Social reward outcompetes drug seeking dopaminergic ensembles to prevent relapse

Domain: biology
Published: 2026-04-03
Editorial outcome: major_revision

Abstract
Drugs of abuse promote substance use disorder (SUD) by hijacking mesolimbic circuits that normally process natural rewards. Among these, social rewards exhibit therapeutic potential, but the underlying neural substrates remain unclear. Using a multimodal approach integrating in vivo single-neuron calcium imaging, optogenetic manipulation, and electrophysiology in male rats, we identified two distinct dopaminergic ensembles in the ventral tegmental area (VTA) that respectively encode social reward and drug seeking. Notably, these antagonistic ensembles exert reciprocal influence through competitive interactions that shape behavioral outcomes. Furthermore, circuit mapping revealed divergent connectivity patterns, with social reward-responsive dopaminergic ensembles receiving preferential input from the dorsal raphe nucleus (DRN). Activation of the DRN-VTA pathway recapitulates the protective effects of social reward against drug seeking. In this study, we uncovered a dynamic competition between functionally specialized dopaminergic ensembles through which social reward attenuates drug seeking, offering insights that may inform development of novel strategies for SUD treatment.

Introduction
Substance use disorder (SUD) is a chronic, relapsing brain disorder that poses a significant global health challenge1. Extensive research has demonstrated that drugs of abuse hijack brain reward systems that typically regulate the pursuit of natural rewards, resulting in maladaptive drug-seeking behaviors2,3. However, recent evidence suggests that natural rewards, particularly social rewards4, can counteract the impact of drugs and provide protection against SUD. Positive social interactions are increasingly recognized as effective alternatives to addictive substances, offering potential interventions in critical phases of SUD, including initiation5, extinction6, and incubation7. While social-based approaches are cost-effective and associated with minimal adverse effects, their neurobiological mechanisms remain elusive.

One region of interest is the ventral tegmental area (VTA), where dopaminergic (DAergic) neurons coordinate the reinforcing properties of both social reward8-10 and drugs of abuse11,12. Growing evidence indicates substantial functional heterogeneity within VTA DAergic subpopulations, characterized by distinct cellular morphologies, neurochemical profiles, electrophysiological properties, and differential responses to rewarding versus aversive stimuli13,14. This diversity raises the possibility of specialized DAergic populations selectively encoding social reward versus drug-related behaviors. Paradoxically, there is evidence of significant overlap among VTA DAergic neurons in response to food and social interaction10, as well as in nucleus accumbens (NAc) medium spiny neurons (MSNs) responding to both food/drink and addictive substances2. These contradictory observations create a fundamental tension in addiction neuroscience: Are there segregated DAergic ensembles in the VTA dedicated to social versus drug-related behaviors, or do overlapping neuronal populations mediate both?

In this work, we perform in vivo calcium imaging to identify distinct DAergic ensembles in the VTA that are responsive to social reward (Social-reward-EnsDA) and drug seeking (Drug-seeking-EnsDA). Multimodal interrogation combining optogenetic manipulation with electrophysiology reveals dynamic, reciprocal inhibition between these two functionally segregated populations. Circuit tracing and chemogenetic perturbation further delineate an organized circuit from the dorsal raphe nucleus (DRN) to VTA that selectively modulates the protective effect of social reward against drug seeking. Together, these findings establish both cellular and circuit-level mechanisms by which socially engaged DAergic ensembles constrain drug seeking-associated neural plasticity, revealing potential therapeutic targets for preventing relapse.

Results
VTA DAergic neurons as a central nexus of social reward and drug seeking

Previous studies have shown that interacting with juvenile conspecifics can serve as an experimental manipulation of social reward in mice15, with mice developing a preference for a compartment associated with the presence of two juvenile conspecifics16. In the present study, we investigate whether this similar social paradigm can serve as a social reward manipulation in rats. Behavioral assays revealed that rats exhibited a robust conditioned place preference for the context paired with juvenile conspecifics (Supplementary Fig. 1a-d) and successfully acquired the operant response required to access juvenile conspecifics (Supplementary Fig. 1e,f). Furthermore, activating the neurons activated by social interaction with juvenile conspecifics induces intracranial self-stimulation behavior (Supplementary Fig. 1g-j). Collectively, these findings confirm that interaction with juvenile conspecifics can serve as a valid experimental manipulation of social reward in rats. Next, we assessed whether social reward could suppress cocaine (a psychostimulant) or heroin (an opioid) seeking during the relapse test. As in previous work17, rats underwent a cocaine or heroin relapse test following 10 days of self-administration training (cocaine or heroin was injected intravenously and paired with a light-tone cue after the rats nose-poked in the active hole), 14 or 7 days of self-administration extinction training (only a light-tone cue was delivered after the rat nose-poked in the active hole), and 4 weeks of home cage recovery (Fig. 1a-c and Supplementary Fig. 2a-c). The relapse test was manifested by cue-induced active nose-pokes for drug seeking. Before the drug relapse test, rats were divided into two groups: a social reward group and a no reward group. Rats in the social reward group were placed in a box and allowed to freely interact with two juvenile conspecifics for 30 minutes before the relapse test, while rats in the no reward group were placed alone in the same box for 30 minutes (Fig. 1a and Supplementary Fig. 2a). Social reward significantly reduced cue-induced active nose-pokes both in the cocaine (Fig. 1d) and heroin relapse tests (Supplementary Fig. 2d), with no effect on inactive pokes, suggesting a selective suppression of drug seeking. To ensure that the observed effect was due to social reward, rather than novelty exploration, two control groups were included: rats were exposed to either two toy rats or anesthetized conspecifics for 30 minutes prior to the relapse test (Supplementary Fig. 2e-g, i-k). Neither of these manipulations affected nose-poke behaviors (Supplementary Fig. 2h,l), confirming that the suppression of drug seeking was driven by social reward, not novelty per se.

The VTA DAergic neurons mediate the rewarding properties of both social interaction8-10 and drugs of abuse11,12. Thus, we attempted to dissect the role of VTA DAergic neurons in mediating the effect of social reward against drug seeking. To achieve this, we used adeno-associated virus (AAV)-tyrosine hydroxylase (TH)-GCamp6s to specifically target VTA DAergic neurons (Fig. 1e). GCamp6s expression was restricted to the VTA, with no spread to adjacent regions (Fig. 1f). Calcium activity was monitored via fiber photometry during both social interaction and the cocaine relapse test (Fig. 1g). Body contact during social interaction reliably evoked robust calcium transients in VTA DAergic neurons (Fig. 1h-j). Similarly, drug seeking during the relapse test was associated with a marked increase in calcium signal (Fig. 1k-m). Notably, this drug-seeking associated activity was significantly attenuated in rats that had prior exposure to social interaction (Fig. 1n-r), suggesting that VTA DAergic neurons contribute to the suppressive effects of social reward on drug seeking.

To further examine changes in DAergic neurotransmission, we assessed DA release in the NAc, the primary target of monosynaptic output from VTA DAergic neurons18. A DA sensor was injected into the NAc to enable fiber photometry recordings (Supplementary Fig. 3a-c). Body contact during social interaction induced a notable increase in DA release within the NAc (Supplementary Fig. 3d-f). Similarly, drug seeking during the relapse test reliably increased DA release in the NAc (Supplementary Fig. 3g-i). However, this drug-seeking induced increase in DA release was significantly attenuated after social interaction (Supplementary Fig. 3j-n), suggesting that social reward decreased DA release following drug seeking.

Distinct DAergic ensembles respond to social reward and drug seeking

While classic work on DAergic neurons in the VTA has highlighted their apparent homogeneous responses to various stimuli19, recent research has uncovered notable heterogeneity at the single-cell level20. For instance, 15 distinct DAergic subpopulations have been identified based on molecular expression profiles, with these subpopulations displaying diverse responses to rewards and aversive events13.

To determine whether distinct or overlapping DAergic subpopulations respond to social reward and drug seeking, we employed an activity-dependent dual-labeling approach. Specifically, we used an AAV expressing ERCreER under the control of an activity-dependent promoter (Label 1, AAV-cfos-ERCreER-PEST, hereafter referred to as TRAPed neurons) and immunofluorescent detection of cfos expression (Label 2) to label neurons activated by social interaction and/or cocaine seeking during the relapse test (Fig. 2a,b). To validate the TRAPing paradigm in rats, we first characterized the temporal dynamics of neuronal labeling. Rats were subjected to 30 min of social interaction at varying time intervals relative to the administration of 4-hydroxytamoxifen (4-OHT). Analysis of mCherry expression patterns demonstrated that the majority of TRAPed neurons were labeled within a 6-hour time window centered on 4-OHT injection. Notably, mCherry expression levels peaked when 4-OHT was administered 30 min prior to the initiation of social interaction (Supplementary Fig. 4a-d). To control for potential confounding effects of viral infection, rats were assigned to four groups: Social-reward (Label 1) + Drug-seeking (Label 2), Drug-seeking (Label 1) + Social-reward (Label 2), Drug-seeking (Label 1) + Drug-seeking (Label 2), and Social-reward (Label 1) + Social-reward (Label 2) (Fig. 2c, and Supplementary Fig. 5a,b). DAergic neurons were identified by immunostaining for tyrosine hydroxylase (TH) (Fig. 2d, and Supplementary Fig. 5c). Quantification revealed that co-labeled neurons—responsive to both drug seeking and social reward—comprised only ~13.35% of all Drug-seeking-responsive neurons and ~15.58% of all Social-reward-responsive neurons (Supplementary Fig.5d-i), and co-labeled DAergic neurons—responsive to both drug seeking and social reward—comprised only ~8.93% of all Drug-seeking-responsive DAergic neurons and ~10.55% of all Social-reward-responsive DAergic neurons (Supplementary Fig. 5d-i). Notably, the proportion of co-labeled DAergic neurons across social reward and drug seeking conditions was significantly lower than that observed in control groups subjected to the same stimulus twice (Fig. 2e,f), suggesting largely distinct neuronal populations for each condition.

Neuronal ensembles are defined as populations of neurons that encode specific information through coordinated activity patterns induced and shaped by learning21. To determine whether the TRAPed neurons in the VTA constitute a neuronal ensemble, we examined whether neurons labeled during the first episode of drug seeking or social interaction could be reactivated during a second, temporally separated episode of the same stimulus. Indeed, re-exposure to the same condition robustly reactivated TRAPed neurons, as evidenced by their overlap with cfos expression, confirming the presence of stable Drug-seeking- and Social-reward-responsive ensembles within the VTA (Supplementary Fig. 5j-o). Finally, spatial mapping of DAergic ensembles revealed that both Social-reward- and Drug seeking-responsive ensembles were preferentially localized to the middle VTA (AP -5.4 to -5.8 mm), with no significant spatial segregation between the two (Supplementary Fig. 6a-c). Moreover, the number of DAergic neurons activated during social reward versus drug relapse sessions did not differ significantly; both populations accounted for approximately 30% of total DAergic neurons in the VTA (Supplementary Fig. 6d,e). Collectively, these findings demonstrate the existence of functionally distinct DAergic ensembles that selectively encode social reward and drug seeking.

To further confirm that distinct ensembles encode social reward and drug seeking, we tracked VTA DAergic neurons' activity at single-cell resolution using single-photon calcium in free-moving rats (Fig. 2g). AAV-TH-GCamp6s was injected into the VTA, and gradient-index (GRIN) lenses were implanted after a 3-week recovery (Fig. 2h). This approach enabled direct comparison of neuronal responses to both stimuli within the same imaging field. We identified discrete populations of VTA DAergic neurons responsive to either cocaine seeking, social interaction, or both (Fig. 2i-t). Across all animals, 948 neurons were extracted, of which 380 were successfully aligned across the two sessions (Fig. 2u). Among these, 22% (84 out of 380 neurons, n = 6 animals) responded selectively to cocaine seeking during the relapse test (Fig. 2i-k and Fig. 2v), 17% (65 out of 380 neurons, n = 6 animals) responded selectively to body contact during social interaction (Fig. 2l-n and Fig. 2v), and only 9% (34 out of 380 neurons, n = 6 animals) responded to both (Fig. 2o-q and Fig. 2v). Statistical analysis revealed no significant overlap between Drug-seeking- and Social-reward-responsive neurons (Fig. 2w). If social reward and drug seeking are encoded by distinct neuronal populations, their response magnitudes across the population would be expected to exhibit no correlation or a negative correlation. Consistent with this, we observed a significant inverse correlation between neuronal responses to social interaction and cocaine seeking across the VTA DAergic population (Fig. 2x, r = -0.48, p < 0.01). Together, these results indicate that most VTA DAergic neurons responsive to cocaine seeking are unresponsive to social interaction, supporting the existence of distinct DAergic ensembles encoding social reward and drug seeking.

To rule out the possibility that the segregation was merely due to stimulus presentation at distinct time points, we tracked VTA DAergic neuronal activity across two separate sessions of cocaine seeking and/or social interaction conducted on different days (Supplementary Fig. 7a,b and Supplementary Fig. 8a,b). We found a significant overlap between the neuronal populations responsive to the same stimulus across sessions. Specifically, 62% of VTA DAergic neurons responsive to the first cocaine seeking session were reactivated during the second drug seeking session (Supplementary Fig. 7c-r), and 56% of those responsive to the first social reward session were reactivated during the second social reward session (Supplementary Fig. 8c-r). In addition, given the substantial array of confounding variables inherent to the social interaction versus drug relapse sessions, one might hypothesize that the observed segregation of neuronal populations stem from divergent sensory and behavioral modalities. To address this alternative explanation, we trained rats to perform operant responses for both social self-administration and cocaine self-administration, then assessed whether distinct neuronal ensembles shaped by learning were engaged when rats executed goal-directed behaviors to obtain these two discrete reward-related stimuli (Supplementary Fig. 9a,b). Our results demonstrated that only 10% of recorded neurons (26 out of 261 neurons; n = 2 animals) exhibited dual responsiveness to both social and cocaine cues (Supplementary Fig. 9c-p). Subsequent statistical analyses confirmed a lack of significant overlap between social-seeking- and drug-seeking-responsive neuronal populations (Supplementary Fig. 9q). Collectively, these findings establish that VTA DAergic populations exhibit stable, stimulus-specific identities and that their segregation is not a consequence of temporal factors or behavioral confounds, consistent with the properties of learning‑induced neuronal ensembles.

Reciprocal inhibition of Social-reward-EnsDA and Drug-seeking-EnsDA

Next, we investigated the relationship between Social-reward-EnsDA and Drug-seeking-EnsDA. Previous studies have shown that midbrain DAergic neurons can exert inhibitory effects on downstream targets via non-canonical release of γ-aminobutyric acid (GABA)22,23. Based on this, we hypothesized that Social-reward-EnsDA may inhibit Drug-seeking-EnsDA through GABAergic transmission.

To test this hypothesis, we expressed the light-activated cation channel channelrhodopsin-2 (ChR2) in Social-reward-EnsDA using Flp recombinase-dependent AAVs (Label 1) and performed whole-cell voltage-clamp recordings from Drug-seeking-EnsDA (Label 2) in sagittal brain slices of VTA (Fig. 3a-d). Optogenetic stimulation of Social-reward-EnsDA with brief (1 ms) flashes of blue light reliably evoked large outward currents in ~72% of tested Drug-seeking-EnsDA (n = 4 animals, 18/25 neurons; Fig. 3e). These currents were abolished by the voltage-gated Na⁺ channel blocker tetrodotoxin (TTX) and rescued by co-application of TTX and the K⁺ channel blocker 4-aminopyridine (4-AP), consistent with monosynaptic connectivity (n = 4 animals, 18 neurons; Fig. 3e). Furthermore, pharmacological profiling revealed that these light-evoked currents were blocked by GABA receptor antagonist picrotoxinin (PTX; n = 3 animals, 13 neurons, Fig. 3f), but were unaffected by the AMPA receptor antagonist NBQX (n = 3 animals, 11 neurons; Fig. 3g) or D1 receptor antagonist SCH-23390 (n = 3 animals, 10 neurons; Fig. 3h), confirming that the inhibitory postsynaptic currents (IPSCs) were mediated by GABA receptors. This inhibitory influence was highly specific. Non-specific optogenetic activation of random DAergic neurons failed to elicit IPSCs in ~87% of Drug-seeking-EnsDA in the VTA (n = 3 animals, 13/15 neurons; Supplementary Fig.10a-e). To test whether the synaptic effects of Social-reward-EnsDA terminals could be sustained at high frequency, we performed cell-attached recordings from Drug-seeking-EnsDA. High-frequency photostimulation (40 Hz, 1 minute) of Social-reward-EnsDA terminals persistently decreased the firing rate of ~64% of tested Drug-seeking-EnsDA (n = 3 animals, 9/14 neurons; Fig. 3i), suggesting a net inhibitory influence. These results establish a specific, functional inhibition from Social-reward-EnsDA to Drug-seeking-EnsDA.

Given that previous studies demonstrated that Aldehyde Dehydrogenase 1a1 (ALDH1a1) but not the glutamate decarboxylase (GAD) mediates a GABA synthesis pathway in midbrain DAergic neurons22,23, we sought to determine whether ALDH1a1 drives functional GABA co-release by Social-reward-EnsDA. To address this, we examined GAD and ALDH1a1 expression in Social-reward-EnsDA via immunohistochemical co-labeling with TH. Our results revealed that only ~10% of Social-reward-EnsDA expressed GAD, whereas ~37% of these neurons expressed ALDH1a1 (Supplementary Fig. 11a-d). Moreover, treatment with the ALDH inhibitor 4-(diethylamino)-benzaldehyde (DEAB, 10 µM) but not the GAD inhibitor 3-mercaptopropionic acid (3-MPA, 500 µM) dramatically reduced light induced IPSC amplitude (Supplementary Fig. 11e-i). Collectively, these findings establish that ALDH1a1 is required for functional GABA co-release by Social-reward-EnsDA.

Based on this circuit-level inhibition, we hypothesized that social interaction engaging Social-reward-EnsDA would reduce the responsiveness of Drug-seeking-EnsDA. We tested this by longitudinally imaging the same DAergic populations across different social conditions (social reward or no social reward) (Fig. 3j). Indeed, following a 30-minute free social interaction, the response of Drug-seeking-EnsDA was markedly attenuated, with both the magnitude and proportion of DAergic neurons responsive to drug seeking significantly diminished (n = 5 animals, 110 neurons; Fig. 3k-o). This effect was specific to the Drug-seeking-EnsDA, as neurons responsive to social reward or those responsive to both categories were unaffected (Fig. 3p,q). Importantly, control animals without prior social interaction showed stable Drug-seeking-responsive DAergic activity across sessions, ruling out extinction effects (Supplementary Fig. 7p-r) and indicating that the observed reduction in Drug-seeking-EnsDA requires prior social reward experience.

Given that drug pursuit often occurs at the expense of natural rewards, particularly social reward24, we investigated the reciprocal possibility: whether Drug-seeking-EnsDA inhibits Social-reward-EnsDA. Using a similar dual-labeling strategy to selectively target Drug-seeking-EnsDA (Label 1) and Social-reward-EnsDA (Label 2) (Supplementary Fig. 12a-d), optogenetic stimulation of Drug-seeking-EnsDA elicited robust outward currents in ~78% of tested Social-reward-EnsDA (n = 4 animals, 18/23 neurons; Supplementary Fig. 12e). These currents were abolished by TTX and rescued by co-application of TTX and 4-AP (Supplementary Fig. 12e), indicating monosynaptic connectivity. Furthermore, the currents were also eliminated by PTX (n = 3 animals, 11 neurons; Supplementary Fig. 12f), but were unaffected by NBQX (n = 3 animals, 10 neurons; Supplementary Fig. 12g) or SCH-23390 (n = 3 animals, 10 neurons; Supplementary Fig. 12h), confirming that these currents represent GABA receptor-mediated IPSCs. Consistently, photostimulation of Drug-seeking-EnsDA terminals produced a sustained reduction in the firing rate of ~62% of tested Social-reward-EnsDA (n = 3 animals, 8/13 neurons; Supplementary Fig. 12i), supporting a net inhibitory influence.

To assess whether cocaine seeking experience alters Social-reward-EnsDA responses, we longitudinally tracked these neurons under varying drug-seeking conditions (cocaine relapse test vs. no relapse test; n = 3 animals, 319 neurons; Supplementary Fig. 12j,k). Following a 1-hour drug seeking during the cocaine relapse test, the proportion of DAergic neurons responsive to social stimuli was significantly reduced, although response magnitude remained unchanged (Supplementary Fig. 12l-n). This reduction was not due to social familiarity, as Social-reward-EnsDA responses remained stable across repeated social interactions without cocaine seeking (Supplementary Fig. 8p-r), suggesting that prior drug-seeking experience is required. Together, these findings support a model of reciprocal inhibition, wherein Social-reward-EnsDA and Drug-seeking-EnsDA dynamically compete to shape behavioral output.

Social-reward-EnsDA mimics the protective effect of social reward

Next, we tested whether activation of Social-reward-EnsDA is sufficient and/or necessary to prevent drug seeking during the relapse test. We bilaterally injected AAV-cfos-ERCreER-PEST and AAV-TH-DIO-hM3Dq-mCherry into the VTA to label Social-reward-EnsDA, and assessed whether chemogenetic reactivation via clozapine-N-oxide (CNO) attenuates cue-induced cocaine seeking (Fig. 4a,b). Electrophysiological recordings confirmed virus efficacy, with CNO increasing spontaneous firing of Social-reward-EnsDA (Fig. 4c). Behaviorally, CNO-mediated Social-reward-EnsDA activation reduced cue-induced active nose-pokes during the cocaine relapse test, without affecting inactive nose-pokes (Fig. 4d-f), indicating that Social-reward-EnsDA reactivation mimics the therapeutic effect of social reward against drug seeking. Optogenetic activation of Social-reward-EnsDA further validated this observation (Supplementary Fig. 13a-f). Additionally, control experiments activating random VTA DAergic neurons using AAV-hSyn-Cre and AAV-TH-DIO-hM3Dq-mCherry, following the methodology of a previously published study25, showed no impact on drug seeking (Supplementary Fig. 13g-l), confirming the specificity of Social-reward-EnsDA.

Given that activation of Social-reward-EnsDA attenuated drug seeking, we hypothesized that inhibiting these neurons would negate the protective effects of social reward. To test this, rats received bilateral injections of AAV-cfos-ERCreER-PEST and AAV-TH-DIO-hM4Di-mCherry into the VTA to label and silence Social-reward-EnsDA. CNO was administered 30 minutes prior to social interaction (Fig. 4g,h). Electrophysiological recordings confirmed reduced spontaneous firing of Social-reward-EnsDA following CNO treatment (Fig. 4i). The behavioral data showed that silencing Social-reward-EnsDA during social interaction increased cue-induced active nose-pokes, without affecting inactive nose-pokes (Fig. 4j-l), suggesting that the inhibition of Social-reward-EnsDA activity undermined the therapeutic effects of social reward against drug seeking during the relapse test.

Social-reward-EnsDA and Drug-seeking-EnsDA exhibit distinct inputs

Rabies virus-based ensemble-specific monosynaptic tracing enables selective labeling of monosynaptic inputs to defined neuronal ensembles26. To compare the whole brain map of the inputs to Social-reward-EnsDA and Drug-seeking-EnsDA, rats were infected with AAV-cfos-ERCreER-PEST and Cre-dependent helper viruses (AAV-TH-DIO-TVA-mCherry and AAV-DIO-RVG) into the VTA. Three weeks after labeling Social-reward-EnsDA and Drug-seeking-EnsDA, rats were infected with the modified rabies virus RV-EvnA-△G-EGFP in the VTA. One week after infection, brains were collected for fluorescence imaging to identify EGFP-labeled input neurons (Fig. 5a,b). Starter ensembles expressing mCherry were restricted to the VTA (Fig. 5c). Notably, additional EGFP-positive neurons within the VTA, not co-labeled with mCherry, suggested local projections from Social-reward-EnsDA to Drug-seeking-EnsDA (Fig. 5c). The normalized input index, which represents the ratio of EGFP-positive neurons to all input neurons, showed that Social-reward-EnsDA receives more inputs from the ventral striatum (VS), ventral pallidum (VP) and dorsal raphe nucleus (DRN) compared to the Drug-seeking-EnsDA, while Drug-seeking-EnsDA receives more inputs from the dorsal medial striatum (DMS) and central amygdala (CeA) compared to the Social-reward-EnsDA (Fig. 5d-f), which suggested that Social-reward-EnsDA and Drug-seeking-EnsDA establish different connections with brain regions.

The DRN-VTA pathway regulates the therapeutic effect of social reward

Our findings that Social-reward-EnsDA bidirectionally regulates drug seeking and preferentially receive input from the DRN, along with selective cfos activation in DRN-projecting but not VS- or VP-projecting VTA DAergic neurons during social interaction (Supplementary Fig. 14a-n), led to a clear hypothesis that activation of VTA DAergic neurons that receive input from the DRN would be sufficient to suppress drug seeking. To test this, we combined an anterograde monosynaptic virus (AAV2/1-TH-flpo) with a flpo-dependent DREADD virus (AAV-FDIO-hM3Dq-EGFP) to selectively activate VTA DAergic neurons that receive input from the DRN (Fig. 6a,b and Supplementary Fig. 15a,b). Electrophysiological validation confirmed that CNO administration robustly increased the firing frequency of targeted VTA DAergic neurons (Fig. 6c and Supplementary Fig. 15c). Crucially, at the behavioral level, activation of VTA DAergic neurons that receive input from the DRN reduced cue-induced active nose-pokes in both the cocaine and heroin relapse tests, without influencing inactive nose-pokes (Fig. 6d-f and Supplementary Fig. 15d-f), suggesting that activating VTA DAergic neurons that receive input from the DRN mimic the therapeutic effect of social reward in preventing drug seeking. In contrast, parallel activation of VTA DAergic neurons that receive input from VP or VS using the same approach failed to alter drug seeking (Supplementary Fig. 16a-l), confirming the circuit specificity of the DRN input. To rule out the effect of potential retrograde transport of AAV2/1-Cre27 and make our conclusion more convincing, we established control groups to dissect the functional roles of DRN-VTA projections versus VTA-DRN projections. The results showed that activation of the DRN-VTA serotonergic projections, rather than VTA-DRN dopaminergic projections, mimicked the therapeutic effect of social reward against drug seeking (Supplementary Fig. 17a-j), confirming the critical role of DRN serotonergic inputs.

To further investigate the functional relevance of VTA DAergic neurons that receive input from the DRN, we used the same virus strategy to inhibit this projection. AAV2/1-TH-flpo was injected into DRN, and AAV-fDIO-hM4Di-EGFP was injected into VTA (Fig. 6g,h and Supplementary Fig. 15g,h). Electrophysiological recordings confirmed that CNO administration reduced the spontaneous firing of EGFP-labeled DAergic neurons (Fig. 6i and Supplementary Fig. 15i). At the behavioral level, inhibiting VTA DAergic neurons that receive input from the DRN during the social interaction significantly increased cue-induced active nose-pokes both in the cocaine and heroin relapse tests, without affecting inactive nose-pokes (Fig. 6j-l and Supplementary Fig. 15j-l), suggesting that inhibition of VTA DAergic neurons that receive input from the DRN could reverse the therapeutic effect of social reward against drug seeking during the relapse test.

Methods
Animals
Male Sprague-Dawley rats (280–300 g; Beijing Vital River, China) were group-housed (5/cage) for one week, then singly housed post-surgery. Rats were kept on a 12 h light/dark cycle (lights on at 20:00) with ad libitum food and water. Behavioral tests were conducted during the dark phase. Only male rats were used in the present study, and sex was not included as a variable in the study design or statistical analysis. This choice was made to minimize potential confounding effects related to sex hormones and estrous cycle fluctuations, which could introduce additional variability in the behavioral and physiological measures under investigation. All procedures followed the guidelines set forth by the Regulation for the Administration of Affairs Concerning Experimental Animals (China, 1988) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with approval by the Biomedical Ethics Committee for Animal Use and Protection of Peking University (LA2021168).

Jugular Vein Catheterization
Rats were anesthetized with isoflurane (4–5% induction, 1.5–2% maintenance). A silicone catheter (OD 1.0 mm, ID 0.55 mm) was inserted into the jugular vein and secured near the right atrium. After a 5-day recovery, catheters were flushed daily with 0.4 mL heparinized saline (100 U/mL) and penicillin (40,000 U/mL) until the end of cocaine self-administration training.

Behaviour
Self-administration Operant Apparatus
Cocaine self-administration, extinction, and relapse tests were conducted in 20 sound-attenuated operant chambers (AniLab, Ningbo, China), each equipped with a house light, two nose-poke holes with infrared sensors, and a buzzer for auditory cues. Catheters were connected to pump-driven syringes. Data were recorded via AniLab software.

Cocaine/Heroin Self-administration Training
Based on our previous studies17,57, rats were trained to self-administer intravenous cocaine (0.75 mg/kg/inf) or heroin (0.05 mg/kg/inf) in 3-hour daily sessions for 10 days under a fixed ratio-1 schedule. Sessions began with the house light on. An active nose-poke triggered a 5-s tone-light cue (CS), followed by drug infusion and a 20-s timeout (house light off), during which responses were recorded but not reinforced. Inactive nose-pokes had no consequence. To prevent overdose, infusions were capped at 21 per hour (max 63/session).

Extinction Training
Under identical conditions (3 h/day), extinction sessions omitted drug delivery while maintaining CS presentation after active nose-pokes. Training continued until active responses dropped to <20% of the average from the last 3 days of self-administration, sustained for 2 consecutive days.

Relapse Test
Four weeks after extinction, rats underwent a 1-hour relapse test. As in extinction, active nose-pokes triggered the CS without drug delivery.

Social Reward Treatment
Social reward treatment was conducted based on a previous study15,58, two juvenile male rats (4 weeks old, 200-220 g) were placed in a box lined with soft bedding to acclimate to the new environment for 5-10 minutes. The test rat was then introduced into the box for a 30-minute period, during which the test rat and juvenile rats were allowed to freely interact.

No Reward Treatment (Exposure Box Control)
Rats were placed alone in the same box for 30 minutes, without any conspecifics.

Toy Rat Interaction Treatment (Novelty Control)
Two lifelike toy rats were placed in the box, and the test rat was allowed to interact freely for 30 minutes.

Anesthetized Rat Interaction Treatment (Novelty Control)
Two juvenile male anesthetized rats (4 weeks old, 200-220 g) were placed in the same box. The test rat was then introduced and allowed to interact freely with the anesthetized rats for 30 minutes.

Sucrose Water Reward Treatment (Other Natural Reward Control)
Adapted from a previous study2, rats were water-deprived for 16–24 hours to induce a thirst state. After 5-10 minute of habituation in the box, they were given free access to sucrose water for 1 hour.

Social Conditioned Place Preference
The social conditioned place preference (CPP) procedure was performed based on a previous study16. On Day 1, rats were placed in the center of a three-chamber CPP apparatus and allowed to explore freely for 15 minutes to assess baseline preferences. Animals showing strong initial side preference were excluded. On Day 2, rats underwent 24 h of social conditioning (with two juvenile males) in one chamber and 24 h of isolation in the opposite chamber. Chamber assignments were counterbalanced. On Day 4, a 15-minute post-conditioning test was conducted. The CPP score was calculated as the time spent in the social-paired chamber minus that in the isolate-paired chamber.

Social Self-Administration
A social self-administration paradigm was conducted in sound-attenuated operant chambers (AniLab, Ningbo, China) for 8 days. Rats were trained to self-administer to gain access to social targets (two juvenile conspecifics) during daily 60-minute sessions. An active nose poke triggered the automated gate to open for 60 s, allowing the rat access to the social targets in a smaller chamber. The social target chamber had an open side covered with holes spaced 1 cm apart, enabling the rats to freely interact, smell, and investigate each other without either animal entering the other chamber. An inactive nose poke, located opposite the active nose poke, had no consequences. Data were recorded via AniLab software.

Optical Intracranial Self-Stimulation (oICSS)
To investigate whether interaction with juvenile conspecifics is rewarding, optogenetic techniques were employed to specifically activate social reward-Ens. oICSS training was conducted in operant chambers (AniLab, Ningbo, China) equipped with two nose pokes (active and inactive). Two weeks following stereotaxic surgery, the animals were placed in the chambers for daily behavioral training. A laser generator was triggered via a Master-9 pulse stimulator (Thinkerbiotech, Nanjing, China). During each 60-minute oICSS training session, an active nose poke elicited a 5-s tone and laser stimulation (1 s, 10 mW, 40 Hz, 5 ms pulse width); an inactive nose poke had no consequences. The number of active and inactive nose pokes was recorded per session. Starting on day 5 of testing, the active nose poke delivering laser stimulation was switched to the opposite location (previously the inactive nose poke) and maintained until testing concluded on day 8. Scheduling of experimental events and data collection was performed using AniLab software.

AAV Injection
Procedures followed our previous protocol59, Rats were anesthetized with isoflurane (4-5% induction; 1.5-2% maintenance) and placed in a stereotaxic frame. AAVs were injected at 30 nL/minute using a glass pipette connected to a microsyringe via mineral oil-filled tubing. The pipette was left in place for ≥10 minutes post-injection to facilitate diffusion before slow withdrawal.

Calcium Imaging in VTA: AAV-TH-GCamp6s (AAV2/9, ≥2×10¹² vg/mL, 300 nL) was unilaterally injected into the VTA.

DA Release Detection in NAc: AAV-hSyn-GRANB-DA3.3 (AAV2/9, 5×10¹² vg/mL, 300 nL) was injected into the NAc.

DRN-VTA Imaging: AAV-TH-Cre (AAV2/R, ≥5×10¹² vg/mL, 250 nL) was injected into VTA; AAV-DIO-GCamp6s (AAV2/T9, 5×10¹² vg/mL, 250 nL) into DRN.

IEG Labeling of Social-reward-EnsDA and Drug-seeking-EnsDA: AAV-cfos-ERCreER (AAV2/9, 5×1011 vg/mL) and AAV-TH-DIO-mCherry (AAV2/8, 2×1013 vg/mL) were mixed 1:1 and injected into VTA (250 nL).

Chemogenetic Manipulation of Social-reward-EnsDA: Activation: AAV-cfos-ERCreER (AAV2/9, 5×1011 vg/mL) + AAV-TH-DIO-hM3Dq-mCherry (AAV2/8, 2×1013 vg/mL); Inhibition: AAV-cfos-ERCreER (AAV2/9, 5×1011 vg/mL) + AAV-TH-DIO-hM4Di-mCherry (AAV2/8, 2×1013 vg/mL); All mixed 1:1 (250 nL/side).

Optogenetic activation of Social-reward-EnsDA: AAV-cfos-ERCreER (AAV2/9, 5×1011 vg/mL) + AAV-TH-DIO-ChR2-mCherry (AAV2/8, 2×1013 vg/mL); All mixed 1:1 (250 nL/side).

Monosynaptic Input Tracing: AAV-cfos-ERCreER (AAV2/9, 5×1011 vg/mL), AAV-TH-DIO-TVA-mCherry (AAV2/8, 2×1013 vg/mL), and AAV-TH-DIO-RVG (AAV2/8, 2×1013 vg/mL) were mixed 1:1:1 and injected into VTA (250 nL). Three weeks later, RV-EnvA-ΔG-EGFP (≥2×10⁸ IFU/mL, 250 nL) was injected at the same site.

Electrophysiological Recording: AAV-cfos-rtTA (AAV2/9, 5×1012 vg/mL), AAV-TH-Flp (AAV2/8, 5×1012 vg/mL), and AAV-TRE-fDIO-ChR2-EGFP (AAV2/8, 5×10¹² vg/mL) were mixed 1:1:1 and injected into VTA (250 nL). Two weeks later, AAV-cfos-ERCreER (AAV2/9, 5×1011 vg/mL) and AAV-TH-DIO-mCherry (AAV2/8, 2×1013 vg/mL) were injected into VTA (1:1, 250 nL).

VTA DAergic Neurons that Receive Input from the DRN Manipulation: Activation: AAV-TH-Flp (AAV2/1, 5×10¹³ vg/mL, 300 nL) into DRN (AP −7.0, ML ±0.0, DV −6.0 mm); AAV-TRE-fDIO-hM3Dq-EGFP (AAV2/9, 5×10¹² vg/mL, 250 nL) into VTA. Inhibition: same coordinates; AAV-TRE-fDIO-hM4Di-EGFP (AAV2/9) into VTA.

DRNSERT-VTA Manipulation: AAV-SERT-Cre (AAV2/9, ≥2×10¹² vg/mL, 250 nL) into DRN; AAV-DIO-hM3Dq-mCherry (AAV2/9, 5×10¹² vg/mL, 250 nL) into VTA.

Activity-dependent Neuronal Labeling
Activity-dependent neuronal tagging followed previous studies60-62, with suitable modifications. 4-hydroxytamoxifen (4-OHT; Sigma, H6278) was dissolved at 20 mg/mL in ethanol (37 °C, 15 minutes), then diluted with corn oil (Sigma, C8267) to 10 mg/mL. Ethanol was removed by vacuum centrifugation. 4-OHT solution was stored at 4 °C for up to 24 h before use. Doxycycline (Dox; Sigma, D9891) was dissolved in saline at 5 mg/mL. Both were administered via intraperitoneal injection.

To minimize immediate-early gene activation from transport stress, rats were moved to an adjacent room ≥3 h before injection. Social-reward-responsive DAergic ensembles were tagged by 4-OHT (50 mg/kg, i.p.) 30 minutes before the social interaction test or Dox (50 mg/kg, i.p.) 1 h before the social interaction test. Drug-seeking ensembles were tagged by 4-OHT (50 mg/kg, i.p.) 30 minutes before the relapse test.

Chemogenetic Activation or Silencing
Procedures followed previous studies61,63. To activate the activity of Social-reward-EnsDA, at least two weeks subsequent to the virus injection surgery, the rats underwent an intraperitoneal injection of 4-OHT 30 minutes prior to the social interaction. Roughly another 4 days later, the same group of rats received an intraperitoneal injection of clozapine N-oxide hydrochloride (CNO, 5mg/kg, dissolved in saline; MCE, HY-17366) 30 minutes before the drug relapse test. To silence the activity of Social-reward-EnsDA, at least two weeks after the virus injection surgery, the rats were intraperitoneally injected with 4-OHT 30 minutes before the social interaction. Approximately another 4 days later, the same rats were intraperitoneally injected with CNO 30 minutes before the social interaction, and then they immediately took a drug relapse test.

To activate the activity of VTA DAergic neurons that receive input from the DRN, at least two weeks after the virus injection surgery, rats received an intraperitoneal injection of CNO 30 minutes before the drug relapse test. To silence the activity of VTA DAergic neurons that receive input from the DRN, at least two weeks after the virus injection surgery, rats were intraperitoneally injected with CNO 30 minutes before the social interaction, and then they immediately took a drug relapse test.

Optogenetic Activation
The experimental procedures were performed as described in a previous study32. To activate Social-reward-EnsDA, at least two weeks after virus injection surgery, fiber optic probes were implanted at stereotaxic coordinates targeting the VTA (AP -5.9, ML ±0.5, DV -8.1 mm). Following a recovery period after probe implantation, the rats received an intraperitoneal injection of 4-OHT 30 minutes prior to the social interaction session on day50. Approximately 4 days later, the same rats underwent photo-stimulation (473 nm, 10-ms pulse duration, 20 Hz, 10 mW) delivered in 3 cycles: 5 minutes on followed by 10 minutes off for the first two, with the final cycle consisting of 5 minutes of stimulation immediately followed by the cocaine relapse test.

Fiber Implantation Surgery for Calcium Imaging
Rats were anesthetized with isoflurane (4-5% induction; 1.5-2% maintenance) and secured in a stereotaxic frame. The scalp was shaved, disinfected, and a midline incision made to expose the skull. Fiber optic probes were implanted at stereotaxic coordinates targeting the VTA (AP -5.9, ML ±0.5, DV -8.1 mm) and NAc (AP +2.1, ML ±1.4, DV -6.6 mm). Probes were lowered through craniotomies to the target depth and fixed with dental cement. The scalp was sutured in layers. After recovery, calcium imaging was performed during social interaction, relapse, or stimulus exposure.

GRIN Lens Implantation for Single-neuron Calcium Imaging
Rats were anesthetized with isoflurane (4-5% induction; 1.5-2% maintenance) and placed in a stereotaxic frame. After scalp incision and periosteum removal, the skull was cleaned with sterile saline and thinned using a microsurgical drill. A small dural incision was made. A 1 mm diameter, 0.5 NA GRIN lens (CLHS100GFT130, Gofoton) was inserted above the VTA (AP -5.9, ML ±0.5, DV -8.1 mm; ipsilateral to viral injection, referenced to bregma). The lens was lowered 0.8 mm and raised 0.4 mm to minimize tissue damage and set final depth. It was secured with cyanoacrylate and sealed with dental cement.

After ~3 weeks of recovery, a baseplate was aligned and fixed with dental cement. Subsequent single-photon imaging sessions were conducted during behavioral tasks, including social interaction and drug relapse tests.

Immunocytochemistry
Rats were deeply anesthetized with isoflurane (4-5%) and transcardially perfused with 4% paraformaldehyde. Brains were post-fixed for 24 h, cryoprotected in 20% and 30% sucrose in PBS (pH 7.2), and coronally sectioned at 40 μm.

Sections were washed (3 × 15 minutes in PBS), permeabilized with 0.1-0.5% Triton X-100 for 30 minutes at room temperature, and blocked with 5% BSA or serum in PBS for 1.5 h. Primary antibodies, mouse anti-cfos (1:1,000, Abcam, ab208942) or rabbit anti-tyrosine hydroxylase (1:2,000, Millipore, AB152), were applied overnight at 4 °C. After washing, slices were incubated for 2 h at room temperature with Alexa Fluor-conjugated secondary antibodies: goat anti-rabbit Alexa Fluor 647 (1:500, Invitrogen, A-21245) or donkey anti-mouse Alexa Fluor 488 (1:500, Invitrogen, A-32766).

Slices were counterstained with DAPI (Abcam, ab104139), mounted, and stored at 4 °C. Images were acquired with a fluorescence microscope (Olympus, 20× objective). Fluorescence-positive cells were manually quantified using ImageJ (v1.8.0).

Electrophysiology
Cell-attached and whole-cell recordings were performed 2 weeks after labeling Social-reward-EnsDA and Drug-seeking-EnsDA. Following anesthesia, brains were rapidly extracted, and 300-μm horizontal VTA slices were prepared using a vibratome (Leica VT1200S) in ice-cold cutting solution (80 mM NaCl, 3 mM KCl, 26 mM NaHCO₃, 1.3 mM MgCl₂, 1 mM NaH₂PO₄, 20 mM glucose, 1 mM CaCl₂, 75 mM sucrose; bubbled with 95% O₂/5% CO₂). Slices were transferred to ACSF (124 mM NaCl, 3 mM KCl, 26 mM NaHCO₃, 1.3 mM MgCl₂, 1.5 mM CaCl₂, 1 mM NaH₂PO₄, 20 mM glucose; 95% O₂/5% CO₂) at 33 °C for 30 minutes and then maintained at room temperature.

Patch pipettes (2–4 MΩ, TW150F-3, WPI) were filled with Cs-based low Cl⁻ internal solution (110 mM Cs-methanesulfonate, 15 mM CsCl, 10 mM HEPES, 0.5 mM EGTA, 4 mM QX-314, 4 mM Mg-ATP, 0.3 mM GTP, 5 mM Na₂-phosphocreatine; pH 7.3 with CsOH, 270–280 mOsm). ChR2-expressing Social-reward-EnsDA neurons were optically stimulated via a 473 nm laser (1 ms pulses, 6.5-10 mW) through the back aperture of the objective. Light pulses were delivered at 30 s intervals.

VTA DAergic neurons were voltage-clamped at -70 mV (EPSCs) or 0 mV (IPSCs). Pharmacological agents (MCE unless noted): NBQX (10 μM), picrotoxin (10 μM), TTX (1 μM), 4-AP (0.1-1 mM) were bath-applied. Opto-evoked EPSCs/IPSCs were recorded for 1 minute before and after drug application. Spontaneous activity was recorded in the cell-attached mode, with baseline, opto-stimulation (~1 minute), and post-stim epochs.

Signals were acquired using a MultiClamp 700B amplifier (Axon Instruments), filtered at 2 kHz, digitized at 5 kHz (PCI-MIO-16E4, National Instruments), and recorded with pClamp 10 (Molecular Devices). Analysis methods followed those in our previous study64. Briefly, all electrophysiological data were analyzed using Mini Analysis software (Synaptosoft, USA). After automated detection, each trace was manually inspected to exclude miscounting, double selections, or noise-induced artifacts. Recordings were discarded if the series resistance (Rs) changed by more than 20% or exceeded 30 MΩ to ensure data stability and reliability.

Data Analysis for Monosynaptic Input
The method of data analysis for whole-brain tracing followed the approach outlined in previous literature18. The 19 regions chosen for analysis included the major inputs to the VTA that fell outside of the excluded region near the injection site. For VTA retro-transsynaptic tracing, GFP-positive input neurons were manually counted from every third 60-μm section throughout the entire brain, excluding the region near the VTA (5.2 mm to 6.0 mm from bregma). As the total number of input neurons varied among brains, we normalized the neuronal count for each area by the total number of input neurons counted in the respective brain. For each of the 19 brain regions, every third section was analyzed. If the total number of inputs in a region was fewer than five, every section was counted, and the counts were then multiplied by a scale factor. We did not adjust for the potential double-counting of cells in our quantifications, which likely led to slight overestimates, the extent of overestimation varied depending on the size of the cells within each quantified region.

Fiber Photometry Recording and Analysis
Fiber photometry recordings were conducted during social interaction and cocaine relapse tests using a 405/470-nm multi-channel system (Inper Technology, Hangzhou, China) to monitor calcium activity in TH⁺ VTA neurons and dopamine release in the NAc. Excitation light intensity at the fiber tip was maintained at 20–40 μW, and signals were sampled at 50 Hz.

Fluorescence data were analyzed using commercial software (Inper Technology, Hangzhou, China). To correct for motion artifacts and baseline drift, the 405 nm control signal was fitted via least-squares regression and subtracted from the 470 nm signal. ΔF/F was calculated as (470 signal – fitted 405 signal)/fitted 405 signal.

For relapse tests, signals were aligned to the onset of active nose pokes (first and last 10 s), while for social interaction, data were aligned to the onset of body contact. Significant calcium transients were defined as epochs in which ΔF/F exceeded 2.91 median absolute deviations (MADs) above baseline and remained at least 2 MADs above baseline for a sustained period. Event-related activity was quantified by computing the area under the curve (AUC), focusing on windows spanning 10 s before to 10 s after behavioral events. Traces were trial-aligned and averaged, with shaded error bands representing the SEM.

Single-neuron Calcium Imaging Recording and Data Analysis
Calcium imaging acquisition
A miniature fluorescence microscope system (Thinkerbiotech, Nanjing, China) was used to image calcium dynamics in freely moving rats. The system consisted of a compact lens, integrated optical and excitation modules (LED light source), and a camera for fluorescence acquisition. The excitation wavelength was selected based on the calcium indicator used. Acquisition parameters (e.g., frame rate, exposure time) were optimized to balance signal quality and minimize phototoxicity. During recording sessions, rats engaged in behavioral tasks such as nose-poking and social interaction.

The system was interfaced with an acquisition card via USB 3.0 and SMA coaxial cables, allowing synchronization with the operant behavior apparatus. The Sync output was connected to the behavioral control system to align neural activity with behavioral events. The microscope baseplate was affixed to the rat’s skull using miniature screws. Imaging was performed after opening the main interface, loading the configuration file (User Config Example_V4_BNO_Miniscope.json), and verifying experimental parameters (e.g., file path, subject ID, LED power, frame rate, gain, focal plane). The recording was initiated by clicking the “Run” and “Record” buttons.

Calcium Imaging Pre-processing and Fluorescence Extraction
Calcium imaging data were processed using commercial software (Thinkerbiotech, Nanjing, China) according to established procedures10,65. Raw videos (608 × 608 pixels, 30 Hz) were downsampled to 304 × 304 pixels and 15 Hz. Motion correction was conducted using a template-matching plugin. Cellular fluorescence signals were extracted using the CNMFE algorithm in MATLAB (https://github.com/zhoupc/CNMF_E) and custom scripts (ROImap.m). ROIs were visually inspected to exclude non-cellular components based on morphological features.

Cell Registration across Sessions
To identify the same neurons across experimental sessions, the CellReg toolbox (https://github.com/zivlab/CellReg) and custom MATLAB code (aligned_cell_activity.m) were used. Calcium traces were aligned to the onset of behavioral events (time 0 s). The [-2 s to -0 s] window served as the baseline, and cellular activity was z-scored across a [-10 s to +20 s] window.

Definition of Responsive Neurons
Responsive neurons were identified using custom MATLAB code (responsive_neurons.m). Mean activity in the [0-20 s] post-event window across the first four trials was calculated. Neurons were classified as responsive to a given stimulus if their mean z-score exceeded 1.5.

Overlap Analysis
To assess whether social-reward- and drug-seeking-responsive DAergic subpopulations significantly overlapped, we compared the observed overlap rate to a null distribution. Responsive cell labels were randomly shuffled across all imaged neurons while maintaining the original number of cells responsive to each stimulus. For each of 10,000 permutations, the proportion of double-responsive neurons was computed. The empirical overlap rate was then compared to this null distribution. A significantly higher overlap than chance suggested a shared subpopulation activated by both stimuli. This analysis was conducted using custom MATLAB code (Comparing_the_overlap_fraction_with_shuffled_distribution.m).

Statistical Analysis
Following the experiments, all animals were euthanized for histological verification of viral expression and fiber placement. Only rats with correct injection and implant locations were included in the analyses. Behavioral data (e.g., number of infusions during cocaine/heroin self-administration and extinction) were analyzed using two-way ANOVA. A three-way ANOVA was used to assess active vs. inactive nose-pokes across sessions. For relapse data, a 2 × 2 repeated-measures ANOVA was performed. Significant ANOVA results were followed by simple effects analysis. All statistical data are presented as mean ± SEM. Analyses were conducted using GraphPad Prism v7.00 and IBM SPSS Statistics v24.00. Immunofluorescence quantification was performed in ImageJ. Statistical significance was set at *p < 0.05, **p < 0.01 and ***p < 0.001. Detailed statistical information is provided in Supplementary Table 1-15.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors investigated a very interesting effect of acute social experience decreasing cocaine relapse pressing in rats. The authors used many different tools to produce a very large amount of interesting data about the possible role of distinct groups of neurons (ensemble or cell types) in the VTA mediating this effect. While I think there are significant strengths in this paper, I do have some questions about the methods and data in the paper.\n1-From the data shown in Figure 1, it appears that pressing during drug relapse decreases when social interaction was immediately before drug relapse, but not when social interaction was 4 days before drug relapse. I did not see any mention of this in Results or Discussion.\n2-The “social reward treatment” they used is not a common way of assessing social behavior, which is fine but it requires some explanation and validation. It is not clear why they used juvenile rats as the social partners. Juvenile mice are used sometimes to get C57 mice to be more interested in social partners, but this is not an issue for rats that are already very social. Why not use peer rats that are age matched? The specific CPP procedure is also non-standard for social behavior and seems to be based again on what has been done previously in mice. They give a single 24 hour conditioning session with 2 juvenile male rats versus 24 hours of isolation in the opposite chamber (although I appreciate their counterbalancing the order of conditioning sessions). However, the results from this procedure could also be interpreted as avoiding the 24 hours of isolation rather than finding exposure to juvenile rats “rewarding”. This issue was a problem in some previous studies examining social CPP.\n3-The authors need to validate the accuracy of the TH promoter in their AAV to obtain expression in only dopamine neurons. To my knowledge, the viral TH promoter is only 80-85% accurate most likely due to many of the restriction elements not included in the very short regulatory sequence used in this virus. For this reason, genes in these TH promoter viruses could be expressed in non-DA cells in the VTA as well. Since half the neurons in VTA are GABAergic, the TH promoter viruses could have been expressing in a significant number of non-DA GABAeric neurons.\n4-Similar to the results for behavior (mentioned in comment 1), relapse-induced calcium increases assessed by photometry were attenuated following immediate exposure to social interaction, but 'not' when delayed 4 days after social. Please interpret and comment on this timing issue in paper.\n5-Again, it seems that social interaction immediately before drug relapse attenuated drug-induced DA in NAc, but I don’t think the authors examined whether this happens 4 days after social interaction. Please interpret and comment in paper.\n6-I am well acquainted with the Fos promoter viruses but I have not read about the ER-Cre-ER, which I found is similar to the more common Cre-ERT2 gene but with an extra ERT2 component upstream of Cre-ERT2. I can see it being useful; however, the use of this virus, at least for its current purpose, needs to be validated and characterized better. For example, in Figure 2a-f, they administered 4-OHT 30 minutes ‘prior’ to testing for all of their many “TRAP” virus experiments. First of all, this is not a typical time point for TRAPping ensembles activated by a behavior; e.g. the original DeNardo paper uses 1 hour ‘after’ (not prior to) activity to inject 4-OHT and others have found even later time points are optimal, at least in mice. They don’t necessarily need a full time course, but they should provide some evidence that there is both 4-OHT-dependent activation, and labeling that is induced by behavior, at 30 min prior to behavioral activation.\n7-Again, there is little to no validation and characterization of most of their Fos promoter viruses in this study. These are extremely finicky viruses to get to work in brain. For example, the effectiveness of the viral Fos promoter technique is extremely sensitive to the ratio of Fos promoter-TRAP virus to its DIO-effector gene virus. Indeed, work from the van den Oever lab indicates that copy numbers of Fos ‘TRAP’ virus has to be much lower than copy numbers of the DIO-effector virus. Overall, the viral Fos promoter methods used here have not worked well for most people who used it to examine ensembles in higher activity neurons such as in cortex and hippocampus and even nucleus accumbens. In general, the highly variable viral copy numbers of Fos-CreERT2 virus and effector virus (e.g. Dio-mCherry) in neurons around the injection site, combined with the fact that there is always some degree of Fos promoter activation in most neurons of the above brain areas, tends to make this technique difficult at best for use. This is why I am having a great deal of trouble believing that everyone of their Fos promoter experiments worked as well as they did.\nNevertheless, I am trying to see how these Fos promoter ‘TRAP’ viruses might work surprisingly well in VTA neurons despite the very low rate of success in cortex or striatum. The only way that I can see these Fos promoter ‘TRAP’ viruses working so well here is maybe (1) because of relatively low Fos promoter activity in DA neurons inducing very low levels of Cre-ERT2 combined with (2) very different sets of DA neurons to the point of being distinctly different subtypes of DA neurons being activated by social interaction versus drug relapse. These factors could create an 'on versus off' activation between the the social versus drug relapse ensembles. One would not see this clear distinction between ensembles in regions such as cortex, hippocampus or striatum, which are made of multiple cell types.\n8-I fully appreciate that they used all combinations of ensemble activation when looking at double-labeling experiments with the two different types of Fos promoter viruses. However, the fundamental problem with their comparison of social versus drug relapse ensembles here is that they are comparing two means of neural activation that cannot be directly compared: drug seeking test versus social reward, which here is just “body contact with a social partner”. For drug relapse, rats have gone through extensive operant training for the drug reward, while social reward is just a single session of exposure to younger animals in a cage. Too many variables are different between the two types of reward for a fair comparison at the ensemble level using calcium imaging. For example, do we see separate ensembles because of the different sensory modalities, i.e. smelling and having contact with a social partner vs. lever pressing for a drug cue? A better comparison would be to train them for both types of reward and then see if ensembles are different when they are performing an operant response to access each separate cue. Then it would be a true test of whether we have separable social and drug ensembles, which I don’t think we can really conclude that from the calcium imaging study done in Figure 2.\n9-The authors also observed very distinct ensembles in VTA using single neuron calcium imaging. I can see this being true only if they are looking at the equivalent of social vs drug relapse in functionally (or perhaps genetically) distinct cell types within all DA neurons in VTA. In which case, I would more accurately call them cell types rather neuronal ensembles which are more flexible and pluripotent than cell types. None of this would have worked out this way in cortex or hippocampus where any given neuron can be involved in encoding many different memories.\n10-The idea of the social ensemble suppressing the drug ensemble is very intriguing and I think there are some really interesting data in this study that support it. However, it’s a bit of a problem that the TH promoter virus could be expressing in GABAergic neurons as well since its only 80-85% accurate. Cell type differences here, rather than ensemble (selected by cues) differences, could explain some of their observed differences.\n11-Minor comment: The authors say they used head-fixed rats in the methods section for single neuron calcium imaging but say they used freely moving animals in page 9 of the Results section.\n12-The description of viral mechanism and transport direction in the results and captions and in the diagram in panels 6a,g are the opposite of what these viruses are actually doing. AAV2/1-TH-Cre or AAV2/1-SERT-Cre is retrogradely transported from the injection site to afferent brain areas that innervate the injection site; not the other way around (not in the prograde direction). The description and figure mechanism is incorrect. Its most likely that the wording and associated figures are incorrect rather than their experiments. They need to state that AAV2/1-TH-flpo was 'retrogradely' transported from the DRN to the VTA to activate AAV-fDIO-hM4Di-EGFP in the VTA TH neurons. They need to change the direction of the arrow in their diagrams in their figures, and state that they are looking at TH projections 'from' VTA to DRN, and NOT projections from DRN to VTA. And then revise the associated description of results and interpretation throughout the manuscript.\n13-In the Discussion section: “Thus, we speculate that social reward and drug seeking may represent distinct motivational states, each engaging specialized DA circuits”. I actually think their data is likely saying this. However, it seems more to do with cell types or DA subtypes in VTA rather than actual ensembles selected by cues. The paper would have been amazing if they showed distinct transcriptomes or cell-type markers with ISH for the two DA cell types activated by social interaction versus drug relapse.\n14- On page 21: “This antagonistic architecture” might explain why the sharp distinctions they observed with all the Fos promoter-driven viruses. But these are distinctions that you would more likely observe with distinct cell types, not with neuronal ensembles made of largely the same cell types selected by cues.\n15-I think there is a typo in the caption for panel 3n: they mention ‘food-responsive neuron’ here and I did not see food seeking anywhere else.", "reviewer_2": "Zheng et al. identify two distinct dopaminergic ensembles in the VTA: one that processes social reward and another that governs drug-seeking behavior, with functional competition between the two shaping behavioral outcomes. They further delineate an organized DRN-to-VTA circuit that selectively underlies the protective effect of social reward against drug-seeking behaviors. The study’s major conceptual advance lies in the identification of these two mutually inhibiting VTA dopaminergic ensembles, which not only elucidates the neuronal ensemble and circuit mechanisms through which social reward suppresses drug relapse, but also opens promising avenues for developing novel neuromodulation-based interventions for addiction. The authors skillfully employ cutting-edge methodologies to dissect the underlying neural mechanisms. Overall, the study presents compelling findings, and the manuscript is logically structured and clearly articulated. The following issues, however, should be addressed in a revision.\n1.While social interaction with two juvenile conspecifics has been demonstrated to induce rewarding effects in the social conditioned place preference (sCPP) paradigm, the rigor of the present study would be significantly improved by explicitly verifying whether the 30-minute social interaction used here indeed elicits a reward state. A robust approach would be to investigate whether the identified \"Social-reward-EnsDA\" neurons support intracranial self-stimulation (ICSS) behavior.\n2.In the Discussion, the authors cite literature suggesting that the behavioral consequences of social interaction in substance use disorder critically depend on the drug exposure history of the social partners. The present study clarifies the dopaminergic mechanisms by which interaction with drug-naïve conspecifics exerts a protective effect against drug seeking. However, the Discussion should be expanded to incorporate research on how social interaction with drug-experienced conspecifics can promote addiction. Given the growing body of evidence on the dual roles of social context in addiction—both protective and risk-promoting—the authors should integrate these perspectives to provide a more comprehensive and balanced discussion.\n3.Regarding the network mapping of the ensembles: the connectivity of the drug-seeking–responsive ensemble is consistent with established evidence, as it receives inputs from the central amygdala—a structure well known to mediate Pavlovian associations of reward-predictive cues. Notably, it is surprising that neither ensemble exhibits robust inputs from the prefrontal cortex (PFC). The authors should identify and discuss prior research that supports or contextualizes this specific observation, and clarify whether this finding aligns with or challenges current models of reward and addiction circuitry.\n4.Regarding the result presentation of fiber photometry recording and miniscope imaging, inconsistencies exist in the descriptions of DAergic neuron responses to social reward. Standardization of these descriptions is recommended to ensure clarity and comparability. For instance, the results from fiber photometry recording experiments are stated as: “Body contact during social interaction reliably evoked robust calcium transients in VTA DA neurons (Fig. 1h–j).” In contrast, the results from miniscope imaging experiments are described as: “17% (65 out of 380 neurons, n = 6 animals) responded selectively to social reward during the free interaction session (Fig. 2l–n and Fig. 2v).”\n5.The manuscript exhibits inconsistencies in terminology formatting: the capitalization of the first letters in “Social-reward-EnsDA” and “Drug-seeking-EnsDA” is not uniform across the text, and “VTA DAergic neurons” is used interchangeably with “VTADA neurons.”\n6.In Fig. 1d, the label \"No rewrad\" is misspelled.", "reviewer_3": "Previous studies have shown that social rewards can help inhibit drug-seeking behavior, although the mechanisms behind this are still unclear. This study provides an intriguing perspective by identifying two populations of VTA DA neurons that are selectively activated by drug-seeking behavior and social reward, respectively. Activation of the Social-reward-Ens-DA neurons can suppress activity in the Drug-seeking-Ens-DA neurons. Additionally, these Social-reward-Ens-DA neurons receive upstream input from the DRN, a region widely recognized for its role in social behavior. Overall, this research offers a valuable mechanism to further elucidate the interaction between social and drug-related rewards in this field. Below are some concerns I have:\n1) For Fig 1f, many green fluorescent-labeled neurons do not appear to co-label with TH. The viral vector based on the TH promoter used here is not commonly employed. Could the authors provide evidence to ensure its specificity?\n2) In Fig 2d, the histological results only present a merged image, making it difficult to verify the co-labeling claims. It would be better to show separate channels for clarity. Additionally, why does TH expression appear so weak in D+D and S+S? Were the VTA locations for the representative images in these groups chosen consistently? Furthermore, the co-labeling observed via the cfos trap and fos staining for the same behavior seems to be quite low in the representative images.\n3) What proportion of VTA dopamine neurons do these two populations represent? Do their downstream projections differ in a way that might explain their functional divergence?\n4) For Fig 3e, the authors hypothesize that Social-reward-Ens-DA neurons inhibit Drug-seeking-Ens-DA neurons via non-canonical release of GABA and demonstrated a monosynaptic inhibitory connection between these two groups of dopamine neurons. However, this evidence alone is insufficient. Additional experiments such as IF or in situ should be performed to confirm co-expression of GABA markers (e.g., GAD or vGAT) and TH within Social-reward-Ens-DA neurons. Furthermore, the authors should ablate/inhibit all VTA GABAergic neurons to test whether the IPSCs still persist. Lastly, could the authors explain the observed latency in the light-evoked IPSCs?\n5) I'm curious about your experiment being done under social isolation conditions. Would social interaction still inhibit relapse in group-housed mice?\n6) For Figure 6, considering that AAV1 is both anterograde and retrograde, previous study (Lin et al., 2021) indicates VTADA also projects to DRN, albeit at a low proportion. Could this viral strategy inadvertently label DRN-projecting VTA DA neurons? Please clarify.\n7) What major types of neurons project from DRN to the Social-reward-Ens-DA neurons? Are they DA or 5-TH neurons?\n8) How do these two populations of neurons respond to other types of rewards, such as tasty food or addictive drug injections?\nMinor Issues:\n1) For Fig 2e, 2f, the statistical analysis should be presented as percentages rather than as raw numbers.\n2) For the miniscope data, the n-value should not only represent the number of neurons but also indicate how many animals these neurons are from.\n3) In Fig 5c, the starting cell is unclear, and the background appears messy."}, "author_response": "Response letter to manuscript NCOMMS-25-79000-T\nThe authors express their gratitude to the Reviewers for providing valuable feedback. Detailed responses addressing each comment are provided below. Comments from the Reviewers are highlighted in bold, while author responses are presented in plain blue text. Modifications made to the manuscript are indicated by italicized text on a yellow background. Page and line numbers referenced in the responses correspond to those found in the revised manuscript.\nComments to the Author:\nThe authors investigated a very interesting effect of acute social experience decreasing cocaine relapse\npressing in rats. The authors used many different tools to produce a very large amount of interesting\ndata about the possible role of distinct groups of neurons (ensemble or cell types) in the VTA mediating\nthis effect. While I think there are significant strengths in this paper, I do have some questions about the\nmethods and data in the paper.\nResponse: We would like to express our sincere appreciation to the Reviewer for recognizing the value of this\nmanuscript. We have carefully revised the manuscript and included our responses below.\nXiaoxing, L. (2026) https://BioRender.com/hqs0eg6.\n1. From the data shown in Figure 1, it appears that pressing during drug relapse decreases when social\ninteraction was immediately before drug relapse, but not when social interaction was 4 days before\ndrug relapse. I did not see any mention of this in Results or Discussion.\nResponse: We sincerely appreciate the Reviewer’s insightful observation that acute social interaction exerts a\ndifferential effect on drug relapse behavior depending on the interval between social exposure and the relapse\ntest. Specifically, that lever pressing during drug relapse was reduced when social interaction was\nadministered immediately before the test, but no such effect was observed when the interval was extended to 4\ndays. We apologize that this critical time-dependent characteristic of the protective effect of social rewards\nwas not addressed in the Results and Discussion sections of the original manuscript. To rectify this oversight,\nwe have supplemented additional experiments to systematically characterize the temporal dynamics of this\nprotective effect, and found that the protective effect of acute social reward is transient, disappearing after 24\nhours (Supplementary Fig. 18a,b).\nThe following text and image have been added to the manuscript:\nNotably, we found that the protective effect of acute social reward (with drug-naïve partners) is transient,\ndisappearing after 24 hours (Supplementary Fig. 18a,b). Consistently, fiber photometry recordings from the\nVTA and NAc revealed that drug seeking-induced calcium signal and DA release elevations were attenuated\nimmediately following social interaction, but this attenuation was no longer observed 24 hours later (i.e., 4\ndays after the initial social interaction). This time-limited protective phenotype may parallel the contingency\nmanagement in clinical addiction intervention30: in the absence of non-drug reward reinforcers, drug-seeking\nbehavior is highly prone to relapse31,32. (page 20-21, lines 419-427 in the revised manuscript)\nSupplementary Fig.18 | The protective effect of acute social interaction against drug seeking is transient. a,\nExperimental design to investigate the duration of the suppressive effect of acute social reward on cocaine-seeking\nbehavior. b, Number of nose pokes during cocaine self-administration training, extinction, and relapse tests. The\nprotective effect of acute social reward against cocaine seeking was absent 24 h after social interaction (n = 8-9,\nthree-way ANOVA with simple simple effect analysis: F =36.033, p < 0.001, more statistics see Supplementary\n(13,3)\nTable13A-C).\n3\n2. The “social reward treatment” they used is not a common way of assessing social behavior, which is\nfine but it requires some explanation and validation. It is not clear why they used juvenile rats as the\nsocial partners. Juvenile mice are used sometimes to get C57 mice to be more interested in social\npartners, but this is not an issue for rats that are already very social. Why not use peer rats that are age\nmatched? The specific CPP procedure is also non-standard for social behavior and seems to be based\nagain on what has been done previously in mice. They give a single 24 hour conditioning session with 2\njuvenile male rats versus 24 hours of isolation in the opposite chamber (although I appreciate their\ncounterbalancing the order of conditioning sessions). However, the results from this procedure could\nalso be interpreted as avoiding the 24 hours of isolation rather than finding exposure to juvenile rats\n“rewarding”. This issue was a problem in some previous studies examining social CPP.\nResponse: We thank the reviewer for this insightful comment. As noted, the use of juvenile conspecifics as\nthe social reward paradigm was indeed adapted from established protocols in mice. In preliminary\nexperiments, we compared the efficacy of age-matched peer rats versus juvenile conspecifics in inducing\nsocial CPP. While age-matched social interaction did elicit CPP in rats, the magnitude of preference was\nsubstantially weaker than that induced by juvenile conspecifics (Supplementary Fig. 1a-d). To further\nvalidate that our paradigm reflects social reward, we supplemented two complementary experiments:\n1) Social self-administration assay: Rats successfully acquired the operant response to gain access to\njuvenile conspecifics within the first day of training (Supplementary Fig. 1e,f). Rapid acquisition of\noperant responding for a stimulus is a hallmark of its rewarding properties in behavioral neuroscience.\n2) Optogenetic modulation of Socia-Ens activity during intracranial self-stimulation (ICSS): Activation of\nthe Social-reward-Ens was found to enhance ICSS behavior (Supplementary Fig. 1g–j). This result\ndirectly links the neural ensemble encoding social reward to the brain’s core reward circuitry,\nconfirming the rewarding nature of juvenile conspecific interaction.\nThe following text and image have been added to the manuscript:\nIn the present study, we investigate whether this similar social paradigm can serve as a social reward\nmanipulation in rats. Behavioral assays revealed that rats exhibited a robust conditioned place preference for\nthe context paired with juvenile conspecifics (Supplementary Fig. 1a-d) and successfully acquired the operant\nresponse required to access juvenile conspecifics (Supplementary Fig. 1e,f). Furthermore, activating the\nneurons activated by social interaction with juvenile conspecifics induces intracranial self-stimulation\nbehavior (Supplementary Fig. 1g-j). Collectively, these findings confirm that interaction with juvenile\nconspecifics can serve as a valid experimental manipulation of social reward in rats. (page 5-6, lines 88-96 in\nthe revised manuscript)\nL. (2026) https://BioRender.com/hqs0eg6.\nSupplementary Fig.1 | Interacting with two juvenile partners is rewarding. a, Experimental timeline for social\nconditioned place preference (CPP) induced by interaction with two juvenile partners. On Day 1, rats were put in\nthe CPP apparatus's center chamber for a 15-minute pre-conditioning test. Then, they had 24-hour social\nconditioning with two juvenile males in one side chamber, followed by 24-hour isolation in the other. On Day 4,\nthey were placed in the center chamber again for a 15-minute post-conditioning test. b, Social CPP scores for\nbaseline preference and the expression test. Rats exhibit a preference for the compartment associated with two\njuvenile partners following social CPP training (n = 8, paired two-tailed t test, t = 9.554, p < 0.001). c,\n(7)\nExperimental timeline for social CPP induced by interaction with an age-matched partner. The experimental\nprocedure was identical to that described above, except that the social stimulus was replaced with an age-matched\nconspecific. d, Social CPP scores for baseline preference and the expression test. Social CPP scores for baseline\npreference and the expression test. Rats also exhibited a preference for the compartment paired with an age-\nmatched partner following social CPP training (n = 8, paired two-tailed t test, t = 3.200, p = 0.015). e,\n(7)\nExperimental timeline for social self-administration induced by interaction with two juvenile partners. Rats were\ntrained to self-administer to gain access to social targets (two juvenile conspecifics) during daily 60-min sessions.\nAn active nose poke triggered the automated gate to open for 60 s, allowing the rat access to the social targets in a\nsmaller chamber. The social target chamber had an open side covered with holes spaced 1 cm apart, enabling the\nrats to freely interact, smell, and investigate each other without either animal entering the other chamber. An\ninactive nose poke, located opposite the active nose poke, had no consequences. f, Number of nose pokes during\nsocial self-administration sessions (n = 8, three-way ANOVA with simple simple effect analysis: F = 3.781, p =\n(7,8)\n0.041, more statistics see Supplementary Table12A). g, Virus injection strategy. h, Experimental timeline for optical\nintracranial self-stimulation (ICSS). ICSS training was conducted in operant chambers (AniLab, Ningbo, China)\nequipped with two nose pokes (active and inactive). an active nose poke elicited a 5-s tone and laser stimulation (1\ns, 10 mW, 40 Hz, 5 ms pulse width); an inactive nose poke had no consequences. i, Active nose pokes at port A and\nport B during each 60-min ICSS session (n = 8, three-way ANOVA with simple simple effect analysis: day1-4, F\n(3,12)\n= 5.764, p = 0.011, more statistics see Supplementary Table12B; day5-8, F = 21.248, p < 0.001, more statistics\n(3,12)\nsee Supplementary Table12C). j, Mean number of nose pokes during laser and non-laser trials, averaged across all\ntraining days. Elevated mean laser-evoked nose pokes relative to non-laser trials were observed exclusively in\nChR2-expressing rats (n = 8, two-way ANOVA with simple effect analysis: F = 103.654, p < 0.001, more\n(1,14)\nstatistics see Supplementary Table12D). All the data are presented as mean ± SEM.\n5\n3. The authors need to validate the accuracy of the TH promoter in their AAV to obtain expression in\nonly dopamine neurons. To my knowledge, the viral TH promoter is only 80-85% accurate most likely\ndue to many of the restriction elements not included in the very short regulatory sequence used in this\nvirus. For this reason, genes in these TH promoter viruses could be expressed in non-DA cells in the\nVTA as well. Since half the neurons in VTA are GABAergic, the TH promoter viruses could have been\nexpressing in a significant number of non-DA GABAeric neurons.\nResponse: We thank the reviewer for this valuable comment. We acknowledge that the TH promoter may not\nrestrict expression exclusively to dopaminergic neurons. This is indeed a critical issue, as it bears on whether\nthe inhibitory interaction mechanism between different ensembles is mediated by non-specific labeling of\nGABAergic neurons by the TH promoter, or by GABA co-release from TH-positive neurons.\nTo address this, we supplemented the following experiments:\n1) We performed immunohistochemical co-labeling experiments to examine the colocalization of TRAPed\nneurons with Glutamate decarboxylase (GAD, a classic marker for GABAergic neurons). The results\nrevealed that only ~10% of TRAPed neurons colocalized with GAD (Supplementary Fig. 11a,b).\n2) We examined the effects of GAD antagonists on IPSCs induced by optogenetic activation of Social-\nreward-EnsDA. The results revealed that the GAD inhibitor 3-mercaptopropionic acid (3-MPA) had no\neffect on IPSCs (Supplementary Fig. 11h).\nThese lines of evidence indicate that the protective effect of social reward against drug seeking is not\nmediated by non-specific labeling of GABAergic neurons by the TH promoter.\nTo clarify the mechanism underlying the protective effect of social reward against drug seeking, we reviewed\nrelevant literature and found that Aldehyde Dehydrogenase 1a1 (ALDH1a1), but not glutamate decarboxylase\n(GAD), mediates a GABA synthesis pathway in midbrain DAergic neurons (Kim et al., 2015, Science; Tritsch\net al., 2012, Nature). Thus, we sought to determine whether ALDH1a1 drives functional GABA co-release by\nSocial-reward-EnsDA. To this end, we supplemented the following experiments:\n1) We performed immunohistochemical co-labeling experiments to examine the colocalization of TRAPed\nneurons with Aldehyde dehydrogenase 1a1 (ALDH1a1, a marker that colocalizes with TH and mediates a\nGABA synthesis pathway in midbrain DAergic neurons). The results revealed that ~37% of TRAPed\nneurons colocalized with ALDH1a1 (Supplementary Fig. 11c,d).\n2) We examined the effects of the ALDH1a1 inhibitor on IPSCs induced by optogenetic activation of\nSocial-reward-EnsDA. The results revealed that the ALDH1a1 inhibitor 4-(diethylamino)-benzaldehyde (DEAB)\nsignificantly attenuated the amplitude of light-evoked IPSCs (Supplementary Fig. 11i).\nThese lines of evidence indicate that GABA co-release from TH-positive neurons, more precisely from those\nexpressing ALDH1a1, mediates the protective effect of social reward against drug relapse.\nThe following text and image have been added to the manuscript:\nGiven that previous studies demonstrated that Aldehyde Dehydrogenase 1a1 (ALDH1a1) but not the\nglutamate decarboxylase (GAD) mediates a GABA synthesis pathway in midbrain DAergic neurons22,23, we\nsought to determine whether ALDH1a1 drives functional GABA co-release by Social-reward-EnsDA. To\naddress this, we examined GAD and ALDH1a1 expression in Social-reward-EnsDA via immunohistochemical\nco-labeling with TH. Our results revealed that only ~10% of Social-reward-EnsDA expressed GAD, whereas\n~37% of these neurons expressed ALDH1a1 (Supplementary Fig. 11a-d). Moreover, treatment with the ALDH\ninhibitor 4-(diethylamino)-benzaldehyde (DEAB, 10 µM) but not the GAD inhibitor 3-mercaptopropionic acid\n(3-MPA, 500 µM) dramatically reduced light induced IPSC amplitude (Supplementary Fig. 11e-i).\nCollectively, these findings establish that ALDH1a1 is required for functional GABA co-release by Social-\nreward-EnsDA. (page 13-14, lines 265-276 in the revised manuscript)\nXiaoxing, L. (2026) https://BioRender.com/hqs0eg6.\nSupplementary Fig.11 | Aldehyde Dehydrogenase 1a1 Mediates the inhibitory effect of Social-reward-EnsDA on\nDrug-seeking-EnsDA. a, Representative histology images of Social-reward-EnsDA co-label TH or GAD. b, The\nproportion of co-labeled neurons (TH + Social-reward-EnsDA vs GAD + Social-reward-EnsDA) represents the total\nSocial-reward-EnsDA. c, Representative histology images of Social-reward-EnsDA co-label TH or ALDH1a1. d, The\nproportion of co-labeled neurons (TH + Social-reward-EnsDA vs ALDH1a1 + Social-reward-EnsDA) represents the\ntotal Social-reward-EnsDA. e,f, Strategy for Virus injection. g, Schematic of the experimental procedure for\nelectrophysiology recording. h, Left: Representative traces of evoked IPSCs upon bath application of GAD\ninhibitor 3-mercaptopropionic acid (3-MPA, 500 µM). Right: Amplitudes under control conditions (ACSF) or 3-\nMPA, normalized to baseline. i, Left: Representative traces of evoked IPSCs upon bath application of ALDH\ninhibitors 4-(diethylamino)-benzaldehyde (DEAB, 10 µM). Right: Amplitudes under control conditions (ACSF) or\nDEAB, normalized to baseline (DEAB: n = 8 neurons, paired t test, **p < 0.01).\n7\n4. Similar to the results for behavior (mentioned in comment 1), relapse-induced calcium increases\nassessed by photometry were attenuated following immediate exposure to social interaction, but 'not'\nwhen delayed 4 days after social. Please interpret and comment on this timing issue in paper.\nResponse: We sincerely appreciate the Reviewer’s astute observation that the photometry-based calcium\nresponse data aligns with our behavioral findings: relapse-induced calcium elevations were significantly\nattenuated when social interaction was administered immediately prior to the relapse test, whereas this\ninhibitory effect was absent when the interval between social exposure and testing was extended to 4 days\n(Fig.1e-r). This cross-modal consistency (behavioral and neural recordings) corroborates the conclusion that\nthe protective effect of social reward against drug relapse is time-dependent and transient. In response to the\nReviewer’s suggestion, we have incorporated a dedicated interpretation of this temporal specificity into the\nrevised Discussion section, integrating both behavioral and neural data to highlight this timing effect.\nThe following text has been added to the manuscript:\nNotably, we found that the protective effect of acute social reward (with drug-naïve partners) is transient,\ndisappearing after 24 hours (Supplementary Fig. 18a,b). Consistently, fiber photometry recordings from the\nVTA and NAc revealed that drug seeking-induced calcium signal and DA release elevations were attenuated\nimmediately following social interaction, but this attenuation was no longer observed 24 hours later (i.e., 4\ndays after the initial social interaction). This time-limited protective phenotype may parallel the contingency\nmanagement in clinical addiction intervention30: in the absence of non-drug reward reinforcers, drug-seeking\nbehavior is highly prone to relapse31,32. (page 20-21, lines 419-427 in the revised manuscript)\n5. Again, it seems that social interaction immediately before drug relapse attenuated drug-induced DA\nin NAc, but I don’t think the authors examined whether this happens 4 days after social interaction.\nPlease interpret and comment in paper.\nResponse: We sincerely appreciate the Reviewer’s critical comment regarding the need to clarify whether the\nmodulation of NAc activity persists at the 4-day interval following social interaction. As the Reviewer astutely\npoints out, our existing data have established a correspondence between DA release in the NAc, calcium\nactivities in the VTA DAergic neurons, and the behavioral effects of social interaction (Fig.1e-r and\nSupplementary Fig.3d-n). To address the Reviewer’s concern, we have supplemented a targeted interpretation\nin the revised Discussion section, explicitly stating that we did not observe any attenuation of relapse-induced\nDA release elevations when the interval between social interaction and the relapse test was extended to 4 days.\nThis finding further reinforces that the regulatory effect of social reward is time-limited and transient.\nThe following text has been added to the manuscript:\nNotably, we found that the protective effect of acute social reward (with drug-naïve partners) is transient,\ndisappearing after 24 hours (Supplementary Fig. 18a,b). Consistently, fiber photometry recordings from the\nVTA and NAc revealed that drug seeking-induced calcium signal and DA release elevations were attenuated\nimmediately following social interaction, but this attenuation was no longer observed 24 hours later (i.e., 4\ndays after the initial social interaction). This time-limited protective phenotype may parallel the contingency\nmanagement in clinical addiction intervention30: in the absence of non-drug reward reinforcers, drug-seeking\nbehavior is highly prone to relapse31,32. (page 20-21, lines 419-427 in the revised manuscript)\nXiaoxing, L. (2026) https://BioRender.com/hqs0eg6.\n6. I am well acquainted with the Fos promoter viruses but I have not read about the ER-Cre-ER, which\nI found is similar to the more common Cre-ERT2 gene but with an extra ERT2 component upstream of\nCre-ERT2. I can see it being useful; however, the use of this virus, at least for its current purpose, needs\nto be validated and characterized better. For example, in Figure 2a-f, they administered 4-OHT 30\nminutes ‘prior’ to testing for all of their many “TRAP” virus experiments. First of all, this is not a\ntypical time point for TRAPping ensembles activated by a behavior; e.g. the original DeNardo paper\nuses 1 hour ‘after’ (not prior to) activity to inject 4-OHT and others have found even later time points\nare optimal, at least in mice. They don’t necessarily need a full time course, but they should provide\nsome evidence that there is both 4-OHT-dependent activation, and labeling that is induced by behavior,\nat 30 min prior to behavioral activation.\nResponse: We sincerely appreciate the Reviewer’s insightful comments on the validation of the ER-Cre-ER\nviral system and the 4-OHT administration timing for TRAP experiments. We note that the canonical TRAP\nprotocol in mice involves 4-OHT injection at 1 hour after behavioral activation, as documented in the original\nwork by DeNardo et al. (2019, Nat Neurosci). Meanwhile, we have also taken note of the work by Wang et al.\n(2022, Nat Commun), which demonstrated that 4-OHT delivery 30 min prior to behavioral exposure is\neffective for labeling behaviorally activated neuronal ensembles in rodent models. To further validate the\nsuitability of this protocol for our rat model, we have conducted dedicated in-house characterization\nexperiments. Specifically, we systematically tested multiple 4-OHT injection time points relative to behavioral\nstimulation, and these data consistently showed that 4-OHT administration 30 min before behavioral\nactivation yielded the highest efficiency of TRAPed neuronal labeling in our experimental setting\n(Supplementary Fig. 4a–d).\nThe following text and image have been added to the manuscript:\nTo validate the TRAPing paradigm in rats, we first characterized the temporal dynamics of neuronal labeling.\nRats were subjected to 30 min of social interaction at varying time intervals relative to the administration of\n4-hydroxytamoxifen (4-OHT). Analysis of mCherry expression patterns demonstrated that the majority of\nTRAPed neurons were labeled within a 6-hour time window centered on 4-OHT injection. Notably, mCherry\nexpression levels peaked when 4-OHT was administered 30 min prior to the initiation of social interaction\n(Supplementary Fig. 4a–d). (page 8, lines 151-158 in the revised manuscript)\nSupplementary Fig.4 | Time course of targeted recombination in active populations (TRAPing). a, Strategy for\nvirus injection. b, Timeline of social stimulation experiment to determine effective TRAPing window. c,\nRepresentative images of TRAPed cells in the ventral tegmental area (VTA) of rats subjected to the social\nstimulation experiment. Scale bars, 200 μm. d, Quantification of TRAPed cell density in the VTA (n = 4, one-way\nANOVA with bonferroni multiple comparisons test: F = 132.4, p < 0.001; home vs 3h, p < 0.001; home vs 0.5h,\n(5,18)\np < 0.001; home vs 0h, p < 0.001; home vs -3h, p < 0.001; home vs -6h, p = 0.973). All the data are presented as\nmean ± SEM.\n9\n7. Again, there is little to no validation and characterization of most of their Fos promoter viruses in this\nstudy. These are extremely finicky viruses to get to work in brain. For example, the effectiveness of the\nviral Fos promoter technique is extremely sensitive to the ratio of Fos promoter-TRAP virus to its DIO-\neffector gene virus. Indeed, work from the van den Oever lab indicates that copy numbers of Fos\n‘TRAP’ virus has to be much lower than copy numbers of the DIO-effector virus. Overall, the viral Fos\npromoter methods used here have not worked well for most people who used it to examine ensembles in\nhigher activity neurons such as in cortex and hippocampus and even nucleus accumbens. In general, the\nhighly variable viral copy numbers of Fos-CreERT2 virus and effector virus (e.g. Dio-mCherry) in\nneurons around the injection site, combined with the fact that there is always some degree of Fos\npromoter activation in most neurons of the above brain areas, tends to make this technique difficult at\nbest for use. This is why I am having a great deal of trouble believing that everyone of their Fos\npromoter experiments worked as well as they did.\nNevertheless, I am trying to see how these Fos promoter ‘TRAP’ viruses might work surprisingly well\nin VTA neurons despite the very low rate of success in cortex or striatum. The only way that I can see\nthese Fos promoter ‘TRAP’ viruses working so well here is maybe (1) because of relatively low Fos\npromoter activity in DA neurons inducing very low levels of Cre-ERT2 combined with (2) very different\nsets of DA neurons to the point of being distinctly different subtypes of DA neurons being activated by\nsocial interaction versus drug relapse. These factors could create an 'on versus off' activation between\nthe the social versus drug relapse ensembles. One would not see this clear distinction between ensembles\nin regions such as cortex, hippocampus or striatum, which are made of multiple cell types.\nResponse: We sincerely appreciate the Reviewer’s critical and insightful comments regarding the validation\nand characterization of Fos promoter-driven TRAP viral tools, as well as the thoughtful analysis of potential\nfactors enabling robust labeling in VTA dopaminergic neurons. We fully acknowledge that Fos promoter-\nbased viral systems are technically demanding, with their efficacy being highly sensitive to experimental\nparameters, including the viral titer ratio of Fos-TRAP virus to DIO-effector virus, as highlighted by work\nfrom the van den Oever lab. We also agree that this technique often yields inconsistent results in brain regions\nwith high basal neuronal activity (e.g., cortex, hippocampus, and striatum), due to issues such as variable viral\ncopy number integration and non-specific Fos promoter activation in these heterogeneous cell populations.\nIn the present study, we optimized multiple experimental variables to ensure the specificity and efficiency of\nTRAP labeling in VTA dopaminergic neurons, which we believe accounts for the robust outcomes observed:\n1) Optimization of viral titer ratio. The viral ratio used in our experiments was established based on a\nprevious work (Lee et al., 2023, Nat Neurosci). Specifically, AAV-cFos-ERCreER (AAV2/9, 5×10¹¹\nvg/mL) and AAV-TH-DIO-mCherry (AAV2/8, 2×10¹³ vg/mL) were mixed at a 1:1 volume ratio for VTA\ninjection. This ratio is consistent with the principle emphasized by the van den Oever lab that the copy\nnumber of the Fos-TRAP virus should be lower than that of the DIO-effector virus.\n2) Temporal optimization of 4-OHT administration. As detailed in our response to Comment 6, we\nsystematically tested multiple 4-OHT injection time points relative to behavioral stimulation. We\nidentified that administration 30 min prior to behavior initiation yielded the peak efficiency of TRAPed\nneuronal labeling in the VTA (Supplementary Fig. 4a–d).\n3) Stringent validation of behavior-dependent labeling specificity. To rule out non-specific Fos promoter\nactivation, we performed a home cage control experiment where 4-OHT was administered without any\nbehavioral manipulation. This resulted in minimal TRAP labeling in VTA neurons, confirming that the\nobserved labeling was strictly dependent on behavioral stimulation (Supplementary Fig. 4a–d).\n4) Validation of DAergic neuron-specific labeling. Colocalization analysis of TRAP-labeled neurons\n(mCherry⁺) with TH (a specific marker for DAergic neurons) demonstrated high co-expression efficiency,\nwith ~90% of TRAPed neurons expressing TH (Supplementary Fig. 11a-d). This confirms the specificity\nof our labeling strategy for VTA DAergic neurons.\n5) Distinct ensemble separation in VTA DAergic neurons. Consistent with the Reviewer’s astute hypothesis,\nwe observed that social reward- and drug relapse-induced TRAPed neuronal populations were largely\ndistinct, this clear separation of functional ensembles is likely attributable to two key characteristics of\nVTA DAergic neurons: i) relatively low basal Fos promoter activity, which minimizes non-specific Cre-\nERT2 expression; and ii) the functional heterogeneity of DAergic neuron subtypes, where distinct\n10\nsubpopulations are preferentially activated by social reward versus drug-associated cues. This stands in\ncontrast to heterogeneous regions like the cortex or striatum, where overlapping activation of diverse cell\ntypes obscures ensemble specificity.\nIn summary, we do not intend to suggest that Fos-TRAP viral approaches are broadly applicable across brain\nregions, but rather that, under carefully optimized conditions, they can be selectively effective in VTA\ndopaminergic neurons.\nThe following image has been added to the manuscript:\nSupplementary Fig.11 | Aldehyde Dehydrogenase 1a1 Mediates the inhibitory effect of Social-reward-EnsDA on\nDrug-seeking-EnsDA. a, Representative histology images of Social-reward-EnsDA co-label TH or GAD. b, The\nproportion of co-labeled neurons (TH + Social-reward-EnsDA vs GAD + Social-reward-EnsDA) represents the total\nSocial-reward-EnsDA. c, Representative histology images of Social-reward-EnsDA co-label TH or ALDH1a1. d, The\nproportion of co-labeled neurons (TH + Social-reward-EnsDA vs ALDH1a1 + Social-reward-EnsDA) represents the\ntotal Social-reward-EnsDA.\n11\n8. I fully appreciate that they used all combinations of ensemble activation when looking at double-\nlabeling experiments with the two different types of Fos promoter viruses. However, the fundamental\nproblem with their comparison of social versus drug relapse ensembles here is that they are comparing\ntwo means of neural activation that cannot be directly compared: drug seeking test versus social\nreward, which here is just “body contact with a social partner”. For drug relapse, rats have gone\nthrough extensive operant training for the drug reward, while social reward is just a single session of\nexposure to younger animals in a cage. Too many variables are different between the two types of\nreward for a fair comparison at the ensemble level using calcium imaging. For example, do we see\nseparate ensembles because of the different sensory modalities, i.e. smelling and having contact with a\nsocial partner vs. lever pressing for a drug cue? A better comparison would be to train them for both\ntypes of reward and then see if ensembles are different when they are performing an operant\nresponse to access each separate cue. Then it would be a true test of whether we have separable social and drug\nensembles, which I don’t think we can really conclude that from the calcium imaging study done in\nFigure 2.\nResponse: We sincerely appreciate the Reviewer’s incisive critique regarding confounding variables in our\ninitial ensemble comparison. We acknowledge that the divergent paradigms, that is a single social interaction\nsession versus drug seeking behavior following extensive operant training, could attribute ensemble\nsegregation to sensory or behavioral differences. According to your suggestions, we performed\ncomplementary operant conditioning experiments where rats were trained to nose poke for both social and\ncocaine rewards under matched conditions, eliminating task related disparities for a direct comparison.\nSpecifically, we first trained rats in Context A for social self-administration (7 consecutive days), a social\nactive nose poke triggered the automated gate to open for 60 s, allowing the rat access to the social targets in a\nsmaller chamber. One week later, the same cohort of rats was trained in Context B for cocaine self-\nadministration (7 consecutive days), a drug active nose-poke triggered a ~2 s cocaine infusion. Following this\nmatched training paradigm, we performed single neuron calcium imaging during both social seeking and drug\nseeking tests, where active nose pokes did not result in the delivery of any reward reinforcer (Supplementary\nFig. 9 a,b). Our results revealed that only 10% of recorded neurons exhibited dual responsiveness to both\nsocial and cocaine cues (Supplementary Fig. 9c–p). Subsequent statistical analyses confirmed a lack of\nsignificant overlap between social seeking and drug seeking responsive neuronal populations\n(Supplementary Fig. 9q). These data rule out the possibility that ensemble segregation of social-seeking and\ndrug-seeking encoding is an artifact of divergent task designs.\nThe following text and image have been added to the manuscript:\nIn addition, given the substantial array of confounding variables inherent to the social interaction versus drug\nrelapse sessions, one might hypothesize that the observed segregation of neuronal ensembles stems from\ndivergent sensory and behavioral modalities. To address this alternative explanation, we trained rats to\nperform operant responses for both social self-administration and cocaine self-administration, then assessed\nwhether distinct neuronal ensembles were engaged when rats executed goal-directed behaviors to obtain these\ntwo discrete reward-related stimuli (Supplementary Fig. 9a,b). Our results demonstrated that only 10% of\nrecorded neurons (26 out of 261 neurons; n = 2 animals) exhibited dual responsiveness to both social and\ncocaine cues (Supplementary Fig. 9c–p). Subsequent statistical analyses confirmed a lack of significant\noverlap between social-seeking- and drug-seeking-responsive neuronal populations (Supplementary Fig. 9q).\nCollectively, these findings establish that VTA DAergic ensembles exhibit stable, stimulus-specific identities\nand that their segregation is not a consequence of temporal factors. (page 11-12, lines 221-234 in the revised\nmanuscript)\nof the experimental design for microscope calcium imaging. b, Representative raw microscope image. c, f, i, l,\nExample field of view with neurons colored by the stimulus type that they are responsive to (n = 131 neurons).\nd, e, Example of a neuron selectivity responsive to cocaine seeking. g, h, Example of a neuron selectivity responsive to\nsocial seeking. j, k, Example of a neuron responsive to both. m, n, Example of a neuron responsive to neither. o,\nMatching neurons imaged across distinct sessions (n = 261/519 neurons). p, Percentage of neurons responsive to\nsocial seeking, cocaine seeking, both, or neither (n = 261 neurons). q, Overlap compared with null distribution\nassuming Social-seeking- and Drug-seeking-responsive neurons are independent samples. All the data are\npresented as mean ± SEM.\n13\n9. The authors also observed very distinct ensembles in VTA using single neuron calcium imaging. I can\nsee this being true only if they are looking at the equivalent of social vs drug relapse in functionally (or\nperhaps genetically) distinct cell types within all DA neurons in VTA. In which case, I would more\naccurately call them cell types rather neuronal ensembles which are more flexible and pluripotent than\ncell types. None of this would have worked out this way in cortex or hippocampus where any given\nneuron can be involved in encoding many different memories.\nResponse: We sincerely appreciate the Reviewer’s insightful comment regarding the distinction between\nneuronal ensembles and cell types in the VTA. We acknowledge that the two dopaminergic populations\nresponsive to social reward and drug seeking may exhibit distinct genetic profiles, and further validation of\ncell type specificity via techniques such as single cell sequencing would represent an important and interesting\ndirection for future research.\nIn here, our use of the term “neuronal ensemble” in the present study is grounded in two lines of published\nevidence in the field:\n1) The review (Yuste et al., 2024, Neuron) defined a neuronal ensemble as “a population of neurons involved\nin a particular computation”. The notion of ensemble implies that coding is produced by populations of\nneurons whose individual contributions are noisy but that together produce coherent outputs. The term is\nmostly used within systems and computational neuroscience to describe a neural network with a particular\nfunction.\n2) The study (Gimenez-Gomez et al., 2025, Nat Neurosci) validated the existence of binge drinking\nassociated neuronal ensembles in the mouse medial orbitofrontal cortex (mOFC) by combining TRAP\nlabeling with cFos immunofluorescence staining. They demonstrated that neurons TRAPed during the\nfirst binge drinking episode could be reactivated during a subsequent binge drinking challenge, with a\ncolocalization rate of 50%–60%. This stable reactivation confirmed that binge alcohol drinking recruits a\ndedicated neuronal ensemble in the mOFC.\nTo confirm that the TRAP labeled VTA dopaminergic neurons constitute social reward ensembles and drug\nseeking ensembles, we performed analogous experiments. Specifically, we combined TRAP labeling with\ncFos immunofluorescence staining to examine whether neurons labeled during an initial episode of drug\nseeking or social interaction could be reactivated during a second temporally separated exposure to the same\nstimulus. Our results showed that 50%–60% of the labeled neurons were reactivated across the two distinct\ntime points of the same stimulus exposure (Supplementary Fig. 5j-n). Thus, by using the term \"ensemble,\"\nwe intend to describe the coordinated, synchronous activity of these neurons that collectively drives a specific\nbehavioral output (social reward vs. drug seeking).\nFinally, we have revised our Results and Discussion to address this important distinction, we view \"cell type\"\nas a static identity and \"ensemble\" as the active functional state of these neurons during behavior. We hope\nthese clarifications and the revised manuscript adequately address the Reviewer’s conceptual concern.\nThe following text has been added to the manuscript:\nNeuronal ensembles, defined as groups of neurons exhibiting recurring patterns of coordinated activity,\nrepresent a functional unit intermediate between single neurons and broader brain regions21. To determine\nwhether the TRAPed neurons in the VTA constitute a neuronal ensemble, we examined whether neurons\nlabeled during the first episode of drug seeking or social interaction could be reactivated during a second,\ntemporally separated episode of the same stimulus. Indeed, re-exposure to the same condition robustly\nreactivated TRAPed neurons, as evidenced by their overlap with cfos expression, confirming the presence of\nstable Drug-seeking- and Social-reward-responsive ensembles within the VTA (Supplementary Fig. 5l-s).\n(page 9-10, lines 173-181 in the revised manuscript)\nHowever, it remains unclear whether these two ensembles exhibit distinct transcriptomic profiles or cell-type-\nspecific markers, and future studies employing techniques such as single-cell sequencing and spatial\ntranscriptomics to characterize their transcriptional features and molecular markers represent a promising\nand interesting direction for further investigation. (page 22, lines 457-462 in the revised manuscript)\n14\n10. The idea of the social ensemble suppressing the drug ensemble is very intriguing and I think there\nare some really interesting data in this study that support it. However, it’s a bit of a problem that the\nTH promoter virus could be expressing in GABAergic neurons as well since its only 80-85% accurate.\nCell type differences here, rather than ensemble (selected by cues) differences, could explain some of\ntheir observed differences.\nResponse: We sincerely appreciate the Reviewer’s insightful comment regarding the potential off-target\nexpression of the TH promoter-driven virus and its implications for interpreting our ensemble-specific\nfindings. We fully acknowledge that it is necessary to rule out the possibility that TH promoter-based viruses\nmay exhibit non-specific expression in GABAergic neurons. To address this, we performed two\ncomplementary lines of investigation to dissociate the roles of DA-mediated GABA co-release versus\ntraditional GABAergic transmission:\n1) We performed immunohistochemical co-labeling experiments to examine the colocalization of TRAPed\nneurons with Glutamate decarboxylase (GAD, a classic marker for GABAergic neurons). The results\nrevealed that only ~10% of TRAPed neurons colocalized with GAD (Supplementary Fig. 11a,b),\nsuggesting while a small degree of off-target expression is inherent to the TH promoter, this low\npercentage indicates that the majority of our manipulated cells are indeed dopaminergic neurons.\n2) We examined the effects of GAD antagonists on IPSCs induced by optogenetic activation of Social-\nreward-EnsDA. The results revealed that the GAD inhibitor 3-mercaptopropionic acid (3-MPA) had no\nsignificant effect on IPSCs (Supplementary Fig. 11h).\nThese lines of evidence indicate that the protective effect of social reward against drug seeking is not\nmediated by non-specific labeling of GABAergic neurons by the TH promoter.\nGiven that previous studies (Kim et al., 2015, Science; Tritsch et al., 2012, Nature) revealed that Aldehyde\nDehydrogenase 1a1 (ALDH1a1), but not glutamate decarboxylase (GAD), mediates a GABA synthesis\npathway in midbrain DAergic neurons. We sought to determine whether ALDH1a1 drives functional GABA\nco-release by Social-reward-EnsDA. To this end, we supplemented the following experiments:\n1) We performed immunohistochemical co-labeling experiments to examine the colocalization of TRAPed\nneurons with Aldehyde dehydrogenase 1a1 (ALDH1a1, a marker that colocalizes with TH and mediates a\nGABA synthesis pathway in midbrain DAergic neurons). The results revealed that ~37% of TRAPed\nneurons colocalized with ALDH1a1 (Supplementary Fig. 11c,d).\n2) We examined the effects of the ALDH1a1 inhibitor on IPSCs induced by optogenetic activation of\nSocial-reward-EnsDA. The results revealed that the ALDH1a1 inhibitor 4-(diethylamino)-benzaldehyde\n(DEAB) significantly attenuated the amplitude of light-evoked IPSCs (Supplementary Fig. 11i).\nThese lines of evidence indicate that GABA co-release from TH-positive neurons, more precisely from those\nexpressing ALDH1a1, mediates the protective effect of social reward against drug relapse.\nIn summary, by demonstrating that the inhibitory effect remains intact even when GAD-mediated GABA\nsynthesis is blocked, we have reinforced the conclusion that the suppression of the drug-seeking ensemble is a\nspecific functional property of the recruited DAergic ensembles.\nThe following text and image have been added to the manuscript:\nGiven that previous studies demonstrated that Aldehyde Dehydrogenase 1a1 (ALDH1a1) but not the\nglutamate decarboxylase (GAD) mediates a GABA synthesis pathway in midbrain DAergic neurons22,23, we\nsought to determine whether ALDH1a1 drives functional GABA co-release by Social-reward-EnsDA. To\naddress this, we examined GAD and ALDH1a1 expression in Social-reward-EnsDA via immunohistochemical\nco-labeling with TH. Our results revealed that only ~10% of Social-reward-EnsDA expressed GAD, whereas\n~37% of these neurons expressed ALDH1a1 (Supplementary Fig. 11a-d). Moreover, treatment with the ALDH\ninhibitor 4-(diethylamino)-benzaldehyde (DEAB, 10 µM) but not the GAD inhibitor 3-mercaptopropionic acid\n(3-MPA, 500 µM) dramatically reduced light induced IPSC amplitude (Supplementary Fig. 11e-i).\nCollectively, these findings establish that ALDH1a1 is required for functional GABA co-release by Social-\nreward-EnsDA. (page 13-14, lines 265-276 in the revised manuscript)\n15\n11. Minor comment: The authors say they used head-fixed rats in the methods section for single neuron\ncalcium imaging but say they used freely moving animals in page 9 of the Results section.\nResponse: We sincerely appreciate the Reviewer for pointing out this inconsistency in our manuscript. To\nrectify this error, we have revised the relevant content in the Methods section to accurately reflect the freely\nmoving imaging paradigm employed in the study.\nThe following text has been added to the manuscript:\nAfter ~3 weeks of recovery, a baseplate was aligned and fixed with dental cement. Subsequent single-photon\nimaging sessions were conducted during behavioral tasks, including social interaction and drug relapse tests.\n(page 49, lines 853-855 in the revised manuscript)\nXiaoxing, L. (2026) https://BioRender.com/hqs0eg6.\n12. The description of viral mechanism and transport direction in the results and captions and in the\ndiagram in panels 6a,g are the opposite of what these viruses are actually doing. AAV2/1-TH-Cre or\nAAV2/1-SERT-Cre is retrogradely transported from the injection site to afferent brain areas that\ninnervate the injection site; not the other way around (not in the prograde direction). The description\nand figure mechanism is incorrect. Its most likely that the wording and associated figures are incorrect\nrather than their experiments. They need to state that AAV2/1-TH-flpo was 'retrogradely' transported\nfrom the DRN to the VTA to activate AAV-fDIO-hM4Di-EGFP in the VTA TH neurons. They need to\nchange the direction of the arrow in their diagrams in their figures, and state that they are looking at\nTH projections 'from' VTA to DRN, and NOT projections from DRN to VTA. And then revise the\nassociated description of results and interpretation throughout the manuscript.\nResponse: We sincerely appreciate the Reviewer’s critical comment. We apologize for the confusion caused\nby the imprecise wording in the original version.\n1) First, we clarify that the AAV-SERT-Cre we employed is based on the AAV2/9 serotype, which does not\npossess trans-synaptic transport capabilities, and this has been revised in the figures.\n2) Second, our viral approach relies on the anterograde trans-synaptic properties of AAV2/1, which enables\nmonosynaptic tracing and modulation according to previous studies (Zingg B et.al, 2017, Neuron; Qi G\net.al, 2022, Nat Commun). The detailed mechanism is as follows: We first injected AAV2/1-TH-Flpo into\nthe DRN. Adeno-associated virus AAV2/1 has been shown to undergo anterograde monosynaptic\ntransmission; thus, AAV2/1-TH-Flpo is anterograde transported monosynaptically to postsynaptic\ndopaminergic neurons in the VTA, where Flpo is expressed. In the VTA, we co-injected AAV-FDIO-\nhM3Dq-EGFP (a Flpo-dependent vector), ensuring that hM3Dq-EGFP is only expressed in VTA\ndopaminergic neurons receiving monosynaptic inputs from DRN neurons labeled by AAV2/1-TH-Flpo.\n3) Finally, to rule out the effect of potential retrograde transport of AAV2/1, we added a control group where\nwe artificially activated VTA-DRN dopaminergic projections and found no significant effect on drug\nseeking (Supplementary Fig. 17f-j).\nThe following text and image have been added to the manuscript:\nTo test this, we combined an anterograde monosynaptic virus (AAV2/1-TH-flpo) with a flpo-dependent\nDREADD virus (AAV-FDIO-hM3Dq-EGFP) to selectively activate VTA DAergic neurons that receive input\nfrom the DRN (Fig. 6a,b and Supplementary Fig. 15a,b). (page 18, lines 371-374 in the revised manuscript)\nTo rule out the effect of potential retrograde transport of AAV2/1-Cre27 and make our conclusion more\nconvincing, we established control groups to dissect the functional roles of DRN-VTA projections versus VTA-\nDRN projections. The results showed that activation of the DRN-VTA serotonergic projections, rather than\nVTA-DRN dopaminergic projections, mimicked the therapeutic effect of social reward against drug seeking\n(Supplementary Fig. 17a-j), confirming the critical role of DRN serotonergic inputs. (page 19, lines 383-390\nin the revised manuscript)\nSupplementary Fig.17 | Activation of the serotonergic DRN-VTA but not VTA-DRN projections mimicked the\ntherapeutic effect of social reward against cocaine seeking. f, Left: Strategy for Virus injection. Right:\ninvestigate the effect of the VTA-DRN dopaminergic projection on cocaine-seeking behavior. h, Number of nose-\npokes during the 10-day cocaine self-administration training. i, Number of nose-pokes during the 14-day extinction\ntraining. j, Number of nose pokes during the relapse test. Activation of the VTA-DRN dopaminergic projection had\nno effect on cocaine-seeking behavior during the relapse test (n = 8, two-way ANOVA with simple effect analysis:\nF = 1.163, p = 0.299, more statistics see Supplementary Table8J-L). All data are presented as mean ± SEM.\n(1,14)\n18\n13. In the Discussion section: “Thus, we speculate that social reward and drug seeking may represent\ndistinct motivational states, each engaging specialized DA circuits”. I actually think their data is likely\nsaying this. However, it seems more to do with cell types or DA subtypes in VTA rather than actual\nensembles selected by cues. The paper would have been amazing if they showed distinct transcriptomes\nor cell-type markers with ISH for the two DA cell types activated by social interaction versus drug\nrelapse.\nResponse: We sincerely appreciate the Reviewer’s insightful comment. We agree that the two dopaminergic\npopulations responsive to social reward and drug seeking may exhibit distinct genetic profiles, and further\nvalidation of cell type specificity via techniques such as single-cell sequencing and transcriptomic analysis\nwould represent an important and interesting direction for future research.\nAccordingly, we have supplemented the Discussion section with relevant statements to address this point.\nThe following text has been revised to the manuscript:\nHere, we demonstrate that social reward and drug seeking engage minimally overlapping DAergic ensembles,\nand they establish different connections with brain regions. These findings are consistent with previous studies\nand further support the unique role of distinct DAergic population42,43. However, it remains unclear whether\nthese two ensembles exhibit distinct transcriptomic profiles or cell-type-specific markers, and future studies\nemploying techniques such as single-cell sequencing and spatial transcriptomics to characterize their\ntranscriptional features and molecular markers represent a promising and interesting direction for further\ninvestigation. (page 22, lines 454-462 in the revised manuscript)\n19\n14. On page 21: “This antagonistic architecture” might explain why the sharp distinctions they\nobserved with all the Fos promoter-driven viruses. But these are distinctions that you would more likely\nobserve with distinct cell types, not with neuronal ensembles made of largely the same cell types selected\nby cues.\nResponse: We are grateful for the Reviewer’s thoughtful critique regarding the conceptual distinction\nbetween \"cell types\" and \"neuronal ensembles.\" This is a fundamental question in systems neuroscience, and\nthe Reviewer’s point regarding the \"sharp distinctions\" we observed is well-taken. We agree that the line\nbetween a highly specialized cell type and a stable functional ensemble can be nuanced, especially in the VTA\nwhere molecular and circuit heterogeneity is prominent.\nIn light of the Reviewer’s feedback, we have carefully re-evaluated our terminology. We do not intend to\nsuggest that these neurons are randomly recruited from a homogeneous pool. Instead, we propose that Social-\nreward-Ens and Drug-seeking-Ens represent functional units that are \"nested\" within existing molecular and\ncircuit architectures. We have adopted the term \"ensemble\" primarily to emphasize the experience-dependent\nrecruitment and functional synergy that defines their role in behavior.\nWe clarify our use of \"neuronal ensemble\" with two lines of evidence:\n1) We ruled out contributions from non-DA cell types (especially GABAergic neurons) via complementary\nexperiments: Immunohistochemical co-labeling showed only ~10% of TRAPed neurons colocalized with\nGAD (a GABAergic marker), and patch-clamp recordings demonstrated that the GAD inhibitor 3-\nmercaptopropionic acid (3-MPA) had no significant effect on IPSCs induced by optogenetic activation of\nSocial-reward-EnsDA (Supplementary Fig. 11a-i). These results confirm our observed effects are\nmediated by DAergic populations.\n2) Our terminology aligns with recent field definitions. The review (Yuste et al., 2024, Neuron) defined a\nneuronal ensemble as “groups of neurons displaying recurring patterns of coordinated activity”, which is\nconsistent with our framework. Similarly, the study (Gimenez-Gomez et al., 2025, Nat Neurosci) used\nthis approach to describe how specific rewards organize cortical neurons into stable, behaviorally\nrelevant groups.\nOf course, we acknowledge that the two DAergic ensembles may exhibit distinct genetic profiles. Validating\nmore refined cell-type classifications via techniques like single-cell sequencing and spatial transcriptomics\nrepresents an important future direction. To address the Reviewer’s concern, we have removed the phrase\n“This antagonistic architecture” in full and revised the Discussion to explicitly note that these functional\nensembles may be anchored in underlying refined cell-type identities.\nThe following text has been revised to the manuscript:\nHowever, it remains unclear whether these two ensembles exhibit distinct transcriptomic profiles or cell-type-\nspecific markers, and future studies employing techniques such as single-cell sequencing and spatial\ntranscriptomics to characterize their transcriptional features and molecular markers represent a promising\nand interesting direction for further investigation. (page 22, lines 457-462 in the revised manuscript)\n20\n15. I think there is a typo in the caption for panel 3n: they mention ‘food-responsive neuron’ here and I\ndid not see food seeking anywhere else.\nResponse: We sincerely appreciate the Reviewer for pointing out this typo. We have corrected the error in the\ncaption of panel 3n.\nThe following text has been revised to the manuscript:\nn, Changes of magnitude in response to drug-seeking after-before social reward experience (n = 110 neurons)\n(page 32, lines 590-592 in the revised manuscript)\nComments to the Author:\nZheng et al. identify two distinct dopaminergic ensembles in the VTA: one that processes social reward\nand another that governs drug-seeking behavior, with functional competition between the two shaping\nbehavioral outcomes. They further delineate an organized DRN-to-VTA circuit that selectively\nunderlies the protective effect of social reward against drug-seeking behaviors. The study’s major\nconceptual advance lies in the identification of these two mutually inhibiting VTA dopaminergic\nensembles, which not only elucidates the neuronal ensemble and circuit mechanisms through which\nsocial reward suppresses drug relapse, but also opens promising avenues for developing novel\nneuromodulation-based interventions for addiction. The authors skillfully employ cutting-edge\nmethodologies to dissect the underlying neural mechanisms. Overall, the study presents compelling\nfindings, and the manuscript is logically structured and clearly articulated. The following issues,\nhowever, should be addressed in a revision.\nResponse: Thank you for reviewing our manuscript and providing valuable constructive suggestions, which\nhave greatly improved its quality. The questions and comments have been carefully considered during the\nrevision process, and we have provided detailed responses to each inquiry below.\nXiaoxing, L. (2026) https://BioRender.com/hqs0eg6.\n1. While social interaction with two juvenile conspecifics has been demonstrated to induce rewarding\neffects in the social conditioned place preference (sCPP) paradigm, the rigor of the present study would\nbe significantly improved by explicitly verifying whether the 30-minute social interaction used here\nindeed elicits a reward state. A robust approach would be to investigate whether the identified \"Social-\nreward-EnsDA\" neurons support intracranial self-stimulation (ICSS) behavior.\nResponse: We sincerely appreciate the Reviewer’s valuable comment regarding the need to verify the\nrewarding nature of the 30-minute social interaction paradigm. In response, we supplemented two\ncomplementary experiments to address this critical point:\n1) Social self-administration assay: our results revealed that rats successfully acquired the operant response\nto gain access to juvenile conspecifics within the first day of training (Supplementary Fig. 1e,f). Rapid\nacquisition of operant responding for a stimulus is a well-recognized hallmark of its rewarding properties\nin behavioral neuroscience.\n2) Optogenetic modulation of Social-reward-Ens during intracranial self-stimulation (ICSS): we found that\noptogenetic activation of the social engram induced ICSS behavior (Supplementary Fig. 1g–j). These\nfindings directly link the neural ensemble encoding social reward to the brain’s core reward circuitry,\nthereby confirming the rewarding nature of interaction with juvenile conspecifics.\nThe following text and image have been added to the manuscript:\nIn the present study, we first investigate whether this similar social paradigm can serve as a social reward\nmanipulation in rats. Behavioral assays revealed that rats exhibited a robust conditioned place preference for\nthe context paired with juvenile conspecifics (Supplementary Fig. 1a-d) and successfully acquired the operant\nresponse required to access juvenile conspecifics (Supplementary Fig. 1e,f). Furthermore, activating the\nneurons activated by social interaction with juvenile conspecifics induces intracranial self-stimulation\nbehavior (Supplementary Fig. 1g-j). Collectively, these findings confirm that interaction with juvenile\nconspecifics can serve as a valid experimental manipulation of social reward in rats. (page 5-6, lines 88-96 in\nthe revised manuscript)\nSupplementary Fig.1 | Interacting with two juvenile partners is rewarding. e, Experimental timeline for social\nself-administration induced by interaction with two juvenile partners. Rats were trained to self-administer to gain\naccess to social targets (two juvenile conspecifics) during daily 60-min sessions. An active nose poke triggered the\nautomated gate to open for 60 s, allowing the rat access to the social targets in a smaller chamber. The social\ntarget chamber had an open side covered with holes spaced 1 cm apart, enabling the rats to freely interact, smell,\nand investigate each other without either animal entering the other chamber. An inactive nose poke, located\nopposite the active nose poke, had no consequences. f, Number of nose pokes during social self-administration\n23\nsessions (n = 8, three-way ANOVA with simple simple effect analysis: F = 3.781, p = 0.041, more statistics see\n(7,8)\nSupplementary Table12A). g, Virus injection strategy. h, Experimental timeline for optical intracranial self-\nstimulation (ICSS). ICSS training was conducted in operant chambers (AniLab, Ningbo, China) equipped with two\nnose pokes (active and inactive). an active nose poke elicited a 5-s tone and laser stimulation (1 s, 10 mW, 40 Hz, 5\nms pulse width); an inactive nose poke had no consequences. i, Active nose pokes at port A and port B during each\n60-min ICSS session (n = 8, three-way ANOVA with simple simple effect analysis: day1-4, F = 5.764, p =\n(3,12)\n0.011, more statistics see Supplementary Table12B; day5-8, F = 21.248, p < 0.001, more statistics see\n(3,12)\nSupplementary Table12C). j, Mean number of nose pokes during laser and non-laser trials, averaged across all\ntraining days. Elevated mean laser-evoked nose pokes relative to non-laser trials were observed exclusively in\nChR2-expressing rats (n = 8, two-way ANOVA with simple effect analysis: F = 103.654, p < 0.001, more\n(1,14)\nstatistics see Supplementary Table12D). All the data are presented as mean ± SEM.\n24\n2. In the Discussion, the authors cite literature suggesting that the behavioral consequences of social\ninteraction in substance use disorder critically depend on the drug exposure history of the social\npartners. The present study clarifies the dopaminergic mechanisms by which interaction with drug-\nnaïve conspecifics exerts a protective effect against drug seeking. However, the Discussion should be\nexpanded to incorporate research on how social interaction with drug-experienced conspecifics can\npromote addiction. Given the growing body of evidence on the dual roles of social context in\naddiction—both protective and risk-promoting—the authors should integrate these perspectives to\nprovide a more comprehensive and balanced discussion.\nResponse: We sincerely appreciate the Reviewer’s valuable comment. In response to your suggestion, we\nhave expanded the Discussion section to incorporate the dual roles of social context in addiction (protective\nvs. risk-promoting) and integrated relevant research on social interaction with drug-experienced conspecifics,\naiming to provide a more comprehensive and balanced perspective.\nThe following text has been added to the manuscript:\nAccumulating evidence indicates that DAergic neurons may mediate the dual effects of social interaction on\nSUD5. A recent study demonstrated that corticotropin-releasing hormone release from the piriform cortex to\namygdala is selectively enhanced during negative social interaction (with drug-exposed partners)33. In\ncontrast, our study showed that the DRN-VTA pathway is selectively enhanced during social reward (with\ndrug-naive partners); specifically, Drug-seeking-EnsDA receives greater input from the amygdala, whereas\nSocial-reward-EnsDA receives greater input from the DRN. Thus, we hypothesize that the protective effect of\nsocial reward is mediated by activation of the DRN-VTA pathway, whereas the opposing effect of negative\nsocial interaction may arise from excessive activation of the amygdala-VTA pathway. (page 21, lines 432-441\nin the revised manuscript)\n3. Regarding the network mapping of the ensembles: the connectivity of the drug-seeking–responsive\nensemble is consistent with established evidence, as it receives inputs from the central amygdala—a\nstructure well known to mediate Pavlovian associations of reward-predictive cues. Notably, it is\nsurprising that neither ensemble exhibits robust inputs from the prefrontal cortex (PFC). The authors\nshould identify and discuss prior research that supports or contextualizes this specific observation, and\nclarify whether this finding aligns with or challenges current models of reward and addiction circuitry.\nResponse: We sincerely appreciate the Reviewer’s insightful comment regarding the unexpected lack of\nrobust prefrontal cortex (PFC) inputs to the VTA DAergic ensembles and the need to contextualize this\nobservation with prior literature.\nWe share the initial surprise about the relatively sparse inputs from the infralimbic (IL) and prelimbic (PL)\ncortices to the DAergic ensembles identified in our study. To address this, we reviewed relevant literature and\nfound consistent evidence supporting our observation: Watabe-Uchida et al. (2012) performed comprehensive\nwhole-brain circuit tracing of direct inputs to midbrain DAergic neurons and reported that, compared to other\nbrain regions (e.g., amygdala, dorsal raphe nucleus), the number and proportion of inputs from the IL and PL\ncortices to DAergic neurons in the VTA are indeed relatively low. Thus, our finding aligns with this\nestablished literature and does not challenge current models of reward and addiction circuitry. Furthermore,\nwe have added relevant statements in the discussion section. Moreover, we have supplemented the relevant\ndiscussion section to clarify this point.\nThe following image has been added to the manuscript:\nHere, we demonstrate that social reward and drug seeking engage minimally overlapping DAergic ensembles,\nand they establish different connections with brain regions. These findings are consistent with previous studies\nand further support the unique role of distinct DAergic population44,45. (page 22, lines 454-457 in the revised\nmanuscript)\n25\n4. Regarding the result presentation of fiber photometry recording and miniscope imaging,\ninconsistencies exist in the descriptions of DAergic neuron responses to social reward. Standardization\nof these descriptions is recommended to ensure clarity and comparability. For instance, the results from\nfiber photometry recording experiments are stated as: “Body contact during social interaction reliably\nevoked robust calcium transients in VTA DA neurons (Fig. 1h–j).” In contrast, the results from\nminiscope imaging experiments are described as: “17% (65 out of 380 neurons, n = 6 animals)\nresponded selectively to social reward during the free interaction session (Fig. 2l–n and Fig. 2v).”\nResponse: We sincerely appreciate the Reviewer’s careful observation regarding the inconsistent descriptions\nof DAergic neuron responses to social reward across fiber photometry and miniscope imaging results.\nSpecifically, we have updated the description of miniscope imaging results to align with the wording used for\nfiber photometry data, emphasizing body contact during social interaction as the common stimulus for both\nassays.\nThe following text has been added to the manuscript:\n17% (65 out of 380 neurons, n = 6 animals) responded selectively to body contact during social interaction\n(Fig.2l-n and Fig.2v). (page 10, lines 200-201 in the revised manuscript)\n5. The manuscript exhibits inconsistencies in terminology formatting: the capitalization of the first\nletters in “Social-reward-EnsDA” and “Drug-seeking-EnsDA” is not uniform across the text, and “VTA\nDAergic neurons” is used interchangeably with “VTADA neurons.”\nResponse: We sincerely appreciate the Reviewer for pointing out the inconsistencies in terminology\nformatting. We have carefully reviewed the entire manuscript, standardized the capitalization of \"Social-\nreward-EnsDA\" and \"Drug-seeking-EnsDA\" (ensuring uniform capitalization throughout), and unified the\nterminology to \"VTA DAergic neurons\" (replacing all instances of \"VTADA neurons\") for consistency.\n6. In Fig. 1d, the label \"No rewrad\" is misspelled.\nResponse: We sincerely appreciate the Reviewer for pointing out this typo. We have corrected the misspelling\n\"No rewrad\" to \"No reward\" in Figure 1d to ensure accuracy.\nComments to the Author:\nPrevious studies have shown that social rewards can help inhibit drug-seeking behavior, although the\nmechanisms behind this are still unclear. This study provides an intriguing perspective by identifying\ntwo populations of VTA DA neurons that are selectively activated by drug-seeking behavior and social\nreward, respectively. Activation of the Social-reward-Ens-DA neurons can suppress activity in the Drug-\nseeking-Ens-DA neurons. Additionally, these Social-reward-Ens-DA neurons receive upstream input\nfrom the DRN, a region widely recognized for its role in social behavior. Overall, this research offers a\nvaluable mechanism to further elucidate the interaction between social and drug-related rewards in this\nfield. Below are some concerns I have:\nResponse: Thank you for taking the time to review our manuscript and providing valuable feedback, which\nhas greatly enhanced the quality of this paper. We have carefully incorporated your suggestions into the\nrevised version, and our responses are provided below.\nhttps://BioRender.com/hqs0eg6.\nHere are some specific comments:\n1. For Fig 1f, many green fluorescent-labeled neurons do not appear to co-label with TH. The\nviral vector based on the TH promoter used here is not commonly employed. Could the authors provide\nevidence to ensure its specificity?\nResponse: We sincerely appreciate the Reviewer’s critical comment regarding the specificity of the TH\npromoter driven viral vector and the apparent lack of co labeling in Figure 1f. We apologize for the ambiguity\ncaused by not presenting individual fluorescent channels in the original figure, which obscured the true co\nlocalization efficiency. To clarify this point, we have now provided separate fluorescent channel images for\nFig 1f, clearly showing the overlap between green fluorescent labeled neurons and TH positive neurons.\nQuantitative analysis further confirms that over 90% of green fluorescent labeled neurons co express TH,\ndemonstrating the high specificity of our TH promoter based viral vector for targeting VTA DAergic neurons.\nThe following image has been added to the manuscript:\nFig.1 | The VTA DAergic neurons act as a central nexus of social reward and drug seeking. e,f,\nRepresentative photo of GCamp6s expression in the VTA. Green: GCamp6s. Pink: Tyrosine hydroxylase, TH.\n28\n2. In Fig 2d, the histological results only present a merged image, making it difficult to verify the co-\nlabeling claims. It would be better to show separate channels for clarity. Additionally, why does TH\nexpression appear so weak in D+D and S+S? Were the VTA locations for the representative images in\nthese groups chosen consistently? Furthermore, the co-labeling observed via the cfos trap and fos\nstaining for the same behavior seems to be quite low in the representative images.\nResponse: We sincerely appreciate the Reviewer’s detailed and insightful comments regarding Figure 2d. In\nresponse to your suggestions, we have implemented the following revisions to address the concerns raised:\n1) Separate fluorescent channel display: To improve the clarity of co-labeling verification, we have added\nindividual channel images corresponding to Fig 2d in the supplementary materials (Supplementary\nFig.5d-o) of the revised manuscript.\n2) Optimization of representative images and tissue section selection: We carefully reviewed the original\nexperimental data and reselected representative images for the D+D and S+S groups based on multiple\ncriteria including fluorescent intensity of TH expression, co-labeling efficiency and anatomical positional\nconsistency. The previously selected images were not sufficiently representative, leading to the\nmisleading appearance of weak TH expression and low co-labeling. The updated representative images\n(Supplementary Fig.5d-o) more accurately reflect the true experimental results. Moreover, given that\nTRAP labeling and cFos activation exhibit slight positional differences during social interaction and drug\nrelapse sessions, we selected tissue sections from adjacent regions with minimal distance where both\nsignals are robust, which ensures anatomical validity while optimizing the visualization of co-labeling\npatterns.\nThe following image has been added to the manuscript:\ndistinct. d, Representative images of TH staining in the Social-reward-responsive ensembles (Label 1) and Drug-\nseeking-responsive ensembles (Label 2). Red: mCherry-expressed positive neurons (Label 1); Green: cfos-\nexpressed positive neurons (Label 2); Pink: TH-expressed positive neurons; Blue: DAPI. Scale bar: 50 μm. e,\nPercentage of co-labeled neurons in Social-reward-responsive ensembles (Label 1) or Drug-seeking-responsive\nensembles (Label 2) (n = 5). f, Percentage of co-labeled DAergic neurons in Social-reward-responsive ensembles\n(Label 1) or Drug-seeking-responsive ensembles (Label 2) (n = 5). g, Representative images of tyrosine\nhydroxylase (TH) staining in the Drug-seeking-responsive ensembles (Label 1) and Social-reward-responsive\nensembles (Label 2). h, Percentage of co-labeled neurons in Drug-seeking-responsive ensembles (Label 1) or\nSocial-reward-responsive ensembles (Label 2) (n = 4). i, Percentage of co-labeled DAergic neurons in Drug-\nseeking-responsive ensembles (Label 1) or Social-reward-responsive ensembles (Label 2) (n = 4). g, Representative\nimages of tyrosine hydroxylase (TH) staining in the Drug-seeking-responsive ensembles (Label 1 versus Label 2). k,\nPercentage of co-labeled neurons in Drug-seeking-responsive ensembles (Label 1 versus Label 2) (n = 4). l,\nPercentage of co-labeled DAergic neurons in Drug-seeking-responsive ensembles (Label 1 versus Label 2) (n = 4).\nm, Representative images of tyrosine hydroxylase (TH) staining in the Social-reward-responsive ensembles (Label 1\nversus Label 2). n, Percentage of co-labeled neurons in Social-reward-responsive ensembles (Label 1 versus Label\n2) (n = 4). o, Percentage of co-labeled DAergic neurons in Social-reward-responsive ensembles (Label 1 versus Label\n2) (n = 4). All the data are presented as mean ± SEM.\n30\n3. What proportion of VTA dopamine neurons do these two populations represent? Do their\ndownstream projections differ in a way that might explain their functional divergence?\nResponse: We sincerely appreciate the Reviewer’s insightful questions regarding the proportion of VTA\nDAergic neurons represented by the two ensembles and the potential functional implications of their\ndownstream projections. In response, we have supplemented quantitative data on the ensemble proportions\nand expanded the Discussion section to address downstream projection divergence, as detailed below:\n1) We calculated the proportion of Social-reward-EnsDA and Drug-seeking-EnsDA relative to total VTA\nDAergic neurons. The results, consistent with the results of single-cell calcium imaging (Fig.2v, Social-\nreward-EnsDA:31%, Drug-seeking-EnsDA:26%; Supplementary Fig.9p, Social-seeking-EnsDA:33%, Drug-\nseeking-EnsDA:35%), show that both ensembles account for approximately 30% of total VTA DAergic\nneurons (Supplementary Fig. 6d,e).\n2) We agree that divergent downstream projections are likely critical for explaining the functional\ndifferences between the two ensembles. We have therefore added relevant discussion to contextualize this\nwith existing literature and outline future research directions.\nThe following text and image have been added to the manuscript:\nMoreover, the number of DAergic neurons activated during social reward versus drug relapse sessions did not\ndiffer significantly; both populations accounted for approximately 30% of total DAergic neurons in the VTA\n(Supplementary Fig. 6d,e). (page 10, lines 185-187 in the revised manuscript)\nBeyond the inputs to DAergic neurons, understanding how their downstream outputs drive opposing behaviors\nremains a critical question in the field54,55. A recent study has highlighted the role of distinct DAergic\ndownstream projections in mediating the influence of social interaction on addiction susceptibility:\noptogenetic activation of the DAergic pathway from the VTA to prefrontal cortex suppressed drug seeking,\nwhereas activation of the VTA-to-nucleus accumbens (NAc) pathway exerted the opposite effect5. Moreover,\ndistinct VTA DAergic projections into defined NAc subregions also mediate diverse behavioral functions55.\nBuilding on these findings, investigating how distinct downstream dopamine signals functionally connect to\nthe Social-reward-EnsDA and Drug-seeking-EnsDA identified in our study will be a key focus of future research.\n(page 24, lines 489-499 in the revised manuscript)\nSupplementary Fig.6 | Spatial distributions of VTA DAergic ensembles activated by social reward or drug\nseeking. d, Representative images showing activated DAergic neuronal ensembles in the VTA during social\ninteraction, with quantification of the proportion of these ensembles relative to the total dopamine neuron\npopulation in the VTA. e, Representative images showing activated DAergic neuronal ensembles in the VTA during\ndrug relapse, with quantification of the proportion of these ensembles relative to the total dopamine neuron\npopulation in the VTA.\n31\n4. For Fig 3e, the authors hypothesize that Social-reward-Ens-DA neurons inhibit Drug-seeking-Ens-DA\nneurons via non-canonical release of GABA and demonstrated a monosynaptic inhibitory connection\nbetween these two groups of dopamine neurons. However, this evidence alone is insufficient. Additional\nexperiments such as IF or in situ should be performed to confirm co-expression of GABA markers (e.g.,\nGAD or vGAT) and TH within Social-reward-Ens-DA neurons. Furthermore, the authors should\nablate/inhibit all VTA GABAergic neurons to test whether the IPSCs still persist. Lastly, could the\nauthors explain the observed latency in the light-evoked IPSCs?\nResponse: We thank the Reviewer for this valuable comment. We acknowledge that the initial evidence\nsupporting non-canonical GABA release from Social-reward-EnsDA neurons was insufficient. In response to\nyour suggestions, we have supplemented a series of complementary experiments to address this critical\nmechanistic question:\nWe performed immunohistochemical co-labeling experiments to examine the colocalization of TRAPed\nneurons with Glutamate decarboxylase (GAD, a classic marker for GABAergic neurons). The results revealed\nthat only ~10% of Social-reward-EnsDA colocalized with GAD (Supplementary Fig. 11a,b). We further\nexamined the effects of GAD antagonists on IPSCs induced by optogenetic activation of Social-reward-EnsDA.\nThe results revealed that the GAD inhibitor 3-mercaptopropionic acid (3-MPA) had no significant effect on\nIPSCs (Supplementary Fig. 11h).\nThese lines of evidence indicate that the protective effect of social reward against drug seeking is not\nmediated by non-specific labeling of GABAergic neurons by the TH promoter.\nTo clarify the mechanism underlying the protective effect of social reward against drug seeking, we reviewed\nrelevant literature and found that Aldehyde Dehydrogenase 1a1 (ALDH1a1, a marker that colocalizes with TH\nand mediates a GABA synthesis pathway in midbrain DAergic neurons) but not GAD mediates a GABA\nsynthesis pathway in midbrain DAergic neurons (Kim et al., 2015, Science; Tritsch et al., 2012, Nature). We\ntherefore sought to determine whether ALDH1a1 drives functional GABA co-release by Social-reward-EnsDA.\nTo this end, we supplemented the following experiments:\nWe performed immunohistochemical co-labeling experiments to examine the colocalization of Social-reward-\nEnsDA with ALDH1a1. The results revealed that ~37% of Social-reward-EnsDA colocalized with ALDH1a1\n(Supplementary Fig. 11c,d). We also examined the effects of the ALDH1a1 inhibitor on IPSCs induced by\noptogenetic activation of Social-reward-EnsDA. The results revealed that the ALDH1a1 inhibitor 4-\n(diethylamino)-benzaldehyde (DEAB) significantly attenuated the amplitude of light-evoked IPSCs\n(Supplementary Fig. 11i).\nThese lines of evidence indicate that GABA co-release from TH-positive neurons, more precisely from those\nexpressing ALDH1a1, mediates the protective effect of social reward against drug relapse.\nFinally, regarding the observed latency in light-evoked IPSCs, we attribute this to minor systematic errors in\nthe light stimulation delivery system used in the electrophysiology experiments.\nThe following text and image have been added to the manuscript:\nGiven that previous studies demonstrated that Aldehyde Dehydrogenase 1a1 (ALDH1a1) but not the\nglutamate decarboxylase (GAD) mediates a GABA synthesis pathway in midbrain DAergic neurons22,23, we\nsought to determine whether ALDH1a1 drives functional GABA co-release by Social-reward-EnsDA. To\naddress this, we examined GAD and ALDH1a1 expression in Social-reward-EnsDA via immunohistochemical\nco-labeling with TH. Our results revealed that only ~10% of Social-reward-EnsDA expressed GAD, whereas\n~37% of these neurons expressed ALDH1a1 (Supplementary Fig. 11a-d). Moreover, treatment with the ALDH\ninhibitor 4-(diethylamino)-benzaldehyde (DEAB, 10 µM) but not the GAD inhibitor 3-mercaptopropionic acid\n(3-MPA, 500 µM) dramatically reduced light induced IPSC amplitude (Supplementary Fig. 11e-i).\nCollectively, these findings establish that ALDH1a1 is required for functional GABA co-release by Social-\nreward-EnsDA.\n(page 13-14, lines 265-276 in the revised manuscript)\nXiaoxing, L. (2026) https://BioRender.com/hqs0eg6.\nSupplementary Fig.11 | Aldehyde Dehydrogenase 1a1 Mediates the inhibitory effect of Social-reward-EnsDA on\nDrug-seeking-EnsDA. a, Representative histology images of Social-reward-EnsDA co-label TH or GAD. b, The\nproportion of co-labeled neurons (TH + Social-reward-EnsDA vs GAD + Social-reward-EnsDA) represents the total\nSocial-reward-EnsDA. c, Representative histology images of Social-reward-EnsDA co-label TH or ALDH1a1. d, The\nproportion of co-labeled neurons (TH + Social-reward-EnsDA vs ALDH1a1 + Social-reward-EnsDA) represents the\ntotal Social-reward-EnsDA. e,f, Strategy for Virus injection. g, Schematic of the experimental procedure for\nelectrophysiology recording. h, Left: Representative traces of evoked IPSCs upon bath application of GAD\ninhibitor 3-mercaptopropionic acid (3-MPA, 500 µM). Right: Amplitudes under control conditions (ACSF) or 3-\nMPA, normalized to baseline. i, Left: Representative traces of evoked IPSCs upon bath application of ALDH\ninhibitors 4-(diethylamino)-benzaldehyde (DEAB, 10 µM). Right: Amplitudes under control conditions (ACSF) or\nDEAB, normalized to baseline (DEAB: n = 8 neurons, paired t test, **p < 0.01).\n33\n5. I'm curious about your experiment being done under social isolation conditions. Would social\ninteraction still inhibit relapse in group-housed mice?\nResponse: We sincerely appreciate the Reviewer’s insightful question regarding the generalizability of our\nfindings to group-housed animals, given that our experiments were conducted under social isolation\nconditions. We address this concern with three key points:\n1) Firstly, the social isolation paradigm was necessitated by the technical requirements of our intravenous\nself-administration surgery. The indwelling dorsal venous catheter used for drug delivery must remain\nunobstructed throughout the training period, and group housing would pose a high risk of catheter damage\ndue to conspecific interaction. Thus, isolation was a practical necessity to ensure the integrity of our\nexperimental procedures.\n2) Secondly, multiple published studies have demonstrated that group housing itself functions as a social\nreward and exerts a protective effect against drug addiction. For example, Alexander et al. (1978), Raz et\nal. (2010), and Bozarth et al. (1989). reported that group-housed rodents exhibit significantly reduced\nacquisition of drug self-administration compared to socially isolated controls. These findings align with\nthe core conclusion of our study that social context modulates drug-seeking behavior.\n3) Finally, to directly address this question, we conducted supplementary experiments in which rats were\ngroup-housed after cocaine SA training. Our results showed that sustained social interaction with drug-\nnaïve conspecifics accelerated the extinction of drug-associated memories and effectively suppressed drug\nrelapse (Supplementary Fig. 18c,d), further confirming the protective role of social context in addiction.\nThe following text and image have been added to the manuscript:\nFurthermore, social housing environments (with drug-naïve partners), characterized by sustained conspecific\ninteraction, exert a long-term protective influence on drug-seeking behavior. Specifically, such housing\nconditions accelerated the extinction of drug-associated memory and suppressed drug relapse (Supplementary\nFig. 18c,d). (page 21, lines 427-431 in the revised manuscript)\nSupplementary Fig.18 | The protective effect of acute social interaction against drug seeking is transient. c,\nExperimental design to investigate the effect of housing conditions (pair-housed vs. socially isolated) on cocaine-\nseeking behavior. d, Number of nose-pokes during cocaine self-administration training, extinction and relapse tests.\nPair-housed rats exhibited accelerated extinction (n = 9-10, three-way ANOVA with simple simple effect analysis:\nF =4.799, p = 0.047, more statistics see Supplementary Table14B) and reduced cocaine seeking during the\n(13,5)\nrelapse test (n=9-10, two-way ANOVA with simple effect analysis: F =14.861, p = 0.001, more statistics see\n(1,17)\nSupplementary Table14C) compared with socially isolated rats. All the data are presented as mean ± SEM.\nXiaoxing, L. (2026) https://BioRender.com/hqs0eg6.\n6. For Figure 6, considering that AAV1 is both anterograde and retrograde, previous study (Lin et al.,\n2021) indicates VTADA also projects to DRN, albeit at a low proportion. Could this viral strategy\ninadvertently label DRN-projecting VTA DA neurons? Please clarify.\nResponse: We sincerely appreciate the Reviewer’s critical comment regarding the potential off-target\nlabeling of DRN-projecting VTA DAergic neurons due to the dual anterograde/retrograde transport properties of\nAAV2/1. We acknowledge that AAV2/1 may exhibit minimal retrograde tracing activity, which raises the\nconcern that our viral strategy could inadvertently label DRN-projecting VTA DAergic neurons. To rigorously\nrule out this possibility and clarify the functional specificity of the projection pathway, we supplemented a\ncontrol experiment to dissect the respective roles of DRN-VTA versus VTA-DRN projections. Specifically, we\nartificially activated these two pathways separately and examined their effects on drug-seeking behavior.\nThe results, now integrated into the manuscript, showed that activation of DRN-VTA serotonergic projections\nmimicked the therapeutic effect of social reward in suppressing drug seeking, whereas activation of VTA-\nDRN dopaminergic projections had no significant impact (Supplementary Fig. 17a-j). This directly confirms\nthat our observed protective effect is mediated by DRN-VTA projections (the anterograde pathway we\nintended to target).\nThe following text and image have been added to the manuscript:\nTo rule out the effect of potential retrograde transport of AAV2/1-Cre27 and make our conclusion more\nconvincing, we established control groups to dissect the functional roles of DRN-VTA projections versus VTA-\nDRN projections. The results showed that activation of the DRN-VTA serotonergic projections, rather than\nVTA-DRN dopaminergic projections, mimicked the therapeutic effect of social reward against drug seeking\n(Supplementary Fig. 17a-j), confirming the critical role of DRN serotonergic inputs. (page 19, lines 383-390\nin the revised manuscript)\nSupplementary Fig.17 | Activation of the serotonergic DRN-VTA but not the dopaminergic VTA-DRN\nprojections mimicked the therapeutic effect of social reward against cocaine seeking. a, Left: Strategy for Virus\ninjection. Right: Representative photo of hM3Dq-mCherry expression in the VTA. Scale bar: 500 µm. b,\nsocial reward against cocaine seeking. The rats were intraperitoneally injected with CNO 30 minutes before the\ncocaine relapse test. c, Number of nose-pokes during the 10-day cocaine self-administration training. d, Number of\nnose-pokes during the 14-day extinction training. e, Number of nose-pokes during the relapse test, an activation of\nthe DRN-VTA serotonergic projections significantly inhibits cue-induced active nose-pokes, mimicking the effects of\nsocial reward on cocaine seeking (n = 8, two-way ANOVA with simple effect analysis: F = 5.803, p = 0.030;\n(1,14)\nmore statistics see Supplementary Table8G-I). f, Left: Strategy for Virus injection. Right: Representative photo of\nhM3Dq-mCherry expression in the DRN. Scale bar: 500 µm. g, Experimental scheme to investigate the effect of the\nVTA-DRN dopaminergic projection on cocaine-seeking behavior. h, Number of nose-pokes during the 10-day\ncocaine self-administration training. i, Number of nose-pokes during the 14-day extinction training. j, Number of\nnose pokes during the relapse test. Activation of the VTA-DRN dopaminergic projection had no effect on cocaine-\nseeking behavior during the relapse test (n = 8, two-way ANOVA with simple effect analysis: F = 1.163, p =\n(1,14)\n0.299, more statistics see Supplementary Table8J-L). All data are presented as mean ± SEM.\n36\n7. What major types of neurons project from DRN to the Social-reward-Ens-DA neurons? Are they DA\nor 5-TH neurons?\nResponse: We sincerely appreciate the Reviewer’s valuable question regarding the neuronal type of DRN\nprojections to Social-reward-EnsDA (i.e., whether they are DAergic or serotonergic). Based on prior literature\nand our own experimental findings, we speculated that these projections are primarily serotonergic, supported\nby the following two key lines of evidence:\n1) Prior anatomical tracing studies: Lin et al. (2020) demonstrated that DAergic neurons in the DRN mainly\ninnervate the central amygdala (CeA) and bed nucleus of stria terminalis (BNST). In contrast, other\nstudies (Wang et al. (2023), Paquelet et al. (2022)) have confirmed that DRN neurons projecting to the\nVTA are predominantly glutamatergic or serotonergic, rather than DAergic.\n2) Functional relevance of serotonergic DRN-VTA projections: Our experimental results show that activation\nof serotonergic DRN-VTA projections mimics the therapeutic effect of social reward in suppressing drug\nseeking (Supplementary Fig. 17a-e). This aligns with prior works (Li et al., 2021, Miyazaki K et al.,\n2021) showing that s serotonin’s role in mitigating compulsive drug-seeking behaviors.\nThe following text has been added to the manuscript:\nThe DRN has been classically recognized as a nucleus that contains an anatomically and functionally diverse\npopulation of serotonergic neurons47, many of which innervate brain areas involved in reward processing48.\nSocial interaction, a behavior with high reward salience, has been linked to serotonergic activity in the\nDRN49—a link our findings corroborate and extend. Brain-wide monosynaptic mapping revealed that Social-\nreward-EnsDA receives substantial inputs from DRN, and social interaction robustly activates DRN\npopulations that project to the VTA50. Prior work demonstrates that serotonergic DRN-VTA projections\nenhance social interaction time in stress-susceptible mice, while silencing this pathway diminishes social\ninteraction time51. Consistent with this, we found that activation of VTA DAergic neurons that receive input\nfrom the DRN or the serotonergic DRN-VTA projections mimicked the therapeutic effect of social reward\nagainst drug seeking (Supplementary Fig. 17a-e). These findings provide partial mechanistic support for\nmodels positing serotonin’s role in mitigating compulsive drug-seeking52,53. (page 23, lines 475-488 in the\nrevised manuscript)\nXiaoxing, L. (2026) https://BioRender.com/hqs0eg6.\n8. How do these two populations of neurons respond to other types of rewards, such as tasty food or\naddictive drug injections?\nResponse: We sincerely appreciate the Reviewer’s insightful question regarding the responses of Drug-\nseeking-EnsDA and Social-reward-EnsDA to other reward types (e.g., tasty food, addictive drugs). To address\nthis, we conducted supplementary experiments by artificially activating these two ensembles and examining\ntheir effects on feeding behavior. Our results showed that activation of Drug-seeking-EnsDA significantly\ninhibited feeding behavior within 2 hours, while activation of Social-reward-EnsDA had no significant impact\non feeding (Supplementary Fig. 19e-h). This finding highlights the functional specificity of the two\nensembles in processing different rewards.\nThe following text and test have been added to the manuscript:\nMoreover, we found that activation of Drug-seeking-EnsDA significantly inhibited feeding behavior\n(Supplementary Fig. 19e-h), further supporting the competitive functional relationship between natural\nrewards and drug-seeking behavior. (page 24, lines 507-510 in the revised manuscript)\nSupplementary Fig.19 | Comparison of therapeutic effects between social and sucrose reward on cocaine\nseeking. e, Schematic of the experimental design for refeeding following activation of Social-reward-EnsDA. f,\nComparison of cumulative food intake in fasted rats. Rats received i.p. injections of CNO or vehicle 30 min prior to\nbeing given free access to food. g, Schematic of the experimental design for refeeding after activation of Drug-\nseeking-EnsDA. h, Comparison of cumulative food intake in fasted rats. Rats received i.p. injections of CNO or\nvehicle 30 min prior to being given free access to food. Fasted rats administered CNO consumed significantly less\nfood than those administered vehicle during the first 1 and 2 h of refeeding (n=8, two-way ANOVA with simple\neffect analysis: F =5.545, p = 0.018, more statistics see Supplementary Table15B). All the data are presented as\n(2,13)\nmean ± SEM.\n1) For Fig 2e, 2f, the statistical analysis should be presented as percentages rather than as raw numbers.\nResponse: We sincerely appreciate the Reviewer’s constructive suggestion. We have revised the statistical\nanalysis in Figures 2e and 2f, replacing raw numbers with percentages to enhance clarity and interpretability\nof the data.\nThe following text and image have been added to the manuscript:\nNotably, the proportion of co-labeled DAergic neurons across social reward and drug seeking conditions was\nsignificantly lower than that observed in control groups subjected to the same stimulus twice (Fig.2e,f),\nsuggesting largely distinct neuronal populations for each condition. (page 9, lines 169-172 in the revised\nmanuscript)\nFig.2 | Distinct VTA DAergic ensembles respond to social reward and drug seeking. e, Proportion of neurons co-\nlabeled for Label 1 and Label 2 (n = 4–5; one-way ANOVA with Bonferroni multiple comparisons test, **p < 0.01).\nf, Proportion of neurons co-labeled for Label 1, Label 2, and TH (n = 4–5; one-way ANOVA with Bonferroni\nmultiple comparisons test, **p < 0.01).\n2) For the miniscope data, the n-value should not only represent the number of neurons but also\nindicate how many animals these neurons are from.\nResponse: We sincerely appreciate the Reviewer’s constructive suggestion. We have revised the presentation\nof n-values for all miniscope imaging data in the manuscript.\n3）In Fig 5c, the starting cell is unclear, and the background appears messy.\nResponse: We sincerely appreciate the Reviewer’s careful observation. We have revised Figure 5c to clarify\nthe starting cell and clean up the messy background, improving the clarity and readability of the image.\nThe following image has been revised to the manuscript:\nFig.5 | Social-reward-EnsDA and Drug-seeking-EnsDA in the VTA establish different connection patterns. c,\nRepresentative images of the VTA injection site and magnified view of starter cells with tamoxifen injection. Scale\nbar: 200 µm.\nThe authors express their gratitude to the Reviewers for providing valuable feedback.\nDetailed responses addressing each comment are provided below. Comments from the\nReviewers are highlighted in bold, while author responses are presented in plain blue text.\nModifications made to the manuscript are indicated by italicized text on a yellow background.\nPage and line numbers referenced in the responses correspond to those found in the revised\nmanuscript.\nReviewer #1 (Remarks to the Author)\nThe authors provided a very thorough set of good responses to my concerns. I am fine\nwith the revised version now with one exception:\nIn response to my point 9, the authors quote a review by Yuste et al 2024 for their\ndefinition of a neuronal ensemble -“a population of neurons involved in a particular\ncomputation”. This is only a partial definition that does not distinguish between\nneuronal ensemble versus cell type, and is thus insufficient. When talking about\nneuronal ensembles as an encoding unit, Yuste is referring to a set of neurons that are\nselected by a learned set of cues, and others have found are composed of multiple cell\ntypes acting together. Neuronal ensembles are not mere cell types that are involved\ndifferentially in different rewards (e.g. social versus drug reward) or (e.g. nicotine versus\ncocaine reward). For example in cortex, different ensembles encoding different\ninformation often have the same relative percentages of cell types.\nWhat I was trying to say in my initial review is that you are most likely looking at\ndifferent cell types for social versus drug reward that are genetically predetermined\nprior to learning - this means they are not neuronal ensembles in the more meaningful\nand informative sense. Ensembles are about encoding high-resolution learned\ninformation, and not about encoding low-resolution largely innate information that can\nbe handled by mere cell types.\nEquating neuronal ensembles with cell types is due to an incorrect over-generalization of\nthe term neuronal ensembles. Unfortunately, this is an overly common error in the field.\nPlease fix and I am fine with the rest of the paper, which was very interesting and a lot\nof good work.\nResponse: We sincerely appreciate the reviewer’s insightful and constructive comments. We\napologize for our earlier misunderstanding of the reviewer’s point, and we now understand the\nconcern that we are most likely looking at different cell types for social versus drug reward that\nare genetically predetermined prior to learning. We acknowledge the reviewer’s critical distinction\nthat neuronal ensembles encode high-resolution learned information, rather than low-resolution\nlargely innate information that can be accounted for by cell types alone.\n1. Regarding the drug-relapse-ensemble, our paradigm involved extensive operant conditioning\nof nosepoke responses, cue presentations, and drug reward. We defined neurons responsive to\nnosepoke as the drug-relapse ensemble, which explicitly indicates that this ensemble is not\npre-existing prior to learning. For the social-reward-ensemble, based on your suggestion, we\nhave added experiments involving operant conditioning for social nosepoke, social cues, and\nsocial reward in the previous round of revision. These additional data further demonstrate\nthat the social-reward ensemble is also established through learning.\n2. Although our results support that both ensembles are experience-dependent, we agree that we\ncannot completely rule out the contribution of genetically predetermined cell-type differences\nunderlying social versus drug reward. Accordingly, we have revised the relevant\ninterpretations. We thank the reviewer again for this important correction, which has greatly\nimproved the clarity and accuracy of our manuscript.\nThe following texts have been revised to the manuscript:\nNeuronal ensembles are defined as populations of neurons that encode specific information\nthrough coordinated activity patterns induced and shaped by learning21.(page 9, lines 173-174 in\nthe revised manuscript)\nTo address this alternative explanation, we trained rats to perform operant responses for both\nsocial self-administration and cocaine self-administration, then assessed whether distinct\nneuronal ensembles shaped by learning were engaged when rats executed goal-directed behaviors\nto obtain these two discrete reward-related stimuli (Supplementary Fig. 9a,b). Our results\ndemonstrated that only 10% of recorded neurons (26 out of 261 neurons; n = 2 animals) exhibited\ndual responsiveness to both social and cocaine cues (Supplementary Fig. 9c-p). Subsequent\nstatistical analyses confirmed a lack of significant overlap between social-seeking- and drug-\nseeking-responsive neuronal populations (Supplementary Fig. 9q). Collectively, these findings\nestablish that VTA DAergic populations exhibit stable, stimulus-specific identities and that their\nsegregation is not a consequence of temporal factors or behavioral confounds, consistent with the\nproperties of learning‑induced neuronal ensembles. (page 11-12, lines 224-235 in the revised\nmanuscript)\nWe speculate that this reciprocal interaction is progressively established during repeated drug use,\nalthough we cannot fully exclude the existence of genetically predetermined cell types in the VTA;\nfurther investigation will be needed to dissect these possibilities. (page 23, lines 470-473 in the\nrevised manuscript)\nReviewer #2 (Remarks to the Author):\nI have no further comments\nResponse: Thank you for your review and positive feedback on our manuscript. We highly\nappreciate your time and efforts in evaluating our work.\nReviewer #3 (Remarks to the Author):\nThe authors answered all my questions and I have no further comments.\nResponse: Thank you for your review and positive feedback on our manuscript. We are glad that\nwe have addressed all your questions satisfactorily, and we greatly appreciate your time and effort\nin evaluating our manuscript."}]

Decision letter