Reiko Fitzsimonds - US grants

Sodium-dependent high-affinity uptake of glutamate is thought to play a major role in the rapid termination of synaptic activity and the maintenance of very low extracellular concentrations of excitatory amino acids (EAA) in the central nervous system. However, few studies have directly or systematically addressed the role of high-affinity glutamate uptake in excitatory synaptic transmission. This may be due, in part, to the lack of selective and potent inhibitors of the sodium-dependent high- affinity glutamate transport system. The recently developed selective, highly potent uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (L- trans-PDC) will be used in the proposed experiments to test the hypothesis that the high-affinity glutamate transport system plays a major role in modulating excitatory synaptic transmission in cultured hippocampal neurons. The involvement of EAA neurotransmission in rapid information transfer, synaptic plasticity and neurotoxicity make the study of the modulation of normal excitatory synaptic transmission particularly relevant for understanding mechanisms underlying changes associated with neuropathologies such as Parkinson's and Alzheimer's diseases and brain aging. Electrophysiologic approaches will be used to elucidate the effects of pharmacological inhibition of the high-affinity glutamate transporter by L-trans-PDC on both spontaneous and evoked synaptic events in cultured hippocampal neurons. Miniature excitatory postsynaptic currents (mEPSCs) reflect the spontaneous quantal release of neurotransmitter at single presynaptic terminals. Whole-cell patch clamp recording methods allow these currents to be resolved with sufficiently high resolution to allow analysis of their frequencies, distribution of amplitudes and time courses. Examining these parameters of the mEPSCs will provide useful information of any changes in the sensitivity of the postsynaptic EAA receptors and/or changes in presynaptic release resulting from inhibition of high-affinity glutamate uptake. Evoked excitatory postsynaptic currents (EPSCs) may also be examined by recording from isolated pairs of monosynaptically coupled hippocampal neurons maintained in very low density cultures. Various presynaptic stimulus paradigms including single stimulation, paired-pulse stimulation and high-frequency tetanic stimulation will be used to determine the effects of the glutamate uptake inhibitor L-trans-PDC on NMDA and non-NMDA components of the EPSC. The hypothesis is that inhibition of high-affinity glutamate transport will result in an increase in the ambient concentration of glutamate in the extracellular space of the cultured hippocampal neurons; the experiments are designed to determine the effects of the elevated levels or prolonged exposure to glutamate on non-NMDA receptor desensitization, the time course and amplitude of the slower NMDA component of the EPSC , and the involvement of presynaptic metabotropic receptors.

Early neuron-neuron interactions play a crucial role in the localization of postsynaptic receptors and the development and plasticity of functional synaptic connections. Synapses of hippocampal neurons have several characteristics which add complexity to the understanding of the early events in synaptogenesis: (1) synaptic connections may be inhibitory, utilizing the transmitter gamma-amino-butyric acid (GABA), or excitatory, utilizing excitatory amino acids (EAA; primarily glutamate); (2) postsynaptically, at least two receptors subtypes are co- localized for both GABAergic and glutamatergic synapses. The proposed work will answer specific questions concerning cellular and molecular mechanisms of transmitter secretion and postsynaptic receptor localization during early synaptogenesis of hippocampal neurons in culture. Using excised patches of excitable membrane or adult dissociated hippocampal neurons as probes to detect spontaneous neurotransmitter release from growing hippocampal neurons in culture, experiments proposed in part one will characterize the phenotype, distribution, and time course of spontaneous and evoked neurotransmitter (GABA vs glutamate) release of hippocampal neurons in culture. Experiments proposed in part two are aimed at understanding the time course and possible mechanisms of localization/co-localization of postsynaptic receptors, either GABA or EAA receptors. In addition to using iontophoretic mapping techniques, immunocytochemistry and electron microscopy will be used to characterize receptor phenotype and clustering on dendritic, axonal and somatic compartments of developing neurons at different time points before and during synaptogenesis. The results of the proposed experiments will provide the basis for future studies aimed at understanding plasticity of developing synaptic connections of hippocampal neurons. The long-term goal of the proposed and future studies is to understand the time course and sequence of cellular and molecular events involved in central synapse formation and plasticity. An understanding of the formation of synaptic connections in growing hippocampal neurons will provide insights into the necessary components involved in synaptic organization and plasticity of the central nervous system, which are crucial for elucidating neural substrates of both normal and pathological states.

The development and plasticity of the nervous system involve activity- dependent modification of synaptic connections. We have recently demonstrated that long-term depression (LTD) induced by repetitive synaptic activity at one synapse is accompanied by extensive but selective propagation of the depression to other synapses within the network. The goal of this third year proposal is to further examine the spread of synaptic signals within a neural network. Experiments in Aim 1 are designed to extend the present studies to include synaptic modifications associated with the induction of long-term potentiation (LTP). Experiments in Aim 2 will determine whether the influences from multiple outputs/inputs of a neuron can be spatially and/or temporally integrated. To study the propagation of synaptic modification, we have taken advantage of a very low density primary culture system where groups of three or four hippocampal neurons (triplets or quadruplets) that are connected with one another but relatively isolated from other neurons in the culture can be routinely found. Primarily electrophysiological approaches, using multiple whole-cell recording from defined neural networks, will e used in the proposed studies. This research promises to yield novel information concerning the selective distribution of activity-induced synaptic modification within a simple biological neural network, which has broad implications for understanding the cellular mechanisms underlying the learning processes of the central nervous system.

DESCRIPTION: (Applicant's Abstract) The pre- and postsynaptic processes underlying long-term potentiation and depression (LTP and LTD) have been the focus of more than two decades of extensive research and debate. We recently demonstrated that induction of LTD between a pair of neurons within an isolated "triplet circuit" of hippocampal neurons in culture results in long-lasting changes in synaptic strength at non-activated synapses of the triplet circuit. The spread of LTD to neighboring synapses following induction of LTD at glutamatergic synapses was more extensive than following induction at GABAergic synapses. Two critical questions arise from these findings: (1) what is the cellular mechanism underlying the spread of plasticity to non-activated synapses, and (2) is "propagated plasticity" a different and additional form of activity-dependent modification to the extensively studied "induced plasticity"? The goal of this application is to describe and develop this larger picture of synaptic plasticity, to gain an understanding of the distributed effects of activity on the efficacy of synapses in a simplified network of neurons in vitro. Simultaneous patch-clamp recordings from two to four synaptically interconnected neurons in culture will allow examination of changes in efficacy of synaptic connections between the different neurons following induction of LTD and LTP. Specific Aim 1 is to extend our previous findings on the spread of LTD, to examine the distributed effects of the induction of LTP on non-activated synapses in the simple circuits of cultured hippocampal neurons. Specific Aim 2 will determine the cellular mechanism underlying the spread of synaptic plasticity. Specific Aim 3 will examine whether an individual neuron integrates induced and propagated plasticity from activity within the local circuit. An understanding of the fundamental principles governing the distributed activity-dependent modifications of synapses in local neural circuits will be vital to the understanding of the mechanisms of learning, memory, various aspects of mental health, and the pathogenesis of neurological and aging disorders such as epilepsy and Alzheimer's disease.

DESCRIPTION (provided by applicant): This revised research proposal for an NIMH Exploratory/Developmental Grant (R21) will explore the combined use of molecular and cell biological methods with electrophysiological and optical imaging techniques in studies aimed at understanding the cellular mechanisms underlying changes in synaptic efficacy. We propose to apply the use of new molecular tool towards examining the dynamic activity dependent redistribution and insertion of glutamatergic and GABAergic receptors to synapses during development and plasticity. DNA constructs encoding the ionotropic glutamate receptor subunit GluR1 or GABAAalpha 1 receptor subunits fused with the pH-sensitive mutants of enhanced green fluorescent protein ('pHluorins') have been generated, and expressed in cell lines and primary cultures of rat hippocampal neurons. The 'pHluorins' fused to the extracellular NH2-terminal region of the receptors will be examined as sensitive and specific indicators of postsynaptic vesicle fusion-mediated delivery of receptors to the synapse. These experiments designed to optically monitor receptor distributions on the cell surface in living neurons over time are critical for evaluating the hypothesis that redistribution and/ or insertion of postsynaptic ionotropic receptors to activated synapses is a major mechanism underlying changes in synaptic efficacy. Studies proposed are not limited solely to glutamatergic synapses, as we are also interested in whether parallel mechanisms of receptor redistribution play a role in plasticity of GABAergic synapses. The proposed work is a novel and innovative approach to studying the dynamics of synapse formation and synaptic plasticity in living microcircuits of hippocampal neurons in culture. An understanding of fundamental mechanisms underlying synaptic plasticity will provide critical insight to not only physiological processes involved in learning and memory, but also to the etiology of pathophysiological disorders involving the deterioration of cognitive function accompanying mental illness, aging and drug addiction.