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 (following 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.

Introduction

Identifying molecular targets and associated mechanisms of ethanol action are key steps to developing successful treatments for alcohol use disorder (AUD). Ethanol affects the functions of both central and peripheral synapses (Liu and Hunt, 1999, McCool, 2011, Lovinger and Roberto, 2013, Abrahao et al., 2017, Roberto and Varodayan, 2017). Ethanol is known to impact postsynaptic functions by binding to receptors, such as GABA_A (Olsen and Liang, 2017), glycine (Soderpalm et al., 2017), glutamate (Rao et al., 2015) and serotonin (Marcinkiewcz, 2015). However, there is also strong support for ethanol’s effect in the presynaptic compartment (Siggins et al., 1987, Diamond and Gordon, 1997, Roberto et al., 2003, Nie et al., 2004, Roberto et al., 2003). Studies suggested that presynaptic GABA_B and mGlu₂ contribute to ethanol’s presynaptic actions (Lovinger, 2017). Ethanol may also affect synaptic transmission at the presynaptic zone by acting on the active zone proteins associated with synaptic vesicle fusion (Barclay et al., 2010, Xu et al., 2018, Das, 2020). Munc13, syntaxin, Munc18, synaptobrevin, synaptosomal-associated protein 25 kDa (SNAP-25), synaptotagmin and complexin are major proteins that constitute the neurotransmitter release machinery (Südhof, 2013).

The Munc13 family of proteins constitute brain-specific homologs of Caenorhabditis elegans Unc-13 (Maruyama and Brenner, 1991) and Drosophila Dunc-13 (Aravamudan et al., 1999). Munc13-1, Munc13-2, Munc13-3, and Munc13-4 are the mammalian isoforms of Munc13 known to date (Augustin et al., 1999a, Chen et al., 2013) of which Munc13-1 has been studied in more detail as compared to the others. Munc13-1 plays an essential role in synaptic vesicle priming (Betz et al., 1997, Sassa et al., 1999) and neurotransmitter release (Betz et al., 1998, Brose et al., 2000) by connecting the plasma membrane and the synaptic vesicle (Quade et al., 2019). Munc13-2 has two splice variants, ubMunc13-2, which is ubiquitously expressed and bMunc13-2, which is expressed only in the brain. ubMunc13-2 closely resembles the structure of Munc13-1, but bMunc13-2 has one C2 domain less at its N-terminus (Brose and Rosenmund, 2002). While Munc13-1 is primarily expressed in the hippocampus, cerebellum, cortex, and striatum, Munc13-2 is expressed in rostral brain regions, including the CA field of the hippocampus, and the cerebral cortex (Augustin et al., 1999a). Munc13-3 is highly expressed in the cerebellum and only slightly in the brain stem. Munc13-1 has been implicated in modulating short-term presynaptic plasticity (Rosenmund et al., 2002, Lipstein et al., 2013) and long-term potentiation (Yang and Calakos, 2011). Neurotransmitter release is completely lost in the Munc13-1 and 13-2 double-knockout mice (Aravamudan et al., 1999, Augustin et al., 1999b, Richmond et al., 1999, Varoqueaux et al., 2002). It was found that Munc13-1 was necessary for vesicle release in 90% of glutamatergic synapses. The remaining 10% of glutamatergic synapses were normal in the Munc13-1 knockout mice, and most likely were Munc13-2-dependent, as glutamatergic synapses remain silenced in Munc13-1/2 double knockout mice (Augustin et al., 1999b, Rosenmund et al., 2002, Varoqueaux et al., 2002). In hippocampus, while glutamatergic neurons are affected due to the mutant phenotype of the Munc13-1 knockout synapses, GABAergic cells are almost completely unaffected (Augustin et al., 1999b, Rosenmund et al., 2002).

Ethanol acts on multiple molecular targets and neurocircuitries in different brain regions. One of these regions is the hippocampus (Ryabinin, 1998, Zorumski et al., 2014, Abrahao et al., 2017), the brain region that regulates the learning and memory functions (Shors and Matzel, 1997). Numerous studies, both clinical and non-clinical, have shown that the hippocampus is particularly susceptible to alcohol drinking (Zahr et al., 2010, Geil et al., 2014). The detrimental effects of chronic alcohol drinking are believed to contribute to the deficits in cognitive functions seen in human alcoholics (Bartels C et al., 2007). In addition, alcohol abuse in adulthood is associated with premature aging and neurodegeneration in the hippocampus, hallmarks of Alzheimer’s disease (Matthews et al., 2019, Ledesma et al., 2021). The synaptic circuits in the hippocampus include both excitatory and inhibitory transmission. The N-methyl-D-Aspartate glutamate receptor (NMDAR), is a central player in memory function and spatial learning processing in the hippocampus (Newcomer et al., 2000). Multiple studies have reported that ethanol regulated the expression and function of numerous receptors in different fields of the hippocampus (Hu et al., 1996, Grifasi et al., 2019, Drissi et al., 2020, Mira et al., 2020). For example, chronic ethanol exposure upregulates NMDA subunits (GluN1, GluN2A and GluN2B) in the hippocampus (Snell et al., 1996, Devaud and Morrow, 1999, Nelson et al., 2005, Hendricson et al., 2007).

Previously, we showed that alcohol binds with the Munc13-1 C1 domain at the Glu-582 (Das et al., 2013). Physiological levels of alcohol inhibit the Munc13-1 C1 domain’s ability to bind diacylglycerol (DAG) in vitro, which should reduce the activity of Munc13-1 in vivo. In Drosophila, the genetic reduction in Dunc13 activity resulted in flies with significantly greater alcohol self-administration (Das et al., 2013). Moreover, genetically reducing Dunc13 activity further resulted in flies that were behaviorally resistant to sedating alcohol levels (Xu et al., 2018). We also showed that in Drosophila, alcohol inhibits the fusion of synaptic vesicles with the plasma membrane without impacting Ca²⁺ influx into the presynaptic compartment and that genetically reducing Dunc13 protects against this inhibition (Xu et al., 2018). These results support the hypothesis that lowering Dunc13 activity, either genetically or through intoxicating alcohol concentrations, leads to a state of ethanol tolerance.

In this study, we examined how ethanol affects the expression levels of Munc13-1, Munc13-2, and several NMDA receptors after alcohol exposure in hippocampal-derived HT22 cells, primary hippocampal neuronal culture and hippocampus of C57 BL/6J male mice subjected to drinking in the dark paradigm (DID), which is widely considered a model of alcohol binge drinking as the animals voluntarily consume intoxicating concentrations of this drug drinking (Rhodes et al., 2005, Rhodes et al., 2007, Wooden et al., 2020). Importantly, we also examined how reduced Munc13-1 activity impacted the ability of ethanol to regulate the expression of these Munc13-2 and the NMDA receptor subunits in the hippocampus. We found that, in neuronal culture, both Munc13-1 and Munc13-2 expression were increased after ethanol treatment. Munc13-1 expression increased in the CA1 subfield of the hippocampus after alcohol consumption, but Munc13-2 was decreased in this subfield after alcohol consumption. We also found that NMDA receptors GluN1, GluN2A and GluN2B increased expression in the hippocampus and in the neuronal culture after ethanol consumption. Moreover, ethanol’s regulation of Munc13-2, GluN1, and GluN2A was significantly blunted in the hippocampus of mice heterozygous for the munc13-1 KO allele. These data provide additional insights into how neurons may functionally adapt to alcohol’s inhibitory effects on presynaptic activity and illuminate a potential role for Munc13-1 activity in this adaptive process.

Experimental Procedures

HT22 cells

Hippocampal derived HT22 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 2 mM glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml) in a humidified CO₂ incubator (5% CO₂) at 37°C. When cells were 60%−70% confluent, medium was replaced with the differentiation medium containing NeuroBasal fortified with N₂ supplement, cAMP, L-glutamine, penicillin, and streptomycin. Approximately 48 h to 72 h post ‘day 0’ of differentiation, cells were treated with different doses of ethanol (10–100 mM) for different time points (24 or 48 h). Following treatment, cells were either fixed with 4% PFA and processed for immunocytochemistry or processed for immunoblotting.

Primary hippocampal neuronal culture

Primary hippocampal neurons were cultured from the hippocampal tissue of P0-P1 male pups (C57BL/6J) (Pany et al., 2017). Briefly, hippocampal tissue was dissected out and maintained in ice-cold calcium-free Hank’s balanced salt solution (HBSS). The tissue was then dissociated in HBSS plus trypsin 0.25 % EDTA for 20 min at 37 °C. After dissociation, cells were plated at an equal density of 0.08 million cells per well on 12 mm coverslip pre-coated with 50 μg/ml of poly-D-lysine. A neuronal feeding media containing neurobasal media fortified with B-27 supplement, 200 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin and HEPES was used to maintain the neuronal cells in a humidified CO₂ incubator (5% CO₂) for 24 h at 37 °C. About one-half of the media was replaced every three days. One week old culture was used for the experiments. Primary neurons were treated with different doses of ethanol (25 mM or 50 mM) for 24 h. Following treatment, cells were fixed with 4% PFA and processed for immunocytochemistry (Pany et al., 2017).

Immunocytochemistry

The primary neuronal cells were fixed with 4 % PFA in PBS for 15 min, followed by blocking buffer composed of 0.4 % bovine serum albumin (BSA), 0.2 % Triton X-100 and 0.02 % Tween-20 in PBS for 1 h to prevent nonspecific antigen and antibody binding. Cells were then incubated overnight at 4 °C with different primary antibodies, such as Munc13-1 (1:500, Rabbit polyclonal, Synaptic Systems, Germany), Tuj1 (1:1000, Mouse monoclonal, R&D, USA), VGLUT1 (1:500, Guinea pig polyclonal, Synaptic Systems, Germany), ChAT (1:100), GluN1 (1:100), GluN2A (1:100), GluN2B (1:100). The cells were then incubated with appropriate secondary antibodies (1:1000), (Alexa Fluor 488 or 594 or 647; Invitrogen, USA) followed by incubation with DAPI (1:3000), (Sigma, USA) to stain the nucleus. Coverslips containing stained cells were mounted on slides and visualized under a Leica SP8 confocal microscope (Leica Microsystem INC., USA) (Pany et al., 2017).

Animals

In the present study, munc13-1 KO allele heterozygous mice were used as munc13-1 homozygotes die shortly after birth (Augustin et al., 1999b). Munc13-1^+/− heterozygous male mice were received from Dr. Thomas Sudhof and crossed with C57BL/6J female mice (Envigo, Indiana, United States) as previously described (Wooden et al., 2020). The progeny from crosses were either heterozygous for the munc13-1 KO allele or wild-type for munc13-1 based on the genotyping performed by Transnetyx Inc (Cordova, TN, USA). For the present study, only male offspring were used. The mice were housed (5 per cage), maintained in constant temperature (22 °C) and humidity (relative, 30%) with a reversed light/dark cycle (9 A.M. off/9 P.M. on) and were given access to food and water ad libitum. Our facility uses OptiMice housing from Animal Care Systems. The dimension of each mouse cage is 13.5” (34.3 cm) L × 11.5” (29.2 cm) W (front) × 6.1” (15.5 cm) H. Mice were allowed free access to food and water, except during the DID procedure (described below). After genotyping, (8–12 weeks old mice, n=40) were divided into four groups: (1) Wild-type (n=10), (2) Wild-type + ethanol (20%) (n=10), (3) Munc13-1 ^+/− (n=10) and (4) Munc13-1 ^+/− + ethanol (n=10). The animals in the ethanol groups were subjected to the Drinking in the Dark (DID) paradigm. The Institutional Animal Care and Use Committee (IACUC) at the University of Houston approved the animal use protocol. The experiments were conducted with male mice before the National Institutes of Health requirement to include sex as a biological variable. Studies with the female mice will be taken up in future.

Drinking in the Dark paradigm

Individually-housed mice underwent 6 cycles of DID for six consecutive weeks, as we have previously described (Wooden et al., 2020). Briefly, a single cycle of DID consisted of 3 consecutive days during which the water bottle was removed and replaced with an identical bottle containing 20% ethanol. The switch occurred 3 h into the dark phase and lasted for 2 h, after which the water bottle was returned, and consumption of the ethanol solution measured. On the 4^th day, identical procedures were followed, except that mice had 4 h of access to 20% ethanol instead of 2. As we have previously reported, this paradigm results in an average of 2 g/kg intake in male wild-type and heterozygous mice (Wooden et al., 2020). Control mice were also handled in a similar way as the DID mice. The mean intake was expressed as grams of ethanol per kilogram of body weight consumed (g/kg) per 4-hour session. At the end of the last 4-hour ethanol session (week 6, end of day 4), blood was collected from the saphenous vein for determination of blood ethanol concentration (BEC). Blood was collected in heparinized glass capillary tubes, and then centrifuged at 10,000 rpm for 5 minutes in 1.5 mL micro-centrifuge tubes. The blood plasma that collected on the top of the sample was withdrawn and stored at −20°C for later analysis. Samples were then processed using an Analox AM-1 model alcohol analyzer (Analox Instruments, Lunenburg, MA). The brain tissues were collected within 12 h after the termination of DID. The age of the mice that were euthanized was 120–150 days.

Immunoblotting

Immunoblotting of cells or tissues were done as described previously (Blanco F et al., 2019). Briefly, the cells or tissues were resuspended in a modified radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitor cocktail. Whereas cell suspensions were sonicated after resuspension, mouse hippocampal tissues were first homogenized, then sonicated, and centrifuged at 14,000g for 45 min at 4°C. Protein concentration was estimated from the collected supernatant using a BCA kit (Thermo Scientific, Waltham, MA USA). Proteins were separated using SDS-PAGE, transferred to a nitrocellulose membrane, and the membrane was blocked by treating with 2.5% BSA in TBS-T for nonspecific binding sites. Then, the membrane was incubated with appropriate primary antibodies, such as (Munc13-1 (1:1500, Rabbit polyclonal, Synaptic Systems, Germany), Munc13-2 (1:1500, Rabbit polyclonal, Synaptic Systems, Germany), GluN1 (1:500, Rabbit polyclonal), GluN2A (1:1000, Rabbit polyclonal), GluN2B (1:1000, Rabbit polyclonal), VGLUT1 (1:2000, Guinea pig polyclonal) and β-actin (1:1500, rabbit polyclonal) for overnight at 4°C. Next day, secondary antibodies, such as anti-rabbit HRP (1:5000) or anti-mouse HRP (1:3000) were used. The immunoreactive bands were developed and visualized using ECL (enhanced chemiluminiscence) reagent (Pierce, Rockford, IL) and images were captured with image processer (Alpha Imager Gel Documentation system, Alpha Innotech, Santa Clara, CA). The gel bands were quantified using Image-J software (NIH) (Blanco et al., 2019).

Immunohistochemistry

Mice were anesthetized with sodium pentobarbital (130 mg/kg; Sigma, USA) and perfused with 4% paraformaldehyde (PFA) in 0.1 M PBS. Next, brains were post-fixed in 30% sucrose. For performing immunohistochemistry, freezing sliding microtome-cut 50 μm thick free-floating sections encompassing hippocampus were used. Sections were incubated with diluted normal blocking serum for 30 min followed by incubation with appropriate primary antibodies for overnight at 4 °C. The antibodies used were: Munc13-1 (1:100, Rabbit polyclonal, Synaptic Systems, Germany), Munc13-2 (1:50, Rabbit polyclonal, Synaptic Systems, Germany), Tuj1 (1:100, Mouse monoclonal, R&D, USA), VGLUT1 (1:500, Guinea pig polyclonal, Synaptic Systems, Germany), ChAT, GluN1 (1:100), GluN2A (1:100) and GluN2B (1:100). Next, sections were washed thrice with PBS followed by incubation with appropriate secondary antibodies, Alexa Fluor 488 (1:1000) or Alexa Fluor 594 (1:1000) (Invitrogen, USA) for 1 h. Sections were then incubated with DAPI (1:1500) (Sigma, USA) for 5 min at room temperature to stain the nucleus. Following mounting on slides, sections were observed under a Leica SP8 confocal microscope (Leica Microsystem INC, USA). For analysis, a total of 3–4 hippocampal sections per brain and five mice per group were stained and observed with confocal microscopy. Confocal images were acquired with Leica SP8 Microsystem Inc., USA, available in the core facilities of the College of Pharmacy, University of Houston, TX, USA. Immunostained slices were sequentially scanned along the Z-axis. Overall, confocal images were obtained with a 100X objective at 1024 × 1024 pixel resolution. For quantifying Munc13-1, Munc13-2, GluN1 and GluN2A following DID, the signal intensity of each sections was captured, and a single confocal image was used to illustrate each set of co-staining throughout the present study. The expression of Munc13-1, Munc13-2, GluN1, and GluN2A were measured using Image J software (http://rsbweb.nih.gov/ij/docs/faqs.html#cite). (Salio et al., 2014).

Statistical analysis

All data were expressed as box-and whisker plots, with the boxes encompassing the 25^th to 75^th percentiles and the median indicated. The whiskers are at the 5^th and 95^th percentiles. Statistical analyses were carried out using GraphPad Prism version 9 (GraphPad Software Inc., CA, USA). Student’s t-test (Unpaired) was used for in-vitro (HT22 cells), one-way ANOVA (Primary hippocampal neuronal culture) and two-way ANOVA (in-vivo experiments) were used for statistical comparisons and results with p<0.05 were considered to be statistically significant. Tukey’s post-hoc tests were used where appropriate.

Results

Differentiated HT22 cells express Munc13-1 and Munc13-2

It was reported earlier that Munc13-1 and Munc13-2 shows differential expression from embryonic to adult stage (Augustin et al., 1999a). As this study is focused on the hippocampal region of the brain, we first used a simpler model of hippocampal derived HT22 cells to study the effects of ethanol on the expression of Munc13-1. Cholinergic neuronal properties are common in hippocampal neurons and to check if HT22 cells possess these properties we performed immunocytochemistry of cholinergic acetyl transferase (ChAT) with Munc13-1 and Munc13-2. Differentiated HT22 cells expressed both Munc13-1 (Figure 1A) and Munc13-2 (data not shown). We found satisfactory levels of ChAT expression in differentiated HT22 cells compared to undifferentiated HT22 cells (Figure 1A), implying that differentiated HT22 cells neurons are cholinergic. Moreover, ChAT immunoreactive positive neurons also expressed Munc13-1 specifically in cytosol, while negligible levels of Munc13-1 were observed in undifferentiated HT22 cells (t₄ = 10.0, p = 0.0002) (Figure 1B). ChAT expression was analyzed with different ethanol concentration (25–100 mM) at various time points, however there was no significant changes in ChAT expression (data not shown). To further confirm the expression of Munc13-1 in differentiated HT22 cells, we performed immunoblotting of Munc13-1 from undifferentiated and differentiated HT22 cells (Figure 1C). One-way ANOVA was significant (F_2,6 = 8.815, p = 0.016) and post hoc comparisons showed a significant increase 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 (F_{2, 6} =8.815, p=0.016) (Figure 1D). Collectively, these results demonstrate that HT22 cells express Munc13-1 when differentiated into neuronal morphology.

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.

Ethanol upregulates the expression of Munc13-1 in differentiated HT22 cells

After demonstrating that differentiated HT22 cells express Munc13-1 and Munc13-2, we asked whether ethanol alters Munc13-1 and Munc13-2 expression level. Different doses of ethanol (10 mM −100 mM for 24h) were added to the differentiated HT22 cells and levels of Munc13-1 and Munc13-2 were checked by immunoblotting. One-way ANOVA was significant (F_{4, 10} = 13.24, p = 0.0005, indicating a dose-dependent increase in the expression of Munc13-1 by ethanol. 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). Maximum induction was at 25 mM (Figures 2A and 2C). To determine the time point of Munc13-1 induction we did a time course experiment with 25 mM dose of ethanol. Again, one-way ANOVA was significant (F_5,18 = 6.230, p = 0.0016), and post hoc Tukey’s indicated significant upregulation of Munc13-1 expression at 24h (p = 0.002) and 48h (p = 0.018) (Figures 2B and 2D). To further confirm the upregulation of Munc13-1 following ethanol treatment, we performed double-label immunocytochemistry of ChAT and Munc13-1. One-way ANOVA was significant (F_2,13 = 11.08, p =0.0016), and post hoc analysis showed that in the cytosol of ChAT positive neurons, both 24 h (p = 0.0020) and 48 h (p = 0.0065) ethanol upregulated Munc13-1 expression (Figures 2E and 2F). We did not observe any significant changes in the level of Munc13-2 in different doses of ethanol (data not shown). Collectively, these results demonstrate that ethanol increases the expression of Munc13-1, but no changes in Munc13-2 in differentiated HT22 cells.

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.

It is to be noted that the final ethanol concentration in the cell culture media was reduced after 24 h and 48 h incubation due to its evaporation. In our experimental condition, 26% and 28% of ethanol were evaporated in 24 h and 48 h, respectively from a 50 mM ethanol solution. For a 25 mM ethanol solution, 19% and 21% ethanol evaporation was observed in 24 h and 48 h, respectively. These measurements were done using the Ethanol Assay Kit (Sigma, catalog # MAK076) and a SpectraMax i3 microplate reader (Molecular Devices, San Jose, USA).

Ethanol upregulates the expression of Munc13-1 and Munc13-2 in primary hippocampal neurons

After measuring the effects of ethanol on Munc13-1 expression in HT22 cells, we checked ethanol’s effect on the expression of Munc13-1 and Munc13-2 in a more complex system of primary neurons. For this, we cultured hippocampal neurons from male pups (P0-P1) and treated with 25 mM and 50 mM of ethanol for 24 h (Figures 3A–D). Ethanol 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 (Figure 3C). Results were similar for Munc13-2 (F_2,6 = 43.31, p =0.0003), with significant upregulation at 25 mM (p = 0.0003) and 50 mM doses (p = 0.0008) (Figure 3D). However, for both proteins, the expression levels were not different between the 25 mM and 50 mM doses.

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.

Glutamatergic neurons express Munc13-1 and Munc13-2 in primary hippocampal neurons

It was previously reported that two types of synapses were formed with individual glutamatergic hippocampal neurons with varied Munc13-1 dependence. About 90% of synapses were 13-1-dependent and about 10% were dependent on both Munc13-1 and Munc13-2. (Augustin et al., 1999b, Rosenmund et al., 2002). In our study, we wanted to confirm whether glutamatergic neurons expressed Munc13-1 and Munc13-2. Primary hippocampal neurons were treated with 25 mM ethanol and 24 h post treatment cells were fixed and immunostained with glutamatergic neuronal marker, vesicular glutamate transporter1 (VGLUT1) and Munc13-1. We observed co-localization of VGLUT1 and Munc13-1 in primary neuronal culture, however ethanol had no effects on the expression levels of VGLUT1 (Figure 4A–B). Next, we wanted to check whether glutamatergic neurons express Munc13-2 in the primary hippocampal culture. Like Munc13-1, VGLUT1 and Munc13-2 were also colocalized in primary neuronal culture; however, there was no effect of ethanol on the expression levels of VGLUT1, as demonstrated by quantification of fluorescence intensity of VGLUT1 (Figure 4C–D).

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.

Effects of ethanol on the expression of glutamatergic ionotropic NMDA receptors in primary hippocampal neuron

As ethanol-induced upregulation of Munc13-1 could be a triggering step in the release of glutamate from presynaptic neurons, we decided to check the expression of glutamatergic NMDA receptor subunits in the postsynaptic neurons. Primary neurons were treated with 25 mM ethanol for 24 h and triple-labeled with VGLUT1, GluN1, and GluN2A (Figures 5A and 5C). We found a robust increase in the expression level of GluN1 (t₈ = 4.373, p = 0.0012) and GluN2A (t₈ = 4.091, p = 0.0017) in glutamatergic neurons upon ethanol treatment. Similar to this observation, we also detected ethanol-induced augmentation of GluN2B expression in Tuj1 and VGLUT1 positive neurons when we did separate immunocytochemistry of VGLUT1, Tuj1, and GluN2B in primary hippocampal neurons (t₈ = 6.262, p = 0.0001) (Figures 5B and 5C). Overall, NMDA receptor subunits were upregulated following ethanol exposure in the primary hippocampal neuronal culture.

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.

Effects of ethanol on the expression of Munc13-1 and Munc13-2 in the hippocampus

We hypothesized that Munc13-1 levels are homeostatically regulated to help maintain neural activity during the ethanol-induced depression of presynaptic activity. For testing this hypothesis, we measured the levels of Munc13-1 protein in naïve and ethanol exposed animals. Mice were subjected to the DID paradigm, then their hippocampi were checked for Munc13-1/Munc13-2 expression by immunoblotting. Amount of ethanol consumed in this paradigm does not differ between Munc13-1^+/− mice and wild-type mice, as we have previously described (Wooden et al., 2020). In the present study, BEC did not differ significantly between Munc13-1^+/− mice and wild-type mice (52.2 ± 21.8 mg/dl and 46.2 ± 13.2 mg/dl, respectively). Our BEC values are lower than the binge levels (80 mg/dl) likely due to mice consuming alcohol early in the access period, whereas BEC was assessed at the end of the access period. Lower value of BEC after DID was also reported earlier (Sparta et al., 2008) and was rationalized in terms of gene-environment interactions (Crabbe et al., 1999, Wahlsten et al., 2003). Two-way ANOVA showed a significant main effect of treatment (F_1,16 = 109.4, p < 0.0001). This reflects approximately two-fold increase in levels of Munc13-1 in wild-type mice subjected to DID compared to naïve control mice and naïve Munc13-1^+/− mice compared to Munc13-1^+/− that underwent DID. The significant main effect of genotype (F_1,16 = 36.3, p < 0.0001) reflects the decrease in Munc13-1 in the heterozygous mice compared to wild-type controls (Figure 6A). For Munc13-1, the treatment × genotype interaction was not significant (Figure 6C). For Munc13-2 expression, however, the treatment × genotype interaction was significant (F_1,16 = 18.6, p = 0.0005) (Figure 6D). Post hoc comparisons revealed that Munc13-2 expression was significantly lower in wild-type mice that underwent DID, compared to naïve controls (p = 0.0004) (Figure 6D). Interestingly, naïve Munc13-1^+/− mice had significantly lower levels of Munc13-2 expression compared to wild-type naïve mice (p = 0.0001), and this did not change with DID (Figure 6D).

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.

We further examined changes in Munc13-1 and Munc13-2 expression within the specific CA1 subfield of the hippocampus using immunofluorescence (Figure 7). Two-way ANOVA showed a significant main effect of treatment (F_1,16 = 88.4, p < 0.0001), indicating that DID increased Munc13-1 fluorescence in both wild-type and Munc13-1^+/− mice. The significant main effect of genotype (F_1,16 = 12.4, p = 0.0028) reflects the decrease in Munc13-1 in the heterozygous mice compared to wild-type controls (Figures 7A and 7C). For Munc13-1 immunofluorescence, the treatment × genotype interaction was not significant. The treatment × genotype interaction was significant for Munc13-2 immunofluorescence (F_1,16 = 9.7, p = 0.0066). 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) (Figures 7B and 7D). Similar to the immunoblot findings, naïve Munc13-1^+/− mice had significantly lower levels of Munc13-2 compared to wild-type naïve mice (p = 0.0006), and this did not change with DID. We also analyzed the expression of Munc13-1 and Munc13-2 in CA2, CA3, and DG using an immunofluorescence method, but could not find significant changes in expression between the wild-type and heterozygous mice. Furthermore, we could not detect sufficient expression of Munc13-2 in these hippocampal subfields for comparing with Munc13-1.

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

In summary, ethanol increased the expression of Munc13-1 and decreased the expression of Munc13-2 in the CA1 field of hippocampus of wild-type mice. Ethanol also increased Munc13-1 in Munc13-1^+/− mice, but changes in Munc13-2 expression after DID were not seen in the Munc13-1^+/− mice. The protective effect of reducing Munc13-1 on ethanol-induced changes in Munc13-2 expression suggests a coordinated, dependent regulation of these two loci after DID.

Effects of ethanol on the expression of NMDA receptors in the hippocampus

The levels of NMDA glutamate receptors subunits in hippocampal sections were measured by immunoblotting (Figure 8) and immunofluorescence (Figure 9). For GluN1 receptor expression, the treatment × genotype interaction was significant (F_1,16 = 23.7, p = 0.0002). Post hoc comparisons showed that expression was significantly increased in wild-type mice that underwent DID compared to naïve controls (p = 0.0011) (Figure 8B). Expression did not change due to DID in Munc13-1^+/− mice. For GluN2A expression, the treatment × genotype interaction was significant (F_1,16 = 17.0, p = 0.0008) (Figure 8C). Post hoc comparisons showed that, similar to GluN1, expression of GluN2A was significantly increased in wild-type mice that underwent DID compared to naïve controls (p = 0.0002) (Figure 8C). Compared to wild-type controls, expression was significantly lower in naïve Munc13-1^+/− mice (p = 0.0093) and Munc13-1^+/− that underwent DID (p = 0.0050), and DID did not increase GluN2A expression in Munc13-1^+/− mice. 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 (Figure 8D). 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.

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.

To summarize, all three NMDA receptors were found to be upregulated in wild-type mice after DID. However, in the Munc13-1^+/− mice, while the GluN1 and GluN2B expression levels were similar to the wild-type mice, there was a significant decrease in GluN2A expression compared to the wild-type. DID caused an increase in the expression of GluN2B in Munc13-1^+/− mice similar to the wild-type mice.

We confirmed with immunofluorescence, the ethanol-mediated expression changes in GluN1 and GluN2A in the CA1 subfield of the hippocampus (Figure 9). 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), indicating that immunofluorescence was not different between wild-type and Munc13-1^+/−. The main effect of treatment was significant (F_1,16 = 44.2, p < 0.0001), reflecting a DID-induced increase in immunofluorescence in both genotypes (Figures 9A and 9C). For GluN2A, the treatment × genotype interaction was significant (F_1,16 = 23.3, p = 0.0002). Post hoc comparisons showed that immunofluorescence was significantly increased in wild-type mice that underwent DID compared to naïve controls (p < 0.0001) (Figures 9B and 9D). In Munc13-1^+/− mice, levels did not differ from wild-type naïve and did not increase with DID.

Discussion

The current study is a part of our ongoing investigations on the impact of ethanol on the presynaptic Munc13 proteins and the specific role of Munc13-1 inhibition in this process (Das et al., 2013, Xu et al., 2018, You and Das, 2020, Wooden et al., 2020). In this study, we specifically looked into ethanol’s effect on the expression levels of Munc13-1 and Munc13-2 using three different models: hippocampal-derived HT22 cells, hippocampal primary neuronal culture and male mouse hippocampus after DID. In addition, we also examined the expression of several postsynaptic NMDA receptors since Munc13-1 is primarily associated with glutamatergic neurotransmission (Augustin et al., 1999b). We used these three distinct models to test the prediction that ethanol-induced changes in Munc13-1 and Munc13-2 activity would represent a general response to this drug within hippocampal neurons.

As part of our working hypothesis for the role of Munc13-1 inhibition as a precipitating event for the neural adaptation to ethanol, we further examined the ethanol induced expression of Munc13-1, Munc13-2, GluN1, GluN2A, and GluN2B in the hippocampus of mice heterozygous for the munc13-1 KO allele. We predicted that if the inhibition of Munc13-1 activity by ethanol has a role in the neural adaptation to this drug, then a genetic reduction in the activity of this Munc13-1 would alter the ability of ethanol to induced changes in the levels of Munc13-2 and these NMDA receptor subunits. The Munc13-2, GluN1, and GluN2A proteins were differentially regulated by ethanol in the munc13-1 KO heterozygotes, supporting a role for this protein in regulating the adaptive synaptic response to ethanol.

Ethanol treatment significantly also increased Munc13-1 levels across all models tested, which could represent a compensatory mechanism for ethanol’s inhibition presynaptic activity. However, this increase in Munc13-1 expression is not seen in naïve munc13-1 KO heterozygotes, nor is the ethanol-induced increase blocked by the presence of this allele. These data suggest that ethanol-induced increase in Munc13-1 is not a simple feedback loop to balance Munc13-1 activity. Hence, we found some ethanol-induced changes in protein expression were dependent on preexisting Munc13-1 activity, and some were not.

To understand the effects of ethanol during the developing stage, we first used a simpler system of hippocampal-derived HT22 cells that endogenously expresses Munc13-1. HT22 murine hippocampal neuronal cell line is a sub-line cloned from its parent HT4 cell line and has been widely used for in vitro neuronal toxicity and addiction research studies (Aminova et al., 2005, Lin and Takemoto, 2007, Pany et al., 2017). Recently, cholinergic properties of differentiated HT22 cells have been demonstrated (Liu et al., 2009). Immunocytochemistry of ChAT and Munc13-1 clearly indicated that undifferentiated HT22 cells neither satisfactorily express cholinergic properties nor Munc13-1 expression, however differentiated HT22 cells express Munc13-1 in cytosol (Figure 1). Then we studied ethanol’s effect on Munc13 proteins in the primary hippocampal neuronal culture. Our results, however, suggest that both Munc13-1 and Munc13-2 are upregulated in the presence of 25 mM ethanol (Figure 3). We also observed vesicular glutamate transporter-1 (VGLUT1), a marker for glutamatergic neurons, expressed Munc13-1 in both primary hippocampal neuronal culture (Figure 4) and HT22 cells (Figures 1 and 2). However, we didn’t observe any alterations in VGLUT1 expression pattern between ethanol-treated and control cells. Hence, VGLUT expression was not responsive to ethanol in our experiments.

After the in vitro models, we checked the effect of ethanol on Munc13s in the hippocampal tissue of male mice following DID. Our immunoblotting results suggest that while ethanol increases Munc13-1 expression in the hippocampus, Munc13-2 is downregulated in this brain region (Figure 6). To further investigate this, we analyzed the CA1 subfield of the hippocampus by immunohistochemistry and observed the same differential expression in this subfield (Figure 7). Immunocytochemical detection of VGLUT1 in the hippocampal neurons also failed to detect any differences in expression levels in the wild-type and Munc13-1^+/− after ethanol treatment (Figure 4). Hence, VGLUT expression was not responsive to ethanol in our experiments.

There could be several reasons for the differential expression observed in HT22 cells, neuronal culture, and animal model. Mice underwent 6 cycles of DID, and repeated exposure to ethanol may produce very different effects than the acute exposure in cultured cells. It is possible that ethanol could differentially alter Munc13s’ expression in the embryonic stage as compared with the adult stage in the hippocampus. It was reported earlier that there was no expression of Munc13-1 at the embryonic stage until day 16, but both Munc13-1 and Munc13-2 expressed after birth (post-natal day 0) with varied levels of expression. In addition, experimental conditions of ethanol exposure were different in the in vitro and the in vivo models. As stated in the methods, there was no abstinence following ethanol exposure in the primary hippocampal and HT22 cell line, but mice have undergone abstinence each day after drinking and this withdrawal could impact the expression of Munc13-1 and Munc13-2 proteins. Previously, a related study in monkey reported upregulation of both Munc13-1 and Munc13-2 in the basolateral amygdala after long term drinking (Alexander et al., 2018). All these results indicate that effects of ethanol on Munc13 protein expression are dependent on the species, brain regions and the types of drinking paradigms.

A change in the ratio of Munc13-1 and Munc13-2 in the hippocampus after ethanol exposure may profoundly impact glutamate release, neuronal adaptations, and ethanol sensitivity. Comparing the expression of Munc13-1 and Munc13-2 in the basolateral amygdala of the B6 and D2 strains of mice, it was found that synapses of the B6 mice express more Munc13-2 and are more sensitive to the inhibitory effects of ethanol than the D2 mice, highlighting the importance of the Munc13-1 and Munc13-2 ratio in determining the glutamate release and ethanol sensitivity (Gioia et al., 2016, Gioia et al., 2017). Even complete knockdown of Munc13-2 in B6 mice facilitated glutamate release (Gioia et al., 2016).

It is interesting to consider the loss of ethanol-induced changes in Munc13-2 in the heterozygous Munc13-1^+/− given these interactions between Munc13-1 and Munc13-2 (Figures 6B and 7B). The reduction of Munc13-1 levels in the Munc13^+/− heterozygotes leaves Munc13-2 levels in the CA1 resistant to the effects of ethanol. This resistance to ethanol’s effect on Munc13-2 may be dependent on a homeostatic measure of Munc13-1 activity. For example, since Munc13-1 expression in the Munc13^+/− heterozygotes only reaches wild-type levels after ethanol exposure, the regulatory impact of ethanol on Munc13-2 expression may not be “perceived”. In this scenario, an artificial increase in Munc13-1 within these glutamatergic neurons, as seen after DID, would be predicted to drive a decrease in Munc13-2 levels, and potentially alter presynaptic activity.

Although it would be premature to predict the mechanism by which Munc13-1 and Munc13-2 could contribute to the global glutamate release in the hippocampus, structural and functional differences between these two Munc13 isoforms could be responsible for tuning glutamate release. For example, both Munc13-1 and bMunc13-2 have conserved C-terminal regions with a C2 domain but the lack of a C2 domain of bMunc13-2 at the N-terminal regions may alter their function (Andrews-Zwilling et al., 2006). Munc13-1 uses its C2A domain to bind with RIM1 proteins and bring voltage gated Ca²⁺ channels into proximity of the primed vesicles (Betz A et al., 2001). Munc13-2, on the other hand, lacks the C2A domain and acts as a negative modulator through competitive complex formation, thus opposing Munc13-1’s role in synaptic vesicle release (Shin et al., 2010, Chen et al., 2013). Munc13-1 and Munc13-2 also differ in diacylglycerol binding (Rosenmund et al., 2002). Most presynaptic terminals of cortical and hippocampal neurons expressing Munc13-1, with only ~10% express both Munc13-1 and Munc13-2. At the active zone, whereas recruitment and activation of Munc13-1 depend on Rab3-interacting proteins (RIMs), ELKS1 protein recruits Munc13-2. These recruitments determine basal synaptic vesicle priming and short-term plasticity (Kawabe et al., 2017). Furthermore, in excitatory hippocampal neurons, following high-frequency stimulation, Munc13-2-dependent synapses show augmentation of synaptic amplitudes in contrast to Munc13-1-dependent synapses (Rosenmund et al., 2002). In glutamatergic neurons, there are two types of synapses, one is solely Munc13-1 dependent and the other employs Munc13-2 as priming factor, whereas both Munc13-1 and Munc13-2 are found to be redundant in GABAergic cells, suggesting synapse-specificity of these two isoforms (Varoqueaux et al., 2002).

Glutamatergic signaling has a major role in neuronal growth, activity-dependent signaling, and synaptic plasticity (Lynch and Guttmann, 2001, Song and Huganir, 2002). Given Munc13’s major role in glutamate release, we predict that upregulation of Munc13-1 will also promote increased glutamate release into the synaptic cleft. Therefore, in addition to the Munc13-1/2 proteins, we also investigated the expression of several postsynaptic NMDA receptors, including GluN1, GluN2A, and GluN2B, which have been shown to play a role in mediating ethanol dependence, tolerance, and withdrawal (Lovinger et al., 1989, Darstein et al., 2000, Hoffman et al., 2003). Our results show the upregulation of GluN1, GluN2A, and GluN2B expression in the mouse hippocampus after DID and with both GluN1 and GluN2A upregulation in the CA1 subfield (Figure 9). Our results are consistent with the previous observation of similar upregulation of the NMDA receptors in the hippocampus with chronic ethanol treatment (Trevisan et al., 1994, Snell et al., 1996, Devaud and Morrow, 1999, Hendricson et al., 2007) and similar upregulation of these receptors in the CA1 subfield of the hippocampus (Nelson et al., 2005). So, we expect that the glutamatergic neurotransmission will be augmented in the CA1 subfield. However, the overall effect of ethanol on glutamatergic neurotransmission in this subfield of the hippocampus will reflect on the time-dependent integral of its action on all glutamate receptors in both presynaptic and postsynaptic regions, including those that have not been studied here. We also see differential regulation of NMDARs in Munc13-1^+/− mice after DID. The levels of the GluN2A subunit within the CA1 region of these heterozygotes were found to be insensitive to ethanol exposure after DID by both immunoblotting and immunofluorescence analysis. These results are consistent with a reduction in Munc13-1 activity culminating in neural adaptations that reduce the impact of ethanol in the CA1 region of the hippocampus.

In conclusion, our results show that ethanol differentially regulates the expression of Munc13-1 and Munc13-2 in the CA1 subfield of the hippocampus of male mice, and ethanol’s ability to change the ratio of Munc13-1 and Munc13-2 in the hippocampus may have a profound impact in the synaptic release and synaptic adaptations. Of particular significance is the strong correlation among ethanol, Munc13-1 and glutamate in the CA1, known for its role in memory formation (Volianskis et al., 2015, Hadipour et al., 2018) and contextual fear conditioning (Murawski et al., 2012). Our studies also highlighted that ethanol’s effect on Munc13 expression was dependent on drinking paradigms. Further functional studies are required to characterize the role of the Munc13 protein isoforms in neuro-adaptations, drinking behavior, and memory.

Highlights.

• Munc13-1 was upregulated, but Munc13-2 was downregulated in the CA1 subfield of the hippocampus following drinking in the dark paradigm in mice.

• The expression of both Munc13-1 and Munc13-2 increased after ethanol exposure in the primary hippocampal neuronal culture.

• The NMDA receptor subunits, GluN1 and GluN2A were upregulated in the CA1 subfield of the hippocampus and also in the primary culture.

• Ethanol augments glutamatergic neurotransmission via Munc13 proteins.