The long-term objective of the proposed research is to study the role and properties of electrical synaptic transmission via gap junctions in the CNS, in particular in the auditory system. The experimental model involves identified mixed electrical and chemical, (glutamatergic) synapses between eighth nerve auditory primary afferents and the goldfish Mauthner (M-) cell. While most studies of gap junction function utilize exogenous expression systems, this preparation uniquely allows continuous monitoring and quantification of changes in junctional conductance in vivo. Both components of the synaptic response exhibit activity-dependent modifications on their strength that is mediated via activation of NMDA receptors. Paired intradendritic and single afferent recordings, molecular biology techniques, and immunocytochemistry, will be used to test specific hypotheses and mechanisms underlying modifications of electrical transmission induced by eighth nerve tetani, determinants of bi-directional communication and the identity of specific gap junction proteins.
Aim 1 explores the cellular and molecular mechanisms underlying activity-dependent modification (potentiation and depression) of gap junctional conductance. It is based on data suggesting that changes in electrical coupling at single terminals following brief tetani can be in the form of both depressions and potentiations. I will explore the roles of elevated levels of postsynaptic calcium/calmodulin-dependent kinase II (CamKIl), protein phosphatases and agents interfering with postsynaptic exofendocytosis on unitary and population synaptic responses.
Aim 2 is to investigate the possibIe role of somatostatin in activity-dependent plasticity of these junctions. This peptide is co-localized with glutamate at presynaptic terminals and preliminary data shows that its application enhances both components of the synaptic response. Since both somatostatin and glutamate are likely to be co-released during tetani, I propose to explore their possible functional interactions and underlying intracellular mechanisms.
Aim 3 concerns identification of the neuron-specific gap junction proteins at these connections. Sub-cellular distributions of antibodies specific to various connexins will be analyzed with immunocytochemistry, using confocal and freeze-fracture electron microscopy, and single cell RT-PCR of the coupled cells. The proposed research addresses the concept that intercellular coupling through gap junction channels is dynamic, based on its functional interaction with neighboring glutamatergic synapses and peptidergic transmission. These modulatory phenomena could constitute a widespread property of electrical synapses in general, relevant not only to normal brain function in structures such as the retina, inferior olive, and neocortex where both forms of transmission co-exist, but also to numerous health-related issues such as epilepsy.
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