Ethanol Regulates Presynaptic Activity and Sedation through Presynaptic Unc13 Proteins in Drosophila

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.

Keywords: Drosophila; Munc13-1; ethanol; presynaptic; resistance; tolerance.

Figure 1.

Ethanol binding to the C1 domain inhibits DAG binding. A, Spectral emission after excitation at 290 nm reveals FRET between the Munc13-1 C1 domain and dansyl-DAG (red trace). The C1 domain emission peaks at ∼335 nm (cyan trace), while the dansyl-DAG emission peak is found at 500 nm (black trace). This FRET is disrupted by 50 mM ethanol (green trace). B, Spectral emission of the Munc13-1 E₅₈₂A mutation after excitation by 290-nm light is shown. C, The binding of dansyl-DAG as revealed by FRET emission at 500 nm is reduced at both 25 and 50 mM ethanol concentrations. The E₅₈₂A C1 domain mutation fails to bind ethanol (Das et al., 2013). Ethanol does not inhibit dansyl-DAG binding to the E₅₈₂A Munc13-1 C1 domain (n = 3 each; *p < 0.05, ***p < 0.001). Error bars are standard error of the mean (SEM).

Figure 2.

Ethanol specifically inhibits presynaptic vesicle release at excitatory synapses. A, B, Presynaptic activity was elicited by ethyl acetate delivered to the antennae and imaged within the DM3 glomeruli. Flies were imaged with ArcLight to measure membrane depolarization, which leads to a decrease in the maximal fluorescence (ΔFmax). Intoxication due to ethanol injection did not impact measured depolarization as measured by % ΔFmax/Fo (p > 0.05, n = 17). C, D, Flies were also imaged with G-CaMP5 to indicate the intracellular Ca²⁺ concentration. Increasing Ca²⁺ leads to an increase in fluorescence. Intoxication due to ethanol injection did not impact measured Ca²⁺ influx as measured by % ΔFmax/Fo (p > 0.05, n = 15). E, F, Flies were imaged with pHluorin to exhibit the presynaptic vesicle release. Intoxication due to ethanol injection significantly reduced synaptic vesicle fusion as measured by % ΔFmax/Fo (***p < 0.001, n = 17). G, H, In vehicle-injected flies, Dunc13^P84200/+ heterozygotes do not show a significant reduction in synaptic vesicle release (p > 0.05, n = 19); however, they showed less ethanol-induced depression of synaptic release (*p < 0.05, n = 25), after 15% ethanol was injected. All error bars are SEMs.

Figure 3.

A reduction in Dunc13 activity leads to an increased resistance to ethanol sedation. A, The Dunc13^P84200/+ heterozygotes require a greater time to reach 50% LOR (T1/2 LOR) reflex levels (***p < 0.001, n = 17). B, The concentration of ethanol was determined in Dunc13^P84200/+ and control flies exposed to 50% ethanol vapor for 0, 15, 30, or 45 min; no significant differences were found (p > 0.05, n = 6). C, The ability of Dunc13^P84200/+ and control flies to metabolize ethanol was determined by first exposing flies to ethanol vapor for 45 min, and then by measuring the ethanol remaining in the flies 0, 30, 60, and 120 min after the exposure. No significant differences in ethanol metabolism were detected at each time point (t = 0.037, p > 0.05, n = 6). D, The neural expression of the Dunc13^KK101383 RNAi transgene led to significantly slower T1/2 LOR compared to the genotype controls (***p < 0.05, n = 10). E, The induced neural expression of the Dunc13^JF02440 RNAi transgenes also led to a significantly slower T1/2 LOR as compared to the within genotype control (p < 0.01, n = 8). Induction was accomplished with a 24 h, 30°C heat treatment, followed by a 3-h recovery period at room temperature. F, Inducing the expression of a wild-type Munc13-1 cDNA for 48 h led to a significant decrease in LOR for the Dunc13^P84200/+ flies (***p = 0.001, N = 9). Induction was accomplished with a 48 h, 30°C heat treatment, followed by a 3-h recovery period at room temperature. G, Munc13-1::EGFP is colocalized with Bruchpilot, a protein localized to presynaptic active zones, in the presynaptic compartment of the larval neural muscular junction. All error bars are SEMs.