Ethanol Mediated Inhibition of Synaptic Vesicle Recycling at Amygdala Glutamate Synapses Is Dependent upon Munc13-2

Abstract

Chronic exposure to alcohol produces adaptations within the basolateral amygdala (BLA) that are associated with the development of anxiety-like behaviors during withdrawal. In part, these adaptations are mediated by plasticity in glutamatergic synapses occurring through an AMPA receptor mediated form of post-synaptic facilitation in addition to a unique form of presynaptic facilitation. In comparison to the post-synaptic compartment, relatively less is understood about the mechanisms involved in the acute and chronic effects of ethanol in the presynaptic terminal. Previous research has demonstrated that glutamatergic terminals in the mouse BLA are sensitive to ethanol mediated inhibition of synaptic vesicle recycling in a strain-dependent fashion. Importantly, the strain-dependent differences in presynaptic ethanol sensitivity are in accordance with known strain-dependent differences in ethanol/anxiety interactions. In the present study, we have used a short-hairpin RNA to knockdown the expression of the presynaptic Munc13-2 protein in C57BL/6J mice, whose BLA glutamate terminals are normally ethanol-insensitive. We injected this shRNA, or a scrambled control virus, into the medial prefrontal cortex (mPFC) which sends dense projections to the BLA. Accordingly, this knockdown strategy reduces the expression of the Munc13-2 isoform in mPFC terminals within the BLA and alters presynaptic terminal function in C57BL/6J mice in a manner that phenocopies DBA/2J glutamate terminals which are normally ethanol-sensitive. Here, we provide evidence that manipulation of this single protein, Munc13-2, renders C57BL/6J terminals sensitive to ethanol mediated inhibition of synaptic vesicle recycling and post-tetanic potentiation. Furthermore, we found that this ethanol inhibition was dose dependent. Considering the important role of Munc13 proteins in synaptic plasticity, this study potentially identifies a molecular mechanism regulating the acute presynaptic effects of ethanol to the long lasting adaptations in the BLA that occur during chronic ethanol exposure.

Keywords: lateral/basolateral amygdala; medial prefrontal cortex; presynaptic; shRNA; vesicle priming.

Figure 1

Ethanol inhibits glutamate synaptic vesicle recycling in a Munc13-dependent manner. (A) Ethanol does not inhibit BLA synaptic responses to a 40 Hz, 100 stimulus train delivered to the stria terminalis of B6 mice expressing scrambled shRNA control virus in mPFC inputs. Baseline traces (no ethanol) are indicated in blue while traces recorded in the presence of 80 mM ethanol are indicated in red (A₁,A₂). All responses to the entire 40 Hz stimulus (dots above the traces) are shown in (A₁) while individual responses corresponding to various sections of the stimulus train are illustrated in (A₂). A cumulative amplitude plot obtained during the 40 Hz stimulation (A₃) shows that ethanol did not alter any portion of the stimulus train. Responses to the stimulus train can be used to quantify relative contributions by the readily releasable pool (RRP) and recycling pool of synaptic vesicles (see Methods). Neither the RRP nor the recycling pool contributions (A₄) were affected by ethanol. (B) Expression of Munc13-2 in the mPFC was knocked down by microinjection of a AAV-viral construct expressing a shRNA to bMunc13-2. mPFC provides robust glutamatergic input to the BLA via the stria terminalis (Gioia et al., 2016). Representations of the entire 40 Hz train (B₁) and individual traces within the train (B₂) are illustrated using the same notations as in (A). Munc13-2 knockdown significantly increased ethanol inhibition of both the cumulative response amplitude measured across the entire stimulus train (B₃, paired t-test, ^***p < 0.001) as well as the size of the recycling vesicle pool (B₄, paired t-test, ^***p < 0.001) but, importantly, not the size of the RRP (paired t-test, p = 0.93).

Figure 2

Munc13-2-dependent ethanol inhibition of the recycling pool of synaptic vesicles is concentration-dependent. (A) Concentration-response relationship for inhibition of synaptic vesicle recycling pool size in BLA neurons from B6 animals injected with scrambled control virus. Ethanol did not significantly inhibit recycling pool size (one-way ANOVA, p = 0.51). (B) Concentration-response relationship in BLA neurons injected with virus expressing shRNA against bMunc13-2. Ethanol inhibition of recycling pool size was both significant (one-way ANOVA, ^***p < 0.001) and concentration-dependent (Dunnett's multiple comparison post-test, #p < 0.05, ##p < 0.01, ###p < 0.001 vs. 0 mM ethanol).

Figure 3

Recovery of the RRP following tetanic stimulation is Munc13-2 dependent and ethanol sensitive. (A) Illustration of the stimulation protocol used to measure RRP recovery. To measure recovery of the RRP, a single 40 Hz, 100 stimulus train (①) was followed by a second 40 Hz, 100 stimulus train (②) with increasing inter-train intervals of 1, 5, and 10 s. The effects of ethanol on recovery were measured 10 min after acute application to the slice. (B) Effects of ethanol on RRP recovery measured in BLA neurons from animals injected with the scrambled control virus. Representative traces corresponding to the first response in each train are shown (B₁) with baseline and ethanol condition represented by blue and red traces, respectively, for each recovery interval. Area-under-the-curve measures (B₂) for the first five responses were used as a proxy for the RRP. There was a significant main effect of recovery interval (two-way ANOVA, p < 0.05) but no significant effect of ethanol and no interaction between these main factors. (C) Effects of ethanol on RRP recovery measured in BLA neurons from animals injected with the virus expression shRNA against bMunc13-2. As in (B), representative traces from the first response of each train across the various recovery intervals are shown (C₁). There were significant main effects for both recovery interval (two-way ANOVA, p < 0.001) and ethanol (p < 0.01) with recovery at the 10 s interval being significantly inhibited by ethanol relative to baseline (Bonferroni's post-test, ^*p < 0.05).

Figure 4

Post-tetanic potentiation is Munc13-2 dependent and ethanol sensitive. (A) Post-tetanic potentiation expressed as the percent amplitude of the first response in the second 40 Hz, 100 stimulus train relative to the first response in the first train. In BLA neurons from B6 mice injected with scrambled control virus, increasing recovery intervals significantly increased the initial second train response amplitude relative to the first train percent first response values (two-way ANOVA, p<0.01), but there was no significant effect of 80 mM ethanol on the development of post-tetanic potentiation at the longer recovery intervals. (B) In BLA neurons from B6 mice injected with virus expressing shRNA against bMunc13-2, interval significantly also increased the expression of post-tetanic potentiation (two-way ANOVA, ^**p < 0.01) but this was significantly suppressed by ethanol exposure (## p < 0.01). This effect was specifically significant at the 10 s interval where post-tetantic potentiation was maximal over this interval range (Bonferroni's multiple comparison post-test, ^*p < 0.05).