Kern, H. | bioRxiv Science

bioRxiv2025

Monoamine-induced diacylglycerol signaling rapidly accumulates Unc13 in nanoclusters for fast presynaptic potentiation

Neuromodulators control mood, arousal, and behavior by inducing synaptic plasticity via G-protein coupled receptors. Long-term potentiation of presynaptic neurotransmitter release requires structural changes, but how fast potentiation is achieved within minutes remains enigmatic. Using the Drosophila melanogaster neuromuscular junction, we show that on the timescale of one minute, octopamine, the invertebrate analog of nor-epinephrine, rapidly potentiates evoked neurotransmitter release by a G protein coupled pathway involving presynaptic OAMB receptors and phospholipase C. No changes of presynaptic calcium influx were seen, but confocal signals of the release factor Unc13A and the scaffolding protein Bruchpilot increased within one minute of octopamine treatment. On the same timescale, live, single-molecule imaging of endogenously tagged Unc13 revealed its instantly reduced motility and its increased concentration in synaptic nanoclusters with potentiation. Presynaptic knockdown of Unc13A fully blocked fast potentiation and removal of its N-terminal localization sequence delocalized the protein fragment to the cytosol, but it was rapidly recruited to the plasma membrane by DAG analog phorbol esters and octopamine, pointing to a role in C-terminal domains. Point mutation of endogenous Unc13 disrupting diacylglycerol-binding to its C1 domain blocked plasticity-induced nanoscopic enrichment and synaptic potentiation. The mutation increased basal neurotransmission but reduced Unc13 levels, revealing a gain of function and potential homeostatic compensation. The mutation also blocked phorbol ester-induced potentiation, decreased the calcium-sensitivity of neurotransmission and caused short-term synaptic depression. At the organismal level, the mutation reduced locomotion and survival while enhancing reproduction. Thus, the Unc13 C1 domain mediates acute subsynaptic compaction of Unc13 under monoamine-induced potentiation and influences short-term plasticity, locomotion, reproduction, and survival.

neuroscience↗

bioRxiv2024

Specific presynaptic functions require distinct Drosophila Cav2 splice isoforms

The multiplicity of neural circuits that accommodate the sheer infinite number of computations conducted by brains requires diverse synapse and neuron types. At many vertebrate synapses release probability and other aspects of presynaptic function are tuned by different combinations of Cav2.1, Cav2.2, and Cav2.3 channels. By contrast, most invertebrate genomes contain only one Cav2 gene. The one Drosophila Cav2 homolog, cacophony (cac), localizes to presynaptic active zones (AZs) to induce synaptic vesicle release. We hypothesize that Drosophila cac functional diversity is enhanced by two specific exon pairs that are mutually exclusively spliced and not conserved in vertebrates, one in the voltage sensor and one in the intracellular loop containing the binding site(s) for Ca{beta} and G-protein {beta}{gamma} subunits. We test our hypothesis by combining opto- and electrophysiological with neuroanatomical approaches at a fast glutamatergic model synapse, the Drosophila larval neuromuscular junction. We find that alternative splicing in the voltage sensor affects channel activation voltage and is imperative for normal synapse function. Only the isoform with the higher activation voltage localizes to the presynaptic AZ and mediates evoked release. Removal of these cac splice isoforms renders fast glutamatergic synapses non-functional. By contrast, alternative splicing at the other alternative exon that encodes the intracellular loop between the first and the second homologous repeats does not affect cac presynaptic AZ localization, but it tunes multiple aspects of presynaptic function. While expression of one exon yields normal transmission, expression of the other exon reduces channel number in the AZ and thus release probability. This also abolishes presynaptic homeostatic plasticity. Moreover, reduced channel number upon selective exon excision increases paired pulse ratios and the variability of synaptic depression during low frequency stimulation trains (1 and 10 Hz), and thus affects short term plasticity. Effects on short term plasticity can be rescued by increasing the external calcium concentration to match release probability to control. In sum, in Drosophila alternative splicing provides a mechanism to regulate different aspects of presynaptic functions with only one Cav2 gene.

neuroscience↗