Mu opioid receptors in the medial habenula contribute to naloxone aversion

Abstract

The medial habenula (MHb) is considered a brain center regulating aversive states. The mu opioid receptor (MOR) has been traditionally studied at the level of nociceptive and mesolimbic circuits, for key roles in pain relief and reward processing. MOR is also densely expressed in MHb, however, MOR function at this brain site is virtually unknown. Here we tested the hypothesis that MOR in the MHb (MHb-MOR) also regulates aversion processing. We used chnrb4-Cre driver mice to delete the Oprm1 gene in chnrb4-neurons, predominantly expressed in the MHb. Conditional mutant (B4MOR) mice showed habenula-specific reduction of MOR expression, restricted to chnrb4-neurons (50% MHb-MORs). We tested B4MOR mice in behavioral assays to evaluate effects of MOR activation by morphine, and MOR blockade by naloxone. Locomotor, analgesic, rewarding, and motivational effects of morphine were preserved in conditional mutants. In contrast, conditioned place aversion (CPA) elicited by naloxone was reduced in both naïve (high dose) and morphine-dependent (low dose) B4MOR mice. Further, physical signs of withdrawal precipitated by either MOR (naloxone) or nicotinic receptor (mecamylamine) blockade were attenuated. These data suggest that MORs expressed in MHb B4-neurons contribute to aversive effects of naloxone, including negative effect and aversive effects of opioid withdrawal. MORs are inhibitory receptors, therefore we propose that endogenous MOR signaling normally inhibits chnrb4-neurons of the MHb and moderates their known aversive activity, which is unmasked upon receptor blockade. Thus, in addition to facilitating reward at several brain sites, tonic MOR activity may also limit aversion within the MHb circuitry.

Fig. 1

Deletion of Mu Opioid Receptors (MORs) in B4 neurons of the medial habenula (MHb). a MOR is expressed in B4-positive neurons (or B4 neurons). Left. Strategy. The AAV2-FlexGFP was injected in the MHb of MORmcherry^KI/KIB4-Cre⁺ mice in order to visualize both the MOR protein and B4 neurons. Right. MOR and eGFP signals partially overlap. Representative images show coronal and sagittal sections with B4 neurons labeled in green and MOR labeled in red, at low and high magnification (see scales bars). FR: fasciculus retroflexus. b Anatomical specificity of the MOR deletion in B4MOR^−/− mice. mRNA levels were quantified using RT-qPCR in microdissected habenula (Hb), striatrum (Str), ventral tegmental area/substantia nigra (VTA/SN), interpeduncular nucleus (IPN), and medulla (MA) samples from B4MOR^−/− mutant mice (blue) and B4MOR^+/+ controls (black). Data are expressed as mean of FOLD ± SEM (n = 10, in triplicates, t-test, *p < 0.05). Analysis of the Oprm1 mRNA shows significant reduction of MOR expression in the Hb sample only. c Cellular characterization of the conditional Oprm1 gene deletion using in situ hybridization (ISH, ACDbio^®). Left. Representative images show both Oprm1 (red) and Cnrb4 (green) transcripts in the MHb of mutant and control mice. The Oprm1 signal is strongly reduced in the MHb of B4MOR^−/− mice, mainly in the ventromedial part. Right. Quantification of single Oprm1-positive and double Oprm1/B4-positive cells in the MHb of mutant (blue) and control (black) mice. VS_DESKTOP measurement tools were applied to RNAscope results. Data are expressed as mean ± SEM number of neurons per area (manually defined in VS-DESKTOP) and show a reduction (49.85 ± 11.1%) and almost complete ablation (99.39 ± 0,21%) of single and double Oprm1-positive in mutant mice, respectively (n = 3 per genotype with 5 slides per animal and 5 slices per slide, ***p < 0.001). d Locomotion in the open field. Data are expressed as mean ± SEM total distance in cm, and show that basal locomotor activity (first 30 min), or locomotor activity after saline (next 30 min), and morphine (40 mg/kg) injection (last 120 min) do not differ between the two genotypes (n = 14–17, two-way RM-ANOVA, ns). e Tail immersion to assess morphine analgesia. Pain sensitivity was measured at 52 °C after morphine (10 mg/kg) injection in mutant and control mice, and the experiment was repeated for 5 days. The effect of saline injection on the tail immersion was also measured. Data are expressed as mean ± SEM withdrawal latency in seconds. Statistical analysis showed no difference between the two genotypes (n = 9; three-way RM-ANOVA, ns)

Fig. 2

Preserved reward-related responses in B4MOR^−/− mice. a Mice were tested in a morphine-conditioned place preference (CPP) paradigm. Data are expressed as mean ± SEM % CPP score defined as % of time spent in drug-paired compartment during post-conditioning − % of time spent in drug-paired compartment during preconditioning. Morphine-conditioned B4MOR^−/− and B4MOR^+/+ mice showed significant morphine CPP, which did not differ across genotypes. Saline-saline groups from both genotypes showed no place conditioning (n = 10–11; performed on compartment per treatment (morphine or saline) per genotype, **p < 0.01). b Mice were tested in an autoshaping test in the Touchscreen system, and the protocol is shown. Trial is initiated when the mouse inserts its head in the food tray. A stimulus is presented on one of the two sides of the screen. One side is always followed by reward presentation (positive Conditioned Stimulus or CSp) whereas the other is not (negative Conditioned Stimulus or CSn). If CSp is displayed and the mouse collects the reward, an inter-trial interval (ITI) starts, at the end of which another trial starts, until mice reach the criterion (40 trials per 1 h session). c, Left panel. Number of trials for the training phase (mice learn the association between the food tray and the reward apparition) was similar for the two genotypes (n = 8/group, t-test, nonsignificant—ns). c, Right panel. Number of trials for the testing phase (mice associate the apparition of a CSp with the apparition of a reward) was also similar for the two genotypes (n = 8/group, t-test, ns). d Number of CSp and CSn approaches during the testing phase. CSp, but not CSn, approaches increased across sessions for both genotypes, with no significant difference across genotypes. Data are expressed as mean ± SEM number of trials per session (n = 8, two-way RM-ANOVA performed on genotype per session, ns). All data are expressed as mean ± SEM

Fig. 3

Reduced aversion to naloxone in naïve and morphine-dependent B4MOR^−/− mice. Mice were tested in a naloxone-conditioned place aversion paradigm. a Naïve mice. Avoidance is expressed as a CPA score defined as % of time spent in drug-paired compartment during post conditioning − % of time spent in drug-paired compartment during preconditioning. Both B4MOR^+/+ controls and B4MOR^−/− mutant mice showed naloxone (10 mg/kg) CPA (n = 12–14, two-way ANOVA performed on genotype per treatment (saline or naloxone), however naloxone CPA was lower in mutant mice as observed by lower significance level and a strong genotype effect. Stars, naloxone effect: ***p < 0.001 B4MOR^+/+ saline versus B4MOR^+/+ Naloxone; *p < 0.05 B4MOR^−/− saline versus B4MOR^−/− Naloxone. Squares, genotype effect, ^###p < 0.001 B4MOR^+/+ Naloxone versus B4MOR^−/− Naloxone). b Morphine-dependent mice. Again, both B4MOR^+/+ controls and B4MOR^−/− mutant mice showed naloxone (0.25 mg/kg) CPA. However, as for naïve mice, the CPA score of morphine-dependent B4MOR^−/− mice conditioned with naloxone (M-N) was significantly lower compared to B4MOR^+/+ mice, and there was a significant genotype effect. Morphine Saline (M-S) groups did not develop any place preference or avoidance compared to Saline–Saline (S–S) group (n = 11, 12, three-way ANOVA performed on genotype per treatment (saline or naloxone) per drug (saline or morphine)). All data are expressed as mean ± SEM. Stars, naloxone effect: ***p < 0.001 B4MOR^+/+ M-N versus B4MOR^+/+ S–S, *p < 0.05 B4MOR^−/− M-N versus B4MOR^−/− S–S; Squares, genotype effect, ^#p < 0.05 B4MOR^+/+ M-N versus B4MOR^−/− M-N

Fig. 4

Reduced somatic withdrawal from chronic morphine in B4MOR^−/− mice. a Naloxone-precipitated withdrawal. Withdrawal was precipitated in morphine-dependent mice by 1 mg/kg of Naloxone. Upper left inset. The global withdrawal score was significantly lower in B4MOR^−/− compared to B4MOR^+/+ control mice across blocks of 5 min and over the entire session (n = 16). Other panels: Scores for each withdrawal symptom monitored during the 25 min session. First row. Paw tremor, body tremor, wet dog shakes, and sniffing were significantly reduced in mutant mice compared to controls. Second row. Activity, ptosis, jumps, teeth chattering, and piloerection did not differ across genotypes. b Mecamylamine-precipitated withdrawal. Upper left inset. The global withdrawal score was significantly lower in B4MOR^−/− compared to B4MOR^+/+ control mice across blocks of 5 min and over the entire session (n = 7, 8). Other panels. Body tremor and ptosis were significantly reduced in mutant mice compared to controls. Paw tremor, wet dog shakes, sniffing, activity, jumps, teeth chattering, and piloerection did not differ across genotypes. All data are expressed as mean ±  SEM; a, b Upper right inset. t-test, **p < 0.01 ***p < 0.001. a, b Upper left inset and Other panels. Two-way RM-ANOVA performed on genotype per block of 5 min, ***p < 0.001 and *p < 0.05; **p < 0.01; ***p < 0.001