Ethanol Regulates Presynaptic Activity and Sedation through Presynaptic Unc13 Proteins inDrosophila

Abstract

Ethanol has robust effects on presynaptic activity in many neurons, however, it is not yet clear how this drug acts within this compartment to change neural activity, nor the significance of this change on behavior and physiology in vivo. One possible presynaptic effector for ethanol is the Munc13-1 protein. Herein, we show that ethanol binding to the rat Munc13-1 C1 domain, at concentrations consistent with binge exposure, reduces diacylglycerol (DAG) binding. The inhibition of DAG binding is predicted to reduce the activity of Munc13-1 and presynaptic release. In Drosophila, we show that sedating concentrations of ethanol significantly reduce synaptic vesicle release in olfactory sensory neurons (OSNs), while having no significant impact on membrane depolarization and Ca2+ influx into the presynaptic compartment. These data indicate that ethanol targets the active zone in reducing synaptic vesicle exocytosis. Drosophila, haploinsufficent for the Munc13-1 ortholog Dunc13, are more resistant to the effect of ethanol on presynaptic inhibition. Genetically reducing the activity of Dunc13 through mutation or expression of RNAi transgenes also leads to a significant resistance to the sedative effects of ethanol. The neuronal expression of Munc13-1 in heterozygotes for a Dunc13 loss-of-function mutation can largely rescue the ethanol sedation resistance phenotype, indicating a conservation of function between Munc13-1 and Dunc13 in ethanol sedation. Hence, reducing Dunc13 activity leads to naïve physiological and behavioral resistance to sedating concentrations of ethanol. We propose that reducing Dunc13 activity, genetically or pharmacologically by ethanol binding to the C1 domain of Munc13-1/Dunc13, promotes a homeostatic response that leads to ethanol tolerance.

Studying alcohol use disorder using Drosophila melanogaster in the era of ‘Big Data’

“…This hypothesis has been supported by the findings in genomic survey studies that identify synaptic signaling and synaptic structural proteins play a role in alcohol behavior [43, 44, 49, 143]. Synaptic reorganization is necessary for the development of tolerance and conditioned place preference in the fly [31, 51, 141, 142, 144]. Subtle underlying changes in Draper function may make synaptic reorganization, which facilitates behavioral extinction, harder following initial cue-reward learning.…”

“…Draper codes for a cell surface receptor found on ensheathing glia in the adult brain, and is involved in the glial phagocytic response following injury or cellular damage [139]. Draper is also important for synapse reorganization (including in the mushroom body) [140], which is hypothesized to underlie tolerance and alcohol conditioned place preference [16, 106, 124, 141, 142]. This hypothesis has been supported by the findings in genomic survey studies that identify synaptic signaling and synaptic structural proteins play a role in alcohol behavior [43, 44, 49, 143].…”

“…Additionally, enrichment for olfactory genes is found when animals are exposed to alcohol vapor (either from a food source or air stream) [123]. A number of studies have demonstrated a role for olfactory genes in alcohol sensitivity and tolerance [49, 142, 150, 169]. Lush , one of the most characterized among these genes, encodes an odorant-binding protein known to bind to alcohols [169].…”

“…It is down-regulated in response to alcohol and is implicated in alcohol behavior [43, 49]). Additionally, recent work has shown that synaptic alterations driven by Dunc13 (homolog of mammalian Munc13 ) in olfactory receptor neurons of the fly play a role in mediating tolerance [142]. Alcohol vapor-induced changes in olfactory genes were addressed by Kong et al, who explained that alcohol is toxic to the antennae cilia causing degeneration of the cilia [49].…”

Identification of alcohol‐binding site(s) in proteins using diazirine‐based photoaffinity labeling and mass spectrometry

“…To aid to better understanding the principle and concept, the photolabeling data for the C1 domain of a presynaptic protein, Munc13‐1, are presented. Recent studies suggest that Munc13‐1 is an important target of alcohol …”