Binge ethanol drinking associated with sex-dependent plasticity of neurons in the insula that project to the bed nucleus of the stria terminalis

Abstract

Modifications in brain regions that govern reward-seeking are thought to contribute to persistent behaviors that are heavily associated with alcohol-use disorder (AUD) including binge ethanol drinking. The bed nucleus of the stria terminalis (BNST) is a critical node linked to both alcohol consumption and the onset, maintenance and progression of adaptive anxiety and stress-related disorders. Differences in anatomy, connectivity and receptor subpopulations, make the BNST a sexually dimorphic region. Previous work indicates that the ventral BNST (vBNST) receives input from the insular cortex (IC), a brain region involved in processing the body's internal state. This IC-vBNST projection has also been implicated in emotional and reward-seeking processes. Therefore, we examined the functional properties of vBNST-projecting, IC neurons in male and female mice that have undergone short-term ethanol exposure and abstinence using a voluntary Drinking in the Dark paradigm (DID) paired with whole-cell slice electrophysiology. First we show that IC neurons projected predominantly to the vBNST. Next, our data show that short-term ethanol exposure and abstinence enhanced excitatory synaptic strength onto vBNST-projecting, IC neurons in both sexes. However, we observed diametrically opposing modifications in excitability across sexes. In particular, short-term ethanol exposure resulted in increased intrinsic excitability of vBNST-projecting, IC neurons in females but not in males. Furthermore, in females, abstinence decreased the excitability of these same neurons. Taken together these findings show that short-term ethanol exposure, as well as the abstinence cause sex-related adaptations in BNST-projecting, IC neurons.

Keywords: BNST; Binge ethanol drinking; Electrophysiology; Insula; Sex difference.

Figure 1:

The IC is connected to both the vBNST and NAc, but a significantly larger population of IC neurons projects to the vBNST. A) Cartoon of viral injection of pAAV-CAG-GFP in the NAc and pAAV-CAG-tdTomato in vBNST. B) Representative image of a coronal section (5X magnification) showing pAAV-CAG-GFP expression (green) in NAc (Top) and pAAV-CAG-tdTomato expression (red) in vBNST (Bottom). C) Schematic and representative image (10X magnification) of the IC showing pAAV-CAG-GFP (green) and pAAV-CAG-tdTomato (red) expression. D) Top: representative image (10X magnification) of IC depicting viral expression of pAAV-CAG-GFP (left), PAAV-CAG-tdTomato (middle), merged (right). Bottom: representative image (20X magnification) depicting viral expression of pAAV-CAG-GFP (left), PAAV-CAG-tdTomato (middle), merged (right). E) Top left: Pie-chart showing the % of IC neurons projecting exclusively to the vBNST (red), to the NAc (green) and to both (blue). Bottom right: graph showing the % of neurons IC-BNST only (red), IC-NAc only (green) and both (blue) in females and males. Data expressed as Mean±SEM. (**) denotes P<0.01; (***) denotes P<0.001.

Figure 2:

Short-term ethanol exposure leads to modest escalation in binge drinking. Mice underwent the drinking in the dark (DID) paradigm and were either exposed to water or ethanol during their 4-hour drinking session that starts 3 hours into their dark cycle. A) Consumption of either water or ethanol (g/kg) for each session over the 4 DID sessions. B) Daily ethanol consumption (g/kg) in the 4-day DID paradigm. We found a significant increase in ethanol consumption in mice after 4 sessions of DID. C) Ethanol consumption over 4 DID sessions in male and female mice. D) 20X Nissl stain of the injected retrograde CAG-GFP in the vBNST of a C57/Bl6 mouse (top) and a 20X nissl stain of the IC indicating labeled GFP containing cell bodies (bottom). E) Two 40X magnification DIC images of the same IC neuron from a CAGvBNST-IC::Retrograde-GFP expressing mouse under Left: wide-field fluorescent illumination to excite GFP in IC expressing neurons; Right: Infrared illumination of the same neuron. Data expressed as Mean±SEM. (*) denotes P<0.05; (**) denotes P<0.01.

Figure 3:

Short-term ethanol exposure differentially modulates neuronal excitability in IC-vBNST neurons in male and female mice. A) Representative traces of vBNST projecting, IC neurons obtained from females (H₂O t=0 red) and males (H2O t=0 blue) in response to 120 pA/s current ramp. B) Representative traces of vBNST projecting, IC neurons obtained from EtOH t=0 females (red) and EtOH t=0 males (blue) in response to 120 pA/s current ramp. C) Number of spikes in response to 5 steps of current injection (40 pA each). There was a significant increase in the number of spikes fired in the EtOH t=0 female group. D) Correlation between ethanol consumption and spike activity. EtOH t=0 female group shows a positive correlation. E) Resting membrane potential (RMP), F) Rheobase and G) membrane resistance (Rm). Data expressed as Mean±SEM. (***) denotes P<0.001.

Figure 4:

Short-term ethanol exposure increases the frequency of spontaneous postsynaptic currents in both males and females. A) Representative traces of spontaneous excitatory postsynaptic currents (sEPSCs) from vBNST projecting, IC neurons in female mice (H2O t=0 red) and male mice (H2O t=0 blue). B) Representative traces of sEPSCs from vBNST projecting, IC neurons in female mice (EtOH t=0 red) and male mice (EtOH t=0 blue). C) Spontaneous EPSC (sEPSC) amplitude and D) frequency E) Representative traces of spontaneous inhibitory postsynaptic currents (sIPSCs) from vBNST projecting, IC neurons in female mice (H2O t=0 red) and male mice (H2O t=0 blue). F) Representative traces of sIPSCs from vBNST projecting, IC neurons in female mice (EtOH t=0 red) and male mice (EtOH t=0 blue). G) sEPSC amplitude, H) frequency and I) excitation-inhibition ratio (E/I). Data expressed as Mean±SEM. (*) denotes P<0.05; (***) denotes P<0.001.

Figure 5:

Abstinence from a short-term ethanol exposure reduces the intrinsic excitability of IC-BNST neurons in female mice. A) Representative traces of vBNST projecting, IC neurons obtained from females (H₂O t=48 red) and males (H₂O t=48 blue) in response to 120 pA/s current ramp. B) Representative traces of vBNST projecting, IC neurons obtained from females (EtOH t=48 red) and males (EtOH t=48 blue) in response to 120 pA/s current ramp. C) Number of spikes in response to 5 steps of current injection (40 pA each). There was a significant decrease in the number of spikes fired in the EtOH t=48 female group. D) Correlation between ethanol consumption and spike activity. E) RMP, F) Rheobase and G) membrane resistance (Rm). Data expressed as Mean±SEM. (***) denotes P<0.001.

Figure 6:

Abstinence from short-term ethanol exposure increases spontaneous excitatory drive of IC-BNST projecting neurons in both sexes. A) Representative traces of spontaneous excitatory postsynaptic currents (sEPSCs) from vBNST projecting, IC neurons in female mice (H2O t=48 red) and male mice (H2O t=48 blue). B) Representative traces of sEPSCs from vBNST projecting, IC neurons in female mice (EtOH t=48 red) and male mice (EtOH t=48 blue). C) sEPSC amplitude and D) frequency. E) Representative traces of spontaneous inhibitory postsynaptic currents (sIPSCs) from vBNST projecting, IC neurons in female mice (H2O t=48 red) and male mice (H2O t=48 blue). F) Representative traces of sIPSCs from vBNST projecting, IC neurons in female mice (EtOH t=48 red) and male mice (EtOH t=48 blue). G) sEPSC amplitude, H) frequency and I) excitation-inhibition ratio (E/I). Data expressed as Mean±SEM. (*) denotes P<0.05; (**) denotes P<0.01; (***) denotes P<0.001.

Figure 7:

Short-term ethanol exposure and abstinence does not result in synaptic facilitation and changes in AMPA\NMDA ratio. A) Representative traces of pair of electrically evoked excitatory postsynaptic currents (50 ms apart) from vBNST projecting, IC neurons in females (H2O t=0 red), males (H2O t=0 blue) on the left, and females (EtOH t=0 red), males (EtOH t=0 blue) on the right. B) Representative traces of pair of electrically evoked excitatory postsynaptic currents from vBNST projecting, IC neurons in females (H2O t=48 red), males (H2O t=48 blue) on the left, and females (EtOH t=48 red), males (EtOH t=48 blue) on the right. C) Paired pulse ratio (PPR) after short-term ethanol exposure (t=0). D) PPR after abstinence (t=48). E) Representative traces of electrically evoked currents at −70 mV and +40 mV from vBNST projecting, IC neurons in females (H2O t=0 red), males (H2O t=0 blue) on the left, and females (EtOH t=0 red), males (EtOH t=0 blue) on the right. F) Representative traces of electrically evoked currents at −70 mV and +40 mV from vBNST projecting, IC neurons in females (H2O t=48 red), males (H2O t=48 blue) on the left, and females (EtOH t=48 red), males (EtOH t=48 blue) on the right. G) AMPA/NMDA ratio after short-term ethanol exposure (t=0). H) Graph showing AMPA/NMDA ratio after ethanol abstinence (t=48). Data expressed as Mean±SEM.

Figure 8:

Bath application of EtOH does not alter the effect on intrinsic excitability and spontaneous activity observed in IC-vBNST neurons after short term ethanol exposure (t=0) A) Representative traces of spontaneous excitatory postsynaptic currents (sEPSCs) from vBNST projecting, IC neurons in female mice (EtOH t=0) during baseline (top) and bath application of EtOH 44 mM (bottom). B) Representative traces of spontaneous excitatory postsynaptic currents (sEPSCs) from vBNST projecting, IC neurons after short-term ethanol exposure (t=0) male mice during baseline (top) and bath application of EtOH 44 mM (bottom). C) sEPSC amplitude and D) frequency in females. E) sEPSC amplitude and F) frequency in males. G) Representative traces of vBNST projecting, IC neurons obtained from females (EtOH t=0) during baseline (top) and bath application of EtOH 44 mM (bottom) in response to 120 pA/s current ramp. H) Representative traces of vBNST projecting, IC neurons obtained from males (EtOH t=0) during baseline (top) and bath application of EtOH 44 mM (bottom) in response to 120 pA/s current ramp. I) Number of spikes in response to 5 steps of current injection (40 pA each) in females and J) in males. K) Change (%) in the number of spikes across the current injections in males and females.Data expressed as Mean±SEM.