The development of a functional nervous system is critically dependent upon the ability of neurons to acquire an appropriate neurotransmitter identity. It is the release of neurotransmitter that constitutes the majority of neural communication in vertebrates and allows for the construction of complex networks modulated by an array of inhibitory and excitatory phenotypes. This is particularly true for the GABAergic and glutamatergic neurons, the most abundant inhibitory and excitatory neurotransmitter phenotypes in the vertebrate nervous system. While the function of specific transcription factors and signaling molecules involved in GABAergic and glutamatergic specification is well established, recent work has pointed to the role of activity-dependent mechanisms in this process. Thought to be mediated via specific frequencies of calcium transients, the mechanisms underlying this activity and the interplay of calcium activity with """"""""hard-wired"""""""" transcriptional programs remain poorly understood. Data from our laboratory and others suggest that several voltage-gated calcium channels (VGCCs) are expressed during the early stages of neural development and may play a role in neurotransmitter fate specification. Using Xenopus as the model system for both in vitro and in vivo analyses, we propose a series of experiments that addresses the role of voltage-gated calcium channels and tests the hypothesis that specific VGCCs mediate the calcium activity required for neurotransmitter phenotype specification. Using fluorescent in situ hybridization, the initial set of experiments will determine whether the alpha subunits of the voltage-gated calcium channels co-localize with markers for specific neurotransmitter phenotypes at the mRNA level. The second group of experiments will complement the co-expression studies by inactivating the alpha subunits of the voltage-gated calcium channels using a morpholino knock-down strategy to determine the effect on neurotransmitter phenotype specification.
The third aim will attempt to determine if specific patterns of calcium activity assayed by Fluo-4 imaging correlate with particular VGCC alpha subunits and specific neurotransmitter phenotypes. In the final set of experiments, VGCC alpha subunit expression will be manipulated in order to determine its effect on patterns of calcium activity and/or the subsequent neurotransmitter phenotype acquisition. Regardless of whether the hypothesis is fully supported, the proposed experiments will likely lead to significant new findings regarding the role of VGCCs in early neural development. In so doing, the project will engage an eager cadre of undergraduate students in all aspects of the research process early in their careers and prepare these young scientists for productive research careers in graduate school and beyond.
An understanding of how neurons acquire and express a specific neurotransmitter identity and the role of voltage-gated calcium channels in this process is relevant to human health on several levels. Unraveling the mechanisms regulating neurotransmitter fate will provide the knowledge base for the coaxing the directed differentiation and transplantation of stem cells in an effort to replace or repair damaged neurons of a specific phenotype. In addition, the focus of this proposal is on the role of voltage-gated calcium channels which have been widely implicated in a profound range of neurological (and non-neurological) human diseases.
