Facilitating mGluR4 activity reverses the long-term deleterious consequences of chronic morphine exposure in male mice

Abstract

Understanding the neurobiological underpinnings of abstinence from drugs of abuse is critical to allow better recovery and ensure relapse prevention in addicted subjects. By comparing the long-term transcriptional consequences of morphine and cocaine exposure, we identified the metabotropic glutamate receptor subtype 4 (mGluR4) as a promising pharmacological target in morphine abstinence. We evaluated the behavioral and molecular effects of facilitating mGluR4 activity in abstinent mice. Transcriptional regulation of marker genes of medium spiny neurons (MSNs) allowed best discriminating between 4-week morphine and cocaine abstinence in the nucleus accumbens (NAc). Among these markers, Grm4, encoding mGluR4, displayed down-regulated expression in the caudate putamen and NAc of morphine, but not cocaine, abstinent mice. Chronic administration of the mGluR4 positive allosteric modulator (PAM) VU0155041 (2.5 and 5 mg/kg) rescued social behavior, normalized stereotypies and anxiety and blunted locomotor sensitization in morphine abstinent mice. This treatment improved social preference but increased stereotypies in cocaine abstinent mice. Finally, the beneficial behavioral effects of VU0155041 treatment in morphine abstinent mice were correlated with restored expression of key MSN and neural activity marker genes in the NAc. This study reports that chronic administration of the mGluR4 PAM VU0155041 relieves long-term deleterious consequences of morphine exposure. It illustrates the neurobiological differences between opiate and psychostimulant abstinence and points to pharmacological repression of excessive activity of D2-MSNs in the NAc as a promising therapeutic lever in drug addiction.

Fig. 1. Deregulated expression of MSN marker genes in the CPu, NAc and CeA allows to best discriminate between morphine and cocaine abstinence.

A We used qRT-PCR to compare the expression of 30 candidate genes, among which 14 previously identified HTT-related genes (purple characters), 13 additional MSN marker genes and Oxt as a marker transcript of social behavior, in the CPu, NAc, CeA and BNST under morphine and cocaine abstinence conditions compared to their saline counterparts. Hierarchical clustering organized gene expression data in three to six clusters per region. In a majority of these clusters, HTT-related and MSN marker genes displayed down-regulated expression under morphine conditions contrasting with up-regulated or unchanged expression under cocaine conditions. This pattern of gene expression was observed in the CPu (clusters a and c), NAc (cluster c), CeA (cluster c) and, to a lesser extent, in the BNST (cluster a). B Seven MSN marker genes displayed consistent down-regulated expression in the CPu, NAc and CeA under morphine conditions contrasting with no regulation or upregulation under cocaine abstinence, among which Grm4, coding for the mGlu4 receptor. Gene expression data are expressed as fold change versus vehicle abstinent for each condition (mean ± SEM, n = 5 per group). Comparison to vehicle group: p < 0.05 (two-tailed t-test, p value adjusted using Benjamin–Hochberg correction for multiple testing). qRT-PCR data used for clustering are displayed in Table S2.

Fig. 2. Effects of chronic VU0155041 administration on social behaviors in morphine and cocaine abstinent mice.

A In the direct social interaction test, facilitation of mGluR4 activity in morphine abstinent mice (top row, n = 8–14 per group) dose-dependently normalized social interaction parameters. In cocaine abstinent mice (bottom row, n = 8–9 per group), VU0155041 at the dose of 5 mg/kg had no significant effect on social behavior, except for an increase in the number of following episodes. B In the three-chamber test, chronic administration of VU0155041 at 5 mg/kg restored social preference in morphine abstinent mice. In cocaine abstinent mice, the difference between close contact duration with the toy and close contact duration with the mouse was significantly increased by mGluR4 PAM treatment. Results are shown as scatter plots and mean ± sem. Asterisk: abstinence effect (morphine/cocaine versus vehicle), open star: treatment effect (VU0155041 versus vehicle), solid star: abstinence × treatment interaction, dagger: stimulus (toy versus mouse) × abstinence × treatment, comparison to close contact duration with the toy in vehicle abstinent mice (two-way ANOVA or three-way ANOVA with stimulus as repeated measure, followed by Newman–Keuls post-hoc test); p < 0.05.

Fig. 3. Chronic facilitation of mGluR4 signaling relieved motor stereotypies, perseverative behavior, elevated anxiety and lowered nociceptive thresholds in morphine abstinent mice.

A Under morphine versus vehicle conditions (upper row, n = 8–14 per group), morphine abstinent mice displayed spontaneous stereotyped grooming, circling and head shakes that were dose-dependently suppressed by chronic VU0155041 administration. Under cocaine versus vehicle conditions (lower row, n = 8–9 per group), this treatment increased the frequency of circling episodes. B In the Y-maze test, chronic VU0155041 administration in morphine abstinent mice (upper panel) normalized their pattern of exploration by suppressing same arm returns. Under cocaine versus vehicle conditions (lower panel), mGluR4 PAM suppressed cocaine abstinence-induced increase in spontaneous alternation by restoring the number of alternate arm returns to vehicle values. C In the marble burying test, chronic VU0155041 dose-dependently normalized the number of marbles buried by morphine abstinent mice while increasing this number in vehicle abstinent controls; similarly this treatment increased marble burying in vehicle and cocaine abstinent mice. D In the novelty-suppressed feeding test, VU0155041 normalized the latency to eat in the arena and food intake of morphine abstinent mice in the home cage. Latency to feed was reduced in cocaine abstinent mice. E In the tail immersion test, at 48 °C, nociceptive thresholds tended to be decreased in morphine abstinent mice treated with vehicle; VU0155041 was analgesic at this temperature and in morphine abstinent mice only. No effect was detected at 50 and 52 °C. Asterisk: abstinence effect (morphine/cocaine versus vehicle); open star: treatment effect (VU0155041 versus vehicle); solid star: abstinence × treatment interaction (two-way ANOVA followed by Newman–Keuls post-hoc test); p < 0.05. AAR: alternate arm returns, SAR: same arm returns, SPA: spontaneous alternation.

Fig. 4. Chronic VU0155041 treatment inhibits morphine-induced locomotor sensitization.

A Morphine abstinent mice (n = 8–9 per group) displayed greater locomotor response to acute morphine (10 mg/kg) than vehicle abstinent mice and this sensitization was significantly decreased upon chronic VU0155041 (5 mg/kg, left panel) as shown by diminished total distance travelled after morphine injection compared to vehicle-treated mice (right panel). B In cocaine abstinent mice (n = 8–9 per group), acute cocaine (15 mg/kg) induced detectable locomotor sensitization during the first 60 min of the test; chronic VU0155041 (5 mg/kg) delayed the peak of locomotor activity (left panel) without decreasing cocaine-induced locomotion (right panel). Results are shown as scatter plots and/or mean ± sem. Asterisk: abstinence effect (morphine/cocaine versus vehicle); open star: treatment effect (VU0155041 versus vehicle); dagger: time × treatment interaction; solid star: abstinence × treatment interaction (two- or three-way ANOVA followed by Newman–Keuls post-hoc test); p < 0.05.

Fig. 5. Transcriptional regulations induced in the striatum and amygdala of morphine abstinent mice by chronic facilitation of mGluR4 signaling.

We evaluated the effects of chronic VU0155041 treatment (5 mg/kg) on transcriptional changes induced by morphine versus vehicle abstinence in the CPu, NAC, and CeA. We focused on the seven MSN marker genes (blue characters) highlighted in Fig. 1, Oxt and Avp coding for the social neuropeptides oxytocin and vasopressin, Fos as a marker of neuronal activity, and Bdnf and Syp as markers of synaptic plasticity. A Clustering analysis organized gene expression in 4 clusters per region. In the CPu and NAc, chronic VU0155041 restored (CPu: clusters b and c; NAc: clusters a, b,c) or increased (CPu: cluster a) the expression of most genes tested, which clustered with social parameters. In the CeA, rescued expression under VU0155041 treatment was observed in a single cluster (cluster b). B Among MSN marker genes highlighted in Fig. 1B, Drd1a and Hpca displayed significant down-regulation in the NAc, but not Arpp21, Gmr4, or Pde10a. VU0155041 rescued the expression of Drd1a and Hpca and increased the expression of Arpp21 and Grm4 in this region. Similarly, down-regulated expression of Oxt and Avp in the NAc of morphine-vehicle mice was rescued by VU0155041 treatment. Syp expression was increased upon VU0155041 exposure in the three regions tested. C Western blot analysis failed to detect modifications of synaptophysin (SYP) protein levels in the NAc, CPu and VTA but revealed a significant decrease in SYP immunoreactivity in the VP, (D) which represents the main projection area of NAc D2-MSNs. Gene (n = 9–10 per group) and protein (n = 8 per group) expression data are expressed as fold change versus vehicle-vehicle group for each condition (scatter plots and mean ± SEM). Comparison to vehicle-vehicle group: star p < 0.05 (two-tailed t-test, p value adjusted using Benjamin–Hochberg correction for multiple testing). Foll: number of following episodes, GASC: grooming after social contact, TNC: time in nose contact. qRT-PCR data used for clustering are displayed in Table S3.