Michelle Mynlieff, Ph.D. - US grants

9407579 Mynlieff ABSTRACT Regulation of neurotransmitter release into the synaptic cleft is a primary means of achieving synaptic plasticity in the central nervous system. Since influx of calcium through voltage-dependent channels is necessary for neurotransmitter release, modulation of the activity of these channels by neurotransmitters is likely to be a common mechanism of regulating synaptic plasticity. There is currently no consensus on the identity of the calcium channel controlling excitatory or inhibitory neurotransmitter release, thus raising the possibility that different synapses may use different channel types. Future studies in Dr. Mynlieff's laboratory will exploit the well-characterized circuitry of the hippocampus to investigate the identity of the calcium channels by neurotransmitters. The first specific aim is to develop the techniques necessary to dissociate inhibitory interneurons from the hippocampus suitable for patch clamp studies. The second specific aim is to pharmacologically characterize the calcium currents present in different types of inhibitory interneurons using whole cell voltage clamp recording techniques. A thorough knowledge of the calcium channel pharmacology in various presynaptic cells is necessary before any studies are performed on the identity of the calcium channel(s) controlling release of neurotransmitters using the hippocampal slice preparation A thorough characterization of the calcium currents is also necessary before performing any studies on neuromodulation of these channels which may lead to changes in synaptic function. Many different neurotransmitters are known to modulate synaptic function and also to modulate calcium currents but it is not clear that these two events are related. By combining experiments in isolated presynaptic cells where the calcium currents can be easily ident ified with experiments in the hippocampal slice preparation where specific synapses can be stimulated in isolation Dr. Mynlieff hopes to make the connection between modulation of calcium currents and modulation of neurotransmitter release stronger. Although the future plans in Dr. Mynlieff's laboratory includes investigating both excitatory and inhibitory synapses in the hippocampus, the experiments outlined in this proposal concentrate on the inhibitory neurons because much less is known about the inhibitory presynaptic cells than the excitatory presynaptic cells in the hippocampus. Understanding how neurotransmitters regulate specific synapses by modulation of distinct calcium channel types will provide insight into some of the mechanisms of synaptic plasticity.

DESCRIPTION: Regulation of neurotransmitter release into the synaptic cleft is a primary means of achieving synaptic plasticity in the central nervous system. Since influx of calcium through voltage-dependent channels is necessary for neurotransmitter release, modulation of the activity of these channels by neurotransmitters is likely to be a common mechanism of regulating synaptic strength. The projects in the PI's laboratory exploit the well-characterized circuitry of the hippocampus to investigate the function and identity of the calcium channels controlling release in specific inhibitory synapses, and also the modulation of these channels by neurotransmitters. The hippocampus is involved in both memory formation and epileptic seizures. The inhibitory circuitry of the hippocampus, in particular, is extremely important in the control of seizure formation. The experiments outlined in this proposal will provide a solid foundation for future studies on the nature of synaptic transmission. The first specific aim is to optimize the culturing and whole cell recording conditions for hippocampal inhibitory interneurons, in order to obtain comparable excitability in culture as is seen in age-matched hippocampal slices. Tissue will be obtained from animals of various ages and allowed to remain in culture for various times. These experiments are a prerequisite for all the future experiments featured in this proposal. The second specific aim is to determine the feasibility of identifying inhibitory interneurons in culture based on electrophysiological parameters using whole cell patch clamp recording in the current clamp mode. These studies will be performed in parallel with identical experiments in age-matched hippocampal slices. The results from these experiments will validate the use of cultured hippocampal cells for studies on interneuron behavior and function. The third specific aim is to provide the first thorough characterization of the voltage dependent calcium channels in the presynaptic cells of 3 different inhibitory synapses in the hippocampus using whole cell voltage clamp recording in dissociated cells. The data obtained in these experiments will provide a foundation for future studies on neurotransmitter release and modulation of calcium channels. The vertical cells of the stratum oriens/alveus, the basket cells in the stratum pyramidale, and stellate cells in the stratum lacunosum/moleculare will be studied. The vertical and basket cells mediate both feedforward and recurrent inhibition primarily by activation of GABAA receptors. The stellate cells mediate feedforward inhibition by activation of GABAB receptors. Future studies in the PI's laboratory will be aimed at understanding how neurotransmitters regulate specific synapses by modulation of distinct calcium channel types providing insight into some of the mechanisms of synaptic plasticity. In addition, these studies will provide a better foundation for pharmaceutical therapies in diseases such a epilepsy and neurodegeneration where the use of neuromodulators is being actively pursued. Moreover, this information will also provide insights into the cellular pathology of various human neuronal disorders in which synaptic function is altered.

DESCRIPTION: (from applicant's abstract) Modulation of the voltage-dependent calcium channels regulating release by neurotransmitters is likely to be a common mechanism of regulating synaptic plasticity. There is currently no consensus on the identity of the calcium channel controlling neurotransmitter release, thus raising the possibility that different synapses may use different channel types. This project exploits the well characterized circuitry of the hippocampus to investigate the identity of calcium channels controlling release in specific inhibitory synapses, and also the modulation of these channels by neurotransmitter. The inhibitory synapses between the CA1 pyramidal cells and the vertical cells of the stratum oriens/alveus, the basket cells in the stratum pyramidale, and the stellate cells in the stratum laciunosum/moleculare will be studied. The vertical and basket cells mediate both feedforward and recurrent inhibition primarily by activation of GABAA receptors. The stellate mediate feedforward inhibition by activation of GABAB receptors. The first specific aim is to provide thorough characterization of the voltage-dependent calcium channels in the presynaptic cells of each of these synapses, using whole cell voltage-clamp recording in dissociated cells. The data obtained in these experiments will provide a foundation for the subsequent studies in neurotransmitter release and modulation of calcium channels. The second specific aim is to determine which calcium channel type controls release in each of the synapses in question, by application of specific calcium channel antagonists during whole cell voltage clamp recording in the postsynaptic cell in hippocampal slice preparation. The final specific aim is to determine if a decrease in calcium influx can account for the decrease of inhibitory synaptic transmission produced by activation of presynaptic GABAB and mu opioid receptors. The effect of GABA and opiates on calcium currents will be studied using whole cell voltage clamp recording in dissociated vertical, basket and stellate interneurons and the involvement of specific G proteins in this process will be explored. Understanding how neurotransmitter regulate specific synapses by modulation of distinct calcium channels types will provide insight into some of the mechanisms of synaptic plasticity. In addition, these studies will provide a better foundation for pharmaceutical therapies in diseases such as epilepsy and neurodegeneration where the use of neuromodulators is being actively pursued. Moreover, this information will also provide insights into the cellular pathology of various neuronal disorders in which synaptic function is altered.

[unreadable] DESCRIPTION (provided by applicant): The long range goal of the laboratory is to enhance our understanding of how neurotransmitters modulate calcium channel activity and the functional consequences of this modulation. The main inhibitory neurotransmitter in the mammalian central nervous system, gamma-aminobutyric acid (GABA) is known to attenuate N-type calcium currents by activation of GABA-B receptors. A unique finding in our laboratory is that L-type current is facilitated in response to GABA-B receptor activation in the neonatal neurons of the rat hippocampus. Understanding the mechanisms and functional significance of this facilitation will have significant consequences for the treatment of diseases such as drug addiction, epilepsy, insomnia and anxiety that are routinely treated with compounds interacting with the GABAergic system. The central hypothesis to be tested is that GABA-B receptor mediated facilitation of L-type current in the superior region of the hippocampus is medfy the signal transduction mechanism of the facilitation of L-type calcium current by activation of GABAB receptors. Specific aim #2 is to determine the specific subcellular location of the L-type calcium channels facilitated by GABAB receptors. Specific aim #3 is to determine whether or not activation of GABAB receptors contributes to the increase in potassium coupled chloride transporter expression seen early in neonatal development. The experiments outlined in this proposal will use whole cell and single channel recording, ratiometric calcium imaging, immunocytochemistry and Western blot analysis of proteins to accomplish the aims of the proposal. The experiments outlined in this proposal will provide knowledge about the signal transduction mechanism underlying the facilitation of L-type current and will begin to investigate the functional significance of this effect by identifying the subcellular location as well as the role in up-regulation of chloride transporter expression in the first two postnatal weeks in the rat hippocampus. A better understanding of the GABA-B receptor function may allow more subtle manipulation of the GABAergic system allowing for better design of pharmaceuticals with fewer side effects. [unreadable] [unreadable]