Shifted balance between ventral striatal prodynorphin and proenkephalin biases development of cocaine place avoidance

Cocaine use is rising and effective pharmacotherapies are lacking. Cocaine produces both rewarding and aversive states: euphoria drives positive reinforcement, whereas withdrawal and, in some individuals, acute intoxication can elicit negative affect (panic, paranoia, anxiety) that may reduce use or motivate opioid co-use. While models of cocaine reward are well established, modeling the acutely aversive state is less developed. Trace (delay) conditioning, where drug is administered immediately after removal from a context, has produced conditioned place aversion (CPA) for ethanol, nicotine, and amphetamine, capturing transient drug-induced aversive states distinct from withdrawal. The neurobiology of cocaine aversion, especially during acute intoxication, is incompletely characterized. Dynorphin/kappa-opioid receptor (KOR) signaling in ventral striatum is implicated in negative affect and stress-induced reinstatement after withdrawal, whereas enkephalin/mu-opioid receptor (MOR) signaling in ventral striatum supports cocaine reward. The study addressed two questions: (1) What trace conditioning parameters best elicit cocaine CPA in mice? (2) Does the balance between striatal prodynorphin (Pdyn) and proenkephalin (Penk) predict or bias development of cocaine CPA versus preference? The work aims to clarify mechanisms governing individual differences in acute cocaine aversion, with implications for vulnerability/resilience to cocaine use disorder and potential treatment targets.

Prior work shows simultaneous CS–US pairing yields conditioned place preference (CPP) for many drugs including cocaine, while timing shifts (e.g., administering cocaine 15 minutes before context) can yield CPA, attributed to pairing the chamber with falling brain cocaine levels (withdrawal-like state). Trace conditioning (context exposure followed by immediate drug injection after removal) produces CPA for ethanol, nicotine, and amphetamine at doses that produce CPP under simultaneous pairing, indicating capture of distinct, bivalent affective states. Dynorphin/KOR signaling increases after repeated cocaine and during withdrawal, promotes stress-induced reinstatement, and is intrinsically aversive within ventral striatum subregions. In contrast, MOR activity in ventral striatum is necessary for cocaine CPP, and enhancing enkephalin facilitates CPP. These lines of evidence suggest that within ventral striatum, dynorphin/KOR may facilitate aversion (especially withdrawal or possibly intoxication-associated), whereas enkephalin/MOR potentiates reward. However, cocaine CPA based on acute intoxication has not been established in mice using trace conditioning, and the contribution of endogenous opioid balance to acute aversion remains unclear.

Animals: Adult male and female mice (8–20 weeks) on C57BL/6J background. For parameter optimization (Experiment 1), 103 wild-type mice from transgenic lines were used: Adora2a-Cre⁻ (n=41), MORfl/fl (n=2), VGat:Cre (n=19), VGluT2:Cre (n=41). A subset (Adora2a-Cre⁻ n=24 plus 6 saline MORfl/fl) were later used for qPCR. For causal testing (Experiment 2), D2-PenkKO mice (Penk deleted in striatal D2-MSNs; n=17) and Adora2a-Cre; Penkfl/fl littermate controls (n=17) were used. Group-housed, standard conditions; experiments during light cycle; procedures approved by UT Austin and Rutgers IACUCs. Drugs: Cocaine HCl in 0.9% saline, i.p., 10 ml/kg. Doses tested: 15, 20, 25 mg/kg, selected based on doses producing CPP in prior work. Conditioned Place Avoidance (Trace Conditioning): Apparatus: 2-chamber (Med Associates; grid vs hole floors) or 3-chamber (Stoelting; smooth vs textured floors with distinct visual cues plus neutral center). Procedure Experiment 1: Between-subjects manipulations of apparatus (2- vs 3-chamber), conditioning session length (15 min in 2-chamber; 30 min in 3-chamber), and cocaine dose (15, 20, 25 mg/kg). On conditioning days, mice confined to one side for the session duration, then removed and immediately injected with cocaine or saline and returned to home cage; drug and saline sessions alternated for 8 sessions total (4 cocaine, 4 saline) across two weeks. Unbiased assignment and counterbalancing of drug-context pairing, side, and order. Preference tests (30 min) conducted at Pre-Test and after conditioning days 4 (Post-Test 1) and 8 (Post-Test 2); saline given post-test. Preference/avoidance quantified as percent time on cocaine-paired floor. Experiment 2: D2-PenkKO and controls underwent the same trace procedure in the 2-chamber apparatus with 15-min sessions at 25 mg/kg. qPCR: After Post-Test 2, a subset of mice (cocaine-conditioned Adora2a-Crefl n=24; saline-only MORfl/fl n=6) were euthanized. Dorsal and ventral striatum dissected; RNA extracted (Qiagen RNeasy), cDNA synthesized (BioRad iScript). TaqMan assays quantified Penk (Mm01212875_m1) and Pdyn (Mm00457573_m1) relative to Actb (Mm01205647_g1) on a CFX384 (95°C 20 s; 40 cycles 95°C 1 s, 60°C 20 s). Triplicates, negatives included; ΔΔCt used for relative expression. Statistics: For Experiment 1, percent time on cocaine floor analyzed with 3-way RMANOVA (Test as repeated measure; Chamber type and Dose between-subjects). Additional 2-way ANOVAs assessed neutral-zone time in 3-chamber; sex effects assessed after collapsing across dose/apparatus. Linear regressions evaluated dose–preference relationships. Post hoc tests Sidak-corrected. Preference categories defined using ±1 SD from Pre-Test mean to classify Preferers, Avoiders, Neutral; proportions compared by Chi-square or Fisher’s exact tests. Correlations between Penk/Pdyn expression and preference assessed by Pearson’s r; Penk vs Pdyn across groups by 2-way RM ANOVA for dorsal and ventral striatum. Experiment 2 preference change analyzed with a Generalized Linear Model (inverse Gaussian distribution) due to kurtosis; genotype effects and stratified proportions compared as above. Alpha 0.05. Software: GraphPad Prism, IBM SPSS.

• Parameter optimization: Across apparatus (2- vs 3-chamber), conditioning time (15 vs 30 min), and dose (15, 20, 25 mg/kg), mean percent time on the cocaine-paired floor was stable with only modest effects (Test × Chamber × Dose: F4,230 = 2.14, p = 0.077). Chamber × Test interaction (F2,230 = 7.1, p < 0.01) was driven by a Pre-Test difference (20 mg/kg), indicating sampling bias rather than conditioning differences.
• Dose effects: Main effect of dose (F2,115 = 3.43, p < 0.05) without significant post hoc differences. In the 3-chamber apparatus at Post-Test 1, higher cocaine dose predicted greater avoidance (F1,58 = 6.6, p < 0.05; R² = 0.10), but no relationship at Post-Test 2 and none in the 2-chamber.
• Neutral zone: Avoidance reflected reduced time in cocaine zone and increased time in saline zone, not increased neutral-zone occupancy (Test × Zone: F4,66 = 5.7, p < 0.001).
• Sex: No sex differences (F1,95 = 1.34, p = 0.25) and no Sex × Dose interaction (F2,190 = 1.00, p = 0.37).
• Polarization: Variability increased from Pre-Test (CV 20.8%) to Post-Test 2 (CV 32.5%). Stratification at Post-Test 2 (n = 103) showed Preference 25.2%, Avoidance 29.1%, Neutral 45.6%; significant shift from Pre-Test (χ²(2, n=103) = 51.08, p < 0.0001). Proportions did not differ by apparatus (χ²(2, n=103) = 1.08, p = 0.58).
• qPCR correlations: In ventral striatum, Pdyn expression positively associated with cocaine preference (R² = 0.2004, p < 0.05); Penk not correlated. Penk:Pdyn ratio in ventral striatum was negatively correlated with percent time on cocaine floor (reported R² = -0.52, p < 0.001), indicating higher Penk relative to Pdyn associated with avoidance. No correlations in dorsal striatum.
• Grouped gene expression: Ventral striatum Gene × Group interaction (F2,17 = 9.7, p < 0.01); Avoiders showed higher Penk relative to Pdyn (t17 = 4.32, p < 0.01). In dorsal striatum, Pdyn slightly higher than Penk across groups (Gene main effect F1,21 = 7.03, p < 0.05) without group differences.
• Genetic test (Experiment 2): D2-PenkKO mice (higher Pdyn:Penk tone) were resistant to developing CPA compared to Penkfl/fl controls. GLM showed Genotype × Test interaction (Wald χ² = 5.73, df=1, p < 0.05). Controls developed significant CPA from Pre to Post-Test 2 (p < 0.05), D2-PenkKO did not (p = 0.12); genotypes differed at Post-Test 2 (p < 0.05). Binary stratification: Avoidance at Post-Test 2 in controls 64.7% vs D2-PenkKO 35.3% (χ²(1, N=34) = 2.9, p = 0.08). Preferer proportions increased in both genotypes without significant genotype differences.

Trace conditioning revealed that acute cocaine at rewarding doses induces bivalent motivational states, producing both preference and avoidance in roughly equal proportions, and that this outcome is not reliably determined by dose (15–25 mg/kg), apparatus type, or session duration. The increased polarization across conditioning suggests latent individual differences in sensitivity to cocaine’s immediate negative affective effects. The opioid milieu in ventral striatum appears to bias these outcomes: lower Pdyn (and higher Penk:Pdyn ratio) associated with avoidance, whereas higher Pdyn tone relative to Penk was protective against CPA and, in D2-PenkKO mice, mitigated avoidance development. These findings refine the role of striatal dynorphin: while KOR signaling promotes negative affect and relapse following withdrawal, during acute intoxication a higher dynorphin tone may buffer against the transient aversive state or shift motivational valence away from avoidance. Conversely, enkephalin/MOR signaling supports cocaine reward and, when dominant over dynorphin, may enhance sensitivity to acute aversion captured by trace conditioning. Together, the results indicate that the balance between enkephalin and dynorphin in ventral striatum regulates the expression of conditioned aversion versus preference to acute cocaine intoxication, offering mechanistic insight into vulnerability or resilience to problematic cocaine use and potential targets for intervention.

The study establishes trace conditioning as a viable approach to capture individual differences in acute cocaine-induced place avoidance in mice and identifies the ventral striatal balance between prodynorphin and proenkephalin as a key correlate and modulator of this behavior. Cocaine CPA did not depend strongly on dose (15–25 mg/kg), apparatus type, or session duration, but responses became polarized with conditioning. Lower ventral striatal Pdyn relative to Penk was associated with CPA, and genetically elevating relative Pdyn tone (via Penk deletion in D2-MSNs) reduced CPA development. These results suggest dynorphin and enkephalin exert opposing influences on motivational valence during acute intoxication versus withdrawal and highlight endogenous opioid balance as a candidate mechanism for predicting vulnerability to cocaine aversion, subsequent use trajectories, and potential co-use of opioids. Future work should dissect ventral striatal subregions, measure peptides and in vivo dynamics directly, acutely manipulate KOR/MOR signaling during expression, and test broader dose ranges and histories of cocaine exposure.

• Regional specificity: Dissections separated ventral vs dorsal striatum but could not resolve finer subregions (e.g., NAc core vs shell subterritories) known to differentially mediate aversion.
• Molecular measures: qPCR measured mRNA (Penk, Pdyn), which may not fully reflect peptide levels; complementary peptide quantification (e.g., immunostaining, mass spectrometry) is warranted.
• Causality and timing: Post hoc mRNA measures cannot distinguish pre-existing differences from conditioning-induced changes; although D2-PenkKO data support a causal role for peptide balance, direct real-time measures (e.g., peptide release, KOR/MOR activity, MSN calcium dynamics) are needed.
• Design constraints: Session duration was confounded with apparatus type in Experiment 1, limiting attribution of small effects; Experiment 2 had smaller sample size and stronger CPA in controls than Experiment 1, raising potential sampling variance concerns.
• Generalizability: Only three doses (15–25 mg/kg) were tested; higher doses or different cocaine histories could yield different CPA magnitudes and persistence.