Differential Expression of Presynaptic Munc13-1 and Munc13-2 in Mouse Hippocampus Following Ethanol Drinking

Abstract

The Munc13 family of proteins is critically involved in synaptic vesicle priming and release in glutamatergic neurons in the brain. Munc13-1 binds to alcohol and, in Drosophila, modulates sedation sensitivity and self-administration. We examined the effect of alcohol consumption on the expression of Munc13-1 and Munc13-2, NMDA receptor subunits GluN1, GluN2A and GluN2B in the hippocampus-derived HT22 cells, hippocampal primary neuron culture, and wild-type and Munc13-1^+/- male mouse hippocampus after ethanol consumption (Drinking in the Dark (DID) paradigm). In HT22 cells, Munc13-1 was upregulated following 25 mM ethanol treatment for 24 h. In the primary neuronal culture, however, the expression of both Munc13-1 and Munc13-2 increased after ethanol exposure. While Munc13-1 was upregulated in the hippocampus, Munc13-2 was downregulated following DID. This differential effect was found in the CA1 subfield of the hippocampus. Although Munc13-1^+/- mice had approximately 50% Munc13-1 expression compared to wild-type, it was nonetheless significantly increased following DID. Munc13-1 and Munc13-2 were expressed in vesicular glutamate transporter1 (VGLUT1) immunoreactive neurons in the hippocampus, but ethanol did not alter the expression of VGLUT1. The NMDA receptor subunits, GluN1, GluN2A and GluN2B were upregulated in the hippocampal primary culture and in the CA1. Ethanol exerts a differential effect on the expression of Munc13-1 and Munc13-2 in the CA1 in male mice. Our study also found that ethanol's effect on Munc13 expression is dependent on the experimental paradigm, and both Munc13-1 and Munc13-2 could contribute to the ethanol-induced augmentation of glutamatergic neurotransmission.

Keywords: Munc13; alcohol addiction; ethanol; glutamate receptors; hippocampus; neurotransmitter; presynaptic.

Figure 1:. HT22 cells in differentiation stage express Munc13-1.

A, Double-label immunocytochemistry of ChAT and Munc13-1. B, Representative Western blot illustrating the expression of Munc13-1 in undifferentiated and differentiated HT22 cells. C, Quantitative intensity analysis of Munc13-1 in differentiated and undifferentiated cells shown in A. D, Quantitative densitometry analysis of Munc13-1/β-actin ratio in HT22 shown in B. Box-and-Whiskers plot: the “box” depict the median and the 25^th and 75^th quartiles and the “whisker” show the 5^th and 95^th percentile. Unpaired student’s t-test revealed a significant difference in Munc13-1 expression between differentiated and in undifferentiated HT22 cells [t (4) = 10.0, p = 0.0002] shown in C. One-way ANOVA with Tukey’s multiple comparison test showed significant increase [F (2, 6) = 8.815, p = 0.016)] in Munc13-1 expression in both day 3 (p < 0.0191) and day 5 (p < 0.0195) of differentiated HT22 cells in comparison to undifferentiated HT22 cells shown in D. Un, undifferentiated, Differ, differentiated.

Figure 2:. Ethanol induces Munc13-1 expression in differentiated HT22 cells.

Differentiated HT22 cells were treated with different doses of ethanol for different time points. A, Representative immunoblot illustrating the expression of Munc13-1 in differentiated HT22 cells treated with different doses of ethanol ranging from 10 mM to 100 mM for 24 h. B, Representative immunoblot illustrating the expression of Munc13-1 in differentiated HT22 cells treated with 25 mM dose of ethanol for different time points ranging from 1 h to 48 h. C&D, Quantitative densitometry analysis of Munc13-1/β-actin ratio in differentiated HT22 cells shown in A and B, respectively. Box-and-Whiskers plot: the “box” depict the median and the 25^th and 75^th quartiles and the “whisker” show the 5^th and 95^th percentile. E, Double-label immunocytochemistry of ChAT and Munc13-1 with 25 mM dose of ethanol for 24 h and 48 h. F, Quantitative intensity analysis of Munc13-1 expression shown in E. One-way ANOVA analysis shows significant Munc13-1 expression [F (4,10) = 13.24, p = 0.0005], indicating a dose-dependent increase in the expression of Munc13-1 by ethanol shown in C. Tukey’s post hoc comparisons indicated significant increases at 25 mM (p = 0.0004), 50 mM (p = 0.0344) and 100 mM (p = 0.0076). Significant Munc13-1 expression [F (5,18) = 6.230, p = 0.0016] in time-dependent changes was observed and post hoc Tukey’s indicated significant upregulation of Munc13-1 at 24 h (p = 0.002) and 48 h (p = 0.018) shown in D. Significant Munc13-1 expression changes in dose-dependent changes was observed [F (2,13) = 11.08, p =0.0016] and post hoc analysis showed that in the cytosol of ChAT positive neurons, both 25 mM (p = 0.0020) and 50 mM (p = 0.0065) ethanol upregulated Munc13-1 expression. Con, control.

Figure 3:. Ethanol induces dose dependent changes in Munc13-1 and Munc13-2 expression in primary hippocampal neuronal culture.

A, Double-label immunocytochemistry of Tuj1, Munc13-1 with 25 mM and 50 mM doses of ethanol for 24h. B, Double-label immunocytochemistry of Tuj1 and Munc13-2 with 25 mM and 50 mM doses of ethanol for 24h. C&D, Quantitative intensity analysis of Munc13-1 and Munc13-2 in the primary hippocampal neuronal culture shown in A and B, respectively. Box-and-Whiskers plot: the “box” depict the median and the 25^th and 75^th quartiles and the “whisker” show the 5^th and 95^th percentile. One-way ANOVA revealed significant upregulated Munc13-1 [F (2,6) = 44.52, p =0.0003] at 25 mM (p = 0.0003) and 50 mM doses (p = 0.0006) in Tuj1 (pan-neuronal marker) positive neurons shown in C. Munc13-2 was also significantly upregulated [F (2,6) = 43.31, p =0.0003] at 25 mM (p = 0.0003) and 50 mM doses (p = 0.0008) shown in D. Con, control.

Figure 4:. Glutamatergic neurons express Munc13-1 and Munc13-2 in primary hippocampal neurons.

A, Double-label immunocytochemistry of vesicular glutamate transporter 1 (VGLUT1) and Munc13-1 i.r. neurons. B, Double-label immunochemistry of VGLUT1 and Munc13-2 expression in the primary hippocampal neurons. C&D, Quantitative intensity analysis of VGLUT1 in the primary hippocampal neurons shown in A and B, respectively. Box-and-Whiskers plot: the “box” depict the median and the 25^th and 75^th quartiles and the “whisker” show the 5^th and 95^th percentile. There are no significant changes between groups. Con, control.

Figure 5:. Ethanol upregulates NMDA receptors subtypes GluN1, GluN2A and GluN2B expression in primary hippocampal neurons.

A, Triple-label immunocytochemistry of VGLUT1, GluN1 and Glu2A. B, Triple label immunocytochemistry of VGLUT1, Tuj1 and GluN2B. C, Quantitative intensity analysis of GluN1, Glu2A and GluN2B in primary hippocampal neurons as shown in A and B. Box-and-Whiskers plot: the “box” depict the median and the 25^th and 75^th quartiles and the “whisker” show the 5^th and 95^th percentile. Unpaired student’s t-test demonstrated significant expression level of GluN1 [t (8) = 4.373, p = 0.0012], GluN2A [t (8) = 4.091, p = 0.0017] and GluN2B [t (8) = 6.262, p = 0.0001] shown in C. Con, control.

Fig.6.. Effect of ethanol on the expression of Munc13-1 and Munc13-2 in the hippocampus of C57BL/6J mice.

A, Representative immunoblot illustrating the expression of Munc13-1 in the hippocampus. B, Representative immunoblot illustrating the expression of Munc13-2 in hippocampus. C&D, Quantitative densitometry analysis of Munc13-1/β-actin and Munc13-2/β-actin ratio in hippocampus, respectively. Box-and-Whiskers plot: the “box” depict the median and the 25^th and 75^th quartiles and the “whisker” show the 5^th and 95^th percentile. Two-way ANOVA showed a significant main effect of treatment [F (1,16) = 109.4, p < 0.0001] and genotype [F (1,16) = 36.3, p < 0.0001] reflecting the decrease in Munc13-1 in the heterozygous mice compared to wild-type controls (C). For Munc13-2 expression, the treatment × genotype interaction was significant [F(1,16) = 18.6, p = 0.0005] shown in D. * p = 0.0004, ** p = 0.0001, WT, wild-type.

Figure 7:. Immunohistochemistry analysis of the effect of ethanol on the expression of Munc13-1 and Munc13-2 in the hippocampus of C57BL/6J mice.

Representative of Munc13-1 (A) and Munc13-2 (B) triple-label immunohistochemistry confocal imaging in CA1 subfield of hippocampus. C&D, Quantitative intensity analysis of Munc13-1 and Munc13-2 in CA1. Box-and-Whiskers plot: the “box” depict the median and the 25^th and 75^th quartiles and the “whisker” show the 5^th and 95^th percentile. Two-way ANOVA showed a significant main effect of treatment [F (1,16) = 88.4, p < 0.0001] and genotype [F(1,16) = 12.4, p = 0.0028] shown in C. The treatment × genotype interaction was significant for Munc13-2 immunofluorescence [F(1,16) = 9.7, p = 0.0066] and Tukey’s post hoc comparisons revealed that Munc13-2 immunofluorescence was significantly lower in wild-type mice that underwent DID compared to naïve controls (p < 0.0001) shown in D. WT, wild-type

Figure 8:. Ethanol alters NMDA receptor expression in hippocampus of C57BL/6J mice.

A, Representative immunoblot illustrating the expression of GluN1, Glu2A and GluN2B in hippocampus. B, C and D, Quantitative densitometry analysis of GluN1, Glu2A and GluN2B in hippocampus. Box-and-Whiskers plot: the “box” depict the median and the 25^th and 75^th quartiles and the “whisker” show the 5^th and 95^th percentile. Two-way ANOVA revealed that for GluN1 receptor expression, the treatment × genotype interaction was significant [F(1,16) = 23.7, p = 0.0002] shown in B. For GluN2A expression, the treatment × genotype interaction was also significant [F(1,16) = 17.0, p = 0.0008] shown in C. Tukey’s post hoc multiple comparisons showed that expression was significantly increased in wild-type mice that underwent DID compared to naïve controls (GluN1, p = 0.0011; GluN2A, p = 0.0002). For GluN2B receptor expression the treatment × genotype interaction was not significant [F(1,16) = 0.338, p > 0.05], nor was the main effect of genotype [F(1,16) = 0.727, p > 0.05], indicating that expression was not different between wild-type and Munc13-1^+/− mice shown in D. The main effect of treatment was significant [F(1,16) = 39.7, p < 0.0001], reflecting a DID-induced increase in expression in both genotypes. WT, wild-type.

Figure 9:. Ethanol modulates NMDA receptor subtypes (GluN1 and GluN2A) expression in the CA1 subfield of the hippocampus of C57BL/6J mice.

A, Representative of GluN1 (A) and GluN2A (B) double-label immunohistochemistry confocal imaging in CA1. C and D, Quantitative intensity analysis of GluN1 and GluN2A in CA1. Box-and-Whiskers plot: the “box” depict the median and the 25^th and 75^th quartiles and the “whisker” show the 5^th and 95^th percentile. Two-way ANOVA revealed that for GluN1 the treatment × genotype interaction was not significant [F (1,16) = 0.06, p > 0.05], nor was the main effect of genotype [F (1,16) = 1.2, p > 0.05] shown in C. The main effect of treatment was significant [F (1,16) = 44.2, p < 0.0001]. For GluN2A, the treatment × genotype interaction was significant [F (1,16) = 23.3, p = 0.0002] shown in D. Tukey’s post hoc multiple comparisons showed that immunofluorescence was significantly increased in wild-type mice that underwent DID compared to naïve controls (p < 0.0001). WT, wild-type.