Oxycodone self-administration and oxycodone-seeking after short and prolonged abstinence.

A: Experimental Timeline. PVT intracranial injections of AAV5-CAMKIIɑ-ChR2-EYFP, followed by intravenous catheter surgery and 22 days of oxycodone self-administration. Rats then received short (24 hours) or long (14 days) abstinence from oxycodone self-administration and tested for cue-induced oxycodone relapse test. Rats were euthanized, and brain slices were prepared for ex-vivo electrophysiological recordings. B: Number of infusions across short and long-access oxycodone self-administration in male and female rats. Mixed-effects model: main effect of days (increased infusions over time): p> 0.0001, main of treatment: p < 0.0001, no effect of sex: p = 0.84. C: Active lever presses across short- and long-access oxycodone self-administration. Mixed-effects model: main effect of days (increased infusions over time): p> 0.0001, main of treatment: p < 0.0001, no effect of sex: p = 0.53. D: Inactive lever presses across short- and long-access oxycodone self-administration. No significant differences between males and females or treatments were observed. E: Somatic withdrawal signs increased in both males and females. Two-way ANOVA; main effect of treatment: ***p = 0.002. F: Oxycodone-seeking (relapse test) after acute abstinence. Similar oxycodone-seeking behaviors between males and females but increased oxycodone-seeking compared to saline after acute abstinence in males and females. Two-way ANOVA; Sidak’s multiple comparisons females saline vs. oxycodone *p = 0.01; males saline vs. oxycodone *p = 0.02. G: Oxycodone-seeking after prolonged abstinence. Both males and females exhibited increased drug-seeking compared to saline. Two-way ANOVA: main effect of drug: F(3,47) = 36.3 *** = p < 0.0001. Females exhibited significantly higher oxycodone-seeking behaviors compared to males: Sidak’s multiple comparisons ### p = 0.006. Data is shown as mean ± SEM.

Synaptic Properties in PVT-to-NAcSh Projections.

A: Representative images showing expression of ChR2-EYFP at the injection site in PVT and ChR2-EYFP-expressing projecting fibers in NAcSh. B: Schematic of optical stimulation of PVT terminals in NAcSh and recording of optically-evoked EPSCs in MSNs. C: Schematic representation of the local circuit within NAcSh with PVT afferents forming monosynaptic glutamatergic contacts on both MSNs and GABAergic interneurons, resulting in of feedforward inhibitory responses in MSNs. D: Photostimulation-induced (10.5 mW/mm2; 5 ms) EPSC (orange) and IPSC (cyan) recorded from NAcSh MSN at holding potentials of -70 mV or 0 mV, respectively. Recordings were performed under control conditions first (left; baseline) and 10 min after NBQX (10 μM) and D-APV (50 μM; right) were added to the bath solution. The inset shows a delayed onset (synaptic latency) of the IPSC recorded at 0 mV. E-F: Summary plot of the amplitude of EPSC (panel E) and IPSC (panel F) recorded in MSNs under control conditions (baseline) and after bath application of NBQX and D-APV. The symbols represent individual experiments. G: Rescue of light-induced and TTX-blocked EPSCs at PVT-NAcSh projections by 4-AP. Left, an example of recordings shows EPSC (average of 10 traces) recorded at -70 mV under control conditions (baseline; 1), the EPSC was blocked by TTX (1 μM, 2), and application of 4-AP (1 mM) in the continuing presence of TTX restored the EPSC (3), thus confirming the monosynaptic nature of the PVT-NAcSh projections. Right, the time course of the EPSC amplitude changes. H: Summary plot of the experiments showing the EPSC amplitudes in NAcSh MSNs under three conditions (baseline, TTX, and TTX + 4-AP. I: Feed-forward IPSCs in NAcSh MSNs, recorded at 0 mV, were blocked by the GABAA receptor antagonist bicuculline (20 μM). J: IPSC amplitudes recorded from NAcSh under control conditions (baseline) and after application of bicuculline.

Prolonged abstinence from oxycodone self-administration is associated with increased synaptic strength in glutamatergic PVT projections to MSNs in NAcSh in male and female rats.

A: Right, 1-day of abstinence from oxycodone self-administration (acute abstinence) had no effect on the efficacy of glutamatergic synaptic transmission in PVT projections to MSNs in NAcSh, as assessed with synaptic input-output curves for light-induced EPSCs which were triggered by the pulses of blue light of increasing intensity. There was no significant difference between saline (n = male: 5 rats, 18 cells; females: 7 rats, 26 cells) and oxycodone (n = male: 8 rats, 36 cells; females: 6 rats, 20 cells) groups. Three-way ANOVA no effect of treatment: p = 0.2, no effect of sex: p = 0.5. Right, example traces of EPSCs triggered by light pulses of increasing intensity from different experimental groups. B: Left, the magnitude of the paired-pulse ratio remained unchanged in the oxycodone (n = males: 10 rats, 25 cells; females: 6 rats, 22 cells) compared to saline control (n = males: 5 rats, 28 cells; females: 7 rats, 30 cells) groups, indicating that the probability of glutamate release was not affected after acute abstinence from oxycodone self-administration. Three-way ANOVA no effect of drug, p = 0.3, no effect of sex, p = 0.1. Right, representative traces of EPSCs evoked by paired-pulses of blue light (10.5 mW/mm²) at different interpulse intervals (50, 70, 100 and 200 ms). C: Left, the efficacy of glutamatergic synaptic transmission in PVT projections to MSNs in NAcSh, assessed as in A with synaptic input-output curves, was enhanced in both male and female rats in the oxycodone group (n = males: 7 rats, 20 cells; females: 5 rats, 21 cells) compared to saline control group (n = males: 5 rats, 16 cells; females: 7 rats, 21 cells) after 14-days of abstinence from oxycodone self-administration (prolonged abstinence). Three-way ANOVA main effect of drug ****p < 0.0001. Right, example traces of EPSCs from different experimental groups. D: Left, the magnitude of the paired-pulse ratio decreases in the oxycodone groups (n = males: 7 rats, 33 cells; females: 6 rats, 22 cells) compared to saline control groups (n = males: 5 rats, 23 cells; females: 7 rats, 33 cells). Three-way ANOVA, main effect of drug **** p < 0.0001. E: Example traces of IPSCs from different experimental groups triggered by photostimuli of increasing intensity. The IPSCs were recorded at a holding potential of 0 mV. F: Left (males), linear mixed-effects modeling revealed a significant main effect of light intensity on IPSC amplitude (F(1, 158) = 43.49, p = 6.1 × 10⁻¹⁰), indicating robust recruitment of inhibitory synaptic responses with increasing stimulation. No significant main effect of condition was observed in males (F(1, 158) = 0.00023, p = 0.988), and no Light × Condition interaction was detected (F(1, 158) = 0.543, p = 0.462), indicating that prolonged abstinence from oxycodone does not alter baseline inhibitory strength or recruitment dynamics in male NAcSh MSNs. Right (females), linear mixed-effects modeling revealed a significant main effect of light intensity on IPSC amplitude (F(1, 164) = 21.26, p = 8.0 × 10⁻⁶), indicating robust recruitment of inhibitory synaptic responses with increasing stimulation. No significant main effect of condition was observed in females (F(1, 164) = 0.025, p = 0.875), and no Light × Condition interaction was detected (F(1, 164) = 1.298, p = 0.256), indicating that prolonged abstinence from oxycodone does not alter baseline inhibitory strength or recruitment dynamics in female NAcSh MSNs. Together, these data indicate that the efficacy of feed-forward inhibition in the PVT–NAcSh pathway, as assessed by input-output curves for light-induced IPSCs, is unaffected by prolonged oxycodone abstinence in either sex. Saline (n = males: 5 rats, 11 cells; females: 7 rats, 14 cells) and oxycodone (n = males: 7 rats, 16 cells; females: 5 rats, 14 cells).

Abstinence from oxycodone self-administration does not change AMPAR subunit composition or AMPAR/NMDAR EPSC amplitude ratio in glutamatergic PVT projections to MSNs in NAcSh.

A-B: Rectification index and current-voltage relationship for AMPAR EPSCs for short (A; 1-day) and long (B; 14-days) abstinence periods. A: Left, representative traces of AMPAR EPSCs recorded at holding potentials of -70, 0 and +40 mV during acute abstinence. Right, current/voltage relationship of AMPAR EPSCs recorded at holding potentials of -70, 0 and +40 mV of saline (n = males: 9 rats, 23 cells; females: 7 rats, 14 cells) vs. oxycodone (n = males: 13 rats, 28 cells; females: 4 rats, 10 cells) rats. Inset: Rectification index for EPSCs (calculated as the ratio of peak EPSC amplitudes at +40/-70 mV [EPSC+40/EPSC-70]). B: Left, representative traces of AMPAR EPSCs recorded at holding potentials of -70, 0 and +40 mV during prolonged abstinence. Right, current/voltage relationship of AMPAR EPSCs recorded at holding potentials of -70, 0 and +40mV of saline (n = males: 4 rats, 14 cells; females: 5 rats, 15 cells) vs. oxycodone (n = males: 5 rats, 13 cells; females: 4 rats, 14 cells) rats. The recordings were performed in the presence of the NMDA receptor antagonist D-APV (50 μM) in the external medium and spermine (200 μM) in the pipette solution. There were no significant differences between groups. Data is shown as mean ± SEM. Comparisons were made using two-way ANOVAs p > 0.05. C-D: AMPA/NMDA ratios of AMPAR EPSCs for short (C) and long (D) abstinence periods. C: Left, representative traces of light-induced EPSCs in projections from PVT to MSNs in NAcSh at +40mV (light-colored traces) and -70mV (dark-colored traces) in slices from male and female rats during acute abstinence. Right, AMPAR/NMDAR EPSC amplitude ratios for saline (n = males: 5 rats, 14 cells; females: 7 rats, 24 cells) and oxycodone (males: 8 rats, 24 cells; females: 6 rats, 18 cells) groups during acute abstinence. D: Left, representative traces of light-induced EPSCs in projections from PVT to MSNs in NAcSh at +40mV (light-colored traces) and -70mV (dark-colored traces) in slices from male and female rats during prolonged abstinence. Right, AMPAR/NMDAR EPSC amplitude ratios for saline (n = males: 5 rats, 12 cells; females: 5 rats, 15 cells) and oxycodone (males: 5 rats, 12 cells; females: 5 rats, 15 cells) groups. Data is shown as mean ± SEM. Comparisons were made using two-way ANOVA.

Intrinsic excitability of NAcSh medium spiny neurons is largely unaltered following oxycodone abstinence, with a trend toward increased spike output in males after prolonged abstinence.

A–B: Example current-clamp traces from NAcSh MSNs from saline- and oxycodone-treated rats after 1 day (A) and 14 days (B) of abstinence. Sample sizes: 1-day saline (males: 7 rats, 16 cells; females: 4 rats, 7 cells), 1-day oxycodone (males: 6 rats, 18 cells; females: 8 rats, 15 cells), 14-day saline (males: 3 rats, 8 cells; females: 6 rats, 14 cells), 14-day oxycodone (males: 4 rats, 14 cells; females: 6 rats, 15 cells). C, F: Spike count–current relationships in oxycodone-treated animals revealed no significant sex differences in baseline spike output or spike output gain at 1 day (C: condition F(1, 351) = 0.063, p = 0.802; interaction F(1, 351) = 0.105, p = 0.746) or 14 days of abstinence (F: condition F(1, 300) = 0.176, p = 0.675; interaction F(1, 300) = 0.036, p = 0.849). D–E: No significant effect of oxycodone on spike output was detected in males or females after 1 day of abstinence (males: condition F(1, 370) = 0.454, p = 0.501; females: condition F(1, 230) = 0.571, p = 0.451). G: After 14 days of abstinence, oxycodone-treated males showed numerically higher spike output than saline controls (7.46 vs. 4.43 spikes/step; Cohen’s d = 1.18), but this did not reach statistical significance (condition: F(1, 225) = 2.419, p = 0.121; interaction: F(1, 225) = 0.008, p = 0.930); post-hoc power analysis indicated only ∼20% power with the current sample, and 13 rats per group would be required for 80% power. H: No significant effect of oxycodone on spike output was detected in females after 14 days of abstinence (condition: F(1, 306) = 0.025, p = 0.875; interaction: F(1, 306) = 0.366, p = 0.546). Spike count data were analyzed using nested hierarchical Poisson GLMMs fitted separately for each sex and abstinence duration. Data are shown as mean ± SEM.

Cell types in the NAcSh and variation in morphology of MSNs.

A: Stacked confocal images show examples of a medium spiny neuron (left), a sparsely spine neuron (middle) and an aspiny neuron (right), respectively. Inset images show enlarged view of a segment of dendritic branch and its spines from the same neuron. Arrowheads in three panels indicate the initial segment of axons. Arrows in the left panel indicate the axonal collaterals in the vicinity of its soma. B-C: Summary plot shows the percentages of three types of neurons observed in NAcSh in female and male rats. D: There is no difference in cumulative frequency distribution of soma size of examined MSNs between female (red circles) and male (blue circles) rats (p = 0.988, Kolmogorov-Smirnov test). E-F: Correlation analysis of soma sizes versus their dendritic length (left), dendritic volume (middle), or branch points (right) in female (E) and male rats (F). The values of the correlation coefficient (R2) and significance (p) are shown in each panel. The solid line in each panel represents the linear trendline (line of best fit) of the data, and dash lines represent 95% confidence interval.

Dendritic morphology of NAcSh-MSNs remained unchanged at either 1-day or 14-days abstinence from oxycodone self-administration in both female and male rats.

A & E: Representative reconstructed NAcSh-MSNs from saline or oxycodone group in female (left, 8-14 cells from 5-7 rats among four treatment groups) and male (right, 6-16 cells from 3-8 rats among four treatment groups) rats at 1-day (A) or 14-days (E) abstinence, respectively. B & F: There is no difference in soma sizes (left, p = 0.077 or 0.352, respectively, two-way ANOVA) and their cumulative frequency distributions (right, p = 0.270 or 0.598, respectively, Kruskal-Wallis test) among treatment groups at 1 day (B) or 14 days (F) abstinence after oxycodone self-administration. C & G: No difference was observed in the total dendritic length (left, p = 0.504 or 0.972, respectively, two-way ANOVA) and Sholl analysis on dendritic length (right, p = 0.503 or 0.969, respectively, three-way ANOVA) between saline and oxycodone groups at 1-day or 14-days abstinence. D & H: No difference was observed in the total number of branch points (left, p = 0.499 or 0.738, respectively) and Sholl analysis on branch points (right, p = 0.221 or 0.738, respectively) between saline and oxycodone group at 1-day or 14-days abstinence. Red and blue symbols represent data sets from female or male rats, respectively. Open or filled symbols represent the data set of saline or oxycodone, respectively.