J. Victor Nadler - US grants

Excitatory amino acids, such as glutamate and aspartate, are prominent neurotransmitters in the mammalian central nervous system. This project employs the hippocampal formation of the rat brain as a test system with which to investigate the mechanisms of excitatory amino acid-mediated transmission. Electrophysiological studies indicate that L-proline acts as an excitant on hippocampal pyramidal cells. In addition, proline-induced excitations exhibit an excitatory amino acid-like pharmacology and proline is released from rat hippocampal slices in a Ca2+-dependent manner. Thus proline, although lacking the second acidic group possessed by recognized amino acid excitants, may function not only as an endogenous excitant but possibly also as a transmitter or modulator in the hippocampal formation. To identify possible "prolinergic" pathways, biochemical markers for proline (Ca2+-dependent release, Na+-dependent high affinity uptake, synaptosomal content, synthetic enzymes) will be related to specific fiber tracts with use of lesion techniques. Parallel studies will compare the pharmacology of proline-induced excitation in the hippocampal slice to the pharmacology of synaptic transmission. Proline receptors on hippocampal synaptic membranes will be labeled with L-(3H)proline, characterized and localized autoradiographically. The proline binding assay will be employed to study receptor mechanisms and to screen analogues for receptor activity. N-Methyl-D-aspartate-sensitive and quisqualate-sensitive excitatory amino acid receptors will be labeled with L-(3H)-glutamate. The effect of NMDA receptor agonists on adenylate cyclase and polyphosphoinositide phosphodiesterase activities will be tested to determine whether the inhibition of glutamate binding to NMDA-sensitive sites by guanine nucleotides implies a guanine nucleotide-dependent link between receptor and enzyme. Possible alterations in excitatory amino acid receptor density or localization in the kindling model of temporal lobe epilepsy will be assessed autoradiographically. Findings from these studies will be relevant to the treatment of neurological conditions, such as epilepsy, which involve hyperactivity of the hippocampal formation, as well as to the etiology of seizures and mental retardation frequently associated with hyperprolinemia.

The objective of this proposal is to carry out a comprehensive research program focussed on the study of the cerebrovascular disease and its sequelae. The projects inlcude a correlative clinical and radiological study of patients at risk for stroke, a pharmacokinetic study of antiplatelet drugs, a study of capillary proliferation in ischemic brain, a study of dopamine receptors, and a study of catecholamine release from adrenal medullary cells. There are several projects studying neuronal plasticity in the hippocampus, one of noradrenergic neurons, one of adrenergic and cholinergic receptors, one correlating recovery of spatial behavior after septal lesions, one studying the sequelae of kainic acid injection and one studying the role of enhaphalins and other neurotransmitters in the development of seizures after brain injury. A final group of projects focusses on neuronal function of the senory cortex after injury to primary afferent pathways, on cell-cell interactions, and on the membrane biochemistry of cultured neurons. The projects reflect a number of experimental approaches to understanding stroke and continues to emphasize understanding the biological basis of recovery of function after stroke.

9318101 Nadler This is a request for shared instrumentation to image and quantitate ion levels and pH in physiological studies on living cells. The requested Noran Odyssey laser scanning con focal microscope will enhance the research of five members of the Department of Pharmacol ogy by allowing the use of novel and innovative experimental approaches. The Noran instru ment has been optimized for the imaging of living tissue in real time. It will be used to investigate the following issues: (1) effects of regulatory substances on stimulus evoked sodium and calcium accumulation in glutamate nerve terminals, (2) NMDA receptor plastici ty in kindling and ontogenesis, (3) role of axon sprouting in facilitating or inhibiting seizure discharge and propagation, (4) role of calcium transients in regulating the cell cycle, (5) role of calmodulin in regulating insulin secretion from pancreatic SYMBOL 98 \f "Symbol" cells, (6) mechanism by which excess calmodulin leads to cardiac hypertrophy, (7) effects of glutamate and GABAA recep tor activation on intracellular calcium and chloride levels in hippocampal neurons, (8) effects of growth stimuli on intracellular pH, (9) role of protein phosphatase activity in the regula tion of cellular calcium levels and DNA synthesis, (10) induction of the protein phosphatase inhibitor 1 gene by calcium and (11) initiation of seizures by spontaneous action potential firing in axon terminals. These studies are aimed at understanding a variety of signaling mechanisms fundamental to cellular physiology. They will also help explain changes in cellular physiology and cell cell communication that occur in response to excessive or inju rious stimuli. u ~ -H- ~CAL3118TMP U. ~CRD0F04TMP #U. ~CAL3527TMP b5 ~CRD142ATMP . ~CRD0F38TMP b5 ~MF0E37 TMP E: ~DOC085BTMP s9 ~CAL2701TMP T~; ~CRD216FTMP X~; ~CAL3639TMP y< 9318101 Nadler This is a request for shared instrumentation to image and quantitate ion levels and pH in physiological studi ( 0 & ( !` ! F ( 0 ( 3 Times New Roman Symbol & Arial 1 Courier 9 " h % % = abstract Deseree King, BIR Deseree King, BIR

Recurrent mossy fiber sprouting in the hippocampal dentate gyrus is a unique feature of temporal lobe epilepsy. The formation of new mossy fiber-granule cell connection rates circuitry that is presumably capable of supporting reverberating excitation, similar to the recurrent excitatory circuitry normally present in area CA3 of the hippocampus. By analogy with area CA3, the development of new recurrent circuitry in the epileptic brain may facilitate granule cell synchronization and diminish the normally high resistance of the dentate gyrus toward seizure propagation. Pilocarpine-treated rats, which become epileptic and also develop a consistently dense recurrent mossy fiber pathway, will be used to test this hypothesis. In area CA3, epileptiform activity is thought to be initiated by the synchronized firing of a small number of pyramidal cells. These "detonator" cells are considered to be more rapidly excited than other cells in the population. A minority of granule cells have unusually large recurrent mossy fiber EPSCs and a minority also develop a basal dendrite innervated by recurrent mossy fibers. The proposed studies will determine whether these two neuronal populations overlap. If they do, then granule cells with a basal dendrite would be candidates for the role of detonator cell in the dentate gyrus of epileptic brain. In area CA3, the effect of recurrent excitatory circuitry is normally suppressed by among other factors, GABA inhibition and inhibitory K+ conductance. The proposed studies will determine whether and how readily frequency facilitation, elevated extracellular K+, reduced GABA inhibition and block of inhibitory metabotropic glutamate receptors unmask recurrent mossy fiber pathway actually does enhance propagation of epileptiform discharge through the dentate gyrus. In addition, glutamate receptors expressed by the mossy fiber pathway will be evaluated for their involvement in granule cell epileptic activity. These studies will provide pharmacological tools to separate the effect of recurrent mossy fiber growth from that of other possible changes in the epileptic dentate gyrus, and they may also suggest novel approaches to pharmacotherapy.

Recurrent mossy fiber growth in the hippocampal dentate gyrus is a unique feature of temporal lobe epilepsy. The formation of new mossy fiber-granule cell connections greatly expands a monosynaptic recurrent excitatory circuit that is normally very limited. It has been proposed that the circuit contributes to epileptogenesis by diminishing the resistance of the dentate gyrus to seizure propagation. The objective of this project is to discover the conditions under which these synapses are most likely to facilitate seizure propagation and to identify synaptic properties that could serve as therapeutic targets. Little is currently known about the physiology and pharmacology of mossy fiber- granule cell synapses; the proposed project is intended to fill this gap. Pilocarpine-treated rats, which become epileptic and also develop a consistently dense recurrent mossy fiber pathway, will be used to investigate three aspects of synaptic function. First, the characteristics of and the conditions needed to provoke three forms of presynaptic plasticity identified at mossy fiber-CA3 pyramidal cell synapses will be determined for the recurrent mossy fiber pathway. These studies will define the patterns of granule cell activity that are most likely to drive strong monosynaptic feedback excitation. Because preliminary results suggest that pilocarpine-induced status epilepticus enhances the contribution of NMDA receptors to the synaptic response, another study will determine whether mossy fiber-granule cell synapse on the epileptic brain exhibit an NMDA-dependent form of long term potentiation. Second, the involvement of kainate and metabotropic glutamate receptors to the synaptic response will be analyzed. One goal of this study is to evaluate these receptors as potential therapeutic targets of drugs directed against the recurrent mossy fiber pathway. Finally, the ability of zinc released by mossy fiber stimulation to alter GABA/A, NMDA, AMPA and kainate receptor function in granule cells will be assessed with use of agonists uncaged by highly focused ultraviolet light. This study will determine whether recurrent mossy fibers might also influence seizures through modification of postsynaptic glutamate receptors.

Recurrent mossy fiber sprouting in the hippocampal dentate gyrus is a unique feature of temporal lobe epilepsy. The formation of new mossy fiber-granule cell synapses creates complex circuitry that is presumably capable of supporting reverberating excitation and thereby diminishing the normally high resistance of the dentate gyrus to seizure propagation. The overall objective of this project is to characterize properties of mossy fiber-granule cell synapses that regulate circuit function. Pilocarpine-treated rats, which become epileptic and also develop a consistently dense recurrent mossy fiber pathway, will be used to investigate three unusual features of recurrent mossy fiber circuitry about which little information is currently available: namely, hilar ectopic granule cells, expression of GABA and neuropeptide Y, and release of zinc. Pilocarpine- induced status epilepticus increases the production of new dentate granule cells, some of which are found in the hilus. Hilar ectopic granule cells possess several unique features that suggest they play a key role in triggering synchronous granule cell activity. These features include reciprocal connections with other granule cells, abnormal excitatory innervation, apparent paucity of inhibitory innervation, and spontaneous bursting in association with CA3 pyramidal cells. Whole cell patch clamp recording and electron microscopy will be used to characterize the innervation of these cells and test the hypothesis that differences between the innervation of hilar ectopic and normally-situated granule cells accounts in large part for their differences in excitability. GABA and NPY are inhibitory transmitters that are strongly expressed in mossy fibers after a seizure; NPY continues to be strongly expressed during the interictal period. Studies will test the hypothesis that these transmitters are released at mossy fiber-granule cell synapses and serve mainly to reduce the further release of glutamate. In this way, they are proposed to limit the ability of the recurrent mossy fiber pathway to enhance seizure propagation. Release of zinc from recurrent mossy fiber boutons has been proposed to increase granule cell excitability. However, recent data suggest that it may instead act as a brake on reverberating excitation by opposing the activation of postsynaptic NMDA receptors. To investigate this issue, the effects of zinc chelators will be tested on granule cell epileptiform activity that depends, at least in part, on NMDA receptor activation.

DESCRIPTION (provided by applicant): Aspartate is associated with the synaptic vesicles of certain excitatory hippocampal pathways and is released by hippocampal slices and synaptosomes in a calcium-dependent manner. Preliminary release studies and information available from other experimental approaches allowed construction of a neuropeptide-like working model for aspartate release and function that includes accumulation into a distinct vesicle population, vesicle fusion with the plasma membrane predominantly outside the active zone, and diffuse activation of NMDA receptors. Aspartate, rather than glutamate, may produce some of the global or extrasynaptic effects of NMDA receptor activation (apoptosis, regulation of neurogenesis, and regulation of axonal/dendritic growth). The proposed release studies will utilize rat hippocampal synaptosomes. A method will be developed to introduce polypeptides and proteins into the synaptosomes by an electroporation technique that does not disrupt glutamate/aspartate release significantly. Then, to discriminate among the possible mechanisms of aspartate release suggested by previous work, the electroporation method will be used to introduce complexin inhibitory peptide, NSF inhibitory peptide, botulinum toxin A light chain, and botulinum toxin B light chain into the synaptosomes. Whole cell patch clamp recordings in organotypic hippocampal slice cultures will test the hypothesis that aspartate release plays a role in excitatory synaptic transmission under certain conditions. These studies will exploit several apparent differences between aspartate and glutamate in their release mechanisms and receptor actions, including release of aspartate from the Schaffer collateral-commissural pathway but not from the hippocampal mossy fibers, increased aspartate under low glucose conditions, increased aspartate release and reduced glutamate release during repetitive activity, insensitivity of aspartate release to Clostridial toxins, lower sensitivity of aspartate release to a blocker of P/Q-type calcium channels, and agonist activity of aspartate upon NMDA but not upon AMPA receptors. The proposed studies will evaluate a potential role for aspartate as a signaling molecule in the brain. They require, in part, the development of new experimental approaches and involve some risk of failure. For these reasons, the Exploratory/Developmental Grant mechanism of support seems appropriate. This project will eventually shed light on developmental and neurodegenerative processes, including the pathophysiology and pathology associated with epileptic seizures and hypoglycemia.

This project concerns the role that establishment of recurrent excitatory circuitry in the dentate gyrus plays in temporal lobe epilepsy. Granule cell neurogenesis, aberrant granule cell migration, and mossy fiber sprouting create a reverberating network that can reduce the threshold for granule cell synchronization and potentially diminish the normally high resistance of the dentate gyrus to seizure propagation. Pilocarpine-treated rats, which become epileptic and also develop a consistently dense granule cell network, will be used to investigate the properties and potential pathophysiological role of hilar ectopic granule cells. Many of the new granule cells produced as a result of seizures migrate aberrantly into the dentate hilus. Most hilar ectopic granule cells, unlike other granule cells in either normal or epileptic brain, fire action potentials and/or cellular bursts spontaneously at resting membrane potential. Therefore these cells are hypothesized both to facilitate recruitment of the granule cell population into seizure activity and to enhance excitability in area CA3. Whole cell patch clamp recordings will characterize the innervation of hilar ectopic granule cells and assess the possibility that abundant perisomatic excitation and sparse synaptic inhibition contribute to their unusual excitability. We will also determine to what extent spontaneous firing/bursting can be attributed to enhanced T-type calcium current, expression of h current, reduced delayed rectifier potassium current and/or reduced BK- and SK-type calcium-dependent potassium current. Perforated patch recordings will test the possibility that an abnormal chloride or potassium gradient plays a role in cellular hyperexcitability. Additional electrophysiological studies will assess the temporal relationship between the firing of these cells and population bursts of entorhinal cortical neurons, normally-situated granule cells or CAS pyramidal cells and the possibility that these cells receive feedback excitation from CAS pyramidal cells. Pharmacotherapy of temporal lobe epilepsy usually fails to achieve long-term remission. This project will shed light on the pathophysiological role of an anatomical reorganization unique to this disorder and may uncover novel therapeutic targets. In particular, it may be advantageous to target mechanisms of hyperexcitability operative in hilar ectopic granule cells, but not in normal granule cells

This project concerns the role of ectopic dentate granule cells in temporal lobe epilepsy. The dentate gyrus is believed to act as a gatekeeper or filter to inhibit the propagation of synchronized discharge through the limbic circuit. In both temporal lobe epilepsy and the pilocarpine model of temporal lobe epilepsy, granule cell neurogenesis, aberrant granule cell migration, and mossy fiber sprouting create a reverberating network that can reduce the threshold for granule cell synchronization and potentially diminish the normally high resistance of the dentate gyrus to seizure propagation. Pilocarpine-treated rats will be used to investigate one aspect of the novel granule cell network: namely, the properties and potential pathophysiological role of hilar ectopic granule cells. Many of the new granule cells produced as a result of seizures migrate aberrantly into the dentate hilus. Many hilar ectopic granule cells, unlike ather granule cells in either normal or epileptic brain, burst spontaneously at resting membrane potential. Preliminary data suggest that these cells also have little spike frequency adaptation, a high ratio of excitatory to inhibitory innervation, a relatively low resting membrane potential, and high input resistance. Their cellular morphology predicts the existence of abundant pathways by which the hyperactivity of these cells can be transmitted to the rest of the granule cell population. We will determine to what extent the hyperexcitable properties of hilar ectopiC granule cells can be attributed to enhanced T -type calcium current, enhanced BK-type calcium-dependent potassium current, and/or reduced SK-type calcium-dependent potassium current. The potential role of hilar ectopic granule cells in epileptiform activity will be assessed by physically removing them from hippocampal slices, by correlating the number of these cells with population activity, and by pharmacological means. Neurons that generate a high-frequency burst of action potentials as their minimal response to threshold stimulation are considered of central importance to the generation and propagation of epileptiform activity. Because hilar ectopic granule cells meet this criterion, they may be critical to seizure propagation through the dentate gyrus in epileptic brain. Pharmacotherapy of temporal lobe epilepsy usually fails to achieve long-term remission. This project will shed light on the pathophysiological role of an anatomical reorganization unique to this disorder and may uncover novel therapeutic targets. In particular, it may be advantageous to target mechanisms of hyperexcitability operative in hilar ectopic granule cells, but not in normal granule cells. .