Acute Negative Allosteric Modulation of M5 Muscarinic Acetylcholine Receptors Inhibits Oxycodone Self-Administration and Cue-Induced Reactivity with No Effect on Antinociception

Abstract

Opioid use disorder (OUD) is a debilitating neuropsychiatric condition characterized by compulsive opioid use, dependence, and repeated relapse after periods of abstinence. Given the high risk of developing OUD following prescription opioid use, the continued need for opioid-induced analgesia, and the limitations of current OUD treatments, it is necessary to develop novel, non-opioid-based treatments for OUD and decrease abuse potential of prescription opioids. Recent evidence suggests that negative allosteric modulation (NAM) of the M₅ muscarinic acetylcholine receptor (M₅ mAChR) may provide an alternative therapeutic approach for the treatment of OUD. Previous studies demonstrated localization of M₅ mAChR expression within the mesocorticolimbic reward circuitry and that the selective M₅ NAM ML375 attenuates both cocaine and alcohol self-administration in rats. In the present study, the effects of ML375 were evaluated in rats self-administering the μ-opioid agonists oxycodone or remifentanil on a progressive ratio (PR) schedule or on cue reactivity (a rodent model of relapse) in the absence of oxycodone following 72 h of abstinence. ML375 reduced the PR break point for oxycodone and remifentanil self-administration and attenuated cue-elicited responding. Importantly, ML375 did not affect sucrose pellet-maintained responding on a PR schedule or opioid-induced antinociception using the hot-plate and tail-flick assays. We also confirm expression of M₅ mAChR mRNA in the ventral tegmental area and show that this is primarily on dopamine (tyrosine hydroxylase mRNA-positive) neurons. Taken together, these findings suggest that selective functional antagonism of the M₅ mAChR may represent a novel, non-opioid-based treatment for OUD.

Keywords: M muscarinic; ML375; antinociception; negative allosteric modulator; opioid self-administration; oxycodone.

Figure 1.. ML375 attenuated remifentanil self-administration.

ML375 dose-dependently attenuates remifentanil self-administration maintained under a fixed (A,B) and progressive (C,D) ratio schedule of reinforcement in rats. ML375 reduced number of reinforcers/session (A) and response rates (responses/min, [B]) under a FR 10 schedule of reinforcement. (C) 30 mg/kg ML375 attenuates the number of reinforcers earned across a range of oxycodone doses. (D) ML375 dose-dependently reduces the number of reinforcers earned on a single dose of remifentanil (3 μg/kg/injection). Values represent the mean ± S.E.M. of 8–9 (A), 6–9 (B), 6 (C), and 6–13 (D) animals/group. * p<0.05, ** p<0.01; *** p<0.001 vs. respective vehicle-treated groups; # p<0.05; ## p<0.01; ### p<0.001 vs. saline self-administration. BP, breakpoint; Sal, saline.

Figure 2.. ML375 attenuated oxycodone self-administration under a progressive ratio schedule of reinforcement.

ML375 dose-dependently reduces the number of reinforcers earned when oxycodone is self-administered (A) at 56 μg/kg/infusion or (B) across a dose range of 10–100 μg/kg/infusion. Values represent the mean ± S.E.M of 7 animals/group (A, B); * p<0.05, ** p<0.01, *** p<0.001 vs. respective vehicle-treated groups, ^ p<0.05, ^^ p<0.01, ^^^ p<0.001 vs. respective groups treated with 30 mg/kg ML375 i.p.; # p<0.05, ### p<0.001 vs. vehicle-treated saline self-administration group; & p<0.05, t-test between 30 mg/kg ML375 treatment and vehicle on saline self-administration. BP, breakpoint.

Figure 3.. ML375 attenuated responding maintained by oxycodone-associated cues following ~72 hours of abstinence.

ML375 attenuates responding on the previously active lever (solid bars) but not inactive lever (open bars) following ~72 hrs of abstinence from stable self-administration of 0.56 μg/kg/infusion oxycodone. Values represent the mean ± S.E.M 6–8 animals/group for the cue-reactivity groups, * p<0.05, *** p<0.001 vs. respective vehicle-treated groups. The solid and open bars show the 3-day mean of active and inactive lever presses (total of 27 rats), respectively, during oxycodone self-administration (SA) preceding forced abstinence. Nal, naloxone.

Figure 4.. ML375 does not alter the antinociceptive effects of oxycodone in the hot plate and tail flick assays.

Oxycodone dose-dependently increased latency for rats to withdraw their hindpaw from hot plate (A) or to remove their tail from a hot water bath (B). Data are presented as % maximum possible effect (MPE) reflecting a change from baseline and represent the mean ± S.E.M. of 6–8 animals/group (hot plate) and 7–9 animals/group (tail flick). Compared to vehicle-treated animals, ML375 (30 and 56.6 mg/kg, i.p.) administration did not alter the % MPE of oxycodone-treated animals.

Figure 5.. Distribution of M₅ message in the ventral midbrain of rats as revealed by fluorescent in situ hybridization histochemistry.

Low power photomicrographs show similar distribution of M₅ (green [A]) and TH (red [B]) mRNA in the midbrain with higher expression of both mRNA species in the substantia nigra pars compacta (SNc) than in the ventral tegmental area (VTA). Panels C - G show high power photomicrographs of triple labeling for M₅ (green), TH (red), the vesicular GABA transporter (vGAT [blue]) and DAPI stained nuclei (white) in the parabrachial pigmented nucleus (PBP) of the VTA, C) and SNc (D). Noticeable fluorescent signals are absent in the negative control sections through the VTA (E) and nucleus accumbens (NAc [H]). In the SNc (D), and to a lesser degree in the PBP (C), cells co-expressing M₅ and TH (white arrow) are more abundant than single labeled TH cells (white arrowhead). Panels F – H show triple-labeled sections through the shell and core (F) region of the NAc, the dorsomedial striatum (G) as well as a negative control section from the NAc (H). Most cells in the NAc and CP express vGAT (magenta arrowhead), whereas M₅ puncta (yellow arrowhead) are only sporadically encountered in the NAS and not at all in the CP. Scale bar in panel B applies also to panel A and scale bar in panel E applies to panels D – H.

Figure 6.. Co-expression of M₅, TH, or vGAT transcripts in the ventral tegmental area.

Proportion of M₅, (A) TH (B), or vGAT (C) cells in the ventral tegmental area (VTA) that are single labeled or double labeled for TH or vGAT (A), M5 or vGAT (B), or M5 or TH (C) as well as cells triple labeled for these mRNA species (A – C). Data are means of 4 animals per group.