Robert KS Wong - US grants

The objective of this proposal is to identify and examine the cellular processes involved in the synchronization of neuronal activity in the hippocampus. Synchronized discharge consisting of the simultaneous burst firing of a large population of neurons can be readily recorded in the hippocampal slice when the GABAergic synaptic inhibition is blocked by convulsive agents. We have previously identified two processes important in the initiation of synchronized discharge. These are (1) the intrinsic bursting capability of the dendrites and somata of hippocampal pyramidal cells and (2) the reciprocal excitatory synaptic connection between these cells. The immediate objectives of the proposed study are: (1) To analyze the kinetic and pharmacological properties of voltage-dependent ionic channels in the hippocampal pyramidal cell. We will begin by studying the non-inactivating inward current presumed to be important in sustaining burst firing. (2) To examine the anatomical and physiological properties of the recurrent synaptic network within a population of synchronized neurons. We will carry out the study with two experimental approaches. This includes the hippocampal slice preparation and our recently developed dissociated cell preparation. Our preliminary data show that viable, dissociated pyramidal cells can be obtained from adult guinea pigs. In addition we have demonstrated that voltage-clamp analysis of macroscopic membrane currents and patch-clamp approach for single-channel current studies can be carried out using dissociated cells. These technical advances have made it possible to begin a quantitative analysis on the membrane and receptor properties of hippocampal cells. We will use these data to coroborate those derived from the slice preparation to provide a better understanding of the synchronization process. Since the convulsant-induced synchronized discharge that we record in the hippocampus slice can be compared directly to the interictal spikes recorded by electroencephalograph in epilepsy, the results of our study may also contribute to our understanding of the initiation and control of interictal spikes in the diseased brain.

The overall objective of the proposed studies is to understand the activities generated by the large neuronal network in the hippocampus. Our studies address the long-standing question in cortical neurophysiology and EEG concerning how a population of interconnected neurons can general rhythmical extracellular waves. Such activities, arising from synchronized oscillations of neuronal populations, are observed in vivo (theta rhythm, epilepsy) and in vitro. Our experiments have focused on defining functional parameters, including cell excitability and synaptic strengths, that determine the amplitude and frequency of rhythmic activities. Recent results have allowed us to hypothesize the role of synaptic inhibition in synchronized population discharges. The proposed studies will characterize the physiology and pharmacology of the inhibitory circuit in the CA3 region of the hippocampus. The circuit primarily involves local GABAergic neurons. In vitro experiments will be carried out to address three major issues. These are: (1) The mode of synaptic activation of local inhibitory neurons. (2) Physiology and morphology of inhibitory neurons, and (3) properties of postsynaptic GABA receptors and ipsp's produced by inhibitory neurons. Our recent studies showed that inhibitory function was suppressed following tetanic stimulation leading to population oscillation in the CA3 region; the proposed experiments will allow us to identify potential modifiable sites within the inhibitory circuit. Experiments will be carried out using simultaneous intracellular recordings in the CA3 region of the hippocampal slice and patch-clamp recordings of dissociated adult hippocampal cells. Using the slice preparation, inhibitory neurons will be identified directly by their action on postsynaptic cells and they will be marked by intracellular fluorescent dye to allow correlation of physiology and morphology. Patch-clamping of dissociated hippocampal cells will be used primarily to examine postsynaptic receptors activated by GABA, the major inhibitory transmitter in the hippocampus. The generator mechanism of the synchronized oscillations we examine in the hippocampus is directly relevant to that underlying interictal discharges recorded n some simple forms of epilepsy. Thus the proposed experiments will provide information on both normal and abnormal operations of the hippocampus.

The overall objective of the proposed research is to contribute to our understanding of how intracellular Ca2+ regulates the excitability of hippocampal cells. Our interest in this issue is prompted by three recent developments. First, results were obtained showing that NMDA or glutamate, a presumed excitatory neurotransmitters in the hippocampus, caused sustained, significant increases of intracellular Ca2+ in isolated adult hippocampal cells. Secondly, it was discovered that the GABAA receptors in the hippocampal cells can no longer be activated by the ligand when intracellular Ca2+ increased beyond 5x10-6M. This latter finding is particularly interesting since previous data show that a blockade of inhibition mediated by GABAA receptors can led to epileptiform discharge in the hippocampus. Thirdly, we have succeeded in developing a recording system which allowed the changing of the intracellular contents during whole cell recording using isolated hippocampal cells. This technical advancement enables one to directly address questions regarding intracellular function of Ca2+. Proposed experiments will be carried out using acutely dissociated cells from the hippocampus of adult guinea-pigs. The specific objectives are: (1) to examine the role of intracellular Ca2+ in controlling the resting potential of the neurons. Experiments will characterize ionic currents activated by increases in Ca2+ when cells are voltage-clamped at their resting potential. The ionic basis and pharmacology of the currents will be studied. (2) To define the modulatory role of intracellular Ca2+ on voltage(V)-gated Ca2+ and K+ currents and (3) To compare the actions of elevated intracellular Ca2+ resulting from intracellular perfusion with that caused by entry through V- or ligand- gated channels. The study thus directly evaluates the control of cellular and perhaps circuitry (via GABAA receptor modulation) excitation by intracellular Ca2+. The results will contribute to our understanding of the generation and control of normal and abnormal activities in the hippocampus.

The overall objective of the proposed studies is to understand the activities generated by the large neuronal network in the hippocampus. Our studies address the long-standing question in cortical neurophysiology and EEG concerning how a population of interconnected neurons can general rhythmical extracellular waves. Such activities, arising from synchronized oscillations of neuronal populations, are observed in vivo (theta rhythm, epilepsy) and in vitro. Our experiments have focused on defining functional parameters, including cell excitability and synaptic strengths, that determine the amplitude and frequency of rhythmic activities. Recent results have allowed us to hypothesize the role of synaptic inhibition in synchronized population discharges. The proposed studies will characterize the physiology and pharmacology of the inhibitory circuit in the CA3 region of the hippocampus. The circuit primarily involves local GABAergic neurons. In vitro experiments will be carried out to address three major issues. These are: (1) The mode of synaptic activation of local inhibitory neurons. (2) Physiology and morphology of inhibitory neurons, and (3) properties of postsynaptic GABA receptors and ipsp's produced by inhibitory neurons. Our recent studies showed that inhibitory function was suppressed following tetanic stimulation leading to population oscillation in the CA3 region; the proposed experiments will allow us to identify potential modifiable sites within the inhibitory circuit. Experiments will be carried out using simultaneous intracellular recordings in the CA3 region of the hippocampal slice and patch-clamp recordings of dissociated adult hippocampal cells. Using the slice preparation, inhibitory neurons will be identified directly by their action on postsynaptic cells and they will be marked by intracellular fluorescent dye to allow correlation of physiology and morphology. Patch-clamping of dissociated hippocampal cells will be used primarily to examine postsynaptic receptors activated by GABA, the major inhibitory transmitter in the hippocampus. The generator mechanism of the synchronized oscillations we examine in the hippocampus is directly relevant to that underlying interictal discharges recorded n some simple forms of epilepsy. Thus the proposed experiments will provide information on both normal and abnormal operations of the hippocampus.

The overall objective of the proposal is to understand the role of GABAergic interneurons in normal and abnormal signaling processes in the cortex. Our recent studies show that there are networks of GABAergic interneurons within the hippocampus and neocortex in which the neurons are linked by excitatory connections. We have found that two different processes are involved in this recurrent excitation among GABAergic interneurons. In the first, GABA, which has generally been considered an inhibitory transmitter, acts instead as an excitatory transmitter between interneurons, causing a depolarization in the postsynaptic interneuron. In the second, the excitatory interconnections are maintained without chemical synaptic transmission, possibly through electronic junctions. The proposed studies will 1) investigate why GABA is having a depolarizing effect, and specifically, the ionic basis of the GABA-mediated depolarizing current; 2) identify which CA1 interneurons are interconnected by the excitatory GABAergic synapses and the distribution and pharmacological properties of the hyperpolarizing and depolarizing GABAergic synapses onto these cells; and 3) identify the CA1 interneurons which communicate by non-chemical means and examine the possible role of electrotonic junctions in their interconnections. Experiments will be carried out using microelectrodes and whole-cell voltage-clamp recording in hippocampal slices and whole-cell voltage clamp from acutely-dissociated hippocampal neurons taken from mature guinea pigs. In addition, the morphology of electrophysiologically- characterized interneurons in the slice will be identified by introducing the marker neurobiotin into the cells through the intracellular recording electrode. Deficiencies of GABAergic inhibition in the cortex can result in epileptogenesis. The proposed studies will provide valuable information on two previously unknown powerful excitatory processes which interconnect GABAergic interneurons. These processes are considered powerful because they can produce rhythmic synchronized discharge of GABAergic interneurons independent of the glutamate-driven excitatory synaptic events. The proposed studies will contribute to the understanding of the role of GABAergic interneurons in the normal and abnormal signaling processes in the mammalian cortex.

DESCRIPTION (provided by applicant): Our data indicate that pharmacological stimulation of group I metabotropic glutamate receptors (group I mGluRs) elicits irreversible epileptiform activity in the hippocampal slices via stimulation of mRNA translation and de novo protein synthesis. Our recent results suggest that the group I mGluR-stimulated mRNA translation, that underlies epileptogenesis, is normally repressed by the Fragile X mental retardation protein (FMRP). By restricting the translation process, FMRP serves as a safe-guard against the induction of group I mGluR-mediated epileptiform discharges in normal subjects. Fragile X Syndrome (FXS), the most common hereditary form of mental retardation, is caused by a loss of FMRP function. In vivo and in vitro experiments using the FXS model mouse show that the propensity for epileptogenesis, presumably mediated by group I mGluRs, is significantly enhanced. Seizure discharges elicited in FXS model mice are potently suppressed by group I mGluR antagonists. The findings in the FXS mouse model mirror the clinical condition, where FXS patients have increased likelihood of epilepsy compared to the general population. The overall goals of the proposed research are (A) to elucidate the molecular and cellular signaling mechanisms underlying epileptogenesis in the FXS mouse preparation and (B) to identify conditions under which group I mGluR activation can cause epileptogenesis in normal, wild type preparations. Electrophysiological, pharmacological, and biochemical techniques will be used to address three specific aims: (1) To assess the role of group I mGluRs in the synaptic induction of epileptiform discharges in the FXS mouse preparation;(2) To characterize the synaptic and cellular plasticity, elicited by the inducing synaptic stimulation, that is necessary for the maintenance of epileptiform discharges in the FXS mouse preparation;and (3) To define the signaling mechanisms through which epileptiform discharges are elicited in the wild type preparation by group I mGluR stimulation. The relevance of the group I mGluR model of epileptogenesis to epilepsy in Fragile X syndrome will be further explored. Results from the proposed study will provide fundamental information on the role of group I mGluRs in epileptogenesis. Such information will be useful in the design of rational therapeutic approaches to combat epilepsy, particularly in patients with Fragile X syndrome. PUBLIC HEALTH RELEVANCE This study examines how glutamate, the most prominent excitatory neurotransmitter in the brain, can cause epilepsy both in the general population and, in particular, in patients with Fragile X syndrome - Fragile X is the most common hereditary form of mental retardation. The proposed experiments will identify abnormal responses of nerve cells in the brain to glutamate and how these abnormal responses can ultimately cause epilepsy. It is expected that the results of the study will contribute towards the design of more specific drugs to combat epilepsy and improve treatment of patients with Fragile X syndrome.