Stress is a well-known risk factor for subsequent alcohol abuse, but the neural mechanisms underlying interactions between stress and alcohol remain largely unknown. Addictive drug reinforcement and stress signaling involve common neural circuitry, including the mesolimbic dopamine system. We demonstrate in rodents that pre-exposure to stress attenuates alcohol-induced dopamine responses and increases alcohol self-administration. The blunted dopamine signaling resulted from ethanol-induced excitation of GABA neurons in the ventral tegmental area. Excitation of GABA neurons was mediated by GABA A receptor activation and involved stress-induced functional downregulation of the K + , Cl − cotransporter, KCC2. Blocking stress hormone receptors, enhancing KCC2 function, or preventing excitatory GABA signaling by alternative methods all prevented the attenuated alcohol-induced dopamine response and prevented the increased alcohol self-administration. These results demonstrate that stress alters the neural and behavioral responses to alcohol through a neuroendocrine signal that shifts inhibitory GABA transmission toward excitation.

To examine the interaction between stress and ethanol, we exposed drug-naive rats to stress and then measured their subsequent ethanol intake. Concomitant with increases in ethanol self-administration, we show that acute stress attenuates ethanol-induced DA neuron firing in the VTA and DA release at target regions. These stress hormone-dependent effects were mediated by an increase in VTA GABAergic inhibition onto DA neurons in response to ethanol. Stress induced the functional downregulation of KCC2, shifting GABA A receptor signaling toward excitation of some downstream GABA neurons. Pharmacological activation of KCC2 restored the GABAergic circuitry and DA neuron signaling and prevented the escalation in ethanol self-administration induced by stress. These results indicate that a shift toward excitatory GABA signaling within the mesolimbic system is associated with increased drinking after exposure to stress.

Stress-induced changes in alcohol use likely arise from an interaction between the stress and reward systems of the brain (). At the cellular level, both stress hormones and ethanol influence the DA system by direct actions on DA neurons or indirectly via changes in excitatory and inhibitory synaptic inputs (). Stress hormone signaling also may alter midbrain GABAreceptor signaling, but the molecular mechanism underlying this adaptation has not been identified () and may arise from changes in GABA synthesis, in release, or in expression of specific GABAreceptor subunits (). Alternatively, acute stress exposure has been shown to induce a paradoxical shift toward excitatory GABAreceptor signaling within the HPA axis by altering the intracellular anion homeostasis (). Given that the GABAreceptor is a target of ethanol, we postulated that alterations in GABAreceptor transmission could contribute to an interaction between stress and ethanol self-administration.

Excessive alcohol use is among the leading causes of preventable death worldwide (). While many variables contribute to the development of alcohol use disorder (AUD), exposure to stressful life events represents a significant risk factor (). Stress increases alcohol consumption in alcohol-dependent and non-dependent populations (), and stress is thought to underlie a transition to pathological drug use (). Animal studies have revealed interactions between stress and ethanol self-administration under certain stressor and drinking paradigms, but some results have been equivocal (). These discrepancies indicate that the basic neuronal mechanisms linking stress and alcohol use are not well understood.

The impact of stressful life events on excessive alcohol consumption in the French population: findings from the GAZEL cohort study.

Next, we locally microinfused CLP290 bilaterally into the VTA prior to the first ethanol self-administration session and measured ethanol intake over 7 days ( Figure 8 C). Intra-VTA infusion of CLP290 ( Figure S8 F) significantly decreased the average daily ethanol intake in the stressed group back to control levels (0.79 ± 0.05 g/kg) compared to the stressed group that received intra-VTA infusions of vehicle (1.00 ± 0.08 g/kg) ( Figure 8 C; n = 12–13, p < 0.05). These data were indistinguishable from the non-stressed control group ( Figure 8 C, dotted horizontal line). VTA infusions of CLP290 did not alter ethanol consumption in control animals (0.84 ± 0.05 g/kg, data not shown).

Given that stress altered anion homeostasis in VTA GABA neurons, we next determined whether this phenomenon mediated stress-induced alterations in alcohol’s actions. In slices from stressed animals, boosting Clextrusion with the KCC2 activator, CLP290, returned the alcohol-induced firing rate of DA neurons to the control value ( Figure 8 B).

Next, we studied the effect of CLP290 on the stress-induced depolarizing shift in Eand conditional GABAreceptor-mediated excitation of GABA neurons observed after stress (as in Figures 7 C and 5 E, respectively). We found that correcting the Clgradient with CLP290 in slices from stressed animals returned the Eback to the control value ( Figure 8 A) and blocked the stimulation-induced increase in GABA neuron firing ( Figure S8 E).

(C) CLP290 was infused bilaterally intra-VTA (40 μM at 0.5 μL/min). After CLP290 administration, stressed animals consumed significantly less ethanol (blue) compared to vehicle-injected stressed animals (red). Ethanol consumption in unstressed control rats is shown for comparison (dotted horizontal line). ∗ Significantly different from the VTA vehicle group by t test, p < 0.05, n = 12–13 rats/group.

(B) After CLP290 incubation, ethanol-induced VTA DA neuron firing was no longer attenuated after exposure to stress (red) and was similar to the response of the unstressed controls (black). The effect of stress exposure and ethanol application in the absence of CLP290 is shown for comparison (dotted red line), n = 10 cells/group. Note: incubation of VTA slices with CLP290 (before ethanol) did not produce any significant alterations in the mean basal firing rate of DA neurons between control and stress groups.

(A) When brain slices were incubated in the KCC2 activator, CLP290, E GABA was hyperpolarized comparably in VTA GABA neurons from control (black) and stressed (red) animals. The effect of stress exposure on E GABA in the absence of CLP290 is shown for comparison (open red square), n = 6 cells/group.

Based on our findings in Figure 7 , we hypothesized that enhancement of Clextrusion would restore normal GABAreceptor-mediated inhibition in VTA GABA neurons from stressed animals. To enhance Clextrusion specifically, we used recently developed activators of KCC2, CLP257 and CLP290 (). First, we confirmed that CLP257 and CLP290 were effective in VTA GABA neurons by measuring activity-dependent depression of IPSCs (as in Figures 7 D, 7E, and S7 B). After incubation of VTA slices with CLP257 (>1 hr, 5 μM) or CLP290 (>1 hr, 10 μM) the rates of decrease in IPSC amplitude at 0 and −90 mV were similar between control and stressed groups ( Figures S8 A–S8D). Given that CLP257 was shown to be rapidly metabolized in vivo, we further tested CLP290, which was demonstrated to have longer bio-availability ().

To determine whether glucocorticoids mediate the effect of stress on KCC2, VTA slices from control animals were incubated for 1 hr in corticosterone. Similar to in vivo exposure to stress, this treatment did not alter levels of total KCC2 protein in the VTA ( Figure S7 C, blue data). However, the ratio of phosphorylated-S940 KCC2 to total KCC2 protein was significantly lower after corticosterone incubation ( Figure 7 H): 75.5% ± 7.6% for monomer, 78.6% ± 9.4% for dimer. Taken together, these results suggest that stress or corticosterone leads to dephosphorylation of KCC2 protein at S940, which decreases KCC2 function and alters anion homeostasis.

Stress-induced reductions in Clextrusion capacity have been associated with dephosphorylation of the K, Clcotransporter, KCC2, at serine 940 (S940) (). To examine stress-induced alterations in KCC2 protein expression and its phosphorylation in the VTA, we performed western blot analysis using an antibody against total KCC2 protein, as well as a phospho-specific antibody against the KCC2 phosphorylation site S940 (). Immunoblots revealed two prominent bands (∼140 and ∼270 kDa) for both total and S940 KCC2 antibodies, indicating the presence of monomeric and dimeric structures of KCC2 protein ( Figure 7 F) (). No significant differences in the expression of total KCC2 protein between control and stressed groups were observed ( Figure S7 C, red data). In contrast, the ratio of phosphorylated-S940 KCC2 to total KCC2 protein after stress was significantly lower compared to control ( Figures 7 F and 7G): 78.3% ± 5.5% for monomer, 79.6% ± 4.7% for dimer. Importantly, as also reported previously (), immunolabeling analysis in the VTA suggested that KCC2 protein was expressed exclusively on non-DA neurons (data not shown), which is consistent with the presence of another chloride extrusion mechanism in DA neurons ().

A depolarizing shift in Ereflects a higher intracellular chloride concentration, which in adult neurons is often mediated by a decrease in Clextrusion capacity. During prolonged GABAreceptor stimulation, decreased Clextrusion capacity leads to intracellular Claccumulation, culminating in the collapse of the Clgradient and decreased synaptic GABAreceptor inhibition. To test whether exposure to stress weakens Clextrusion in VTA GABA neurons, we applied repetitive GABAreceptor stimulation and measured activity-dependent depression of the IPSCs (). The rate of decrease in IPSC amplitude at the conditions that favor Clinflux (0 mV) depends on Claccumulation and activity-dependent synaptic depression. In contrast, GABAreceptor stimulation at the conditions of Clefflux (−90 mV) only depends on activity-dependent synaptic depression. Upon electrical stimulation at 20 Hz at a holding potential of −90 mV, stress did not affect the rate of synaptic depression in VTA GABA neurons ( Figures S7 A and S7B). In contrast, the decrease of IPSC amplitude at 0 mV occurred significantly faster after stress ( Figures 7 D and 7E): F = 39.9, p < 0.01. The differential effect of stress at −90 mV versus 0 mV indicates that stress increased Claccumulation, suggesting a reduced capacity for Clextrusion in VTA GABA neurons.

We next investigated the causes underlying the increased GABA cell excitation by alcohol. The transition in GABAreceptor signaling ( Figure 5 B) suggests a depolarizing shift in the GABAreversal potential (E) in VTA GABA neurons (). Eis the membrane potential at which evoked IPSCs change their direction from inward to outward. To determine Ein VTA GABA neurons, we performed gramicidin-perforated patch-clamp recordings to preserve the intracellular anion concentrations, and we measured GABAIPSCs at different membrane potentials ( Figure 7 A). VTA GABA neurons from stressed animals showed a significantly more depolarized Evalue compared to controls ( Figures 7 B and 7C): −63.4 ± 3.1 mV after stress (red data) versus −88.2 ± 3.3 mV in controls (black data), n = 10, p < 0.01.

(H) Densiometric analysis revealed a significant reduction in the ratio of pS940 KCC2 to total KCC2 protein in corticosterone-incubated slices from controls. ∗ Significantly different from the control by t test, p < 0.05, n = 16 animals/group.

(G) Densiometric analysis revealed a significant reduction in the ratio of pS940 KCC2 to total KCC2 protein in stressed animals compared to non-stress controls (horizontal dashed line). ∗ Significantly different from the control by t test, p < 0.05, n = 10 animals/group.

(F) Western blot analysis was conducted for total KCC2 and phosphorylated-S940 KCC2 with GAPDH as a loading control. A representative western blot indicates no differences in total KCC2 expression after stress. However, stressed animals showed reduced expression of pS940 KCC2 relative to total KCC2 when compared to non-stressed controls.

(E) At 0 mV, VTA GABA neurons from stressed animals (red) demonstrated a significantly higher rate of evoked IPSC amplitude depression compared to control animals (black). ∗∗ Significantly different from the control by F-test, p < 0.01, n = 10–17 cells/group.

(D) Cl − accumulation was estimated by stimulating repetitive GABA A receptor input. Upon stimulation (20 Hz, V h = 0 mV), a representative GABA neuron from a control (black) animal demonstrated a minor depression of IPSC amplitude compared to the significantly greater depression seen in a GABA neuron from a stressed animal (red).

(C) VTA GABA neurons from stressed animals (red, ∗∗ p < 0.01 by t test) demonstrated a significantly more positive E GABA value compared to neurons from unstressed control animals (black square), n = 10 cells/group.

(B) Representative IPSCs recordings from control (black) and stressed (red) animals at the given holding potentials. The IPSCs reverse direction at the E GABA . For display, the traces were filtered and stimulus artifacts were removed.

(A) GABAergic input onto VTA GABA neurons was recorded using gramicidin perforated patches at different holding potentials to measure stress-induced alterations in anion homeostasis. GABA A IPSCs were evoked by electrical stimulation in the presence of DNQX, AP5, and CGP55845.

To prevent stress from escalating ethanol intake, we bilaterally infused acetazolamide into the VTA prior to each ethanol self-administration session ( Figure 6 E). Intra-VTA infusion of acetazolamide significantly decreased the average daily ethanol intake in the stressed group (0.67 ± 0.06 g/kg) compared to the stressed group that received intra-VTA infusions of vehicle (0.92 ± 0.08 g/kg) ( Figure 6 F; n = 10–14, p < 0.05). These data were indistinguishable from the non-stressed control group ( Figure 6 F, dotted horizontal line). VTA infusions of acetazolamide did not alter ethanol consumption in control animals ( Figure S6 B). All microinfusion sites of acetazolamide in the VTA are shown in Figures S6 C and S6D.

To prevent stress from changing ethanol-induced DA release in the NAc, we infused acetazolamide into the VTA prior to the microdialysis experiments ( Figure 6 C). In contrast to the intra-VTA infusion of vehicle, acetazolamide prevented the inhibitory effect of stress on ethanol-induced [DA] ( Figure 6 D): group × time: F(10,120) = 2.96, p < 0.01. The effect of acetazolamide was indistinguishable from the non-stressed control group ( Figure 6 D, black dotted trace). Microinfusion of acetazolamide outside the VTA did not reverse the inhibitory effect of stress exposure ( Figure S6 A). In unstressed control animals, the microinfusion of acetazolamide in the VTA did not alter the DA response to ethanol (data not shown, n = 6, p > 0.05).

Given that stress promoted excitatory GABA input onto VTA GABA neurons, we tested whether this phenomenon mediated the stress-induced alterations in alcohol’s actions. Based on the results of our repetitive stimulation studies ( Figures 5 D and 5E), we bath applied acetazolamide to prevent the changes in GABA and DA neuron firing to ethanol observed after stress. We found that upon application of ethanol, there was no longer a difference in GABA and DA neuron firing rates between control and stressed groups ( Figures 6 A and 6B , black and red traces compared to the dotted red lines representing stress without acetazolamide).

(F) ACTZ-infused stressed animals consumed significantly less ethanol (blue) compared to vehicle-infused stressed animals (red). Ethanol consumption in unstressed control rats from Figure 1 C is shown for comparison (dotted horizontal line).Significantly different from the VTA vehicle group by t test, p < 0.05, n = 10–14 rats/group.

(E) Stressed animals received bilateral intra-VTA infusions of ACTZ (1 μL at 50 μM) or vehicle prior to the onset of each self-administration session.

(D) In contrast to vehicle injection (red), ethanol-induced DA levels in the NAc following ACTZ infusion were not blunted (blue) and were similar to the control response from Figure 2 B (dotted black line).Significantly different from the VTA vehicle group by ANOVA with repeated measure, p < 0.01, n = 7 rats/group.

(C) Stressed animals received intra-VTA infusion of ACTZ (1 μL at 50 μM) or vehicle prior to the onset of baseline microdialysis sample collection. Subsequent ethanol-induced DA release in the NAc was measured.

(B) In the presence of bath ACTZ, ethanol-induced VTA DA neuron firing was no longer attenuated after exposure to stress (red) and was similar to the response of the unstressed controls (black). The effect of stress exposure and ethanol application in the absence of ACTZ from Figure 3 D is shown for comparison (dotted red line), n = 10–14 cells/group. Note: bath application of ACTZ on VTA slices (before ethanol) did not produce any significant alterations in the mean basal firing rate of DA neurons between control and stress groups.

(A) In the presence of bath-applied acetazolamide (ACTZ), ethanol-induced VTA GABA neuron firing was no longer enhanced after exposure to stress (red) and was similar to the response of the unstressed controls (black). The effect of stress exposure and ethanol application in the absence of acetazolamide from Figure 4 D is shown for comparison (dotted red line), n = 8 cells/group. Note: bath application of acetazolamide on VTA slices (before ethanol) did not produce any significant alterations in the mean basal firing rate of GABA neurons between control and stress groups.

It has been reported () that GABAreceptor-mediated excitation can be prevented by application of acetazolamide, an inhibitor of carbonic anhydrase. Based on these findings, we postulated that acetazolamide would prevent the transition from GABAreceptor-mediated inhibition to excitation of GABA neurons observed after stress. Bath application of acetazolamide (10 μM) did not change basal GABA neuron firing rate between control and stress groups, nor did it change control responses to repetitive stimulation. However, repetitive stimulation in the presence of acetazolamide blocked the increase in GABA neuron firing after stress ( Figures 5 D, bottom, and 5 E, Stress + ACTZ).

Next, we tested whether the stress-induced transition in GABAreceptor signaling was also observed in VTA DA neurons. Upon electrical stimulation of GABAreceptor input (20 Hz, 1 s), DA neurons from control and stressed animals showed decreased firing ( Figures S5 A and S5B), indicating that the direct GABA input onto DA neurons remains inhibitory.

Based on our findings in Figure 4 F, we hypothesized that GABA inputs onto VTA GABA neurons produced excitatory responses. To test this, we measured VTA GABA neuron firing rates in response to repetitive stimulation of synaptic GABAreceptor inputs with ionotropic glutamate receptors inhibited ( Figure 5 A). Upon electrical stimulation (20 Hz for 1 s), GABA neurons from control animals showed decreased firing ( Figures 5 B and 5C, black data), indicative of GABAergic inhibition of the recorded GABA neuron. In marked contrast, slices from stressed animals showed increased GABA neuron firing after GABAreceptor stimulation ( Figures 5 B and 5C, red data): group × time: F(29,464) = 10.03, p < 0.01. This finding directly demonstrates excitation mediated by high-frequency stimulation of GABAreceptors. Importantly, this effect was blocked by picrotoxin ( Figures 5 D, top, and 5 E, Stress + picrotoxin), providing further confirmation that the observed excitation of VTA GABA neurons following stress was mediated by GABAreceptors. In control animals, similar GABAreceptor-mediated excitation was also observed following 1 hr incubation of brain slices in corticosterone ( Figure 5 E, blue bar, Cort), suggesting that prolonged exposure to corticosteroids is sufficient to promote excitatory GABAtransmission onto VTA GABA neurons.

(E) Normalized mean changes in the firing rates of VTA GABA neurons in response to stimulation for each treatment group. Values were averaged over 5 s immediately following termination of the stimulation. In controls, GABA A receptor-mediated increase in the firing rate was observed after slice incubation with corticosterone (blue). Significantly different from the control by t test ( ∗ p < 0.05), n = 8–10 cells/group or ( ∗∗ p < 0.01), n = 6–8 cells/group.

(D) Representative VTA GABA neuron recording from a stressed rat demonstrated that in the presence of picrotoxin (top) or acetazolamide (ACTZ, bottom), repetitive stimulation of GABA inputs failed to increase the firing rate.

(C) Mean changes in VTA GABA neuron firing rate from control (black) and stressed (red) rat slices following repetitive stimulation of synaptic GABA inputs. ∗∗ Significantly different from the control by ANOVA with repeated measures, p < 0.01, n = 8–10 cells/group.

(B) Representative GABA neuron recording from a control animal demonstrated decreased firing rate in response to stimulation of GABAergic input (black). Similar stimulation enhanced the firing rate of VTA GABA neurons from stressed animals (red). For display, the traces were filtered and stimulus artifacts were removed.

(A) VTA GABA neurons were recorded in a cell-attached configuration before and after electrical stimulation of GABA A receptor inputs. To isolate GABA A receptor inputs, we inhibited AMPA, NMDA, and GABA B receptors with antagonists DNQX, AP5, and CGP55845, respectively.

As was previously examined in VTA DA neurons ( Figures 3 E and 3F), we probed for changes in excitatory and inhibitory input onto VTA GABA neurons ex vivo. After application of ethanol, inhibition of ionotropic glutamate receptors with DNQX and AP5 did not prevent the enhanced GABA cell firing rate observed after stress, indicating that this effect was not mediated via changes in glutamatergic neurotransmission ( Figure 4 E): group × time: F(10,110) = 11.19, p < 0.01. However, blocking GABAreceptors with picrotoxin prevented the enhanced firing rate of VTA GABA neurons observed after stress ( Figure 4 F, red trace). This result suggests that, in stressed animals, ethanol increased VTA GABA neuron firing rate via GABAreceptors.

Qualitatively similar effects of stress on ethanol-induced GABA neuron firing were observed during in vivo single-unit electrophysiological recordings in anesthetized rats. Putative GABA neurons in the lateral VTA ( Figure S4 A) were identified based on their electrophysiological and pharmacological properties ( Figures S4 B and S4C). Whereas i.v. administration of ethanol (0.6–1.5 g/kg) slightly decreased the firing rate of VTA GABA neurons in control animals (88.7% ± 5.2%, n = 6), the same doses of ethanol significantly increased spontaneous activity after stress (124.5% ± 8.1%, n = 6, p < 0.01) ( Figures S4 D–S4F).

Patch-clamp recordings indicated that stress induced long-term alterations in GABAergic inputs onto VTA DA neurons. Local VTA GABA neurons can modulate VTA DA neuron activity (). To examine whether stress altered the effects of ethanol on local VTA GABA neurons, we performed electrophysiological recordings of these cells ex vivo ( Figure 4 A). Putative VTA GABA neurons in slices were recorded in the lateral VTA and identified by a combination of factors including small somata size (<20 μm), high pacemaker-like firing rate (>7 Hz), and the lack of I-current ( Figure 4 B) (). Importantly, 47 of 49 VTA neurons with these properties were not immunoreactive for TH ( Figure 4 C). We measured ethanol’s effect on VTA GABA neuron spontaneous firing rate in a cell-attached configuration. Bath-application of ethanol on VTA slices from control animals produced a marginal increase of GABA neuron firing rate ( Figure 4 D, black trace). A significantly higher ethanol-induced increase in GABA neuron firing rate was observed following stress, compared to non-stressed controls ( Figure 4 D, red trace): group × time: F(10,160) = 5.89, p < 0.01.

(F) GABA A receptor antagonist, picrotoxin, prevented the ethanol-induced increase of GABA neuron firing rates after stress (red), and the firing rate was similar to the unstressed controls (black), n = 7–12 cells/group. The basal firing of VTA GABA neurons was not significantly different between control and stress groups (before ethanol): 11.1 ± 1.4 Hz versus 11.3 ± 1.3 Hz, p > 0.05, n = 9.

(E) Glutamatergic receptor antagonists (DNQX and AP5) did not prevent the enhanced firing rate of VTA GABA neurons from stressed animals in response to ethanol. ∗∗ Significantly different from the control by ANOVA with repeated measures, p < 0.01, n = 6–7 cells/group.

(D) VTA GABA neurons from stressed rats (red) showed a greater increase in firing rate following ethanol application (gray horizontal bar) than did GABA neurons from the unstressed controls (black). ∗∗ Significantly different from the control by ANOVA with repeated measures, p < 0.01, n = 9 cells/group.

(A) Spontaneous firing rate of VTA GABA neurons was measured using the cell-attached configuration, and the whole-cell configuration was used to identify GABA neurons electrophysiologically and histochemically upon termination of the recording.

Effects of arousal- and feeding-related neuropeptides on dopaminergic and GABAergic neurons in the ventral tegmental area of the rat.

To demonstrate the direct action of glucocorticoids in mediating the effects of stress, brain slices from control rats were incubated in corticosterone (1 μM) for 1 hr (). DA neurons from control animals treated with corticosterone showed a potentiation of ethanol-induced sIPSC frequency that was indistinguishable from stressed animals ( Figure 3 I, dark blue bar): 182.7% ± 10.7%, n = 8. Importantly, incubation with RU486 prevented this corticosterone-mediated increase in sIPSC frequency ( Figure 3 I, light blue bar): 114.5% ± 5.5%, n = 6. Increased frequency, but not amplitude, of sIPSCs suggests that the change caused by stress and corticosterone resides with the presynaptic neuron (i.e., the GABA neuron) not with the postsynaptic neuron (i.e., the DA neuron) (see Figure 3 G).

Altered inhibitory control of DA neurons after stress could be mediated through changes in presynaptic neurotransmitter release or postsynaptic receptor responses. To determine the site of adaptation for GABAergic neurotransmission, we performed whole-cell patch-clamp recordings of VTA DA neurons and we measured spontaneous inhibitory postsynaptic currents (sIPSC) in the presence of ethanol ( Figure 3 G). In control animals, bath-applied ethanol produced a small increase in sIPSC frequency (116.1% ± 5.0%). In contrast, DA neurons from stressed animals showed significantly greater ethanol-induced potentiation of sIPSC frequency compared to the control response (171.5% ± 7.2%) ( Figures 3 H and 3I, black and red data; n = 8–10, p < 0.01). Systemic injection of RU486 prior to stress prevented the stress-mediated increase in sIPSC frequency observed after ethanol application ( Figure 3 I, gray bar): 110.6% ± 4.5%, n = 8.

Next, we tested the contribution of GABAergic signaling by comparing ethanol-induced VTA DA neuron firing between control and stress groups in the presence of picrotoxin, a GABAreceptor antagonist. Picrotoxin (50 μM) did not significantly alter the basal firing rate between the control and stress groups. However, upon application of ethanol, the presence of picrotoxin prevented the blunted DA responses to ethanol observed after stress ( Figure 3 F, red trace). This finding suggests that stress reduced DA responses to ethanol via changes in GABAergic neurotransmission.

Reduced DA activity after stress could be mediated through decreased excitation or increased inhibition of DA neurons. To probe the relative contribution of excitatory neurotransmission, we recorded ethanol-induced DA activity during inhibition of ionotropic glutamate receptors with DNQX (20 μM) and AP5 (50 μM), which inhibit AMPA and NMDA receptors, respectively. When comparing control and stressed groups, the basal firing rate of DA neurons was not significantly altered by bath application of DNQX and AP5. Upon ethanol application, DA neurons from stressed animals again showed reduced DA activity compared to controls ( Figure 3 E): group × time: F(10,130) = 6, p < 0.01, indicating that this effect of stress was not mediated via changes in glutamatergic neurotransmission.

Bath application of 50-mM ethanol increased the spontaneous firing rate of DA neurons from unstressed control rats ( Figures 3 C and 3D, black data), but DA cells from stressed animals failed to show significant increases in firing rate upon ethanol application ( Figures 3 C and 3D, red data): group × time: F(10,200) = 3.55, p < 0.01.

To examine the cellular mechanisms of the blunted DA signaling after stress, we measured the ex vivo responses of VTA DA neurons to ethanol ( Figure 3 A). Midbrain horizontal slices containing the VTA were cut 15 hr after restraint stress, and patch-clamp cell-attached recordings were performed on DA neurons. Putative DA neurons were recorded from the lateral VTA and were identified based on established electrophysiological criteria ( Figures S3 C–S3E). In 34 of 36 cells displaying these electrophysiological properties, the DA phenotype was confirmed by tyrosine hydroxylase staining ( Figure 3 B).

(I) Mean changes in the sIPSC frequency after ethanol application in VTA DA neurons. DA neurons from stressed animals (red) demonstrated a significantly increased ethanol-mediated sIPSC frequency compared to neurons from unstressed controls (black). Systemic inhibition of glucocorticoid receptors with RU486 (40 mg/kg) prior to stress prevented elevated sIPSC frequency (gray). Incubation of VTA slices from control animals with corticosterone increased ethanol-mediated sIPSC frequency in DA neurons up to stress levels (dark blue). Co-incubation with RU486 prevented this increase (light blue). Incubation of brain slices with RU486 and/or corticosterone did not alter basal parameters of sIPSCs (data not shown). Across all groups, ethanol application did not produce significant changes in the sIPSC amplitudes (data not shown, n = 6–10, p > 0.05). ∗∗ Significantly different from control and RU486-treated groups by t test, p < 0.01, n = 6–10 cells/group.

(H) Representative recordings of sIPSCs before and after ethanol administration in the control (black) and stressed (red) groups.

(G) Spontaneous inhibitory postsynaptic currents (sIPSCs) onto VTA DA neurons were recorded using the whole-cell patch-clamp configuration. No significant differences were detected in the mean basal sIPSC frequency or amplitude between stressed and control groups before ethanol: frequency, 2.4 ± 0.6 Hz in control versus 2.3 ± 0.3 Hz after stress; amplitude, 26.2 ± 4.0 pA in control versus 30.8 ± 3.6 pA after stress, p > 0.05, n = 8–10.

(E) Glutamatergic receptor antagonists (DNQX and AP5) did not prevent ethanol-induced attenuation of DA cell firing after exposure to stress (red) and showed a lower firing rate than unstressed controls (black). ∗∗ Significantly different from the control by ANOVA with repeated measures, p < 0.01, n = 7–8 cells/group.

(D) Normalized spontaneous firing rates of VTA DA neurons following ethanol (gray horizontal bar) in unstressed control group (black) and in rats exposed to stress 15 hr prior to cutting the slice (red). ∗∗ Significantly different from the control by ANOVA with repeated measures, p < 0.01, n = 10–12 cells/group.

(C) Representative recordings from DA neurons before and after bath ethanol administration in the control and stressed (red) groups. No significant differences in mean basal firing rate were detected before ethanol: 2.3 ± 0.2 Hz in control versus 2.0 ± 0.2 Hz after stress, n = 10–12.

(A) Spontaneous firing rate of VTA DA neurons was measured using the cell-attached configuration, and the whole-cell configuration was used to identify DA neurons electrophysiologically and histochemically upon termination of the experiment.

The spontaneous firing rate of VTA DA neurons was measured before and after intravenous infusion of ethanol (0.6–1.5 g/kg). Ethanol administration induced an increase in the spontaneous firing rate of VTA DA neurons in control animals (128.6% ± 7.6%). In contrast, stressed animals failed to demonstrate a significant firing-rate increase upon ethanol administration (102.3% ± 3.1%) ( Figures 2 E and 2F; n = 8–13, p < 0.01). Together with the microdialysis experiments, these data indicate that ethanol-induced DA signaling was blunted after exposure to stress.

Ethanol stimulates DA release in the NAc by increasing the firing rate of VTA DA neurons (). To determine whether restrained stress altered DA neuron firing rate in vivo, we conducted single-unit recordings of VTA DA neurons in anesthetized rats 15 hr after stress ( Figure 2 C). DA neurons were recorded in the lateral VTA ( Figure 2 D) and were identified based on their electrophysiological and pharmacological properties ( Figures S3 A and S3B).

Next, we tested whether glucocorticoid receptor activation mediated the stress-induced decrease in ethanol-evoked DA release. Systemic pretreatment with RU486 prevented the inhibitory effect of stress on ethanol-induced DA release ( Figure 2 B, gray data). The distribution of the microdialysis probe placements within the NAc is shown in Figure S2

Because ethanol self-administration involves DA signaling in the nucleus accumbens (NAc) (), we hypothesized that stress might also alter ethanol-induced DA release in the NAc. To test this hypothesis, we subjected rats to 1 hr restraint stress 15 hr prior to ethanol exposure, and we collected microdialysis samples to measure changes in the extracellular DA concentration in response to ethanol administration ( Figure 2 A). We observed a sustained increase in DA levels in the control group ( Figure 2 B, black trace), but stressed animals showed a blunted DA response to ethanol ( Figure 2 B, red trace): group × time: F(10,160) = 2.79, p < 0.01. No significant differences in baseline DA levels were detected between control and stress groups: 2.0 ± 0.2 nM in control versus 2.1 ± 0.2 nM after stress.

(F) In the control group (black), ethanol increased the firing rate of putative DA neurons. In the stressed group (red), ethanol failed to increase the firing rate of putative DA neurons. ∗∗ Significantly different from the control group by t test, p < 0.01, n = 8–13 rats/group.

(E) Representative recordings from putative DA neurons before and after ethanol administration (0.6–1.5 g/kg) in the control (black) and stressed (red) groups. No significant differences in the mean basal firing rate were detected between control and stressed groups: 6.3 ± 0.7 Hz in control versus 8.0 ± 1.0 Hz after stress, n = 8–13, p > 0.05.

(C) The spontaneous firing rate of VTA DA neurons was measured in vivo using single-unit recordings in anesthetized animals.

(B) Time course of DA release in the NAc following in vivo ethanol administration in control rats (black), in stressed rats (red), and in rats injected with RU486 (i.p.) prior to stress exposure (gray). Ethanol (1.5 g/kg) was injected i.v. during the 5 min period (shaded vertical gray bar). ∗ Significantly different from the control group and from the RU486+Stress group by ANOVA with repeated measures, p < 0.05, n = 7–9 rats/group.

Restraint stress is known to increase circulating glucocorticoid levels (). To determine whether the effect of stress on drinking was mediated by stress hormone signaling, we pretreated a separate group of rats with RU486 (a glucocorticoid receptor antagonist) prior to stress. Pretreatment with systemic RU486 prevented the stress from increasing subsequent ethanol self-administration ( Figure 1 C, dark blue bar; 0.67 ± 0.07 g/kg). To determine whether stress hormone signaling acted locally within the DA system (), we microinfused RU486 bilaterally into the VTA prior to stress exposure. Intra-VTA administration of RU486 ( Figure S1 C) prevented the stress from increasing subsequent ethanol self-administration ( Figure 1 C, light blue bar; 0.55 ± 0.05 g/kg), revealing that the effect of stress requires glucocorticoid receptor activation within the VTA. In non-stressed control rats, RU486 did not significantly influence ethanol intake when administered systemically or by local microinfusion ( Figure 1 C, gray bar on the right; 0.67 ± 0.04 g/kg), demonstrating a selective effect of RU486 on stress-induced drinking. Additional control experiments with saccharin and palatable food suggest that the effect of stress we observe was specific to ethanol self-administration (see Supplemental Experimental Procedures ).

Pre-exposure to stress caused a significant and long-lasting increase in ethanol self-administration compared to the non-stressed control group ( Figure 1 A): group: F(1,21) = 19.32, p < 0.01. Blood-ethanol levels were measured in a subset of animals and were correlated with ethanol intake ( Figure 1 B). Stressed rats showed significantly higher blood-ethanol levels (120.8 ± 13.6 mg/dL, n = 5) than non-stressed rats (61.3 ± 4.3 mg/dL, n = 10, p < 0.01). Mean intake of ethanol over the first 7 days was 0.74 ± 0.03 g/kg for the control group (n = 19) and 0.95 ± 0.03 g/kg for the stressed group (n = 16, p < 0.01) ( Figure 1 C, black and red bars). Elevated drinking after stress was also observed at higher ethanol concentrations (7%–10%) over a 3-week period ( Figures S1 A and S1B). Therefore, acute restraint stress induced robust changes in the acquisition and maintenance of ethanol-drinking behavior.

We first examined how a single episode of restraint stress alters subsequent ethanol intake measured during daily operant self-administration sessions ( Figure 1 A). Stable lever pressing for saccharin (0.125%, w/v) was first established followed by the introduction of ethanol (2%–4%) into the drinking solution (). Animals were subjected to restraint stress (1 hr) approximately 15 hr prior to the first ethanol self-administration session. The 15 hr separation between the stress and ethanol self-administration was chosen to examine the lasting impact on neural circuits, not the immediate proximal influence of the stressor itself ().

(C) Mean daily ethanol intake over the first seven self-administration sessions. Stressed rats (red bar) consumed significantly more ethanol (g/kg) compared to control rats (black bar). Blockade of glucocorticoid receptors with RU486 systemically (dark blue, 40 mg/kg, i.p.) or locally in the VTA (light blue, 40 ng/1 μL) prior to stress prevented increases in ethanol intake, n = 10, 14. RU486 administered systemically or intra-VTA to control animals did not alter ethanol intake, n = 9, gray bar. ∗∗ Significantly different from all groups by t test, p < 0.01.

(B) Ethanol intake (g/kg) versus blood ethanol levels (mg/dL). Blood ethanol was measured immediately after the self-administration session in control (black) and stressed (red) animals. A regression analysis showed a significant and positive correlation between ethanol intake and blood ethanol levels, F(1,13) = 162.7, p < 0.01.

(A) Rats self-administered saccharin prior to fading ethanol into the drinking solution. Rats were subjected to a single restraint stress 15–20 hr before the first ethanol exposure (red arrow). Daily fluid intake was measured in control and stressed rats. Stressed rats showed greater ethanol intake compared to unstressed control rats. ∗∗ Significantly different from the control group by ANOVA with repeated measures, p < 0.01, n = 16–19 rats/group.

Discussion

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Baulieu E.E. RU486 (mifepristone): mechanisms of action and clinical uses. While epidemiological studies consistently report associations between stress and ethanol consumption (), the underlying neuronal effects have not been well delineated. We found that alterations in GABAreceptor responses on GABAergic neurons of the VTA correlate with an increase in ethanol self-administration induced by temporally distant, acute stress. After stress, we detected enhanced VTA GABAergic inhibition of DA neurons and reduced mesolimbic DA release in response to ethanol. Blunted DA signaling was mediated by a transition toward excitatory GABAreceptor signaling and was associated with decreased Clextrusion capacity in VTA GABA neurons. Stress-induced adaptations were prevented by acetazolamide () or by CLP290 (). The effect of stress on GABA transmission was recapitulated in vitro by corticosterone exposure and was prevented by pharmacological blockade of glucocorticoid receptors (). Most importantly, when acetazolamide, CLP290, or RU486 were locally infused in the VTA in vivo, stress no longer increased ethanol self-administration.

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Gonzales R.A. Ethanol preference is inversely correlated with ethanol-induced dopamine release in 2 substrains of C57BL/6 mice. The decreased DA response to ethanol was correlated to the stress event and to the excitatory GABA signaling. Although the DA response was not directly examined as a cause of the increased self-administration, others have reported that enhancing DA signaling exogenously attenuates voluntary drinking in rats (). Furthermore, the correlation between decreased ethanol-induced DA release and increased self-administration has been previously reported in rodent studies ().

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Russo S.J. Peripheral and Central Mechanisms of Stress Resilience. Although animal studies generally support the hypothesis that stress increases ethanol consumption, some results have shown that stress decreases intake or has no effect on it (). These differences arise from a combination of factors, including the type of stressor used, the duration or timing of the stressor, as well as the type of drinking paradigm employed (). An important parameter in our experimental design is that the stress exposure was well separated (15–20 hr) from the ethanol self-administration, which allowed us to examine the lasting neural circuit consequences of the treatment not the proximal effect of stress itself. In our study, we kept the ethanol content of the drinking solutions relatively low during the acquisition phase, which likely resulted in less variability in self-administration. The rats experienced less of the aversive stimulus cues of ethanol while still achieving significant blood-ethanol levels. In addition, some animals show resilience to the effects of stress (), so it is essential to verify that there is a physiological response to the stressor and to exclude animals that do not show that response.