Paradoxical ventral tegmental area GABA signaling drives enhanced morphine reward after adolescent nicotine

Abstract

Background:

An important yet poorly understood risk factor for opioid use disorder is adolescent nicotine use. We investigated the neural mechanisms underlying this understudied interaction.

Methods:

Male and female adolescent mice received two-weeks of nicotine water (Adol Nic) or plain water (Adol Water). In adulthood, mice underwent three morphine tests: conditioned place preference (CPP), locomotor sensitization, and two-bottle choice. Ex vivo ventral tegmental area (VTA) brain slices were assessed via patch clamp for GABA and dopamine (DA) neuron morphine responses. Finally, VTA GABA neurons were chemogenetically inhibited during morphine CPP.

Results:

In adulthood, Adol Nic mice had greater morphine CPP, more morphine locomotor sensitization, and more choice-based oral morphine consumption vs. Adol Water mice. In contrast, adult mice given nicotine vs. water had similar morphine CPP. Patch clamp analysis of VTA neurons from adult Adol Water mice showed canonical cell-type responses to bath-applied morphine: fewer action potentials in GABA neurons and more in DA neurons. Paradoxically, VTA GABA and DA neurons from adult Adol Nic mice did not show these morphine responses. In support of a causal relationship between GABA neuron firing and reward behavior, chemogenetic inhibition of VTA GABA neurons in Adol Water mice during pairing increased morphine CPP. In contrast, inhibition of VTA GABA neurons in Adol Nic mice brought morphine CPP down to control levels.

Conclusions:

These data reveal an electrophysiological mechanism by which adolescent nicotine intake promotes morphine reward later in life, showing that adolescent nicotine exposure alters reward circuitry well into adulthood.

INTRODUCTION

Opioid use disorder (OUD) claims thousands of lives (1,2) and costs the U.S. billions of dollars (3). A key, yet poorly understood, risk factor for OUD is the use of nicotine (4), the main addictive component in tobacco and e-cigarettes (5). After years of decline, cigarette smoking is rising again, driven by e-cigarettes (6). Adolescent nicotine has surged (7,8), with nearly one-third of teenagers reporting use (7,9). Epidemiological and animal studies link prior nicotine to increased intake of other addictive drugs, including opioids (10–21). However, the neural mechanisms underlying this relationship remain unknown.

Adolescence is a critical neurodevelopmental period, marked by major reorganization of limbic regions involved in reward processing (22). Both clinical and preclinical data show that adolescent drug exposure induces persistent behavioral changes (22–24), and adolescent nicotine exposure enhances drug reinforcement in adulthood (19,20,25–27). While adolescent nicotine exposure induces long-term adaptations in rodent brain function (19,28,29), how these neuroadaptations contribute to increased morphine reward remains unclear.

The ventral tegmental area (VTA) is a heterogenous midbrain region essential for addiction-related behaviors (30–37). Morphine infusion into the rodent VTA supports self-administration and place conditioning (38,39). The VTA consists primarily of dopamine (DA) neurons but also contains GABAergic and glutamatergic neurons (40,41). DA projections to the nucleus accumbens form the primary brain “reward” pathway (42–55), but GABA and glutamate neurons also regulate reward (56,57) often by modulating DA activity. VTA GABA neurons inhibit neighboring DA neurons and thus regulate DA release. Morphine’s acute rewarding effects partly stem from inhibiting VTA GABA neurons, leading to DA neuron disinhibition (58–60). Morphine binds to mu opioid receptors, predominantly on GABA neurons in the VTA, causing hyperpolarization and subsequent DA neuron disinhibition. Recent studies show that VTA GABA neurons participate in reward learning (61,62) and are disrupted by drugs of abuse (63–65). Chronic nicotine exposure in rodents dysregulates VTA GABAergic signaling (19,64,66–68), influencing DA signaling and behavioral responses to morphine (66,69). However, no studies have directly linked VTA GABA neuron activity changes to morphine reward, particularly following adolescent nicotine.

We postulate that adolescent nicotine enhances adult morphine reward by altering VTA GABA neuron responses. To test this, we used behavioral, electrophysiological, and chemogenetic approaches in mice. We found that adolescent nicotine increased adult morphine conditioned place preference (CPP), two-bottle choice morphine consumption, and morphine locomotor sensitization. Correspondingly, VTA GABA neurons exhibited a blunted response to morphine, diverging from the morphine-induced decrease seen in non-nicotine mice. VTA DA neurons also showed a blunted response to morphine, contrasting with the expected morphine-induced increase in DA activity (70,71). Finally, chemogenetic manipulations established a causal link between adolescent nicotine-induced VTA GABA dysfunction and adult enhanced morphine reward. These results indicate that reducing VTA GABA neuron firing during adult morphine exposure prevents the heightened morphine preference seen after adolescent nicotine.

MATERIALS AND METHODS

Animals and Ethics

Male and female wildtype C57BL/6J mice (Jackson Laboratory [JAX], #000664) and male heterozygous VGAT-Cre mice (homozygous VGAT-ires-Cre knock-in, JAX#028862 crossed with C57BL/6J mice) were used. Procedures were carried out in compliance with guidelines specified by the University of Pennsylvania Institutional Animal Care and Use Committee. This study adhered to guidelines for data management (randomization, blinding, etc. (72,73) (Supplementary Materials [Supp. Mat.]). For adolescent nicotine exposure, mice were deliberately group-housed despite this preventing individual intake measurements, as social isolation during adolescence produces robust stress effects that can permanently alter addiction-related behaviors and interact with nicotine’s effects on reward circuitry (16,74). Where sample sizes permitted, data were analyzed for sex differences as detailed in Supp. Table 1.

Drugs

Nicotine hydrogen tartrate salt (Glentham Life Sciences) was given as 5–8mg nicotine/kg/mouse/day (75–78). Morphine sulfate (Spectrum) was given/used as 13–20mg/kg/mouse for two-bottle choice (79,80), 10mg/kg IP for CPP and locomotor sensitization (81), and 10uM for electrophysiology (82,83)). Clozapine N-oxide (CNO) dihydrochloride was given as 3 mg/kg IP (Hello Bio; (84,85)) and 50μM for slice electrophysiology experiments. This CNO concentration for slice experiments, while relatively high, was verified to produce reliable inhibition of VTA GABA neurons. Meloxicam as 2 mg/kg SC (Supp. Mat.). Drug details and all other experimental resources are provided in Supp. Table 2.

Behavioral Tests

Testing started at P70 for adolescent nicotine experiments or at P112 for adult nicotine experiments. P70 testing consisted of morphine CPP, two-bottle choice, and locomotor sensitization (86–91). P112 testing consisted of morphine CPP (Supp. Mat.). Statistical analyses used the Sidak multiple comparisons test to control Type 1 error while maintaining reasonable statistical power.

Stereotaxic Surgery

VGAT-Cre heterozygous mice received bilateral VTA infusion of a virus that expressed either an inhibitory DREADD (AAV5-DIO-hM4Di-mCherry, Addgene 44362-AAV5) or a control reporter protein AAV5-DIO-mCherry (Addgene 50459-AAV5) (92–95). For electrophysiology, mice received bilateral infusions of AAV9-CAG-FLEX-tdTomato (Addgene 28306-AAV9; Supp. Mat.).

Ex Vivo Electrophysiology

Slice recordings were performed as previously described (19,21,96–98) (Supp. Mat.) using a Leica VT 1200S vibratome (Leica Microsystems). For DREADD validation experiments, VGAT-Cre mice were exposed to nicotine or water in adolescence, followed by bilateral VTA injection of cre-dependent hM4Di at P49. After three weeks of expression, VTA GABA neuron firing was recorded before and after bath-application of 50 μM CNO (Supp. Mat.).

Tissue collection, Immunohistochemistry (IHC), and Microscopy

Slide-mounted and free-floating IHC were performed as previously described (99,100) (Supp. Mat.).

Data Analysis

Supp. Table 1 provides statistical approaches, data structures, and results. Each figure legend provides a statistical analysis summary (101). Electrophysiological data was analyzed as previously published (21,96,97,102).

RESULTS

Adolescent, but not adult, oral nicotine exposure promotes morphine reward in adulthood

Past work showed male mice given nicotine in adolescence had higher morphine reward learning in adulthood (20). To test how adolescent nicotine influenced adult morphine CPP in both sexes, mice were exposed to nicotine or plain water for two weeks (P28–42; Fig. 1A). Daily fluid consumption was similar between Adol Nic and Adol Water groups, though within the adolescent nicotine group, intake was dose-dependent (Fig. 1B–D). Weight gain was comparable across groups (Supp. Fig. 1). After four weeks drug-free, adult (P70) Adol Nic mice of both sexes showed two-fold greater preference for the morphine-paired chamber vs. Adol Water mice (Fig. 1E–F), despite similar locomotion (Fig. 1G). There was no sex effect (Supp. Fig. 2), indicating adolescent nicotine enhances adult morphine reward independent of locomotion or sex.

Figure 1: Nicotine exposure in the drinking water during adolescence promotes morphine conditioned place preference (CPP) in adulthood.

(A) Experimental design. C57BL/6J male (n=12) and female (n=15) group-housed mice received 24-hour, continuous access to nicotine dissolved in their drinking water or plain water only from postnatal day (P) 28–42. CPP testing began at P70, consisting of 5 days (8 sessions, 30 min each): Pre-test (Day 1), conditioning (Days 2–4; saline session followed by morphine session 4h later), and Post-test (Day 5). (B) Daily fluid consumption was similar between adolescent nicotine-exposed (red) and water (black) groups at both concentrations (two-way RM ANOVA: F(1, 6) = 0.6077, p=0.4653), with a main effect of concentration (F(1, 6) = 13.65, p=0.0102). Nicotine-exposed mice increased consumption at 0.2 mg/mL (Sidak’s post hoc, p=0.0386). Groups: Nicotine (n=14; 6 male, 8 female; 4 cages), Water (n=13; 6 male, 7 female; 4 cages). (C) Average nicotine intake per cage (n=4 cages; 2 male, 2 female cages). Nicotine concentration increased from 0.1 mg/mL to 0.2 mg/mL on Day 6. (D) Nicotine consumption increased at 0.2 mg/mL compared to 0.1 mg/mL (two-tailed paired t-test, ****p<0.0001). (E) At P70, morphine CPP testing (10 mg/kg, i.p. for three conditioning days) revealed 2-fold higher preference for the morphine-paired chamber in nicotine-exposed versus water mice (two-tailed unpaired t-test, **p=0.0052). (F) Representative heatmaps of animal positions during the Post-test. (G) Locomotion was similar between groups during Pre-test and Post-test (two-way RM ANOVA: F(1, 25) = 0.4021, p=0.5318). Data presented as mean ± SEM, *p<0.05, **p<0.01, ****p<0.0001.

To test if nicotine’s effect on morphine CPP was age-dependent, adult mice (P70–84) received nicotine or plain water for two weeks, followed by morphine CPP at P112 (Fig. 2A). Unlike adolescents, Adult Nic and Adult Water mice showed similar preference for the morphine-paired chamber (Fig. 2B) and locomotion (Fig. 2C). Thus, adolescence — but not adult — nicotine enhances morphine reward.

Figure 2: Nicotine exposure in the drinking water during adulthood does not impact adult morphine conditioned place preference (CPP).

(A) Experimental design. C57BL/6J mice (n=16; 8 male, 8 female) received 24-hour continuous access to either nicotine solution or plain water from postnatal day (P) 70–84. CPP testing began at P112. (B) Preference for the morphine-paired chamber was similar between adult nicotine-exposed and water groups (two-tailed unpaired t-test, p=0.6509). (C) Locomotion did not differ between groups during Pre-test and Post-test (two-way RM ANOVA: F(1, 14) = 3.875, p=0.0691). Data presented as mean ± SEM.

Beyond CPP, we tested voluntary morphine consumption in adulthood using a two-bottle choice assay. Following adolescent nicotine or water exposure, adult (P67) mice were single-housed and acclimated to two saccharin bottles (Fig. 3A), showing no baseline preference (Fig. 3B). Beginning at P70, one bottle contained morphine dissolved in saccharin, increasing in concentration from 0.1 to 0.2mg/mL after nine days. Adol Nic mice consumed more morphine on 11 days (Fig. 3C) and sharply increased intake at the higher dose, while Adol Water mice maintained stable intake. Across all days and doses, Adol Nic mice drank more morphine than Adol Water mice (Fig. 3D–E). Notably, dose-dependent morphine intake occurred only among Adol Nic mice (Fig. 3E). These data suggest adolescent nicotine increases both reward learning and voluntary opioid consumption in a sex-independent manner (Supp. Fig. 3).

Figure 3: Adolescent nicotine increases consumption of morphine in adulthood in a two-bottle choice drinking paradigm.

(A) Experimental design. C57BL/6J mice received 24-hour continuous access to nicotine solution or plain water from postnatal day (P) 28–42. At P67, mice were single-housed with access to two bottles containing 0.2% saccharin solution. After 3 baseline days, one saccharin bottle was replaced with morphine (0.1 mg/mL in 0.2% saccharin, Days 1–9; increased to 0.2 mg/mL, Days 10–18). (B) Saccharin intake was similar between groups during baseline days (two-way RM ANOVA: F(2, 52) = 0.5379, p=0.5872). Groups: Water (n=12; 7 male, 5 female), Nicotine (n=16; 7 male, 9 female). (C) Adolescent nicotine mice consumed more morphine than water controls (two-way RM ANOVA: interaction F(17, 442) = 1.991, p=0.0108; main effect of Adolescent Treatment F(1, 26)=9.615, p=0.0046; main effect of Day F(17, 442) = 10.69, p<0.0001). Sidak’s post hoc revealed higher consumption in nicotine group on Days 6, 8–17 (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). (D) Total morphine intake, collapsed across dose and day, was higher in the nicotine group (two-tailed unpaired t-test, p=0.0046). (E) Nicotine-exposed mice showed increased consumption at higher dose (two-way RM ANOVA: interaction F(1, 26) = 2.848, p=0.1035; main effect of Adolescent Treatment F(1, 26)=9.615, p=0.0046; main effect of Dose F(1, 26)=27.24, p<0.0001). Within the nicotine group, consumption increased at 0.2 mg/mL versus 0.1 mg/mL (Sidak’s post hoc, p<0.0001). Data presented as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Adolescent nicotine exposure enhances morphine sensitization

Behavioral sensitization often co-occurs with changes in mesolimbic DA transmission (37). Thus, we tested whether adolescent nicotine affects adult locomotor sensitization to morphine. Mice of both sexes were exposed to nicotine or plain water during adolescence. Four weeks later, mice underwent an 11-day morphine sensitization paradigm (Fig. 4A). Both groups exhibited sensitization, with greater locomotion on the last vs. first day. However, despite similar movement between groups on the first day, Adol Nic mice traveled a greater distance on the last day compared to Adol Water mice (Fig. 4B–C), confirming enhanced morphine sensitization. These findings suggest adolescent nicotine exposure increases morphine addiction-related behaviors in adulthood.

Figure 4: Adolescent nicotine exposure enhances morphine sensitization in adult mice.

(A) Experimental timeline: C57BL/6J mice received either nicotine-containing or plain drinking water from postnatal day (P) 28–42. At P67, mice underwent morphine sensitization testing over 11 days, consisting of 6 sessions (2 saline habituation, 4 morphine) conducted every other day. Each session included a 20-minute baseline period followed by 2-hour post-injection monitoring. (B) Two-way repeated measures ANOVA revealed significant Adolescent Treatment x Day interaction (F(1,9) = 29.08, p=0.0004) and a main effect of Time (F(1,9) = 171.4, p<0.0001). Both adolescent water-treated and adolescent nicotine-treated mice expressed morphine sensitization, as there was more morphine-induced locomotion on the last relative to the first testing day (Sidak’s post hoc, p=0.0006 and p<0.0001, respectively). In addition, there was a significant difference in Total Distance Travelled between Adolescent Water and Adolescent Nicotine on the Last Day (Sidak’s post hoc, p=0.0427). Group composition: Adolescent Water: n=6 (5 males, 1 female); Adolescent Nicotine: n=5 (2 males, 3 females). (C) Sensitization magnitude (last day minus first day locomotion) was significantly higher in adolescent nicotine-treated mice compared to water controls (two-tailed unpaired t-test, p=0.0004). Data shown as mean ± SEM. *p<0.05, ***p<0.001, ****p<0.0001.

Adolescent nicotine exposure alters VTA GABA neuron firing rate in response to morphine

Chronic drug exposure alters VTA GABA transmission (19,103–105), and adolescent nicotine induces lasting changes in VTA GABA neuron function (19). To test adolescent nicotine’s impact on morphine responses ex vivo, VGAT-Cre mice received adolescent nicotine or plain water, followed by AAV9-CAG-FLEX-tdTomato infusions at P56 to label VTA VGAT+ neurons. Patch clamp recordings at P70 identified VTA GABA neurons (Fig. 5A–B, Supp. Fig. 4A–C). Baseline firing rates were similar across groups (Fig. 5C). After bath-application of 10 μM morphine (71,82,83), VTA GABA neurons in Adol Water mice had dramatically reduced firing (58,59,106) (Fig. 5D–E). However, VTA GABA neurons from Adol Nic mice showed little or no inhibition, with some even increasing firing (Fig. 5F). Over time, Adol Nic neurons fired more than Adol Water neurons at multiple time points (Fig. 5G), independent of sex (Supp. Fig. 5). Since baseline rates were unaffected (Fig. 5C), this effect is not due to a floor effect. Thus, in contrast to adolescent water, adolescent nicotine exposure prevents morphine-induced inhibition of VTA GABA neurons in adulthood.

Figure 5: Prior adolescent nicotine exposure alters morphine-induced inhibition of ventral tegmental area (VTA) GABA neuron firing in adult mice.

(A) Experimental timeline: VGAT-Cre mice received nicotine-containing or plain drinking water from postnatal day (P) 28–42. At P56, mice received bilateral infusions of Cre-dependent tdTomato virus to label GABA neurons. At P70, spontaneous firing rates of VTA GABA neurons were recorded in cell-attached configuration before and after morphine (10 μM) bath application. (B) Representative biocytin-filled neuron visualized with streptavidin labeling. Scale bar: 50 μm. (C) Baseline spontaneous firing frequencies did not differ between groups prior to morphine application (two-tailed unpaired t-test, p=0.3939). Measurements taken during second minute of baseline recording. Group composition: adolescent water (n=12 cells from 6 mice; 2 males, 4 females), adolescent nicotine (n=14 cells from 10 mice; 7 males, 3 females). Data shown as mean ± SEM. (D) Representative traces from adolescent water-exposed (black) and nicotine-exposed (red) mice. (E) VTA GABA neurons from adolescent water-exposed mice showed significant reduction in firing rate between baseline (ACSF, minute 2) and morphine treatment (minute 12) (two-tailed paired t-test, p=0.003). (F) VTA GABA neurons from adolescent nicotine-exposed mice maintained firing rates between baseline and morphine treatment (Wilcoxon matched-pairs signed rank test, p=0.3575). (G) Time course of normalized firing rates following morphine application. Two-way repeated measures ANOVA revealed significant group differences over time (F(11,264) = 2.709, p=0.0025). Sidak’s post-hoc analysis showed significant differences between groups at minutes 8, 10, 11, and 12 (p<0.05). Data shown as mean ± SEM. *p<0.05, **p<0.01.

Adolescent nicotine exposure changes VTA DA neuron firing rate in response to morphine

VTA GABA cells regulate local VTA DA neurons and project to other brain regions. To investigate the circuit-level impact of adolescent nicotine, we examined VTA DA neuron responses to morphine in adulthood. VGAT-Cre mice received adolescent nicotine or plain water, followed by AAV9-CAG-FLEX-tdTomato VTA infusions at P56. Patch clamp recording at P70 identified non-fluorescent, TH+ DA neurons (Fig. 6A–B). Baseline DA neuron firing was similar between groups (Fig. 6C). Among Adol Water mice, morphine increased DA neuron firing (70,71) (Fig. 6D–E). However, among Adol Nic mice, morphine did not change DA firing (Fig. 6D–E). Over time, Adol Water DA neurons fire more than Adol Nic neurons at several time points (Fig. 6F), with no baseline differences (Fig. 6C), ruling out a ceiling effect. These data suggest adolescent nicotine exposure prevents morphine-induced excitation of VTA DA neurons in adulthood, implicating local VTA GABA interneurons in this altered response.

Figure 6: Prior adolescent nicotine exposure attenuates morphine-induced excitation of ventral tegmental area (VTA) dopamine neurons in adult mice.

(A) Experimental timeline: VGAT-Cre mice received nicotine-containing or plain drinking water from postnatal day (P) 28–42. At P56, mice received bilateral infusions of Cre-dependent tdTomato virus to label GABA neurons. At P70, spontaneous firing rates of VTA dopamine neurons (identified as VGAT-negative/non-fluorescent cells) were recorded in cell-attached configuration before and after morphine (10 μM) bath application. (B) Representative biocytin-filled neuron visualized with streptavidin labeling. Scale bar: 500 μm. (C) Baseline spontaneous firing frequencies did not differ between groups prior to morphine application (two-tailed unpaired t-test, p=0.0878). Measurements taken during second minute of baseline recording. Group composition: adolescent water (n=7 cells from 5 mice; 2 males, 3 females), adolescent nicotine (n=6 cells from 4 mice; 1 male, 3 females). Data shown as mean ± SEM. (D) Representative traces from adolescent water-exposed (black) and nicotine-exposed (red) mice. (E) Two-way repeated measures ANOVA revealed significant Adolescent Treatment × Wash-on interaction (F(1,11) = 7.511, p=0.0192) and main effects of Adolescent Treatment (F(1,11) = 5.002, p=0.0470) and Wash-on (F(1,11) = 5.623, p=0.0371). VTA dopamine neurons from adolescent water-exposed mice showed increased firing between baseline (minute 2) and morphine treatment (minute 8) (Sidak’s post-hoc, p=0.0063). Firing rates significantly differed between groups at minute 8 (Sidak’s post-hoc, p=0.0326). (F) Time course of normalized firing rates following morphine application. Two-way repeated measures ANOVA revealed significant effects of time (F(7,77) = 4.325, p=0.0004) and Adolescent Treatment (F(1,11) = 8.203, p=0.0154). Sidak’s post-hoc analysis showed significant differences between groups at minutes 4, 6 (p<0.05), and 7, 8 (p<0.01). Data shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

Paradoxical firing of VTA GABA neurons is necessary for adolescent nicotine-induced enhanced morphine CPP

To determine if altered VTA GABA neuron activity contributes to enhanced morphine CPP, we tested if chemogenetic inhibition of VTA GABA neurons prevents this effect. VGAT-Cre mice received adolescent nicotine or water, followed by AAV5-hsyn-DIO-hM4Di-mCherry or control AAV5-hsyn-DIO-mCherry infusions into the VTA (Fig. 7A–B, Supp. Table 3). Mice were divided into 4 groups (Fig. 7C) and underwent morphine CPP.

Figure 7: Chemogenetic inhibition of ventral tegmental area (VTA) GABA neurons prevents enhanced morphine conditioned place preference (CPP) in adult mice with adolescent nicotine exposure.

(A) Experimental timeline: VGAT-Cre mice received nicotine-containing or plain drinking water from postnatal day (P) 28–42. At P49, mice received bilateral VTA infusions of Cre-dependent virus expressing either inhibitory DREADD (hM4Di-mCherry) or control fluorophore (mCherry). Morphine CPP testing began 3 weeks post-surgery. (B) Representative image showing bilateral VTA viral expression. Immunofluorescence for mCherry (red) shows viral expression, and tyrosine hydroxylase (TH, green) defines VTA boundaries. Scale bar: 200 μm. Mice with virus expression outside VTA boundaries were excluded from analysis. (C) Morphine CPP (10 mg/kg, i.p.) was assessed in mice expressing hM4Di-mCherry or mCherry control. CNO (3 mg/kg, i.p.) was administered 30 minutes before each morphine conditioning session to activate DREADDs. Two-way ANOVA revealed significant Virus × Adolescent Treatment interaction (F(1,44) = 33.51, p<0.0001), and main effects of Adolescent Treatment (F(1,44) = 4.575, p=0.038) and Virus (F(1,44) = 4.887, p=0.0323). Sidak’s post-hoc analysis showed significant differences in morphine chamber preference between: Adolescent water-mCherry vs nicotine-mCherry (p=0.0171); Adolescent water-mCherry vs water-hM4Di (p<0.0001); Adolescent water-hM4Di vs nicotine-hM4Di (p<0.0001); Adolescent nicotine-mCherry vs nicotine-hM4Di (p=0.0232). Group composition: Adolescent water-mCherry: n=12 (6 males, 6 females); Adolescent nicotine-mCherry: n=15 (11 males, 4 females). Adolescent water-hM4Di: n=10 (3 males, 7 females); Adolescent nicotine-hM4Di: n=11 (7 males, 4 females). Data shown as mean ± SEM. *p<0.05, ****p<0.0001.

hM4Di, when activated by CNO, suppresses neuronal activity via Gi-coupled signaling. Since Adol Nic VTA GABA neurons resist morphine inhibition (which also occurs through Gi-coupled signaling), we confirmed that hM4Di remained functional in these neurons (Supp. Fig. 6C–E). Adol Nic-mCherry mice expressed greater preference for the morphine-paired chamber than Adol Water-mCherry mice (Fig. 7C), consistent with Fig. 1D. However, chemogenetic inhibition of VTA GABA neurons (Adol Nic-hM4Di + CNO) blocked this enhanced CPP. This suggests abnormal VTA GABA neuron firing is necessary for adolescent nicotine-induced enhancement of morphine reward. Interestingly, Adol Water-hM4Di mice had a higher CPP score than Adol Water-mCherry and Adol Nic-hM4Di mice. Since morphine reward is typically mediated by VTA GABA neuron inhibition and thus DA disinhibition, we propose a model where adolescent nicotine prevents normal GABA suppression, thereby driving heightened morphine CPP. Conversely, inhibiting VTA GABA neurons in drug-naïve mice enhances opioid reward (107). These results indicate that adolescent nicotine disrupts normal morphine reward circuitry, engaging a novel reward mechanism.

DISCUSSION

Nicotine use often begins in adolescence, a uniquely vulnerable period during which drug exposure has long-lasting neural and behavioral ramifications (24). Clinical and preclinical data reveal that nicotine use enhances the rewarding effects of multiple drugs of abuse, including alcohol, cocaine, and opioids (4,14,19, 20). Here we demonstrate that adolescent nicotine exposure fundamentally alters how morphine engages reward circuits in adulthood. Mice exposed to nicotine during adolescence show enhanced morphine reward in adulthood across multiple behavioral measures: increased conditioned place preference, greater voluntary consumption, and heightened locomotor sensitization. This effect is specific to adolescent exposure, as nicotine given in adulthood did not alter subsequent morphine reward. Most strikingly, we identify a novel mechanism whereby adolescent nicotine exposure transforms how VTA GABA neurons respond to morphine, leading to enhanced reward through sustained — rather than reduced — GABA activity.

The canonical model of morphine reward involves inhibition of VTA GABA neurons, which then disinhibits DA neurons to drive reward. Previous work has also found that the inhibition of GABA neurons alone is reinforcing (108), and our control data robustly support this established mechanism. VTA GABA cells from adolescent water-exposed mice show the expected decrease in firing in response to morphine (58) while their DA neurons show increased firing (70,71). Adolescent nicotine exposure fundamentally rewires this circuit, as demonstrated by our comprehensive recordings from both cell types. VTA GABA neurons from nicotine-exposed adolescent mice paradoxically maintain their firing in response to morphine, while their DA neurons consequently fail to show the typical increase in firing. This coordinated transformation in both cell types provides compelling evidence for local circuit reorganization as a key mechanism underlying how sustained GABA activity results in heightened reward. The maintained GABA firing directly prevents the normal morphine-induced increase in DA neuron activity, a relationship we causally validated through chemogenetic manipulations: inhibiting VTA GABA neurons during morphine conditioning specifically prevents the heightened preference seen in nicotine-exposed mice.

The insensitivity of VTA GABA neurons to morphine following adolescent nicotine exposure drives this paradoxical enhancement of reward through multiple mechanisms. While GABA neurons typically increase their firing in response to morphine, our recordings reveal a complete loss of this canonical response, suggesting fundamental alterations in opioid signaling. This GABA neuron insensitivity likely disrupts the balance between tonic and phasic dopamine release, where altered GABA activity could reduce tonic DA firing while enhancing the signal-to-noise ratio of phasic DA bursts. Our dopamine recordings indicate that local VTA GABA interneurons are particularly affected, pointing to adaptations in local circuit connectivity that transform how GABA activity influences DA release. Additionally, distinct populations of VTA GABA neurons may show different adaptations - long-range GABAergic projections to the nucleus accumbens influence associative learning, while rostral VTA GABAergic projections to dorsal raphe nucleus modulate morphine reward (109,110). The differential sensitivity of these GABA populations to adolescent nicotine exposure may orchestrate the enhanced reward phenotype.

Beyond local VTA reorganization, adolescent nicotine exposure likely also induces broader neuroadaptations across the reward circuitry that could amplify these effects. These include alterations in nucleus accumbens synaptic plasticity and receptor expression, changes in prefrontal cortical control over VTA-NAc signaling affecting reward seeking, adaptations in stress-responsive circuits including the extended amygdala, and modifications to cholinergic signaling in VTA-projecting regions. These circuit-wide changes likely work synergistically with our observed VTA adaptations to enhance morphine’s rewarding properties.

At the cellular level, several mechanisms could explain why VTA GABA neurons from nicotine-exposed adolescent mice resist morphine-induced inhibition. Morphine typically inhibits GABA neurons through mu opioid receptor-mediated activation of inhibitory signaling cascades. One possibility is that adolescent nicotine exposure alters this signaling cascade. Alternatively, compensatory circuit-level adaptations may emerge, such as through VTA glutamate neurons that express mu opioid receptors (111) and could excite neighboring GABA neurons. The diversity of VTA GABA neurons likely contributes to these complex adaptations, as these neurons differ in their synaptic targets and molecular composition (40). While canonical markers of interneuron subtypes from forebrain regions do not directly map onto the VTA (40), and calcium-binding proteins often co-express in both GABA and DA neurons (112), this heterogeneity may explain both our observed cell-to-cell variability and the novel reward mechanism we report.

Within our behavioral results, it is important to consider that nicotine exposure during adolescence could alter taste perception in ways that influence our two-bottle choice drinking results. While we attempted to control for bitter taste by dissolving morphine in saccharin, chronic nicotine exposure can modify taste processing in rodents (113–115). However, two aspects of our data suggest that altered taste sensitivity alone cannot explain our results. First, the dose-dependent increase in consumption specifically in nicotine-exposed adolescent mice suggests an interaction with morphine’s rewarding properties rather than just reduced bitter taste detection. Second, our CPP and sensitization findings, where taste is not a factor, support that adolescent nicotine exposure enhances morphine’s rewarding properties through mechanisms beyond taste alone.

These findings challenge the canonical view of VTA GABA neurons in reward processing. While optogenetic activation of VTA GABA neurons typically produces aversion and interrupts reward consumption (116,117), and their inhibition is rewarding (107), these neurons also encode reward expectation (61,118). Our results reveal that adolescent nicotine exposure creates an alternative pathway to reward that operates through sustained, rather than reduced, GABA activity. This fundamentally changes our understanding of how early nicotine exposure affects reward circuitry and may help explain why adolescent nicotine use increases vulnerability to opioid addiction.