Female (Saline, N=22; Oxycodone N=23) and male (Saline, N=15; Oxycodone, N=27) rats were trained to self-administer oxycodone (or saline) under short access (ShA) conditions (1h/day for 8 days), followed by 14 days of long access (LgA) conditions (6 h/day) (Fig. 1A Experimental Design). Males and females escalated their oxycodone intake under LgA conditions: there was an increase in the number of infusions over time (Fig. 1B: Mixed-effects model (REML): days x treatment interaction: F_(63,1463) = 13.14, p < 0.0001; main effect of days: F_(21,1463) = 38.91, p < 0.0001; main effect of treatment: F_(3,71) = 31.29, p < 0.0001; no sex difference: F_(1,71) = 0.04, p = 0.84), and there was an increase in active lever presses (Fig. 1C: Mixed effects model (REML): days x treatment interaction: F_(63,1149) = 5.210, p < 0.0001; main effect of days: F_(21,1149) = 14.59, p < 0.0001; main effect of treatment: F_(3,56) = 12.48, p < 0.0001; no sex differences: F_(1,56) = 0.40, p = 0.53). There was no change in inactive lever presses in either sex and lever pressing did not increase across days (Fig. 1D: Mixed effects model; main effect of treatment: F_(3,56) = 3.909, p = 0.01; no change across days: F_(21,1148) = 1.47, p = 0.08; no sex difference: F_(1,56) = 0.41, p = 0.52). These findings are consistent with published results [33], showing that escalation of oxycodone self-administration behavior did not differ between male and female rats under LgA conditions.

To assess whether our LgA oxycodone SA produced similar levels of dependence in male and female rats, we measured spontaneous somatic withdrawal 1 day after the last LgA oxycodone SA session in females (n = 7 saline, 7 oxycodone) and males (n = 5 saline, 10 oxycodone). Since opioid dependence is characterized by somatic withdrawal signs upon withdrawal, we predicted an increase in somatic withdrawal signs in both sexes. We found a significant increase in expression of somatic withdrawal behaviors (combined into a Total Withdrawal Score) in females and males that received oxycodone compared to saline (Fig. 1E: Two-way ANOVA; main effect of treatment: F_{(1, 25)} = 12.68, p = 0.002). There were no sex differences in the somatic withdrawal score (p = 0.67), consistent with the similar level of escalation of oxycodone SA. This is consistent with previously published studies that have shown increases in somatic withdrawal symptoms after 24-hours in male and female rats [34].

We also assessed cue-induced drug-seeking after acute abstinence from LgA oxycodone SA. Female (n = 6 saline, 8 oxy) and male rats (n = 4 saline, 8 oxy) remained in their home cages without any access to oxycodone (forced abstinence) for 1-day. After 1-day of abstinence, rats returned to the operant box where only the house-cue, lever-cue and lever were available for 2 hrs. No infusions were given. We measured the number of active lever presses during cue presentation without drug delivery. We found that 1-day of abstinence increased cue-induced oxycodone seeking in males and females compared to saline (Fig. 1F: Two-way ANOVA; significant sex x treatment interaction: F_(3,47) = 3.9, p = 0.02; no effect of sex: F_(1,47) = 3.9, p =0.06; main effect of drug: F_(3,47) = 36.3, p < 0.0001). Sidak’s multiple comparison: female saline group vs. oxycodone group, p = 0.01; male saline group vs. oxycodone group, p = 0.02.

Cue-induced drug-seeking was assessed after 14-days of abstinence from LgA oxycodone SA. Female (n = 8 saline, 7 oxycodone) and male (n = 6 saline, 8 oxycodone) rats underwent 14-days of forced abstinence in their home cages. On the 14th day of abstinence, rats were returned to the operant box where only the house-cue, lever-cue and lever were available for 2 hrs. No infusions were given. We found that both male and female rats on oxycodone pressed the active lever more frequently than rats in the saline control group (Fig. 1G: Two-way ANOVA: main effect of drug: F (3,47) = 36.3, p < 0.0001. Additionally, females made significantly more active lever presses than males (Fig. 1G: Two-way ANOVA: significant drug x sex interaction: F (3,47) = 3.9, p = 0.02; Sidak’s multiple comparisons test female oxycodone greater than male oxycodone, p = 0.006). In addition, when comparing oxycodone-seeking in males and females during acute or prolonged abstinence, we found that females but not males show an increase in seeking over time (Fig. 1F-G: Two-way ANOVA: significant day x sex interaction: F (3, 47) = 3.9, p = 0.02. Sidak’s multiple comparisons: oxycodone male acute vs. prolonged: p = 0.9; oxycodone female acute vs. prolonged: p < 0.0001.

These findings suggest that prolonged forced abstinence increased oxycodone-seeking in both males and females, with a stronger effect in females than in males.

To test whether PVT-arising inputs to NAcSh contribute to cue-induced relapse and drug-seeking, we targeted PVT-NAcSh projections optogenetically by expressing ChR2 in the PVT via stereotaxic injections of AAV5-CaMKIIα-ChR2-eYFP viral construct in rats from all experimental groups. 7-8 weeks after viral transfection, eYFP-tagged ChR2 was expressed at the injection site (PVT) and ChR2-eYFP-expressing PVT-arising projections were found in the NAcSh (Fig. 2A). Using whole-cell patch clamp recordings (Fig. 2B), we confirmed that PVT projections form functional synapses on the NAcSh neurons (Fig. 2C, D). Photostimulation-induced (470 nm. 5 ms-long pulses of blue light) excitatory postsynaptic currents (EPSCs) were recorded under voltage clamp recording conditions at a holding potential of –70 mV. EPSCs at the PVT-NAcSh synapses are glutamatergic, as demonstrated by their sensitivity to NBQX (10µM), an AMPA/Kainate receptor antagonist, and D-AP5, an NMDA receptor antagonist (Fig. 2D, E). Glutamatergic EPSCs in PVT-NAcSh projections were monosynaptic in nature as they were rescued by the potassium channel blocker, 4-AP (1 mM) after they were blocked by tetrodotoxin (TTX, 1 µM), a sodium channel blocker (Fig. 2G, H) [35].

Stimulation of glutamatergic PVT projections resulted in activation of GABAergic interneurons in NAcSh (Fig. 2C), triggering feed-forward inhibitory postsynaptic currents (IPSCs) in recorded MSNs (Fig. 2D). The IPSCs, recorded at a holding potential of 0 mV, were GABAergic as they were blocked by the GABA_A receptor antagonist, bicuculline (20 μM) (Fig. 2I, J), and they were disynaptic in nature (Fig. 2C) as they were sensitive to glutamate receptors antagonists NBQX and D-AP5 (Fig. 2F). Based on these findings, we conclude that PVT sends monosynaptic projections to NAcSh, forming glutamatergic synapses on MSNs, as well as triggering feedforward inhibition in the PVT-NAcSh circuits.

To examine the effect of acute abstinence from oxycodone self-administration on synaptic transmission in the PVT-NAcSh projections, we performed whole-cell patch-clamp recordings of light-induced EPSCs in medium spiny neurons in NAcSh in slices from rats which self-administered oxycodone at both abstinence time points. To assay the effects of acute abstinence on the efficacy of excitatory synaptic transmission, we obtained input-output curves for the light-induced EPSCs in all experimental groups (male saline group: 5 rats, 18 cells; male oxycodone group: 8 rats, 36 cells; female saline group: 7 rats; 26 cells; female oxycodone group: 6 rats, 20 cells) by recoding the EPSCs at a holding potential of –80 mV evoked by photostimuli of increasing intensity (Fig. 3A). Although the EPSC amplitude increased with light density (Fig. 3A: Three-way ANOVA: main effect of light intensity F_(5,582) = 52.69, p < 0.0001), we did not find any treatment effect on synaptic efficacy in the PVT-NAcSh pathway (Fig. 3A: Three-way ANOVA: no effect of treatment: F_(1,582) = 1.5, p = 0.2) or sex specificity (Fig. 3A: Three-way ANOVA: no effect of sex: F_(1,582) = 0.5, p = 0.5). This finding indicates that 1-day abstinence from oxycodone self-administration did not affect the efficacy of glutamatergic synaptic transmission in PVT-NAcSh projections in both male and female rats.

Consistent with the lack of changes in synaptic strength, the magnitude of paired-pulse ratio (PPR), and index of presynaptic function [35] of EPSC amplitude at the PVT-NAcSh synapses did not differ between the treatment groups (male saline: 5 rats, 28 cells; male oxycodone: 10 rats, 25 cells; female saline: 7 rats, 30 cells; female oxycodone: 6 rats, 22 cells) (Fig. 3B: Three-way ANOVA: F_(1,443) = 1.19, p = 0.3) or sex specificity (Fig. 3B: Three-way ANOVA: F_(1,443) = 2.20, p = 0.1), indicating that the probability of neurotransmitter release at the PVT-NAcSh synapses was unaffected by acute abstinence.

In analogously designed experiments, we assayed the effects of prolonged abstinence on synaptic transmission in PVT-NAcSh projections. As above, input-output curves for PVT-NAcSh EPSCs were obtained by recording photostimulation-induced EPSCs in MSNs at a holding potential of –80 mV (Fig. 3C) (male saline group: 5 rats, 16 cells; male oxycodone group: 7 rats, 20 cells; female saline group: 7 rats; 21 cells; female oxycodone group: 5 rats, 21 cells). We found that the EPSC amplitude increased with light intensity (Fig. 3C: Three-way ANOVA: main effect of light intensity; F_(5,442) = 40.5, p < 0.0001), and, unlike 1-day abstinence, long abstinence from oxycodone self-administration was associated with increases in the EPSC amplitude in both male and female rats compared to control group (Fig. 3C: Three-Way ANOVA: main effect of treatment; F_(1,442) = 18.3, p < 0.0001). The effects of prolonged abstinence on synaptic strength in the PVT-NAcSh pathway was similar between males and females.

Notably, the magnitude of PPR at the studied synapses was found to be decreased after prolonged abstinence from oxycodone self-administration in both male and female rats (Fig. 3D: Three-Way ANOVA: main effect of treatment: F_(1,388) = 25.8, p < 0.001; male saline: 5 rats, 23 cells; male oxycodone: 7 rats, 33 cells; female saline: 7 rats, 23 cells; female oxycodone: 6 rats, 22 cells). Thus, the observed synaptic potentiation in PVT-NAcSh projections after longer abstinence periods was at least in part due to increased glutamate release probability.

To assess whether prolonged abstinence from oxycodone self-administration alters feed-forward inhibition within the PVT-NAcSh pathway (Fig. 3E-F), light-evoked IPSC amplitudes were analyzed using linear mixed-effects modeling. In both males and females, IPSC amplitude increased robustly with light intensity, indicating preserved recruitment of inhibitory synaptic responses with increasing stimulation (males: F_{(1, 158)} = 43.49, p = 6.1 × 10⁻¹⁰; females: F_{(1, 164)} = 21.26, p = 8.0 × 10⁻⁶). No significant main effect of treatment condition was detected in either sex (males: F_{(1, 158)} = 0.00023, p = 0.988; females: F_{(1, 164)} = 0.025, p = 0.875), indicating that prolonged abstinence from oxycodone did not alter baseline inhibitory synaptic strength within the PVT-NAcSh pathway in males or females. No significant Light × Condition interactions were observed in either sex (males: F_{(1, 158)} = 0.543, p = 0.462; females: F_{(1, 164)} = 1.298, p = 0.256), indicating that the scaling of inhibitory responses with increasing stimulation intensity was not altered by oxycodone exposure in either sex. Together, these results demonstrate that feed-forward inhibition within the PVT-NAcSh pathway is preserved following prolonged abstinence from oxycodone, with no significant changes in either the magnitude or the input-output scaling of inhibitory synaptic responses in males or females.

Taken together with the observed strengthening of excitatory synaptic transmission in PVT–NAcSh projections after prolonged abstinence (Fig. 3C), these results suggest that prolonged abstinence from oxycodone selectively potentiates excitatory but not inhibitory synaptic transmission within this pathway. This relative shift in the excitation–inhibition balance may facilitate MSN output within relapse-related circuitry and contribute to the behavioral effects observed following prolonged abstinence.

To explore whether changes in glutamatergic PVT projections to MSNs in NAcSh correlated with abstinence from oxycodone self-administration, we recorded light-induced AMPA receptor-mediated (AMPAR) EPSCs at the PVT-NAcSh synapses in slices from all experimental groups at holding potentials of −70 mV, 0 mV or +40 mV. In these experiments, we included endogenous polyamine spermine (200 μM), in the internal recording solution. We then calculated the rectification index for AMPAR EPSCs at the studied PVT-NAcSh projections [36] by dividing the peak amplitude of AMPAR EPSC at +40 mV by the EPSC amplitude at -70 mV. The changes in rectification index associated with experimental interventions would indicate that the AMPAR subunit composition was modified, as the GluR1 subunit trafficking to synapses was shown to affect the rectification index [36]. However, after acute abstinence, we did not observe significant changes in the rectification index (male saline: 5 rats, 23 cells; male oxycodone: 8 rats, 28 cells; female saline: 7 rats, 14 cells; female oxycodone: 6 rats, 18 cells) by sex (Fig. 4A, inset bar graph: Two-way ANOVA: F_(1,68) = 0.83, p = 0.4) or treatment (Fig.4A, inset bar graphs: Two-way ANOVA: F_(1,68) = 0.35, p = 0.6).

Similarly, after prolonged abstinence we did not observe significant changes in the rectification index (male saline: 4 rats, 15 cells; male oxycodone: 5 rats, 23 cells; female saline: 5 rats, 22 cells; female oxycodone: 5 rats, 15 cells) by treatment (Fig. 4B (inset bar graph): Two-way ANOVA: F_(1,50) = 2.7, p = 0.1). These results suggest that neither acute nor prolonged abstinence from oxycodone self-administration was associated with changes in the AMPAR subunit composition at synapses formed by PVT projecting fibers on MSNs in NAcSh. Thus, synaptic potentiation in PVT-NAcSh projections, observed after 14 days of abstinence (see Fig. 3C), was unlikely due to increased GluR1 trafficking at the synapses studied.

Consistent with the latter notion, the AMPAR/NMDAR EPSC amplitude ratio was not affected by acute abstinence (Fig. 4C: Two-way ANOVA: no effect of sex: F_(1,76) = 0.06, p = 0.8; no effect of treatment: F_(1,76) = 0.008, p = 0.9; n = male saline: 5 rats, 14 cells; male oxycodone: 8 rats, 24 cells; female saline: 7 rats, 24 cells; female oxycodone: 6 rats, 18 cells). The AMPAR/NMDAR EPSC ratio was assessed by measuring the amplitude of the NMDAR-mediated EPSC at a holding potential of +40mV, 40ms after the peak of AMPAR-mediated EPSC at -70mV. Similarly, the AMPAR/NMDAR EPSC amplitude ratio was also unaffected after prolonged (14-days) abstinence (Fig. 4D: Two-way ANOVA: no effect of sex: F_(1,45) = 0.4, p = 0.5; no effect of treatment: F_(1,45) = 0.3, p = 0.6; (n = male saline: 5 rats, 12 cells; male oxycodone: 5 rats, 12 cells; female saline: 5 rats, 15 cells; female oxycodone: 5 rats, 15 cells), suggesting that acute or prolonged abstinence from oxycodone self-administration might have no detectable postsynaptic effects as assessed by these synaptic measures in PVT-NAcSh projections.

To determine whether abstinence from oxycodone self-administration alters intrinsic excitability of NAcSh medium spiny neurons (MSNs), we performed whole-cell current-clamp recordings from NAcSh MSNs in slices from saline- (males: 7 rats, 16 cells; females: 4 rats, 7 cells) and oxycodone-treated rats (males: 6 rats, 18 cells; females: 8 rats, 15 cells) following acute abstinence (1 day; Fig. 5A, C–E), as well as from saline- (males: 3 rats, 8 cells; females: 6 rats, 14 cells) and oxycodone-treated rats (males: 4 rats, 14 cells; females: 6 rats, 15 cells) following prolonged abstinence (14 days; Fig. 5B, F–H). Spike output was quantified as the number of action potentials fired during depolarizing current steps. Across all conditions, injected current robustly increased spike output, confirming intact spike count–current relationships in all groups (acute abstinence males: F_{(1, 370)} = 50.03, p = 7.6 × 10⁻¹²; acute abstinence females: F_{(1, 230)} = 71.63, p = 3.1 × 10⁻¹⁵; prolonged abstinence males: F_{(1, 225)} = 19.23, p = 1.8 × 10⁻⁵; prolonged abstinence females: F_{(1, 306)} = 30.14, p = 8.4 × 10⁻⁸).

Following acute abstinence, oxycodone exposure did not significantly alter baseline spike output or spike output gain in males (Fig. 5D: condition, F_{(1, 370)} = 0.454, p = 0.501; current × condition, F_{(1, 370)} = 0.236, p = 0.628) or females (Fig. 5E: condition, F_{(1, 230)} = 0.571, p = 0.451; current × condition, F_{(1, 230)} = 0.776, p = 0.379), indicating that acute oxycodone abstinence does not detectably alter MSN intrinsic excitability in either sex.

Following prolonged abstinence, no significant effect of oxycodone exposure on baseline spike output or spike output gain was detected in females (Fig. 5H: condition, F_{(1, 306)} = 0.025, p = 0.875; current × condition, F_{(1, 306)} = 0.366, p = 0.546). In males, oxycodone-treated animals showed numerically higher spike output compared to saline controls (mean spikes per step: oxycodone 7.46 ± SD 2.29 vs saline 4.43 ± SD 2.82), corresponding to a large effect size (Cohen’s d = 1.18); however, this difference did not reach statistical significance (Fig. 5G: condition, F_{(1, 225)} = 2.419, p = 0.121; current × condition, F_{(1, 225)} = 0.008, p = 0.930). A post-hoc power analysis indicated that the current sample size (3 saline rats, 4 oxycodone rats) provided only approximately 20% power to detect this effect, and that 13 rats per group would be required to achieve 80% power. These results should therefore be interpreted with caution, and the possibility of a biologically meaningful increase in MSN excitability in males following prolonged oxycodone abstinence cannot be excluded.

No significant difference in baseline spike output or spike output gain was observed between males and females at either 1 day (Fig. 5C: condition, F_{(1, 351)} = 0.063, p = 0.802; current × condition, F_{(1, 351}) = 0.105, p = 0.746) or 14 days of abstinence (Fig. 5F: condition, F_{(1, 300)} = 0.176, p = 0.675; current × condition, F_{(1, 300)} = 0.036, p = 0.849).

Together, these results indicate that intrinsic excitability of NAcSh MSNs is largely unaltered following oxycodone abstinence in both sexes. The exception is a trend toward increased spike output in males following prolonged abstinence, which, despite a large observed effect size, did not reach statistical significance due to limited sample size in this group.

More than 85% of Neurobiotin-stained cells obtained from the medial division of NAcSh in male or female rats are medium spiny neurons (MSNs) characterized by dense spines on a second or higher order of dendritic branches (Figure 6A-C). The number of MSNs primary dendrites (3.3-3.6 on average for male rats, 2.8-4.1 in average for female rats) and elongated dendritic trees (∼1.5 on average ratios of long axis over short axis of dendritic field among treatment groups) are consistent with the previously described morphological features of NAcSh-MSNs [32]. Axons arising from soma or proximal dendrites can be observed in the majority of reconstructed MSNs (86.8% or 85.7% in male or female rats, respectively). Some of the axons (21% or 37% in male or female rats) were found to give rise to axonal collateral ramifications with terminal-like varicosities in the vicinity of their soma. This observation suggests that some MSNs may contribute to feedforward inhibition in studied projections as manifested by polysynaptic IPSC activated by photostimulation of PVT afferents (Figure 6A, left and Figure 2C,D,F,I and J [37,38]). In addition, less than 15% of Neurobiotin-stained neurons were identified either as sparsely spined neurons (SSNs) characterized by a few spines on thread-like dendrites, or aspiny neurons (ASNs) without visible dendritic spine (Figure 6A-C). These two types of neurons observed in both female and male rats were not included in the subsequent quantitative analysis.

To examine the effects of abstinence from oxycodone self-administration on neuronal structure in NAcSh, we analyzed 45 reconstructed MSNs from 23 female rats (8-14 cells from 5-7 rats among treatment groups) and 41 reconstructed MSNs from 21 male rats (6-16 cells from 3-8 rats among treatment groups). We found that soma sizes of the MSNs, estimated by maximal projection area at a single optical plane, vary in a fivefold range (∼40-200 µm2) in both male and female rats with identical cumulative frequency distributions (p = 0.954, Mann-Whitney test; Figure 6D). The varied soma sizes are correlated with dendritic size (dendritic length or volume), and, to a lesser degree, with the number of branch points (Figure 6E, F). This observation indicates a wide variation in the somatodendritic morphology of NAcSh-MSNs in both male and female rats. We then examined cumulative frequency distributions of soma size among four treatment groups, including male and female rats, and found no differences at 1 day of abstinence (p = 0.2703) or 14 days of abstinence (p = 0.5982, Kruskal-Wallis test, Figure 6-Supplementary Figure 1B and F). We did not detect significant effect of oxycodone abstinence on any parameter assayed in dendritic morphology (Two-way ANOVA, variables: treatment x sex) either at 1-day abstinence (F(1,40) = 0.455, p = 0.504 for dendritic length; F_(1,40) = 0.465, p = 0.499 for branch points), or at 14-days abstinence (F_(1,38) = 0.001, p = 0.972 for dendritic length; F_(1,38) = 0.114, p = 0.738 for branch points, left panels in Figure 6-Supplementary Figure 1C,D,G, H). Consistent with these observations, Sholl analyses (right panels in Figure 6-Supplementary Figure 1C,D,G,H; three-way ANOVA, variables: treatment x sex x radial distance) indicate no changes after 1 day abstinence (F_{(1, 40)} = 0.4568, p = 0.5030 for dendritic length; F _{(1, 40)} = 1.547 p = 0.2208 for branch points), or at 14 days abstinence (F(_{1, 38)} = 0.001, p = 0.9693 for dendritic length; F_{(1, 38)} = 0.1138 p = 0.7378 for branch points). On the other hand, we observed small sex differences in the number of branch points (F_(1,40) = 4.444, p = 0.041; Bonferroni’s multiple comparisons test, p = 0.546 for male versus female saline rats) in NAcSh-MSNs at 1 day abstinence (whereas the effect on dendritic length was not significant, F_(1,40) = 3.396, p = 0.073; left panels in Figure 6-Supplementary Figure 1C, D). At 14 days of abstinence (left panels in Figure 6-Supplementary Figure 1G,H), there were no differences between male and female rats (F_(1,38) = 1.675, p = 0.203 for dendritic length; F_(1,38) = 1.964, p = 0.169 for branch points). No significant interaction between treatment and sex was found on dendritic length and branch points. The Sholl analyses of same data as above showed no sex differences both at 1 day abstinence (F_{(1, 40)} = 3.396, p = 0.0728 for dendritic length; F_{(1, 40)} = 1.848, p = 0.1816 for branch point) and at 14 days abstinence (F_{(1, 38)} = 1.670, p =0.2041 for dendritic length; F_{(1, 38)} = 1.964, p = 0.1692 for branch point). Taken together, our quantitative analyses demonstrate the lack of effect on somatodendritic morphology of NAcSh-MSNs at either 1 day or 14 days of abstinence from oxycodone self-administration in both female and male rats.

Escalation of drug taking in rats is defined as an increase in the amount and frequency of drug intake over time during self-administration protocols. It is used as a model of addiction, specifically to study tolerance to drugs taken and dependence on them. Our findings revealed that escalation of intravenous oxycodone self-administration under long access conditions was similar between female and male rats, indicating that sex does not significantly affect the development of oxycodone addiction using this protocol of self-administration. This is consistent with our previous observations when long-access self-administration was used [28,33]. However, it contrasts with other studies that have found that female rats orally self-administered more oxycodone than males in a short-access (1-hr) FR-1 schedule self-administration paradigm [39], whereas other studies from our lab have shown that males intravenously self-administered more oxycodone compared to females in the first three trials of an FR-1 schedule of reinforcement [25]. These results suggest that specific behavioral measurements and routes of administration may reveal differences in the development and maintenance of oxycodone use in male and female subjects.

Spontaneous withdrawal symptoms from opioids use refer to the symptoms that occur when an individual who has been taking opioids regularly suddenly stops using them or reduces their dose.

These symptoms occur as a result of the body’s physical dependence on the drug. Here, we found that both male and female rats exhibited increased spontaneous withdrawal signs after acute abstinence from oxycodone self-administration. These findings are consistent with the results of previous experiments with non-contingent morphine administration, which showed a similar increase in withdrawal symptoms after 1-day of abstinence in both males and females, with males showing a stronger effect [26]. However, we did not find a significant difference in the magnitude of spontaneous withdrawal signs between sexes following oxycodone self-administration in rats, highlighting the importance of considering the drug’s mechanism of action and administration routes when assessing its potential to affect males and females differently.

In rats, abstinence periods from drug use have been shown to lead to the development of drug-seeking behaviors, which can intensify over time and lead to relapse. It has been shown that the longer abstinence periods result in a greater intensity of drug-seeking [10,40,41]. During this period, there are changes in the brain that can lead to the development of drug cravings, including alterations in synaptic function and changes in the expression of neurotransmitters and their receptors [10,23,42–46]

In addition, a recent study [47] found that male rats that underwent a longer abstinence period (30 days) following oxycodone self-administration showed significantly higher levels of drug-seeking behavior compared to rats that underwent a shorter abstinence period (1-day). This suggests that the incubation of oxycodone craving occurs in a time-dependent manner, with longer abstinence periods leading to increased drug-seeking behavior. Here we found that females but not males have an increased in cue-induced oxycodone-seeking after prolonged abstinence (14-days) compared to acute (1-day) abstinence. Females also exhibited increased drug-seeking during the prolonged abstinence period compared to males. This suggests that cue-induced cravings for oxycodone may develop faster and they are stronger in females compared to males.

Furthermore, we investigated the effects of abstinence on glutamatergic synaptic transmission in PVT-NAcSh projections. Both the PVT and the NAc are highly heterogeneous nuclei with distinctive anatomical subregions (anterior/posterior PVT; core/shell NAc) that contain different types of cells (Type I and Type II in the PVT; dopamine receptor 1 (D1)- and dopamine receptor 2 (D2)-expressing medium spiny neurons (MSNs; D1-MSN or D2-MSN) in the NAc). Recent studies have highlighted the role of the PVT in modulating reward-seeking behaviors, particularly in response to drugs of abuse [48–52]. Several studies have shown that the PVT is involved in retrieving and consolidating drug-associated memories [53,54]. For example, one study found that optogenetic activation of PVT inputs to the NAc was sufficient to reinstate drug-seeking behavior in rats [15]. Another study showed that silencing PVT neurons during the retrieval of opiate-associated memories impaired the expression of drug-seeking behavior [53]. The PVT has also been implicated in the regulation of drug-seeking behavior more broadly. For example, transient inactivation of the posterior PVT can block cocaine-seeking behavior in rats [50], which seems to be dependent on the type of reinstatement model and individual differences [55]. Additionally, the PVT has been shown to play a key role in cue-induced drug-seeking after abstinence [13], suggesting that it serves an important function in the neural circuitry underlying relapse.

Here, we found that acute abstinence from oxycodone self-administration did not appear to affect neurotransmission in the PVT-NAcSh projections, as assessed by the EPSC amplitude input-output curves and paired-pulse ratio. Furthermore, the rectification index for the AMPAR-mediated EPSCs and AMPAR/NMDAR EPSC amplitude ratio at the PVT-NAcSh synapses were not significantly different between treatment groups or sexes, indicating that subunit composition of postsynaptic AMPA receptors or their sensitivity to glutamate were not affected by acute abstinence from oxycodone self-administration. These results suggest that a brief period of abstinence may not be sufficient to induce significant changes in synaptic properties of the PVT-NAcSh circuit.

In contrast, prolonged abstinence from oxycodone self-administration resulted in significant changes in glutamatergic synaptic transmission in the PVT-NAcSh circuits, as measured by EPSC input-output curves and paired-pulse ratio. Specifically, prolonged abstinence resulted in an increase in EPSC amplitude and a decrease in the paired-pulse ratio, suggesting that there is an increase in the probability of neurotransmitter release and/or alterations in presynaptic release mechanisms in the studied pathway. However, prolonged abstinence did not change the rectification index of the EPSCs or the AMPAR/NMDAR EPSC amplitude ratio, indicating that there is no change in the subunit composition of AMPA receptors at the PVT-NAcSh glutamatergic synapses.

These results suggest that long-term abstinence from oxycodone self-administration is associated with significant changes in synaptic properties of the PVT-NAcSh circuit—presynaptically-expressed synaptic potentiation, specifically—potentially contributing to the persistent changes in reward processing and relapse vulnerability that are often observed in individuals with opioid use disorder.

The NAc plays an important role in regulating motivated behaviors such as reward-seeking and cravings [56,57]. Within the NAc, MSN intrinsic excitability regulates NAc responses to afferent inputs and controls the NAc outputs to other components of reward system in the brain. In our study, we investigated changes in NAcSh MSN intrinsic excitability following acute or prolonged abstinence from oxycodone self-administration using nested hierarchical Poisson GLMMs to appropriately account for the hierarchical structure of the electrophysiological data.

One goal of this study was to examine the interplay between PVT-NAcSh glutamatergic transmission and NAcSh MSN intrinsic excitability following oxycodone abstinence. The standing theory suggests that MSN excitability decreases as a homeostatic response to increased glutamatergic input [23,43,58]. Our data do not support a compensatory decrease in excitability in either sex at either abstinence time point. Instead, the trend toward increased excitability in males after prolonged abstinence, if confirmed with adequate sample sizes, would suggest that prolonged oxycodone abstinence may produce maladaptive neuroadaptations that enhance rather than suppress MSN excitability. Such an increase, acting in concert with the strengthening of PVT-NAcSh synaptic transmission observed in the current study, could facilitate signal flow through this circuit and enhance MSN output to downstream targets, potentially contributing to relapse-related behaviors.

Future studies with larger cohorts will be necessary to determine whether this trend in males represents a reproducible and sex-specific neuroadaptation following prolonged oxycodone abstinence.

Our study was designed to target posterior PVT-NAcSh projections, based on previous studies [13,16,21]. The anterior/posterior PVT have been shown to have different subcortical targets [59] and increasing evidence demonstrates substantial functional differences in anterior vs posterior PVT outputs [60]. Posterior projections to the NAc are selectively tuned to aversive stimuli, while the anterior PVT is more involved in reward-seeking behaviors [61]. Furthermore, our goal was to determine the overall effects of acute versus prolonged abstinence on synaptic and neuronal properties in PVT-NAcSh circuits in male and female rats, as opposed to differentiating between the contributions of D1 and D2 receptor-expressing MSNs. However, recent studies demonstrated differences in the roles of PVT-to-D1, PVT-to-D2 MSN, and PVT-to-Interneurons in mediating drug-seeking or withdrawal after either acute or prolonged abstinence from opioid self-administration [13,21]. Thus, it would be interesting to explore, in future studies, the effects of our oxycodone administration regimens on neurotransmission in PVT projections to D1- or D2-MSNs, using ex vivo optogenetic studies analogous to those described in the present work.

Previous work has demonstrated cell-type-specific plasticity at PVT-NAc circuit following heroin self-administration and extinction. It includes strengthening PVT inputs to D2 MSNs and reducing synaptic strength from PVT to parvalbumin-expressing interneurons (PV-INs) [21]. In more detail, stimulation of PVT terminals in the NAcSh has been shown to increase opioid-seeking prior to, but not after, extinction of drug availability[13]. Consistent with this behavioral shift, extinction training can occlude the increase in synaptic strength at PVT to NAc D2 MSNs observed following heroin self-administration in mice [21]. This increase in synaptic strength was reflected by enhanced opto-evoked EPSC amplitude, an increased AMPA/NMDA ratio, and a decreased paired-pulse ratio (PPR), all consistent with potentiated excitatory transmission. PVT projections also synapse onto parvalbumin-expressing interneurons in the NAc (NAc PV-INs; decreased opto-evoked EPSCs and AMPA/NMDA ratios) [21], where an overall decrease in synaptic strength has been reported after both self-administration and extinction [21]. In contrast, PVT-to-NAc D1 MSNs do not show changes in synaptic strength during self-administration or extinction [21]. However, a shift in rectification index following heroin self-administration suggests increased calcium-permeable AMPA receptor (CP-AMPAR) function at these synapses [21]. Importantly, restoring normal synaptic transmission in the PVT-to-NAc PV-IN pathway was sufficient to prevent relapse-like behavior, measured as active lever pressing for heroin [21]. Furthermore, a recent study found that prolonged abstinence period increased CP-AMPAR expression in D1 and D2 MSNs in the NAc of male and female rats [62].

In a morphine conditioned place preference (CPP) model, transient inhibition of the PVT-NAc pathway prevented morphine-primed relapse after both short (4 days) and prolonged (14 days) abstinence without affecting morphine-induced locomotor activity. Importantly, inhibition of this pathway disrupted relapse only when animals were re-exposed to the drug-associated context, indicating that PVT-NAc activity is required for the retrieval and maintenance of opiate-associated memories [53].

Consistent with a circuit architecture in which PVT inputs both directly excite MSNs and recruit local inhibitory microcircuits, our results show that stimulation of glutamatergic PVT terminals in the NAcSh evokes monosynaptic excitation alongside disynaptic inhibition mediated by local GABAergic interneurons. Following prolonged abstinence from oxycodone, excitatory synaptic transmission at PVT-NAcSh projections was strengthened, suggesting enhanced excitatory drive within this pathway. In contrast, inhibitory synaptic transmission was not significantly altered in either males or females, indicating that feedforward inhibitory recruitment and overall inhibitory tone within the PVT-NAcSh circuit remain intact following prolonged abstinence from oxycodone self-administration. Together, these findings suggest that prolonged oxycodone abstinence selectively potentiates excitatory rather than inhibitory synaptic transmission within this pathway, potentially shifting the excitation-inhibition balance in a manner that may contribute to relapse-related behaviors.

In our study, morphological analyses revealed no detectable changes in dendritic length or branching complexity in NAcSh MSNs at either 1 day or 14 days of abstinence following oxycodone self-administration. Additionally, no sex differences were observed in the somatodendritic morphology of NAcSh MSNs between control male and female rats. These findings suggest that the abstinence-dependent adaptations previously reported in this circuit may occur primarily through synaptic or molecular mechanisms, rather than through large-scale structural remodeling of MSN dendritic architecture.

When considered alongside prior work, these results support a model in which the anatomical and cell-type-specific organization of PVT inputs to the NAcSh plays a critical role in relapse-related behaviors. Specifically, PVT projections onto D2 MSNs and PV interneurons may contribute to relapse vulnerability during early or acute abstinence, potentially through mechanisms linked to negative affective states. In contrast, adaptations involving D1 MSNs, including CP-AMPAR recruitment during prolonged abstinence, may underlie the incubation of craving that emerges after extended drug abstinence.

Overall, our findings provide further insight into the circuit mechanisms underlying oxycodone relapse and highlight the importance of considering cell-type specificity, abstinence duration, and behavioral context when interpreting plasticity within the PVT-NAc pathway. Importantly, differences across studies may also reflect variations in drug exposure paradigms, including contingent (self-administration) versus non-contingent drug delivery, as well as whether animals undergo extinction training or remain in forced abstinence. These experimental variables engage distinct learning processes and motivational states that can differentially shape circuit adaptations and relapse-related behaviors.

Future studies should aim to dissect the distinct contributions of anterior versus posterior PVT projections onto D1 and D2 MSNs in the NAcSh and determine how selective manipulation of these pathways influences relapse behaviors across different abstinence states and relapse triggers (e.g., drug-, cue-, or stress-induced). In addition, understanding how thalamic inputs interact with cortical projections to ventral striatal circuits during abstinence and relapse represents an important avenue for future investigation.

Adult male (250-275g; Total N = 42) and female (200-225g; Total N = 45) Sprague Dawley rats (Charles River Laboratory, Wilmington, MA) were used in this study. Upon arrival, rats were group housed (4 rats per cage) and were habituated for 1 week to the animal colony kept on a 12-h light/dark cycle (lights on 7:00AM) with food and water ad libitum. Following surgeries, rats were singly-housed for the rest of the experiment. All animal procedures were conducted in accordance with the guidelines of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee (IACUC) at McLean Hospital/Harvard Medical School (Protocol #2017N0000277, #2018N000090 & #2017N000125).

Coronal slices (300 µm in thickness) containing the NAc were obtained using a vibratome in cold cutting solution containing the following in mM: 252.0 sucrose, 1.0 CaCl₂, 5.0 MgCl₂, 2.5 KCl, 1.25 NaH₂PO₄, 26.0 NaHCO₃ and 10.0 glucose and equilibrated with 95% O₂ and 5% CO₂. Slices were then incubated in artificial cerebrospinal fluid (ACSF) containing the following in mM: 125 NaCl, 2.5 KCl, 2.5 CaCl₂, 1.0 MgSO₄, 1.25 NaH₂PO₄, 26.0 NaHCO₃, and 10.0 glucose at room temperature for at least 1 hr before recordings started. Whole-cell recordings were obtained from the NAcSh neurons with patch electrodes (3-5 MΩ resistance) containing the following in mM: 135.0 Cs-methane-sulfonate, 5.0 NaCl, 1.0 MgCl₂, 10.0 BAPTA, 10.0 HEPES, 2.0 ATP and 0.20 GTP adjusted to pH 7.2 with CsOH. Neurobiotin (0.2%; Vector Laboratories) was also added to the internal solution before the recordings to allow subsequent histochemical localization of the recorded neurons in the NAcSh.

Synaptic responses were induced by photostimulation of ChR2-expressing PVT projecting terminals in the NAcSh with a LED light source (excitation wavelength: 470 nm, 5 ms in duration, Thorlabs). Whole-cell recordings were accepted if the access resistance was ≤20 MΩ and remained stable throughout the recording period (defined as <20% change from baseline). Recordings that exceeded these thresholds were excluded from analysis. All recordings were performed at 30-32°C. After recordings, slices were placed in PBS containing 4% paraformaldehyde and kept in the refrigerator until histological processing.

The pharmacological reagents used in electrophysiological experiments included NBQX disodium salt, D-AP5, NBQX, and (-)-bicuculline methobromide, which were prepared as stock solutions in water at 1000- to 5000-fold concentrations and stored at -20°C.

Male and female rats were randomly assigned to either saline or oxycodone groups. In electrophysiological experiments, ∼2-3 neurons were recorded per animal. The numbers of rats and recorded neurons for the analysis of the different experiments are indicated in the results section. Data are reported as mean +/- SEM. All electrophysiology data were collected using Patch Master (Heka systems). We used Prism 9 (GraphPad) for statistical analysis using Two-tailed t-tests, Mixed effects model (Restricted Maximum Likelihood/REML), and Two- or Three-way ANOVAs with Sidak’s multiple comparisons, as appropriate.

For analyses of intrinsic excitability based on spike count–current relationships, spike output (number of action potentials per current step) was analyzed using nested hierarchical Poisson generalized linear mixed-effects models (GLMMs), fitted separately for each sex and abstinence duration. Each model included injected current (mean-centered), drug condition (Saline vs. Oxycodone), and their interaction as fixed effects. Random effects included random intercepts and slopes for injected current at the animal level, and random intercepts for cells nested within animals, to account for the non-independence of repeated current steps within cells and of multiple cells recorded from the same animal. Unique identifiers were assigned to each animal and cell by concatenating condition labels with animal and cell IDs, ensuring that animals across treatment conditions were treated as independent subjects. Models were fitted using maximum likelihood estimation with Laplace approximation. Overdispersion was assessed using the Pearson chi-squared statistic divided by the residual degrees of freedom; when this ratio exceeded 1.5, a dispersion correction was applied within the Poisson framework. Statistical significance of fixed effects was assessed using marginal F-tests with residual degrees of freedom. Sex comparisons among oxycodone-treated animals were conducted as separate analyses using identical model structures, with sex as the grouping condition. For visualization, marginal model-predicted values were overlaid on each plot as dashed lines, representing the population-level fixed-effects predictions from the fitted GLMM with random effects set to zero, back-transformed from the log scale to spike counts. To assess statistical power, a posthoc power analysis was conducted for each comparison using a two-sample t-test approximation at the rat level, with rat-level mean spike output as the unit of analysis and the pooled between-rat standard deviation as the denominator for effect size estimation (Cohen’s d). This approach was used to estimate the number of animals per group required to achieve 80% and 90% statistical power, given the observed effect sizes. All analyses were performed in MATLAB (R2025b).

IPSC amplitudes were analyzed using linear mixed-effects models (LMEs) to account for the hierarchical structure of the data, with repeated measurements across light intensities nested within animals and multiple cells recorded per animal. Unique identifiers were assigned to each rat and cell by concatenating condition labels with animal and cell IDs, ensuring that animals across treatment conditions were treated as independent subjects. Models included fixed effects of light intensity (mean-centered across the six stimulation levels: 0.5, 1.3, 2.0, 4.7, 7.6, and 10.4 mW), treatment condition (Saline vs. Oxycodone), and their interaction. Random effects included random intercepts and light intensity slopes for individual animals, and random intercepts for cells nested within animals, capturing both between-animal variability in baseline responses and sensitivity to light, as well as within-animal variability across cells. Model parameters were estimated using restricted maximum likelihood (REML). Statistical significance of fixed effects was assessed using marginal F-tests with residual degrees of freedom, applied consistently across male and female datasets. Data are presented as mean ± SEM, computed from rat-level averages, in which responses from multiple cells within the same animal were first averaged to yield one value per animal per light intensity, consistent with the animal-level inference of the LME. Male and female datasets were analyzed separately using identical model structures to assess sex-specific effects. All analyses were performed in MATLAB (R2025b).

The number of animals and cells used per experiment, as well as the results of all statistical analyses, are reported in the text and figure legends.