof the15minutesapplicationof ferricchloride (b),and30minutesaftertheapplicationofferricchloride(C).Therestingmembranepotentialof thisneuronwas-63mV.13418.8.2) High Dose (2.0 mM) of Ferric Chloride:After superfusing slices with 2.0 mM of ferric chloride for 15 minutes, while theEPSP (% of the control slope: 117.55 ± 12.69; n = 5; p <0.05) was enhanced, the fastIPSP (% of the control response: 100.63 ± 16.10; n = 8; p > 0.05), and the slow IPSP(% of the control response: 90.57 ± 11.56; n = 7; p > 0.05) were not alteredsignificantly when measured at the end of the application (Fig. 24D, E, & F). At 30minutes after the removal of iron, the EPSP (% of the control slope: 92.21 ± 7.22; n =8; p > 0.05) recovered. The fast IPSP (% of the control response: 74.56 ± 9.54; n =8), and the slow IPSP (% of the control response: 78.33 ± 9.00; n = 7) recorded at 30minutes post-iron application were not significantly changed (p > 0.05) compared tothe measurements recorded at the end of the iron application (Fig. 22A & B; 24D, E, &F). Like the low dose of ferric chloride, the high dose of ferric chloride also did notchange the membrane potential. The number of depolarization-induced discharge ofaction potentials was marginally suppressed in the presence of 2.0 mM ferric chloride(Fig. 23). Even though the input resistance was not altered by 0.4 mM of ferricchloride, a decrease of 12.23 ± 1.52 % (p <0.05) in the input resistance was causedby 2.0 mM ferric chloride.135abC 50msBabc5mV200msFigure22.Effectsof2.0mMferricchlorideontheevokedsynapticresponsesofrathippocampalCAlneurons.Ferricchloride(2.0mM)wasappliedfor15minutes.Thesynapticresponseswererecordedinresponseto0.05Hzstimulationofthestratumradiatum.A)TheeffectsofferricchlorideontheEPSP.TheEPSPtracesweretakenduringcontrol(a),immediatelybeforetheendoftheapplicationofferricchloride(b),andat30minutesaftertheremovalofferricchloride(c).B)TheeffectsofferricchlorideonbothfastandslowIPSPs.Thesynapticresponseswererecordedduringcontrol(a),immediatelybeforetheendoftheapplicationofferricchloride(b),andat30minutesaftertheremovalofferricchloride(C).Therestingmembranepotentialofthisneuronwas-62mV.136aCl1OmV200msFigure23.Theeffectof2.0mMferricchlorideontheexcitabilityofrathippocampalCAlneurons.Thecellexcitabilitywasmonitoredbythenumberofspikesinducedbyinjecting a0.2nAdepolarizingrectangularpulse(200ms).Adepolarizingpulsewasinjectedintotheneuronsduringcontrol (a),immediatelybeforetheendofthe15minutesapplicationofferricchloride(b), and30minutesafter theapplicationofferricchloride(c).Therestingmembranepotential ofthisneuronwas-68mV.b137Figure24.Timecourseoftheeffectsofferricchlorideonevokedsynapticresponsesofrathippocampalneurons.A)Timecourseoftheeffectof0.4mMferricchloride(15mm)ontheEPSP(n=8).B)Timecourseoftheeffectof0.4mMferricchloride(15mm)onthefastIPSP(n=8).C)Timecourseoftheeffectof0.4mMferricchloride(15mm)ontheslowIPSP(n=7).D)Timecourseoftheeffectof2.0mMferricchloride(15mm)ontheEPSP(n=5).E)Timecourseoftheeffect2.0mMferricchloride(15mm)onthefastIPSP(n=5).F)Timecourseoftheeffectof2.0mMferricchloride(15mm)ontheslowIPSP(n=5).Thesynapticresponseswererecordedinresponseto0.05Hzstimulationofthestratumradiatum.00 -4Figure24100 80 60 40 20 080I0. ..60E n40 20n=8Iron0rIIII01020304050607080The(miniB140HWflffIronfl70102030405060708090Time(miniC100fr.160 400208Iron00102030405060708090Time(miii.)A1601401200 Co100 80n=5Iron20 00102030405060708090Time(miii.)D90a120100 80—60E ii40140120100 80 60n5Iron80900=520Iron010203040506070Time(miii.)0102030405050708090Time(mm.)EF13919) DISCUSSION:In hemorrhagic stroke and other cerebrovascular injuries, blood often accumulatesintracranially for a considerable period of time (Sornás et al., 1972). Slow hemolysis oferythrocytes with release of hemoglobin into the supernatant fluid has been reportedto occur after 2 days of in-vitro incubation of blood (Asano et al., 1980; Barrows et aI.,1955; Osaka, 1977; Miyaoka et al., 1976; Sonobe and Suzuki, 1978; Sasaki et aI.,1979; Okwuasaba, 1981; Duffetal., 1987). After intracranial bleeding or hemorrhagicstroke, erythrocytes, which can remain in the intracranial cavity for days, arehemolysed in a similar fashion as in in-vitro incubation (Barrows et al., 1955; Findlayet al., 1989). After 2 hours of subarachnoid hemorrhage, hemoglobin was releasedfrom erythrocytes (Barrows et al., 1955). Because hemoglobin can remain in thecerebrospinal fluid for weeks (Barrows et al., 1955), neurons may be exposed tohemoglobin. In fact, hemoglobin has been suggested to induce cerebral vasospasmassociated with subarachnoid hemorrhage (Weir, 1987; Osaka et al., 1980).Moreover, its iron content may be related to stroke- or head injury-induced epilepsy(Hammond et al., 1980). To date, little is known about the electrophysiological actions ofhemoglobin. Therefore, in the present investigation, we examined the actions ofhemoglobin on the membrane potential, the input resistance, the ability to dischargeaction potentials, and the evoked synaptic responses, of CAl pyramidal neurons in rathippocampal slices.In the present study, hemoglobin was found to induce a depolarization, to increasethe input resistance and the depolarization-induced discharge of action potentials, as wellas to suppress the EPSP and IPSPs of the CAl neurons.14019.1) Suppressions of the Evoked Synaptic Responses:The hemoglobin-induced suppression of the EPSP, which might attenuate thenetwork activity by decreasing inter-neuronal communications, did not seem to be causedby the depolarization evoked by the drug since current-clamping the membrane potentialto the pre-hemoglobin level did not abolish the suppression of the EPSP. Becausehemoglobin significantly suppressed the glutamate-evoked depolarization even after thehemoglobin-induced depolarization was compensated with a current clamp, it is possiblethat hemoglobin suppressed the EPSP by interfering with excitatory transmission. Thesuppression of the actions of glutamate may be due to blockade of the excitatory aminoacid receptors or the calcium influx at postsynaptic terminals. However, the suppressionof the EPSP may be due to a number of other reasons unrelated to an action at the aminoacid receptor sites as hemoglobin-induced suppression of the EPSP is larger than that ofthe glutamate-induced depolarization. The difference between the time taken for recoveryof the glutamate-induced depolarization and that of the EPSPs from hemoglobin-induceddepression supports the idea that the interference with the excitatory transmission is notthe sole cause of the suppression of the EPSP. An inhibition of the propagation of actionpotentials in the presynaptic axons could also be a cause of the EPSP suppression sinceEPSPs and IPSPs were both suppressed by hemoglobin. Hemoglobin (0.1 mM or 0.5mM) suppressed the presynaptic volley. While the suppression caused by 0.1 mMhemoglobin is minimal, that induced by 0.5 mM hemoglobin is significant. Theseobservations suggest that the suppression of the EPSP induced by 0.1 mM hemoglobinmay be partly attributed to an interference with the propagation of presynaptic actionpotentials.141In this study, the mechanisms which underlie the suppressions of both fast andslow IPSPs were not explored. These actions of hemoglobin may be due to asuppression of the GABA-ergic inhibition or GABA release. Hemoglobin may act at itsown receptors. If an interaction at the receptor level is ruled out, effects of blockingsecond messenger systems such as the inositol phosphate/Ca2system on the actions ofhemoglobin should be examined.19.2) Actions of Hemoglobin on the Membrane Potential, the Input Resistance,and the Depolarization-Induced Discharge ofAction Potentials:The depolarizing action of hemoglobin probably was not caused by activations ofeither NMDA or non-NMDA glutamate receptors and was not secondary to the release ofglutamate since this action of hemoglobin was insignificantly altered in the presence ofAPV nor CNQX but was present in a Ca2-free and TTX-containing medium. In addition,the hemoglobin-induced depolarization probably does not require a Ca2-infIux becausean exclusion of extracellular calcium ions did not suppress the depolarization. In thecalcium-free medium, the enhancement in the depolarizing action of hemoglobin whenapplied in the control medium is not entirely understood but may suggest that the agentmay actually enhance a calcium-activated potassium current. During the hemoglobin-induced depolarization, calcium-activated potassium channels may be activated bycalcium entering through the voltage-dependent calcium channels. As a consequence ofthe hyperpolarization caused by potassium entering through the calcium-activatedpotassium channels, the depolarizing action of hemoglobin is dampened in a calciumcontaining medium.Because the slow IPSP was suppressed by hemoglobin, it is possible that closuresof K channels may partially underlie the depolarization, the increase in the input142resistance, and the loss of spike frequency adaptation during depolarization caused byhemoglobin. In hippocampal pyramidal cells, activation of muscarinic receptor is known todecrease the leak K current, the Ca2-activ ted K current, the noninactivating voltage-dependent K current (IM ), and the transient voltage-dependent K current (IA).Application of acetylcholine and carbachol have been reported to strongly depolarizehippocampal pyramidal cells, to increase the input resistance, and to suppress the spikefrequency adaptation during depolarizing pulses (Bernado and Prince, 1982; Cole andNicoll, 1983; Cole and Nicoll, 1984a; Cole and Nicoll, 1984b). While a decrease in thevoltage-independent leak K current has been suggested to underlie the depolarizationcaused by muscarinic receptors activation, a blockade of the Ca2-activ ted K currentcould lead to a loss of spike frequency adaptation during a depolarizing pulse (Bernadoand Prince, 1982; Benson et al., 1988; Madison et al., 1987b). Because muscarinicreceptors activation and hemoglobin have similar actions on the membrane potential, theinput resistance, and the spike frequency adaptation during a depolarizing pulse, it ispossible that hemoglobin acts either on muscarinic receptors or blocks the same ionchannels via an unknown mechanism. The use of muscarinic antagonists will be useful indetermining if hemoglobin functions at the muscarinic receptor level. If hemoglobin doesnot interact with muscarinic receptors, effects of a non-specific blockade of K channels byintracellular Cs on the depolarization, the increase in input resistance, and the spikefrequency adaptation during depolarizing pulses mediated by hemoglobin should beexamined. It is expected that this nonspecific blockade of K channels will shed somelight on the relationship between closures of IC channels and the actions of hemoglobinon the membrane potential, the input resistance, and spike frequency adaptation during adepolarizing pulse. In the present study, however, it is unclear whether the depolarization143induced by hemoglobin is due to the increase in the input resistance as no attempt wasmade to correlate these two actions of hemoglobin.Besides the closures of potassium channels, closures of chloride channels couldalso be a contributing factor to the hemoglobin-induced increase in the input resistancesince the fast IPSP was suppressed by hemoglobin. However, this could not be the solecause of the hemoglobin-induced depolarization and increase in the input resistancesince the suppression of fast IPSP has a later onset than the depolarization and theincrease in input resistance caused by hemoglobin.19.3) Possible Involvement of Metabotropic Receptors in the Actions ofHemoglobin:In the mammalian central nervous system, trans-ACPD, a selective agonist formetabotropic glutamate receptors, has been suggested to cause depolarization ofthalamic (Hall et al., 1979) and spinal cord (McLennan et al., 1982; McLennan and Liu,1982) neurons, to induced membrane potential oscillations in neurons of rat dorsolateralseptal nucleus (Zheng and Gallagher, 1991), to decrease EPSPs in the striatum(Lovinger, 1991), and to induce calcium mobilization in cultured cerebellar neurons (Irvinget al., 1990). In hippocampal CAl area, trans-ACPD has been reported to decreaseevoked EPSPs at the Schaffer collateral-CAl pyramidal cell synapse (Baskys andMalenka, 1991 a, b), to block synaptic inhibition (Desai and Conn, 1991), and to havedirect excitatory effects on pyramidal cells (Desai and Conn, 1991; Stratton et al., 1989).The direct excitatory effects include pyramidal cell depolarization accompanied by anincrease in input resistance, blockade of spike frequency adaptation, and inhibition of aslow AHP that follows a burst of action potentials (Desai and Conn, 1991; Stratton et al.,1441989). Not only are these effects observed in CAl, but also in CA3 (Charpak et al.,1990).Even though actions of moderate concentrations of trans-AC PD are often assumedto be mediated by the activation of the phosphoinositide hydrolysis-linked glutamatereceptor, recent cloning of multiple metabotropic glutamate receptor subtypes indicatesthat some of the actions of trans-ACPD may not be mediated via the activation of thephosphoinositide hydrolysis-linked glutamate receptor (Tanabe et al., 1992). In area CAlof the hippocampus, the effects of trans-ACPD have been suggested to be mediated by ametabotropic glutamate receptor which is not phosphoinositide hydrolysis-linked becauseAP3, which effectively inhibits trans-ACPD-induced phosphoinositide hydrolysis, does notblock these effects (Desai et al., 1992).Activation of metabotropic receptors has been reported to block a voltage-sensitivepotassium current which is known as an inward rectifier (Nicoll et al., 1990) as well as anon-inactivating and voltage-dependent potassium current (IM) (Brown, 1990). While theblockade of the inward rectifier could increase in the input resistance (Nicoll et al., 1990),the inhibition of ‘M may contribute to the membrane depolarization as well as reducedaccommodation. In addition to potassium conductances, activation of metabotropicreceptors also depresses calcium currents (Lester and Jahr, 1990). This action ofmetabotropic receptors can lead to a decrease in calcium influx at the presynapticterminal and therefore a decrease in neurotransmitter release (Lester and Jahr, 1990).Indeed, a presynaptic rather than postsynaptic mechanism has been suggested tounderlie the suppressions of glutamate and GABA-mediated synaptic potentials in striatalneurons (Calabresi et al., 1992) and suppressions of evoked field potentials in the CAlSchaffer collateral pathway (Baskys and Malenka, 1991a, b) mediated by metabotropic145receptors. For this reason, it is possible that suppressions of both EPSPs and IPSPsreported by Desai et al. (1992) are also mediated through a decrease in calcium influx atthe presynaptic terminal.In comparison to the effects of hemoglobin, the actions of trans-ACPD except theblockade of AHP are almost identical to those of hemoglobin. For this reason, theelectrophysiological actions of hemoglobin may be caused by activation of a subtype ofmetabotropic receptors which is not linked to phosphoinositide hydrolysis or activation ofthe same second messenger system as this metabotropic receptor. However, the linkbetween activation of this metabotropic receptor subtype and the reported actions is yet tobe determined because no specific antagonist is available for this subtype of metabotropicreceptors. Moreover, it appears that the depolarization induced by hemoglobin is not dueto a presynatic action as it is observed in a Ca2-free and TTX-containing medium; and thedepolarization induced by applied glutamate was suppressed by hemoglobin presumablythrough a postsynaptic mechanism of action.194) Implications of the Actions of Hemoglobin:When the GABA-mediated inhibitions are depressed, the non-NMDA receptor-mediated EPSP is prolonged (Collingridge and Lester, 1989). Therefore, NMDAreceptors will be activated as a result of the prolonged depolarization (Collingridge andLester, 1989). Because the NMDA receptor-mediated EPSP has a long duration andincreases in size with membrane depolarization, it may promote repetitive firing andtherefore contributing to abnormal activity at an epileptic focus (Collingridge and Lester,1989). Moreover, agents that suppress GABA-ergic IPSPs can induce epileptiformactivity. For these reasons, the hemoglobin-induced suppressions of both fast and slowIPSPs may lead to an increase in the excitability of CAl neurons.146Even though the underlying mechanisms of the depolarizing action of hemoglobinare not clear, it may have important implications. The hemoglobin-induced depolarizationcould open voltage-dependent calcium channels and therefore leading to neuronaldamage as a result of excessive influx of calcium into neurons. In addition, thisdepolarization may remove voltage-dependent Mg2 blockade of NMDA channels andthus causing excitotoxicity by an excessive influx of sodium and calcium ions throughNMDA channels (Choi, 1985 and 1987; Choi et al., 1988; Garthwaite and Garthwaite,1986; Garthwaite et al., 1986). In order to determine whether excessive calcium influxoccurs during hemoglobin-induced depolarization, Fura-2 dye can be used to trace themobilization of calcium during the hemoglobin-induced depolarization. If excessivecalcium influx actually occurs, the controversial role of NO in excitotoxicity may beexplained since hemoglobin may play a protective role by chelating NO and an offensiverole by increasing calcium entry. The net effect of hemoglobin will then rely on thebalance between the NO scavenging property and the depolarizing action of hemoglobin.Similar to the depolarizing action of hemoglobin, the hemoglobin-induced increasein the input resistance may also cause an increase in the excitability of neurons since anincrease in the input resistance can lead to an increase in the depolarization-induceddischarge of action potentials which may be epileptogenic. The paroxysmaldepolarization shifts observed after the application of hemoglobin during extracellularrecordings suggest that neurons are prone to seizures after exposure to hemoglobin.19.5) Nitric Oxide (NO) Scavenging Property of Hemoglobin:Since 0.1 mM hemoglobin has been suggested to block LTP and excitotoxicity byscavenging NO (Schuman and Madison, 1991), it is possible that the actions ofhemoglobin on the CAl hippocampal neurons are also due to chelation of NO. In the147current investigation, however, neither 100 riM, an effective concentration in blocking LTP(Schuman and Madison, 1991), nor 500 1iM Nw-nitro-L-arginine was capable of removingthe hemoglobin-induced suppression of the evoked synaptic potentials. Thedepolarization, the increase in the input resistance, and the increase in the depolarizationinduced discharge of action potentials caused by hemoglobin in the presence of the NOsynthase inhibitor (100 1iM or 500 jiM) indicate that the reported actions of hemoglobinare mostly independent of its NO scavenging property.19.6) The Iron Component of Hemoglobin:In traumatic brain injuries, seizures as well as deposition of iron derived fromhemoglobin in neurons have been reported (Caveness, 1963; Reid et al., 1979; Willmoreet al., 1978a; Willmore et al., 1978b). Since each molecule of hemoglobin contains 4ferrous ions, it is possible that iron accumulates near the neurons and glia afterhemoglobin is broken down intracranially. Even though hemoglobin is normallymetabolized in the liver, spleen, and kidney, it is possible that under physiologicalconditions hemoglobin is broken down as a result of the ischemia-induced change in pHof the intracranial cavity. For these reasons, it is important to examine the actions of ironon hippocampal CAl neurons. The effects of iron on hippocampal CAl neurons werecompared to those of hemoglobin. The statistically insignificant changes in the evokedsynaptic responses caused by 0.4 mM or 2.0 mM of ferric chloride suggest that theactions of hemoglobin are not due to its iron content. Similarly, the depolarization, theincrease in the input resistance, and the increase in the depolarization-induced dischargeof action potentials caused by hemoglobin are unlikely to be mediated by its iron contentbecause these parameters were not altered significantly by neither 0.4 mM nor 2.0 mMferric chloride. However, ferrous ion may have different actions on CAl neurons and their148actions should be determined before entirely ruling out the involvement of iron in theactions of hemoglobin.19J) Future Studies Required for Determining the Role of Hemoglobinin Neurological Deficits Induced by Cerebrovascular Injuries:In the present investigation, superfusion solution which contains hemoglobin wasoxygenated with carbogen (95% 02 5% C02). However, in hemorrhagic stroke and incerebrovascular injuries, oxygen supply to neurons could be compromised and thereforeleading to hypoxia. For this reason, future experiments which examine the actions ofhemoglobin in ischemia are necessary for further understanding of hemoglobin’s role inhemorrhagic stroke and head injuries mediated neurological disorders. It is known in theliterature that ischemia induces an anoxic depolarization followed by a persistentdepolarization (Garaschuk et al., 1993; Rader and Lanthorn, 1989). While the anoxicdepolarization is seen during ischemia, the persistent depolarization is present during theearly stage of reperfusion. In order to examine the actions of hemoglobin in ischemia,hemoglobin should be applied during ischemia. but not in reperfusion, during reperfusionbut not in ischemia, and during both ischemia and reperfusion so that the actions ofhemoglobin could be isolated from those induced by ischemia.In addition to ischemia, the actions of heme and globulin on CAl hippocampalneurons should also be examined. These experiments will shed some light on themechanisms of action of hemoglobin on hippocampal CAl neurons.14920) CONCLUSION:Although hemoglobin has been suggested to block LTP (Böhme et al., 1991; Haleyet al., 1992; Musleh et al., 1993; O’Dell eta; 1991; Schuman and Madison, 1991) and tosuppress excitotoxicity (lzumi et al., 1992) in literature, the direct electrophysiologicaleffects of hemoglobin still have not been directly examined. In this investigation, theeffects of hemoglobin on rat hippocampal CAl neurons are shown to be broader thanwhat have been reported in literature. Even though chelation of NO by hemoglobin hasbeen suggested as the mechanism of LTP and excitotoxicity suppressions in literature, itis not involved in the electrophysiological actions of hemoglobin reported in thisinvestigation. The iron content of hemoglobin also does not seem to be a contributingfactor for the actions of hemoglobin. While the suppression of the EPSP could be partlyattributed to the suppression of the actions of glutamate and the presynaptic volley, themechanisms for suppressions of both fast and slow IPSP5 are still unknown.Although the underlying mechanisms of the actions of hemoglobin are still notclear, effects such as the depolarization and the increase in the input resistance haveimportant implications. While the hemoglobin-induced depolarization, which isindependent of extracellular calcium and does not interact with the excitatory amino acidreceptors, may cause neuronal death by excessive calcium influx through voltagedependent calcium or NMDA channels, the increase in the input resistance may increasethe excitability of neurons.Effects reported in this investigation such as the change in the membranepotential, the increase in the input resistance, the increase in the depolarization-induceddischarge of action potentials, and suppressions of the evoked synaptic responses, ofCAl hippocampal neurons may play a role in modulating LTP and excitotoxicity. More150importantly, these actions of hemoglobin may contribute to the neurological deficitsassociated with cerebrovascular injuries and hemorrhagic stroke. 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