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: Top, 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. Bottom, the efficacy of feed-forward inhibition in the PVT-NAcSh pathway, as assessed with the input-output curves for light-induced IPSCs, was unaffected by prolonged oxycodone abstinence. There was no significant difference between saline (n = male: 5 rats, 11 cells; females: 7 rats, 14 cells) and oxycodone (n = male: 7 rats, 16 cells; females: 7 rats, 14 cells) groups. Mixed effects model: no effect of treatment: p = 0.9, no effect of sex: p = 0.3. Data is shown as mean ± SEM.

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.

Prolonged abstinence from oxycodone self-administration increases NAcSh MSN intrinsic excitability in both male and female rats.

A: Example traces of current-clamp recordings from NAcSh MSNs from saline- (n = males: 7 rats, 16 cells; females: 4 rats, 7 cells) and oxycodone-treated (n = males: 6 rats, 18 cells; females: 8 rats, 15 cells) rats after 1 day of abstinence. B: Example traces of current-clamp recordings from NAcSh MSNs from saline- (n = males: 3 rats, 8 cells; females: 6 rats, 14 cells) and oxycodone-treated (n = males: 4 rats, 14 cells; females: 6 rats, 15 cells) rats after 14 days of abstinence. C: Number of action potentials fired at increasing current injections (increments of 30pA) in male and female rats after 1 day of abstinence from oxycodone self-administration. No significant difference in excitability between males and females after 1 day of abstinence from oxycodone self-administration. Two-way ANOVA p = 0.39. D: Comparison of number of action potentials fired at increasing current injections between saline-treated vs. oxycodone-treated males after 1 day of abstinence from self-administration. No significant difference in excitability between males that self-administered saline vs. oxycodone after 1 day of abstinence from self-administration. Two-way ANOVA p = 0.67 E: Comparison of number of action potentials fired at increasing current injections between saline-treated vs. oxycodone-treated females after 1 day of abstinence from self-administration. No significant difference in excitability between females that self-administered saline vs. oxycodone after 1 day of abstinence from self-administration. Data shown as mean ± SEM. Two-way ANOVA p = 0.09. F: Number of action potentials fired at increasing current injections in male and female rats after 14 days of abstinence from oxycodone self-administration. No significant difference in excitability between males and females after 14 days of abstinence from oxycodone self-administration. Two-way ANOVA p = 0.14. G: Comparison of number of action potentials fired at increasing current injections between saline-treated vs. oxycodone-treated males after 14 days of abstinence from self-administration. Oxycodone-treated males have greater MSN excitability after 14 days of abstinence from oxycodone self-administration compared to saline males. Two-way ANOVA ****p < 0.0001. H: Comparison of number of action potentials fired at increasing current injections between saline-treated vs. oxycodone-treated females after 14 days of abstinence from self-administration. Oxycodone-treated females have greater MSN excitability after 14 days of abstinence from oxycodone self-administration compared to saline females. Two-way ANOVA *p < 0.05. Data shown as mean ± SEM.