Michael Barish - US grants

Transient potassium currents play an important role in shaping the electrical properties of hippocampal neurons; increases in excitability associated with their inhibition have been linked experimentally to enhancement of synaptic transmission and to epilepsy. Embryonic mouse hippocampal neurons express two transient potassium currents, an A-current and a D-current showing slower activation and inactivation, that can be separated in conventional whole-cell gigaohm-seal voltage clamp recordings based on voltage dependence and pharmacological sensitivities. In dissociated cell cultures relative levels of A- and D-current expression are dependent on the degree of contact between neurons and underlying glial cells, with greater contact favoring A-current at the expense of D-current. Freely diffusible factors do not appear to be involved. We propose here to further investigate the mechanism(s) by which glia influence transient potassium current expression, and the potential significance of transient potassium current variation in modulating hippocampal neuron excitability. Specifically, we propose to: a.Determine the type(s) of glia competent to induce the pattern of transient potassium current expression characteristic of neurons growing on mixed populations of glia. The patterns of potassium current expression induced by monolayers enriched in astrocytes, oligodendrocytes, microglia or fibroblasts (purity assayed with antibodies against characteristic markers) will be assayed electrophysiologically. b.Identify the modulatory signal and/or produce antisera with blocking activity. First, the ability of isolated glial membranes as compared to living cells to support the on-glia pattern of current expression will be determined. Second, panels of modulators and/or cell surface and extracellular matrix components will be screened for activity on potassium current expression. Third, if a modulator is not identified, blocking antisera will be produced using techniques for suppression of response to background antigens if necessary. c.Evaluate the potential functional significance of variation in transient potassium current expression using a computational model of hippocampal neuron excitability. An existing model will be modified to incorporate two transient potassium currents, and used to examine the consequences of variation in A- and D-current expression levels on the action potential wave form, repetitive firing, and consequent calcium entry and accumulation.

The individuality of neurons is determined by the particular complement of functional ion channels present in the neuronal membrane. Different neurons express varying numbers of channels selective for sodium, calcium, potassium and chloride ions, and as a consequence have specific patterns of responses to incoming excitatory and inhibitory stimuli. In order to understand how the individuality of neurons is established, a class of potassium currents whose activity influences both the waveform of single action potentials and the pattern of repetitive action potentials will be studied. Experiments will be performed on cultured embryonic mouse hippocampal neurons, a standard preparation for study of early neural development. It has been previously observed that the expression in neurons of two variant forms of transient potassium currents, termed A-current and D-current, is dependent on contact with non-neuronal glial cells. Since A- and D-currents are involved in neuronal electrogenesis, these observations raise the possibility that glial cells modulate neuronal activity by influencing channel expression. To understand how this process might occur, the precise molecular relationship between A- and D-current channels should be clarified. In the present experiments differences in gating and permeation behavior at the single channel level will be examined. Future experiments will focus on issues of gene expression and post-translational modification that may establish the differences between the ion channels underlying A- and D-currents.

This research is concerned with the dynamic behavior of intracellular Ca2+ in hippocampal neurons. In neurons Ca2+ acts as a second messenger coupling synaptic. activity and action potential generation to multiple intracellular processes. While intracellular mechanisms may shape the spatial and temporal patterns of Ca + signals and thus modulate Ca2+- dependent events, the fate of Ca2+ ions after they enter the cytoplasm is less well understood than the mechanisms by which they cross the cell membrane. We have studied intracellular mechanisms of Ca2+ release in cultured mouse hippocampal neurons. We localized Ca2+ release channels of endoplasmic reticulum in somas and dendrites using specific antibodies and confocal microscopy, and concluded that ryanodine receptor channels (mediating Ca2+-induced Ca2+ release) and inositol 1,4,5-trisphosphate (InsP3-) receptor channels (mediating InsP3-gated Ca2+ release) are not homogeneously distributed. We also used Ca2+-sensitive dyes to image and measure cytoplasmic Ca2+ concentrations ([Ca2+]) while inducing activation of ryanodine receptors (with caffeine) and InsP3 receptors (with muscarinic acetylcholine receptor agonists). We observed that acetylcholine induced [Ca2+] fluctuations in small dendritic regions, while caffeine induced more widely distributed and sustained increases in [Ca2+]. Our three Specific Aims further study of Ca2+ release in hippocampal neurons. 1. To examine the intracellular distributions of InsP3 and ryanodine receptors in comparison to the distributions of endoplasmic reticulum and cytoskeletal elements. These experiments will test hypotheses that Ca2+ release channels may be linked to the cytoskeleton and that.disruption of microtubules and/or microfilaments will distort or inhibit intracellular Ca2+ release. 2. To examine fluctuations in dendritic Ca2+ to determine if the distribute randomly or propagate in an orderly manner. We will image larger portions of dendritic fields at higher temporal resolutions than i our previous experiments to examine regions of elevated (Ca2+] after stimulation with caffeine or acetylcholine and after local dendritic activation with iontophoretically applied glutamate or acetylcholine, or synaptic activation. We will test the hypothesis that the dendritic cytoplasm may support propagating Ca2+ waves. 3. To examine dendrites for restricted domains of high (micromolar) Ca2+ levels associated with aggregations of ryanodine or InsP3 receptors. We will image the distribution of [Ca2+] during stimulation using fluorescent dyes with low Ca2+ affinity, and compare regions of high [Ca2+] to the locations of Ca2+ release channels. If they are in registration, these observations would suggest that one function of intracellular Ca2+ release in neurons is to effect local activation of Ca2+-dependent processes.