Optogenetic Analysis of neuropeptidergic regulation of fast synaptic transmission at the zebrafish neuromuscular junction

• Applicant: Professor Dr. Alexander Gottschalk
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells ; Molecular and Cellular Neurology and Neuropathology
• Term: from 2021 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 459267427

Regulation of synaptic transmission is central to mechanisms of plasticity and homeostasis, and enables learning and adaptation to new conditions. Maintaining the ability to transmit synaptic signals is based on the availability of neurotransmitter-filled synaptic vesicles (SVs). It is achieved through the “SV cycle”, which involves recycling of SVs. Synaptic function can be modulated in the SV cycle by altering 1) the sensitivity of the presynaptic machinery to the increase in Calcium, 2) the rate at which SVs are recruited from the reserve pool of SVs, and 3) the SV neurotransmitter levels. In recent years, detailed studies have addressed the acute mechanisms of SV release and recycling using optogenetic stimulation and electrophysiological or electron microscopic examinations. My laboratory analyzed how the acute cAMP generation at the neuromuscular synapse of C. elegans modulates its function, and was able to show a previously unknown role of neuropeptides in the regulation of synaptic transmission, by influencing SV mobility and filling status. The synaptic transmission is thus regulated in two ways: 1) Depolarization and Calcium control the acute fusion (rate) of SVs, while 2) cAMP and peptidergic signals determine the SV content. Thus, in addition to the network activity of central pattern generators, motor neurons can also integrate neuromodulatory signals, e.g. in various systemic states. We want to investigate whether this dual control is also preserved in vertebrates, especially in the zebrafish. Danio rerio is unique as a vertebrate model system, as it enables these types of analyses in intact animals, in contrast to neuronal cell cultures, like in similar approaches in mice. We established transgenic lines that enable motor neuron photostimulation (channelrhodopsin, photoactivated adenylate cyclase), and can thus induce (and quantify) light-stimulated behavioral changes, as well as electrophysiological analyses of miniature endplate currents (mEPCs). We want to compare the transgenic lines we generated in wild type background, with knockdowns and knockouts that lack components required for neuropeptide biogenesis, or for their regulated release. Furthermore, we want to detect cAMP-induced neuropeptide release, using fluorescence-based reporters, and analyze a small number of candidate peptides that are expressed in zebrafish motor neurons. This is to show whether these neuropeptides enhance effectivity of the synaptic transmission, e.g. by increasing quantal size. We also established methods to analyze the synaptic ultrastructure of photostimulated synapses. Our work addresses whether the dual-mode control of synaptic transmission is conserved in zebrafish, and thus perhaps in vertebrates in general, and will enable future studies toward an understanding of diseases that are characterized by dysregulation of synaptic transmission.

• DFG Programme: Research Grants