MUNC13-1 heterozygosity does not alter voluntary ethanol consumption or sensitivity in mice

Abstract

The role of the munc13-1 presynaptic protein in alcohol-related behaviors has been little-studied, despite being a known site of action for ethanol binding. Munc13-1 is an active zone protein that plays a vital role in vesicle maturation and the release of neurotransmitters in excitatory neurons. Ethanol binds munc13-1, which decreases its functionality. In Drosophila, loss of the homologous protein Dunc13 is associated with an increase in ethanol preference, and is associated with a resistance to sedation following ethanol exposure. The current study assessed the effects of munc13-1 heterozygosity on ethanol sensitivity and consumption in mice, as well as on learning and anxiety-like behaviors, which can influence alcohol intake. Wild-type and mutant mice underwent 6 cycles of drinking-in-the-dark (DID) as well as rotarod testing following ethanol injection, to probe for differences in ethanol consumption and sensitivity, respectively. We did not detect genotype-based differences in our measures of anxiety, spatial learning, ethanol consumption, or ethanol sensitivity. However, heterozygotes showed increased use of a spatial navigation strategy in a dual-solution water maze, as opposed to a stimulus-response strategy. To summarize, although reduction of Dunc13 in flies produces clear effects on ethanol consumption and sensitivity, heterozygosity for munc13-1 does not, potentially due to compensatory adaptation by other munc-13 isoforms.

Keywords: Addiction; Anxiety; Cognition; Spatial learning; Tolerance.

Figure 1.. Visual representation of the dual-solution water maze.

a) The setup of the maze during 6 days of acquisition (left). The extramaze cues are the large simple figures in the background, while the intramaze cue is the striped tube hanging over the hidden platform. The setup of the maze during the probe trial on Day 7 (right). The intramaze cue has been moved to the opposite side of the pool, but the extramaze cues do not change location. The hidden platform has been removed. b) Quadrants and zones of the maze. The pool is divided into four quadrants based on the cardinal directions, colored in light and dark blue. The platform (P) and cue (C) zones occupy a smaller space within the NW and SE quadrants, and are colored in black. The platform zone is where the platform and the intramaze cue are located for all 6 days of acquisition. The cue zone is where the intramaze cue is moved for the probe trial. To assess use of a spatial strategy versus a stimulus-response strategy, time spent in the NW and SE quadrants during the probe trial were compared.

Figure 2.. Performance in the dual-solution water maze.

a) Latency to find the hidden platform across 6 days of acquisition. All mice learned the location of the platform by Day 6, as indicated by the main effect of Day as well as latencies of <10 seconds. Females found the platform significantly faster as indicated by the main effect of Sex. b) Swim velocity of mice in the water maze. Overall, heterozygotes swam faster than wildtypes, and swim speed for all animals decreased over time. c) Swim path length of mice in the water maze. Heterozygotes travelled further than wildtype mice, and swim paths of all mice shortened over time. d) Spatial bias scores across sex and genotype, based on quadrant. Values above 0 indicate bias toward a spatial strategy, while values below 0 indicate bias toward a stimulus-response strategy. There was no interaction, but there were main effects of Sex (p = .004) and Genotype (p = .023), such that males and heterozygotes showed higher scores (more spatial bias) when compared to females and wildtypes. *p < 0.05 main effect of Sex; ^#p < 0.05 main effect of Genotype; ^$p < 0.05 main effect of Day; ⁺p < 0.05 heterozygotes significantly different from wildtype

Figure 3.. Ethanol consumption in the DID paradigm.

a) Consumption in C57BL mice over 6 weekly cycles of DID. Ethanol consumption decreased over time, as indicated by the main effect of Week. b) Consumption of C57BL mice averaged over 6 weeks. Overall, female mice drank more than males. c) Consumption in 129S1 mice over 6 weekly cycles of DID. Females drank more than males did at Week 5 (p = .013) and Week 6 (p = .008), but not on average, as shown in d). *p < 0.05 main effect of Sex; ^$p < 0.05 main effect of Week; ⁺p < 0.05 significant pairwise comparisons

Figure 4.. Rotarod performance following injection of ethanol or saline.

a) Rotarod latency across trials after saline injection (a 0.0 g/kg “dose” of ethanol). Mice significantly improved performance from Trial 1 to Trial 3 (p = .018). b) Rotarod latency across trials after injection of a 0.5 g/kg dose of ethanol. c) Rotarod latency across trials after injection of a 1.5 g/kg dose of ethanol. Mice significantly improved performance from Trial 1 to Trial 3 (p < .001). All values are presented as mean ± standard error.