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: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 459267427

Neuropeptide signaling modulates neuronal activities and can define internal states. It is predominant in the central nervous system, but also used for communication between brain and periphery, as well as within the body and the enteric nervous system. Neuropeptides have also been attributed functions in development and in neuroprotection, including roles for tachykinin / substance P and pituitary adenylate cyclase-activating polypeptide (PACAP), in amyotrophic lateral sclerosis (ALS) and spinobulbar muscular atrophy (SBMA). Studying the mechanistic aspects of the involvement of neuropeptides in the development of such in motor neurons diseases is difficult in mammals, but could be more easily done in a simpler vertebrate. In the previous funding period, we established optogenetic tools and analysis methods for neuromuscular junction (NMJ) function in larval zebrafish. We generated knockout mutants of tachykinin (tac1), as well as the neuropeptide precursor processing enzyme carboxypeptidase E (cpe), which are both expressed in cholinergic neurons in the spinal cord. Optogenetic stimulation using the photoactivated adenylyl cyclase bPAC induced locomotion behavior, and these behaviors were unexpectedly more pronounced in both tac1 and cpe mutants. We assessed NMJ signaling by recording evoked postsynaptic currents (EPSCs) in muscle. bPAC activation induced an increase in the EPSC rate, however, no major phenotype could be detected in the mutants, apart from some imbalance in very large and very small EPSC amplitudes. Thus, we also analyzed postsynaptic acetylcholine receptors and found that these were increased in the mutants. We suggest that a presynaptic imbalance in neurotransmitter output is compensated by postsynaptic upregulation of nAChRs. In the next funding period, we want to analyze the mechanism of how nAChRs were upregulated and the role of additional neuropeptide related mutants in NMJ function. Specifically, we generated mutants in adcyap1b (PACAP2) and capsb, encoding CAPS, the major release factor for neuropeptide containing dense core vesicles. These studies will require implementation of an optogenetic neuropeptide release reporter. Also, we will assess whether neuropeptide malfunction could cause developmental defects, that might be causative for the compensatory nAChR upregulation, and we will probe presynaptic use ultrastructure. Last, we will analyze the expression pattern, and possibly knockdowns, of the receptors for tac1 and adcyap1b neuropeptides, in influencing ACh release, or its detection, to determine the site of action of the peptides. Our work will shed light on the involvement of neuropeptides in motor neuron function and might provide information that could guide future research in motor neuron disease also in humans.

• DFG Programme: Research Grants