VTA dopamine inputs activate accumbal D1 receptors to promote alcohol seeking

Summary

The mesolimbic dopamine system is central to cue-induced alcohol craving, but the role of D1 receptors (D1R) in the nucleus accumbens (NAc) remains unclear. Here, we show that cue-induced, but not context-elicited, alcohol seeking selectively upregulates NAc D1R protein expression in mice, which positively correlates with craving intensity. Systemic D1R antagonist SCH23390 suppresses cue-induced seeking and reverses c-Fos activation in the NAc and ventral pallidum. Chemogenetic inhibition of ventral tegmental area (VTA) dopamine neurons attenuates craving, an effect fully rescued by intra-NAc D1R agonist SKF38393 without affecting locomotion. These findings establish that dopaminergic projections from the VTA to the NAc drive cue-specific alcohol seeking through D1 receptor-dependent manner.

Graphical abstract

Highlights

• •Cue-induced alcohol seeking upregulates D1R expression in NAc
• •D1R antagonist suppresses cue-induced seeking, and reverses NAc c-Fos expression
• •Chemogenetic inhibition of VTA dopamine neurons attenuates cue-induced craving
• •Intra-Nac application of D1R agonist restores the inhibited craving behavior

Zoology; Phylogenetics; Paleobiology; Paleogenetics

Introduction

Alcohol use disorder (AUD) is a chronic relapsing condition, fundamentally characterized by a loss of control over alcohol consumption despite severe negative consequences across social, occupational, or health domains.^1,2,3,4 At the core of AUD’s clinical presentation is alcohol craving—a compelling subjective urge to drink that is now formally recognized as a key diagnostic criterion.⁵ This craving is not merely a symptom but a critical driver of the disorder, serving as one of the strongest predictors of relapse following periods of abstinence.^6,7 Clinical evidence consistently shows that the severity of craving correlates significantly with subsequent drinking behaviors.⁸ Notably, elevated baseline craving can forecast relapse risk,⁸ and cue-induced exacerbations of craving potently amplify vulnerability to resuming alcohol use,^9,10,11,12 as corroborated by neuroimaging studies linking cue reactivity in reward-related brain circuits to relapse outcomes.¹³

The neuropathology of AUD and alcohol craving involves dysregulation across a distributed network of brain circuits. For instance, aberrant signaling between the medial prefrontal cortex (mPFC) and basolateral amygdala (BLA) is closely linked to the loss of behavioral control and compulsive alcohol-seeking behaviors.¹⁴ The orbitofrontal cortex (OFC), a key node in the addiction network, modulates impulsive decision-making, and its dysfunctional communication with the dorsomedial striatum (DMS) contributes to impaired goal-directed behavioral control.^15,16 Parallelly, the ventral hippocampus (vHip) influences reward-seeking motivation through its projections to the nucleus accumbens (NAc), a pathway that demonstrates hyperactivity in alcohol dependence.¹⁷ Furthermore, the anterior insular cortex (aIC)→BLA circuit is implicated in processing the aversive properties of alcohol, with its plasticity critically shaping motivational states.¹⁸ As AUD progresses, craving becomes more habitual and automated, a shift associated with dorsal striatal hyperactivation. Neuroimaging evidence consistently correlates alcohol cue reactivity in the dorsal striatum and related networks with elevated craving scores on clinical assessments like the clinical alcohol craving scale (CAS-A).¹⁹

While the neural substrates of craving are diverse, the mesolimbic dopamine system—particularly the dopaminergic projections from the ventral tegmental area (VTA) to the NAc—is considered the cornerstone pathway for reward processing and, critically, for cue reactivity.²⁰ Within this circuit, the dopamine D1 receptor (D1R) emerges as a pivotal molecular switch for craving behaviors. Evidence from psychostimulant models illustrates that a shift in the balance between D1R and D2R signaling—characterized by upregulated D1R expression and function alongside D2R downregulation—creates a neuroadaptive excitatory imbalance that drives drug-seeking and reinstatement.^21,22 This D1R-mediated mechanism appears to be a convergent mechanism in addiction neuroplasticity, as evidenced by findings across diverse drug classes—including methamphetamine, morphine, and cocaine—where manipulations of NAc D1R signaling consistently dictate the expression of drug-seeking behaviors.^21,23,24 Conversely, interventions that normalize this imbalance, such as electroacupuncture therapy, have been shown to mitigate craving by downregulating NAc D1R and upregulating D2R expression, thereby suppressing VTA→NAc hyperexcitability.²⁵ Despite this compelling evidence for a trans-substance role of D1R signaling, a critical and substance-specific question remains: Does the VTA→NAc dopaminergic pathway drive cue-induced alcohol craving specifically through the activation of D1Rs on NAc medium spiny neurons (D1-MSNs), analogous to its role in psychostimulant models? To address this, the present study tests the hypothesis that cue-specific alcohol seeking is dependent on D1 receptor activation within the NAc, downstream of VTA dopaminergic inputs.

Results

Successful establishment of a cue-induced alcohol seeking model in mice

To investigate the neural mechanisms underlying alcohol craving, we first established a robust operant self-administration model in C57BL/6J mice. Mice were trained to press an active lever for a 10% alcohol solution reward under progressively increasing fixed-ratio (FR) schedules. As the response requirement escalated from FR1 to FR3 over 16 days, the number of active lever presses significantly increased and stabilized, demonstrating efficient acquisition of the alcohol-seeking behavior. On the final day of training (FR3), mice exhibited 108.7 ± 7.70 presses on the active lever, compared to only 10.5 ± 4.48 on the inactive lever (Figure 1A). This was associated with the retrieval of 30.33 ± 1.50 rewards, corresponding to an alcohol intake of 1.97 ± 0.09 g/kg (Figure 1B), confirming stable operant performance.

Figure 1.

Establishment of an operant alcohol self-administration and craving model

(A) The time course of active and inactive lever presses over time during fixed-ratio (FR) training sessions (FR1, FR2, and FR3).

(B) Number of rewards earned and corresponding alcohol intake (g/kg) during FR training.

(C) Lever presses across daily extinction sessions, during which lever presses no longer delivered rewards or cues.

(D) Reinstatement of alcohol-seeking behavior following re-exposure to the alcohol-predictive cue. Data are represented as mean ± SEM; n = 6 mice per group. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Subsequently, mice underwent extinction training, during which lever presses no longer resulted in reward or cue presentation. Active lever presses progressively decreased across extinction sessions (Figure 1C), from 88.5 ± 5.74 on day 1–17.33 ± 3.53 on day 7. Critically, from extinction day 5 onward, no significant difference was observed between active and inactive lever presses (two-way ANOVA, F(1,10) = 109.8, p < 0.0001; day 5: 27.33 ± 3.02 vs. 16.00 ± 3.45, p = 0.3775; day 7: 17.33 ± 3.53 vs. 10.33 ± 1.84, p > 0.9999), indicating successful extinction of the learned behavior.

To assess cue-induced craving, mice were re-exposed to the alcohol-associated cue light in the absence of reward. This cue presentation robustly reinstated alcohol-seeking behavior, with active lever presses increasing to 70.83 ± 6.27 (Figure 1D), a significant increase over the final extinction session (one-way ANOVA, F(2,(15) = 54.07, p < 0.0001). These data demonstrate that re-exposure to alcohol cues reinstated alcohol-seeking behavior.

Systemic D1 receptor antagonism suppresses cue-induced alcohol seeking

We next sought to determine the contribution of D1 receptor (D1R) signaling to cue-induced alcohol seeking. Following the establishment of the reinstatement model, mice received systemic administration of the selective D1R antagonist SCH23390 at varying doses (0.1, 1, 3, and 10 mg/kg, i.p.) prior to the cue-induced craving test. SCH23390 pretreatment produced a significant suppression of active lever presses at all doses tested during reinstatement (One-way ANOVA, F(4,(25) = 7.599, p = 0.0004; Figure 2A). Post-hoc analysis revealed that all tested doses significantly suppressed seeking behavior compared to the saline control group (Saline: 67.67 ± 6.83 vs. 0.1 mg/kg: 43.5 ± 3.667, p = 0.0193; vs. 1 mg/kg: 37.6 ± 5.116, p = 0.0027; vs. 3 mg/kg: 39.83 ± 3.807, p = 0.0057; vs. 10 mg/kg: 31 ± 5.354, p = 0.0003), with no significant differences observed between individual doses (p > 0.05 for all pairwise comparisons).

Figure 2.

Systemic D1 receptor antagonism suppresses cue-induced alcohol seeking

(A) Suppressive effect of the D1R antagonist SCH23390 (0.1–10 mg/kg, i.p.) on active lever presses during cue-induced reinstatement.

All doses significantly reduced responding compared to saline control, with no significant differences between doses. (two-way ANOVA, F (4, 25) = 7.599, p = 0.0004; Tukey’s post hoc test: Saline vs. 0.1 mg/kg SCH23390: 67.67 ± 6.83 vs. 43.5 ± 3.667, p = 0.0193; Saline vs. 1 mg/kg SCH23390: 67.67 ± 6.83 vs. 37.6 ± 5.116, p = 0.0027; Saline vs. 3 mg/kg SCH23390: 67.67 ± 6.83 vs. 39.83 ± 3.807, p = 0.0057; Saline vs. 10 mg/kg SCH23390: 67.67 ± 6.83 vs. 31 ± 5.354, p = 0.0003).

(B) Total distance traveled in the open field test following administration of saline or SCH23390 (10 mg/kg), indicating no general locomotor impairment (unpaired t test, t = 0.1473, p = 0.8858).

Data are represented as mean ± SEM; n = 6 mice per group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

To rule out nonspecific motor effects, locomotor activity was assessed in the open field test following administration of the highest dose of SCH23390 (10 mg/kg). No significant difference was observed compared to saline controls (Figure 2B, unpaired t test, t = 0.1473, p = 0.8858), indicating that the suppression of alcohol seeking behavior was not due to general motor impairment. Lower doses (0.1–3 mg/kg) were not tested in the open field, as they were behaviorally effective but below the threshold typically associated with sedative effects.

Cue-induced reinstatement selectively activates the NAc and VP in a D1R-dependent manner

To identify the neural circuits engaged during cue-induced alcohol seeking, we analyzed the expression of the immediate-early gene product c-Fos, a marker of neuronal activation, across seven addiction-related brain regions (Figures 3A–3G). Western blot analysis revealed that cue-induced reinstatement of alcohol seeking significantly increased c-Fos protein levels specifically in the ventral pallidum (VP; F(2,(6) = 10.14, p = 0.0119), nucleus accumbens (NAc; F(2,(6) = 22.03, p = 0.0017), and ventral tegmental area (VTA; F(2,(6) = 11, p = 0.0098), compared to alcohol-naïve control mice (CON vs. reinstatement (R): VP, p = 0.0114; NAc, p = 0.0025; VTA, p = 0.0087). No significant changes were observed in the central amygdala (CeA), bed nucleus of the stria terminalis (BNST), ventral hippocampus (vHip), or prefrontal cortex (PFC).

Figure 3.

Cue-induced reinstatement increases neuronal activation in specific brain regions via D1 receptor signaling

(A–G) c-Fos protein expression in the central amygdala (CeA), bed nucleus of the stria terminalis (BNST), ventral hippocampus (vHip), ventral pallidum (VP), nucleus accumbens (NAc), prefrontal cortex (PFC), and ventral tegmental area (VTA) under three conditions: control (CON, alcohol-naive), reinstatement (R, cue-induced seeking), and reinstatement with SCH23390 (R + SCH). Insets: representative western blot bands. (One-way ANOVA with Tukey’s post hoc: ∗p < 0.05, ∗∗p < 0.01). Data are represented as mean ± SEM; n = 3 mice per group.

Critically, the increased c-Fos expression in the NAc and VP was reversed by systemic administration of the D1R antagonist SCH23390 (R vs. R + SCH: NAc, p = 0.0036; VP, p = 0.0435). In the VTA, SCH23390 administration induced a strong trend toward a reduction in c-Fos expression that did not reach statistical significance (R vs. R + SCH: p = 0.052). These findings highlight the NAc and VP as key nodes where D1 receptor signaling is necessary for the neuronal activation associated with cue-induced alcohol craving, while also suggesting a potential, more modest role for D1R-mediated regulation of VTA activity.

NAc D1R expression correlates positively with cue-induced but not context-elicited alcohol seeking

Our data indicate that D1 receptor antagonism reverses neuronal activation in both the NAc and VP during cue-induced seeking (Figure 3). Given the established roles of the NAc as a central hub for motivational drive generation²⁶ and the VP in translating motivation into action,²⁷ we hypothesized that the initial impetus for cue-induced craving is encoded by D1R signaling within the NAc. To test this, we first identified that the mice used for molecular analyses exhibited typical behavioral profiles during self-administration, extinction, and reinstatement. As shown in Figures S1A–S1C, all mice acquired stable lever press during SA (Figure S1A), with consistent reward retrieval and alcohol intake (Figure S1B), and successfully extinguished responding during E (Figure S1C). We then investigated whether D1R protein expression levels are altered in the NAc during reinstatement. Western blot analysis revealed a significant upregulation of D1R protein in the NAc following cue-induced reinstatement, compared to extinction or control levels (One-way ANOVA, F(3,(36) = 4.065, p = 0.0138; post-hoc: CON vs. R, p = 0.0091; Figure 4A). Moreover, NAc D1R expression levels showed a strong positive correlation with the intensity of seeking behavior (R² = 0.8768, p = 0.0059; Figure 4B), suggesting that D1R abundance may scale with motivational strength.

Figure 4.

NAc D1R expression actively correlates with cue-induced alcohol seeking but not with context-elicited alcohol seeking behavior

(A) D1R protein expression in the NAc across different behavioral phases: naive control (CON), self-administration training (SA), extinction (E), and cue-induced reinstatement (R). Inset: representative western blot bands. (One-way ANOVA, F(3,36) = 4.065, p = 0.0138; followed by post hoc Tukey’s test, CON vs. R, p = 0.0091, with n = 10 mice per group).

(B) Positive correlation between active lever presses and NAc D1R expression levels during cue-induced reinstatement (Pearson’s r = 0.936, R² = 0.8768, p = 0.0059; n = 6).

(C) Active and inactive lever presses during the context exposure test (CE). (two-way ANOVA, F (1, 10) = 5.898, p = 0.0355; Bonferroni’s post hoc test: SA vs. CE, p = 0.0037).

(D) NAc D1R protein expression is unaltered after context exposure (CE).

(One-way ANOVA, F(3, 20) = 0.9766, p = 0.4235, with n = 6 mice per group). Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.

To determine the specificity of this mechanism, we employed a context exposure paradigm, in which mice were re-exposed to the operant chamber without cues or rewards (context exposure test, CE). Behavioral performance of these mice during self-administration was comparable to that of the SA group shown in Figure 4A (Figure S1D). Context re-exposure successfully provoked significant seeking behavior (two-way ANOVA, F(1,10) = 5.898, p = 0.0355; post-hoc: SA vs. CE, p = 0.0037; Figure 4C), but did not alter NAc D1R protein expression (one-way ANOVA, F(3,20) = 0.9766, p = 0.4235; Figure 4D). This double dissociation demonstrates that NAc D1R upregulation is a specific molecular adaptation driving cue-induced, but not context-elicited alcohol seeking.

Chemogenetic inhibition of VTA dopamine neurons suppresses cue-induced alcohol seeking via NAc D1R activation

The specific upregulation of NAc D1R during cue-induced seeking prompted us to identify the afferent pathway responsible for its activation. We focused on the dopaminergic projections from the VTA to the NAc, a primary source of striatal dopamine. To investigate the role of VTA dopamine neurons in this process, we employed a combination of chemogenetics and site-specific pharmacology (Figures 5A and 5B). We bilaterally injected the VTA of mice with an AAV vector enabling the specific expression of inhibitory DREADDs (hM4Di) in dopaminergic neurons (AAV-TH-hM4Di-mCherry). Control mice received a virus expressing only mCherry. While this approach inhibits all VTA dopamine neuron projections, the subsequent intra-NAc D1R agonist rescue experiment allowed us to specifically interrogate the VTA→NAc pathway’s functional significance.

Figure 5.

Chemogenetic inhibition of VTA→NAc dopaminergic projections suppresses alcohol seeking via D1R

(A) Schematic of the experimental strategy: AAV encoding hM4Di-mCherry or mCherry control was injected into the VTA, and a cannula was implanted in the NAc for drug microinjection.

(B) Representative immunofluorescence image showing mCherry+ dopaminergic terminals in the NAc. Scale bars: 20 μm.

(C–E) Baseline behavioral measures were unaffected by viral expression: (C) Lever presses during self-administration training, (D) number of alcohol rewards earned, and (E) lever presses during extinction training. (two-way ANOVA for C, D, and E, with n = 6 mice per group).

(F) Effects of chemogenetic inhibition (CNO, 1 mg/kg i.p.) and/or intra-NAc D1R agonism (SKF38393, 1 μg/0.5 μL/side) on active lever presses during cue-induced reinstatement. (two-way ANOVA, F(4, 25) = 24.51, p < 0.0001, Tukey post-hoc test, CON vs. CNO, p < 0.0001; CON vs. SKF38393, p < 0.0001; Vehicle vs. CNO, p < 0.0001; Vehicle vs. SKF38393, p < 0.0001; CNO vs. SKF38393, p < 0.0001; CNO vs. CNO+SKF38393, p = 0.007; SKF38393 vs. CNO+SKF38393, p < 0.0001, n = 6 mice per group).

(G) Locomotor activity (total distance) in the open field test across treatment groups. Inset: representative locomotion traces. (One-way ANOVA, F(4, 25) = 0.1625, p = 0.9553, with n = 6 mice per group). Inset: locomotion traces for each group. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

We first verified that viral expression alone did not alter baseline behaviors. During self-administration and extinction training, no significant differences were observed between the mCherry and hM4Di groups in active lever presses, rewards earned, or extinction performance (Figures 5C–5E), suggesting that viral manipulation per se did not affect these processes.

To assess the role of VTA dopamine neurons in cue-induced reinstatement, we focused exclusively on the hM4Di group, as the mCherry group lacks DREADD expression and thus cannot respond to CNO. The hM4Di group was subdivided into four treatment conditions for the reinstatement test: vehicle, CNO (1 mg/kg, i.p.), intra-NAc SKF38393 (1 μg/side), and CNO + SKF38393 (Figures 5F and 5G). This design allowed us to isolate the specific contributions of VTA dopamine neuron activity and NAc D1R signaling to cue-induced alcohol seeking.

Chemogenetic inhibition of VTA dopamine neurons via CNO significantly suppressed cue-induced alcohol seeking behavior compared with vehicle-treated controls (Figure 5F; two-way ANOVA, F(4,(25) = 24.51, p < 0.0001; CON vs. CNO, p < 0.0001). To test whether activating NAc D1Rs was sufficient to drive seeking and to determine if they act as downstream effectors of VTA dopamine signaling, we performed a rescue experiment. While intra-NAc infusion of the D1R agonist SKF38393 (1 μg/0.5 μL/side) alone potentiated craving (CON vs. SKF38393, p < 0.0001), co-administration of SKF38393 fully rescued the seeking behavior suppressed by chemogenetic inhibition (CNO vs. CNO+SKF38393, p = 0.007). This rescue effect demonstrates that activation of NAc D1Rs is not only sufficient to promote seeking but is also necessary for the behavioral output driven by VTA dopaminergic inputs. Finally, open-field tests confirmed that none of these treatments affected general locomotor activity (Figure 5G; one-way ANOVA, F(4,(25) = 0.1625, p = 0.9553), ruling out motor confounds.

Collectively, these data establish a direct causal chain wherein cue-induced alcohol seeking is driven by VTA dopamine neuron activity and is mediated through the activation of D1 receptors in the NAc. The combination of chemogenetic inhibition and intra-NAc pharmacological rescue implicates the VTA→NAc dopaminergic pathway, although future studies using projection-specific approaches will be needed to further establish its necessity.

Discussion

This study systematically elucidates the central neural mechanisms by which VTA→NAc dopaminergic projections drive cue-induced alcohol craving through D1 receptor (D1R)-dependent mechanisms, employing integrated approaches including behavioral analysis, molecular biology, and circuit-specific intervention techniques. The key findings are: (1) Cue-induced alcohol craving significantly upregulates D1R protein expression in NAc, with expression levels positively correlating with behavioral intensity, while context-induced craving shows no difference in D1R alteration; (2) systemic administration of D1R antagonist SCH23390 significantly suppresses alcohol craving behavior while selectively reversing c-Fos activation in both NAc and ventral pallidum (VP); (3) chemogenetic inhibition of VTA dopaminergic projections significantly reduces the craving behavior, an effect completely reversible by intra-NAc D1R agonist SKF38393 administration and independent of motor function interference. These results suggest that cue-induced alcohol craving follows a complete causal chain: “VTA dopamine input → D1R activation in NAc → motivational behavioral output.”

Our finding that c-Fos expression level selectively elevated in the VTA, NAc, and VP, but not in other regions such as the mPFC or ventral hippocampus, during cue-induced reinstatement warrants discussion. This pattern does not necessarily contradict the established roles of these regions in broader aspects of AUD.^14,17 Instead, it likely reflects the behavioral specificity in our experimental paradigm. The mPFC and ventral hippocampus are critically implicated in contexts requiring executive control over compulsive seeking or in processing contextual associations, respectively.^14,15,16,17 Our model, which emphasizes a discrete, alcohol-predictive cue, may preferentially recruit the core incentive motivation circuitry of the mesolimbic dopamine system (VTA→NAc) and its primary output node for motivated behavior (VP). Thus, the absence of significant c-Fos activation in the mPFC and vHip underscores the selectivity of the VTA→NAc pathway and its primary output target, the VP, in mediating cue-triggered, as opposed to context- or stress-triggered, relapse behavior.

The positive correlation between NAc D1R expression and active lever pressing intensity (Figure 4B), along with the efficacy of SCH23390 in suppressing seeking (Figures 2A and 5F), matches the incentive sensitization theory proposed by Berridge and Robinson, which posits that NAc dopamine signaling mediates cue-evoked “wanting” motivation primarily through D1R.²⁸ Our evidence of a molecular correlation—the upregulation of NAc D1R protein—provides a potential mechanism for the persistent neural hypersensitivity to cues that characterizes addiction. The necessity of VTA dopaminergic projections is further validated by the reduction in craving following their chemogenetic inhibition (Figure 5F), aligning with previous reports of phasic dopamine neuron firing encoding reward-predictive cues.²⁹

The critical role of NAc D1R signaling in relapse-related behaviors extends beyond alcohol to other categories of abused substances, suggesting a convergent neural mechanism across drug classifications. In heroin self-administration models, D1R blockade in the NAc shell attenuates context-induced reinstatement, whereas core blockade selectively blocks discrete-cue-induced reinstatement.³⁰ Similarly, D1R antagonism in either NAc core or shell suppresses context-induced reinstatement of ethanol seeking,³¹ and in alcohol-preferring rats, D1R activity in the shell, but not core, mediates spontaneous recovery of seeking.³² Furthermore, D1R blockade in both subregions attenuates renewal of alcohol seeking after punishment-imposed abstinence,³³ extending the role to abstinence models that mimic human relapse triggers. In psychostimulant models, DREADD-mediated inhibition of D1-MSN projections to the VP, but not to the ventral mesencephalon, blocks cue-induced cocaine seeking,³⁴ resonating with our observation that D1R antagonism reversed c-Fos activation in both NAc and VP (Figures 3C and 3E). Moreover, cell-type-specific studies have revealed opposing roles of D1-and D2-MSNs in cocaine reward,³⁵ and epigenetic regulation within D1-MSNs during heroin use limits future relapse by controlling MSN excitability.³⁶ Collectively, these findings establish NAc D1R signaling as a common substrate for relapse-related behaviors across alcohol, heroin, and cocaine, with subtle variations in subregional specificity depending on the relapse trigger. Our study extends this literature by demonstrating that cue-specific alcohol seeking selectively upregulates NAc D1R protein expression, providing a molecular issue that may contribute to the persistent neural hypersensitivity characteristic of addiction.

While the functional specificity of D1-MSNs in addictive behaviors has been widely documented, our experiment, wherein intra-NAc D1R agonist administration fully restored seeking behavior suppressed by chemogenetic inhibition of VTA inputs (Figure 5F), further clarifies its pivotal and necessary role as a important downstream effector in alcohol craving. This finding positions D1R activation in the NAc as a critical convergence point for the VTA→NAc pathway to drive alcohol-seeking behavior.

Our data that D1R antagonism reversed c-Fos activation in both the NAc and VP (Figures 3C and 3E) suggests that D1R signaling in the NAc may regulate behavioral output through its projections to the VP while Smith et al. emphasized the dominance of the VP in habitual drug seeking.²⁷ This discrepancy likely stems from differences in experimental models: our study focused on the early motivational stages of cue-induced craving, a process that critically depends on the NAc for the attribution of incentive salience to alcohol-predictive cues, whereas the VP may exert a more dominant influence in the “habitualization” of seeking behavior observed after prolonged training or in chronic addiction models.³⁷ Future research employing projection-specific manipulations is required to dissect the distinct contributions of the direct NAc→VP pathway versus other VP afferents to the different stages of alcohol seeking.

Notably, our chemogenetic inhibition results (Figure 5F) are consistent with the work by Nguyen et al., who demonstrated that optogenetic activation of VTA→NAc dopaminergic projections enhances real-time place pref.³⁸ These studies reciprocally validate the rewarding effects mediated by this circuit. In this experimental design, we did not include a mCherry+CNO control group, because CNO (1 mg/kg) has been shown to lack significant off-target effects in the absence of DREADD expression.³⁹ Thus, the observed behavioral suppression in the hM4Di+CNO group can be confidently attributed to specific inhibition of VTA dopamine neurons. There is an interpretive consideration regarding the rescue experiment: while the CNO+SKF38393 group showed equivalent responding to vehicle controls, this pattern is statistically compatible with both functional compensation and additive effects of opposing drug actions. Although the biological plausibility of D1R as a downstream effector of VTA dopamine signaling (as supported by the well-established role of D1-MSNs in reward processing)^26,28 favors the rescue interpretation, further definitive proof requires additional approaches, such as selective D1R knockdown in the NAc during VTA intervention. Future studies employing such strategies will be necessary to further establish the necessity of NAc D1R activation for VTA-driven cue-induced seeking. However, it is crucial to note that the pharmacological mechanisms engaging the VTA→NAc circuit differ across substance types. For instance, while opioids enhance NAc dopamine release primarily via disinhibition of VTA dopamine neurons through μ-opioid receptors on local GABAergic interneurons,^40,41 the reinforcing properties of nicotine are mediated by the activation of β2-subunit-containing nicotinic acetylcholine receptors (β2-nAChRs) on VTA dopamine neurons. Such mechanistic variations suggest that inhibition of the VTA→NAc pathway might differentially affect substance-specific cravings and could even produce divergent affective consequences, such as potentially exacerbating anxiety-related behaviors in the context of nicotine withdrawal.³⁸

The interpretation of our findings is informed by several methodological considerations. While c-Fos imaging provides a snapshot of population-level activation, it cannot resolve real-time neural dynamics such as phasic dopamine release or spatiotemporal firing patterns during cue presentation.⁴² Similarly, although our residual volume measurements indicated that >85% of delivered alcohol was consumed, finer-grained analysis of consummatory behavior (e.g., lick frequency) would further clarify the relationship between seeking and drinking.⁴³ Additionally, we did not directly record neuronal activity; however, the hM4Di construct and CNO dose used here have been extensively validated in previous electrophysiological studies.^44,45 Future work incorporating fiber photometry or in vivo recordings will be valuable to complement our findings.

In conclusion, this study delineates a mechanism for cue-induced alcohol craving, establishing that dopaminergic projections from the VTA to the NAc drive this behavior through the specific activation of D1 receptors in NAc. By integrating behavioral, molecular, and circuit-level approaches, we provide a cohesive causal chain from circuit input to molecular effector and behavioral output. These findings not only advance the theoretical understanding of AUD but also highlight the VTA→NAc D1R pathway as a precise target for future therapeutic strategies aimed at mitigating relapse.

Limitations of the study

Our study has several limitations. First, only male mice were used to control for sex hormone fluctuations; whether the identified VTA→NAc D1R mechanism operates similarly in females remains unknown, given known sex differences in alcohol-seeking behavior.^28,46,47,48 Second, systemic CNO administration inhibits all VTA dopamine projections, not exclusively the VTA→NAc pathway. Although the intra-NAc D1R agonist rescue experiment strongly supports NAc D1R as a critical downstream effector, contributions from other VTA targets (e.g., prefrontal cortex, amygdala) cannot be fully excluded.^{49,50,51,52,53} Third, the VTA contains heterogeneous cell types (dopaminergic, GABAergic, and others)^54,55; the potential roles of non-dopaminergic neurons in our model await future investigation using cell-type-specific tools.^56,57,58

Resource availability

Lead contact

Further information and requests for resources should be directed to the lead contact, Yanzhong Guan (guanyz@mdjmu.edu.cn).

Materials availability

• •This study did not generate new unique reagents.
• •All experimental procedures were conducted in accordance with the National Guidelines for Experimental Animal Welfare and Ethics and were specifically approved by the Animal Ethics Committee of Mudanjiang Medical University (approval number: IACUC-20240222-41).

Data and code availability

• •The lead contact will share all data reported in this paper upon request.
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This work was supported by Mudanjiang Key R&D Program Project (XDHD25CR018 to Y.G.) and the Key Project of the Heilongjiang Natural Science Foundation (ZD2022H007 to Y.G.).

Author contributions

Conceptualization, Y.G., J.X., and M.D.; methodology, J.X., M.D., Y.L., T.Z., X.L., H.D., C.D., X.S., X.X., X.W., X.Z., J.W., and D.C.; investigation, J.X., M.D., J.W., D.C., H.D., and C.D.; formal analysis, J.X. and M.D.; data curation: J.X. and M.D.; visualization: J.X. and M.D.; resources: Y.G.; supervision: Y.G.; project administration: Y.G.; funding acquisition: Y.G.; writing – original draft: J.X. and M.D.; writing – review & editing: Y.G. and J.X.

Declaration of interests

The authors report no biomedical, financial, or other potential conflicts of interest.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the author(s) did not use any AI or AI-assisted technologies.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit anti-D1R	Abcam	Cat# ab279713
Rabbit anti-c-Fos	MedChemExpress	Cat# HY-P80616
Rabbit anti-GAPDH	Abcam	Cat# ab181602
HRP-conjugated goat anti-rabbit IgG	Beyotime	Cat# A0208
Rabbit anti-RFP	Proteintech	Cat# pabr1
Alexa Fluor® 594-conjugated goat anti-rabbit IgG	MedChemExpress	Cat# HY-P8003
Bacterial and virus strains
AAV-TH-hM4Di-mCherry	BrainVTA (Wuhan) Co., Ltd.	Cat# PT-3960
AAV-TH-mCherry (control)	BrainVTA (Wuhan) Co., Ltd.	Custom order
Chemicals, peptides, and recombinant proteins
SCH23390 (D1R antagonist)	Sigma-Aldrich	Cat# D054
SKF38393 (D1R agonist)	Sigma-Aldrich	Cat# D047
RIPA lysis buffer	Beyotime	Cat# P0013B
BCA assay kit	Beyotime	Cat# P0012
BeyoECL Plus chemiluminescence kit	Beyotime	Cat# P0018S
DAPI	Beyotime	Cat# C1006
Experimental models: Organisms/strains
Mouse: C57BL/6J male, 8 weeks old	Mudanjiang Medical University Comparative Medicine Center	SYXK2019-006
Software and algorithms
GraphPad Prism 8.0	GraphPad Software	RRID: SCR_002798
ImageJ	National Institutes of Health	RRID: SCR_003070
VisuTrack	Shanghai Xinruan	XR-VT
Other
Operant conditioning chambers	Shanghai Xinruan	Model XR-XS109
Stereotaxic frame	RWD Life Sciences, Shenzhen, China	Model 68030
Open field automated tracking system	Med Associates Inc., Fairfax, VT	Model MED-OFA-510

Method details

Animals

Male C57BL/6J mice (8 weeks old) were obtained from the Comparative Medicine Center of Mudanjiang Medical University (SYXK2019-006). All mice were specific pathogen-free (SPF) grade and individually. The housing environment was maintained under standard conditions: temperature at 22 ± 2°C, relative humidity of 50–60%, and a 12-h/12-h light/dark cycle (lights on at 7:00 a.m.). Standard rodent chow and sterilized water were provided ad libitum, and cage bedding was changed weekly. All experimental procedures were conducted in accordance with the National Guidelines for Experimental Animal Welfare and Ethics and were specifically approved by the Animal Ethics Committee of Mudanjiang Medical University (approval number: IACUC-20240222-41).

Reagents and drug preparation

The selective D1 receptor antagonist SCH23390 and the D1 receptor agonist SKF38393 were purchased from Sigma-Aldrich. The composition of the artificial cerebrospinal fluid (ACSF) was as follows (in mM): 126 NaCl, 2.5 KCl, 1.2 NaH₂PO₄, 1.2 MgCl₂, 2.4 CaCl₂, 25 NaHCO₃, and 11 glucose (pH adjusted to 7.4; osmolarity ∼300 mOsm). All drug solutions were freshly prepared on the day of use. Alcohol solutions (10% v/v) for operant self-administration were prepared daily by diluting 95% alcohol (Henan Hanyang Alcohol Co., Ltd., China) with sterile distilled water.

Operant self-administration training

The protocol was adapted from previous studies.^59,60,61,62 All behavioral training and testing were conducted in standard operant conditioning chambers (Model XR-XS109, Shanghai Xinruan Information Technology Co., Ltd.). Each chamber was equipped with two retractable levers, a liquid reward delivery system, and a stainless-steel grid floor. Responses on the designated active lever resulted in the delivery of a 10 μL aliquot of 10% (v/v) alcohol solution (or 10% sucrose during initial training), accompanied by the illumination of a white cue light above the lever for 2000 m s. Each correct response was followed by a 5-s timeout period, during which lever presses were recorded but had no programmed consequences. Responses on the inactive lever were recorded throughout all sessions but never resulted in reward delivery or cue presentation. All behavioral data, including lever presses, rewards earned, and cue activations, were automatically recorded and timestamped by VisuTrack software (XR-VT).

Overnight adaptation

Mice underwent a single overnight training session (∼16 h) under an FR1 schedule with 10% sucrose reward, with food chow provided ad libitum.

Fixed-ratio schedule training

Daily 45-min sessions began with FR1 for 8 days, then FR2 for 4 days, then FR3 for 4 days. Stable responding was defined as <20% variation in daily active lever presses over three consecutive sessions and discrimination accuracy (active/total presses) ≥0.63.

Validation of alcohol consumption

Evaporation controls showed <5% fluid loss over 45 min. In a subset of mice (n = 6), residual fluid was collected after each FR3 session; mean residual volume per reward was 1.41 ± 0.05 μL, indicating >85% consumption.

Forced abstinence

After FR training, mice underwent 7 days of forced abstinence in home cages with no access to alcohol or the operant context.

Extinction training

Daily 45-min sessions where lever presses produced no reward or cue. Extinction was considered complete when active lever presses were not significantly different from inactive presses for three consecutive sessions (p > 0.05, paired t test).

Reinstatement tests

(1) Context exposure test: 45-min session in the operant chamber under extinction conditions without cue presentation. (2) Cue-induced reinstatement: Each press on the previously active lever produced the conditioned cue light (2000 m s) according to the last completed FR schedule (FR3), but no alcohol reward. This test specifically assessed the capacity of the discrete alcohol-predictive cue to reinstate seeking behavior.

Stereotaxic surgeries and microinjections

Mice were anesthetized via intraperitoneal injection of a xylazine and ketamine mixture (10 mg/kg and 80 mg/kg, respectively) and securely positioned in a stereotaxic frame (RWD Life Sciences, Shenzhen, China). Body temperature was maintained throughout the procedure at 37°C–38°C using a feedback-controlled heating pad. Bilateral guide cannulas were implanted targeting the nucleus accumbens (NAc) according to coordinates from the Franklin and Paxinos mouse brain atlas.⁶³ anterior-posterior (AP) +1.3 mm, medial-lateral (ML) ±0.7 mm, dorsal-ventral (DV) −4.4 mm. Cannulas were affixed to the skull using dental acrylic anchored with 2–3 stainless-steel screws. Dummy cannulas of matching length were inserted post-surgery and sealed with dust caps to maintain patency; subsequent infusion cannulas extended 0.1 mm beyond the guide tip. Following the procedure, mice received an intraperitoneal injection of penicillin sodium (80,000–150,000 U) to prevent infection and were allowed to recover for at least 7 days before further experimentation.

For chemogenetic inhibition of dopaminergic terminals, an adeno-associated virus carrying a Cre-dependent inhibitory DREADD construct (AAV-Th-Gi-mCherry; titer ≥1 × 10¹³ μg/mL, BrainVTA (Wuhan) Co., Ltd., China) was microinjected into the ventral tegmental area (VTA) using the following stereotaxic coordinates: AP –3.2 mm, ML ±0.5 mm, DV –4.3 mm. A total volume of 0.4 μL per side was delivered via a 33-gauge Hamilton syringe at a rate of 0.1 μL/min using a microinfusion pump. The injection needle remained in place for an additional 5 min post-infusion to ensure adequate diffusion and limit backflow. Behavioral tests were conducted following a 3-week viral expression period.

For pharmacological activation of D1 receptors in the NAc, the D1R agonist SKF38393 (1 μg/0.5 μL/side)^64,65 was prepared in a vehicle of 0.1% dimethyl sulfoxide in normal ACSF. Mice were permitted to re-establish stable lever-pressing performance on an FR schedule prior to drug testing. On test days, SKF38393 or vehicle was bilaterally infused into the NAc via dual infusion cannulas at a rate of 0.1 μL/min. The injector was left in place for an additional 2–5 min after administration to minimize reflux. Behavioral testing commenced 30–60 min after recovery from handling and microinjection.

To verify injection sites, an equal volume of Coomassie Brilliant Blue dye was microinfused into the VTA following the conclusion of all behavioral procedures. Histological verification (Figure S2) was performed by comparing dye locations with the corresponding plates in the Franklin and Paxinos mouse brain atlas.⁶³

Western blot

Following final behavioral tests, mice were anesthetized with isoflurane and decapitated. For c-Fos studies, mice were sacrificed 60 min after reinstatement tests; for D1R studies, immediately after final sessions. All collections occurred between 2:00 p.m. and 5:00 p.m. Target brain regions were microdissected in ice-cold ACSF. Tissue was homogenized in RIPA lysis buffer (P0013B, Beyotime, China) with protease inhibitors, centrifuged at 12,000 ×g for 20 min at 4°C. Protein concentration was determined by BCA assay (P0012, Beyotime, China). Proteins (20 μg per lane) were separated by 8% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk and incubated overnight at 4°C with primary antibodies: rabbit anti-D1R (1:1000, ab279713, Abcam), rabbit anti-c-Fos (1:1000, HY-P80616, MedChemExpress, China), and rabbit anti-GAPDH (1:1000, ab181602, Abcam). After TBST washes, membranes were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:1000, A0208, Beyotime, China) for 2 h at room temperature. Bands were visualized using BeyoECL Plus chemiluminescence kit (P0018S, Beyotime, China) and imaged with a gel documentation system (AI600, GE Healthcare, USA). Band intensities were quantified using ImageJ (NIH, USA), normalized to GAPDH, and expressed as percentage of control group mean.

Immunofluorescence staining

Mice were transcardially perfused with ice-cold PBS followed by 4% paraformaldehyde (PFA). Brains were post-fixed in 4% PFA for 1 h at 4°C, then cryoprotected in 30% sucrose in 4% PFA overnight at 4°C. After sinking, brains were embedded in OCT compound and frozen. Coronal sections (4 μm) were cut on a cryostat (Leica CM 1850). Sections were fixed in pre-cooled acetone for 10 min, air-dried, washed with PBS, and subjected to antigen retrieval in 1 mM sodium citrate buffer (pH 7.(4) at 92°C–98°C for 15 min. After blocking with 5% BSA for 1 h at room temperature, sections were incubated with rabbit anti-RFP primary antibody (1:500, pabr1, Proteintech, China) overnight at 4°C, then with Alexa Fluor® 594-conjugated goat anti-rabbit IgG (H&L) secondary antibody (1:500, HY-P8003, MedChemExpress, China) for 30 min at 37°C in the dark. Nuclei were counterstained with DAPI (C1006, Beyotime, China). Images were acquired with a fluorescence microscope (Nikon, Japan) and processed with ImageJ.

Open Field Test (OFT)

General locomotion and anxiety-like behavior were assessed using the open field test following established procedures.⁶⁶ Briefly, each mouse was placed in the center of a square arena (50 × 50 cm) and allowed to explore freely for 10 min. The test was conducted under diffuse lighting, and all sessions were recorded using an automated tracking system (Med Associates Inc., Fairfax, VT). The total distance traveled (cm) was quantified as a measure of locomotor activity. To assess anxiety-like behavior, the time spent in the central zone (a 25 × 25 cm area) was recorded. The arena was thoroughly cleaned with a 10% alcohol solution between trials to eliminate any olfactory cues.

Quantification and statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, USA). Data are presented as mean ± standard error of the mean (SEM). The exact value of n (number of mice) is indicated in each figure legend. Comparisons between two groups were performed using unpaired Student’s t test. For multiple groups, one-way or two-way analysis of variance (ANOVA) was used, followed by Tukey’s or Bonferroni’s post hoc test as appropriate. A p-value <0.05 was considered statistically significant. In figures, asterisks denote significance levels: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Statistical details (test names, n, and exact p-values or ranges) are provided in the figure legends. All experiments were conducted with a minimum of three biological replicates. Animals were allocated to each experimental group using stratified randomization. To ensure blinded group allocation during data collection, animal preparation and the experiments were conducted by different investigators. The numbers and individual dots refer to the number of animals.