Inhibition of PSD95-nNOS protein-protein interactions decreases morphine reward and relapse vulnerability in rats

Abstract

Glutamate signalling through the N-methyl-d-aspartate receptor (NMDAR) activates the enzyme neuronal nitric oxide synthase (nNOS) to produce the signalling molecule nitric oxide (NO). We hypothesized that disruption of the protein-protein interaction between nNOS and the scaffolding protein postsynaptic density 95 kDa (PSD95) would block NMDAR-dependent NO signalling and represent a viable therapeutic route to decrease opioid reward and relapse-like behaviour without the unwanted side effects of NMDAR antagonists. We used a conditioned place preference (CPP) paradigm to evaluate the impact of two small-molecule PSD95-nNOS inhibitors, IC87201 and ZL006, on the rewarding effects of morphine. Both IC87201 and ZL006 blocked morphine-induced CPP at doses that lacked intrinsic rewarding or aversive properties. Furthermore, in vivo fast-scan cyclic voltammetry (FSCV) was used to ascertain the impact of ZL006 on morphine-induced increases in dopamine (DA) efflux in the nucleus accumbens shell (NAc shell) evoked by electrical stimulation of the medial forebrain bundle (MFB). ZL006 attenuated morphine-induced increases in DA efflux at a dose that did not have intrinsic effects on DA transmission. We also employed multiple intravenous drug self-administration approaches to examine the impact of ZL006 on the reinforcing effects of morphine. Interestingly, ZL006 did not alter acquisition or maintenance of morphine self-administration, but reduced lever pressing in a morphine relapse test after forced abstinence. Our results provide behavioural and neurochemical support for the hypothesis that inhibition of PSD95-nNOS protein-protein interactions decreases morphine reward and relapse-like behaviour, highlighting a previously unreported application for these novel therapeutics in the treatment of opioid addiction.

Keywords: PSD95; nNOS; nitric oxide; protein-protein interaction; relapse; reward.

FIGURE 1

IC87201 and ZL006 block morphine‐induced reward without intrinsically producing conditioned place preference or aversion. (A) The schematic shows the timeline of the CPP study. Rats are assessed for the lack of a pre‐conditioning chamber bias on day 3, and the time spent in each side chamber is recorded as a baseline. The black vertical arrows show the days when rats received an i.p. drug injection prior to placement in the drug‐paired chamber whereas the grey vertical arrows show the days when rats received an i.p. vehicle injection prior to placement in the vehicle‐paired chamber. Post conditioning, on day 12, rats were assessed for the development of CPP in a drug‐free state. (B) Vehicle (VEH)–vehicle (VEH) pairings did not result in preference or aversion for any chamber. (C) Morphine (6 mg/kg i.p.) increased the time spent in the drug‐paired chamber relative to the vehicle‐paired chamber on the test day indicative of CPP, consistent with the development of opioid‐induced reward. (D) IC87201 (10 mg/kg i.p.) treatment in the absence of morphine did not result in preference or aversion for any chamber. (E) ZL006 (10 mg/kg i.p.) treatment in the absence of morphine did not result in the development of preference or aversion for any chamber. (F) Co‐administration of IC87201 (10 mg/kg i.p.) with morphine (6 mg/kg i.p.) blocked morphine‐induced CPP. Data are expressed as mean ± SEM (n = 7–11 per group). (G) Co‐administration of ZL006 (10 mg/kg i.p.) with morphine (8 mg/kg i.p.) blocked morphine‐induced CPP. **p < 0.01 morphine versus vehicle‐paired chamber, two‐way repeated measures ANOVA followed by Bonferroni's post hoc test.

FIGURE 2

ZL006 attenuates morphine‐induced potentiation of electrically evoked DA efflux but does not alter evoked DA efflux when administered alone. (A) A carbon‐fibre microelectrode was lowered into the nucleus accumbens (NAc) shell. A triangular waveform was applied to the carbon‐fibre microelectrode from −0.4 V to +1.3 V and back at a scan rate of 400 V/s. A bipolar stimulating electrode (SE) was lowered over the ipsilateral medial forebrain bundle (MFB) to evoke endogenous DA release by application of a biphasic current (60 Hz, 0.4 s, 300 μA). (B) Morphine (6 mg/kg i.p.) elicited a robust time‐dependent effect on the maximal concentration of electrically evoked DA ([DA]_max) (black squares), while neither ZL006 (10 mg/kg i.p.) alone (filled grey circles) nor vehicle (VEH) (empty black circles) produced changes in stimulated DA signal. Co‐administration of ZL006 (10 mg/kg i.p.) + morphine (6 mg/kg i.p.) prevented the effect of morphine alone (empty squares). (C) Representative scans of electrically evoked resulting current converted to DA concentration. The evoked DA signals elicited by electrical stimulation are shown (black arrows) in pre‐drug condition (top left panel) and after co‐administration of ZL006 (10 mg/kg i.p.) + morphine (6 mg/kg i.p.) (bottom left panel). Insets show their corresponding cyclic voltammograms confirming peak of DA oxidation potential (~+6 V) after the electrical stimulation. Colour plots display cyclic voltammograms from pre‐drug (right top panel) and after co‐administration of ZL006 (10 mg/kg i.p.) + morphine (6 mg/kg i.p.) (right bottom panel). Data are expressed as mean ± = SEM (vehicle [VEH] n = 6, ZL006 n = 5, morphine n = 6 and ZL006 + morphine = 4). ⁺ p < 0.05 versus morphine baseline (−15 min) and 60–90 min post‐injection time points; ***p < 0.001, **p < 0.01, *p < 0.05 ZL006 + morphine group versus morphine alone group, two‐way repeated measures ANOVA followed by Bonferroni post hoc test.

FIGURE 3

ZL006 did not affect the outcome of the acquisition phase of morphine self‐administration. (A) Schematic showing the order of the experimental procedures. Rats were trained to lever press for food before and after the intrajugular catheter implantation. Then animals completed 5 days in FR1 and 5 days in FR3 to accomplish the acquisition phase of morphine self‐administration. (B) The average number of infusions in the FR1 stage of the acquisition of morphine self‐administration was transiently lower in the ZL006 (10 mg/kg i.p.) treated group (grey circles/bars) compared with the vehicle (VEH) group (black circles/bars). These differences were no longer present at the end of the acquisition when the animals were subjected to FR3. (C) The average number of active lever presses in the FR1 was transiently lower in the ZL006 (10 mg/kg i.p.) group (grey squares/bars) in comparison with vehicle (VEH) group (black squares/bars), but these differences were no longer present when animals were subjected to FR3. (D) No differences were found in the number of inactive lever presses at any stage of the acquisition of morphine self‐administration (ZL006, grey triangles/bars; vehicle [VEH], black triangles/bars). Data are expressed as mean ± SEM (vehicle [VEH] n = 5, ZL006 n = 6). *p < 0.05, t test, one‐tailed.

FIGURE 4

ZL006 does not alter the maintenance phase of morphine self‐administration. (A) Schematic showing the order of the experimental procedures. Rats were trained to lever press for food before and after the jugular catheter implantation. After the completion of the acquisition phase of morphine self‐administration (5 days on FR1 and 5 days on FR3), rats were allowed to self‐administer morphine until showing stable behaviour and reaching the maintenance phase of morphine self‐administration (FR3; infusion variance <20% for three consecutive days) (i.e., baseline, empty bars). Animals received 5 days of i.p. injections of vehicle (VEH) or ZL006 (10 mg/kg i.p.) before each of the five consecutive daily drug self‐administration sessions during the maintenance phase of morphine self‐administration (i.e., treatment, line pattern bars). (B) The number of infusions during maintenance remained stable during baseline and treatment with vehicle (VEH) or ZL006 (10 mg/kg i.p.). No changes in the number of active (C) and inactive (D) lever presses were detected in the groups receiving vehicle (VEH) or ZL006 (10 mg/kg i.p.) when comparing baseline versus treatment. Data are expressed as mean ± SEM (n = 5 per group).

FIGURE 5

ZL006 decreases the relapse of morphine seeking behaviour after forced abstinence. (A) Schematic showing the order of the experimental procedures. Rats were trained to lever press for food before and after the jugular catheter implantation. After the completion of the acquisition phase of morphine self‐administration (5 days on FR1 and 5 days on FR3), rats were subjected to 21 days of forced abstinence in their home cage. Rats were exposed to a single relapse session where they received a vehicle (VEH) or ZL006 (10 mg/kg i.p.) injection. (B) Rats treated with ZL006 (10 mg/kg i.p.) before the relapse test showed fewer lever presses on the previously designated active lever than animals that received vehicle (VEH). (C) No differences in lever pressing behaviour on the inactive lever were found between groups receiving vehicle (VEH) or ZL006 (10 mg/kg i.p.) during the relapse test. Data are expressed as mean ± SEM (n = 7 per group). *p < 0.05, t test, one‐tailed.