Sex-dependent effects of an Hnrnph1 mutation on fentanyl addiction-relevant behaviors but not antinociception in mice

Abstract

Opioid Use Disorder (OUD) and opioid-related deaths remain a major public health concern in the United States. Both environmental and genetic factors influence risk for OUD. We previously identified Hnrnph1 as a quantitative trait gene underlying the stimulant, rewarding, and reinforcing properties of methamphetamine. Prior work shows that hnRNP H1, the RNA-binding protein encoded by Hnrnph1, post-transcriptionally regulates Oprm1 (mu opioid receptor gene)-the primary molecular target for the therapeutic and addictive properties of opioids. Because genetic variants can exert pleiotropic effects on behaviors induced by multiple drugs of abuse, in the current study, we tested the hypothesis that Hnrnph1 mutants would show reduced behavioral sensitivity to the mu opioid receptor agonist fentanyl. Hnrnph1 mutants showed reduced sensitivity to fentanyl-induced locomotor activity, along with a female-specific reduction in, and a male-specific induction of, locomotor sensitization following three, daily injections (0.2 mg/kg, i.p.). Hnrnph1 mutants also required a higher dose of fentanyl to exhibit opioid reward as measured via conditioned place preference (CPP). Male Hnrnph1 mutants showed reduced fentanyl reinforcement. Hnrnph1 mutants also showed reduced sucrose motivation, suggesting a reward deficit. No genotypic differences were observed in baseline thermal nociception, fentanyl-induced antinociception, physical or negative affective signs of opioid dependence, or in sensorimotor gating. In the context of our prior work, these findings suggest that Hnrnph1 dysfunction exerts a selective role in reducing the addiction liability to drugs of abuse (opioids and psychostimulants), which could provide new biological pathways to improve their therapeutic profiles.

Keywords: addictive; analgesia; opiate; pain; psychostimulant.

Figure 1.. Sex-dependent modulation of fentanyl-induced locomotor activity and sensitization in H1+/− mice.

(A): In response to saline (i.p.), Hn1+/− females showed greater saline-induced locomotor activity than their WT controls following the 2^nd saline injection (Day 2; *p = 0.028). In response to fentanyl, Hn1+/− females showed reduced fentanyl-induced locomotor activity following the 3^rd injection of 0.2 mg/kg fentanyl (i.p.; Day 5; *p = 0.014). (B): In contrast no genotypic differences in saline- or fentanyl-induced locomotion were observed in males. (C): In WT mice, the locomotor response to repeated saline (Day 1 – Day 2) habituated to a lesser extent in males versus females (+p = 0.0008), while no sex difference was apparent in Hn1+/− mice. Further, saline-induced locomotion showed greater habituation in Hn1+/− males relative to WT males (*p = 0.047). No genotypic difference in habituation was observed for females. (D): When taking into account genotypic differences in baseline locomotor activity (i.e. response to the 2^nd saline injection), Hn1+/− mice showed less robust acute fentanyl-induced locomotor activity (0.2 mg/kg, i.p.) compared to WT mice (*p = 0.039). (E): A marked Genotype x Sex interaction was apparent with respect to the change in fentanyl-induced locomotion across the 3 injections, with Hn1+/− females showing less (*p = 0.04) and Hn1+/− males showing more (*p = 0.02) sensitization compared to their female and male WT counterparts, respectively. Data represent the mean ± S.E.M. Ns are included in the figure legends and the sample sizes employed in Panels D and E are the same as Panel C. *p < 0.05 vs. WT, +p < 0.05 vs. other sex.

Figure 2.. Modulation of fentanyl reward but not antinociception in H1+/− mice.

(A): When assessed in a drug-free state, WT mice conditioned with 0.05 mg/kg fentanyl exhibited a significantly greater conditioned place preference (CPP) than mice conditioned with saline (versus 0 mg/kg; #p = 0.044), while significant CPP was evident in Hn1+/− mice only with the 0.2 mg/kg dose (versus 0 mg/kg; #p = 0.011). (B): When tested under the influence of their conditioning dose, both WT and Hn1+/− mice exhibited a significant CPP at the 0.2 mg/kg fentanyl dose (versus 0 mg/kg; #p = 0.033, < 0.0001, respectively ). (C): No genotypic difference was apparent for the latency to lick the hind paw at baseline or in fentanyl antinociception following treatment with a cumulative fentanyl-dosing regimen. (D): Likewise, no genotypic difference was noted in the precent maximum possible effect (%MPE) of fentanyl antinociception. Data represent the mean ± S.E.M. of the number of mice indicated in the figure legends. #p < 0.05 versus 0 mg/kg fentanyl.

Figure 3.. Blunting of operant-responding for sucrose reinforcement in male Hn1+/− mice.

When trained to lever-press for 10% sucrose, Hn1+/− males exhibited fewer active (A: *p = 0.008) and inactive (B: *p = 0.02) lever-presses, than WT male controls. However, both genotypes directed a similar percentage of their responses toward the sucrose-reinforced lever, indicating that the mutation did not alter fentanyl-directed responding (C). (D): While sucrose intake was lower in Hn1+/− males versus WT males, this genotypic difference was not statistically significant. Data represent the mean ± S.E.M. Ns are indicated in the figure legends and in Table #. *p < 0.05 vs. WT

Figure 4.. Sex-dependent effects of Hn1+/− on fentanyl reinforcement during acquisition of self-administration in sucrose-naïve mice.

(A): When assessed across a range of low fentanyl doses (0.03–3.0 mg/L), no genotypic differences were apparent in experimentally-naïve females for the allocation of responding in the active, fentanyl-reinforced hole. (B): Hn1+/− males directed more nose-pokes toward the fentanyl-appropriate active lever, overall, than did their WT controls (*p = 0.004). (C): For females, there was no significant genotypic difference in low-dose FEN intake. (D): In contrast, Hn1+/− males consumed less FEN than WT males at 3 mg/L (*p = 0.05). Data represent the mean ± S.E.M. of the number of mice indicated in the figure legends. *p < 0.05 vs. WT; * denotes main effect of Genotype.

Figure 5.. Sex-dependent effects of Hn1+/− on behavior in assays of anxiety-like behavior in naïve and fentanyl-withdrawn mice.

(A): In the light-dark shuttle-box test, Hn1+/− males exhibited a greater number of light-side entries than WT males (*p = 0.04). (B): In the novel object encounter, fentanyl (FEN) withdrawal decreased the latency to first make contact with the object compared to saline (SAL) controls, but this effect was observed only in Hn1+/− males (right panel, versus WT males: *p = 0.002). (C): Irrespective of sex, fentanyl withdrawal reduced the number of novel object contacts in WT mice (#p = 0.01), but this effect was not apparent in Hn1+/− mice. (D): Hn1+/− males exhibited a higher number of contacts with the novel object than WT males (*p = 0.005), with no genotypic difference observed in females. (E): Hn1+/− mice trended towards spending more time burying marbles than WT mice and fentanyl withdrawal tended to increase the time spent burying. Neither of these main effects were statistically significant. (F): FEN withdrawal increased marble burying, irrespective of Genotype (#p = 0.01). (G): Hn1+/− males buried more marbles than WT males, irrespective of Treatment (*p = 0.01), with no genotypic difference observed in females. Data represent the mean ± S.E.M. of the number of mice indicated in the figure legends. Note that the sample sizes in Panel E are the same as in Panel C and the sample sizes in Panel G are the same as in Panel D. Likewise, the sample*p < 0.05 vs. WT, #p < 0.05 vs. repeated SAL.

Figure 6.. Buspirone pretreatment reverses the increase in swimming behavior in the forced swim test exhibited by WT and Hn1+/− mice during fentanyl withdrawal.

Irrespective of genotype, fentanyl (FEN)-experienced mice exhibited (A) a longer latency to first float, (B) reduced time spent floating and (C) reduced number of floats, relative to their saline (SAL)-injected controls. In a separate cohort of mice, we replicated the effect of repeated fentanyl treatment upon swimming behavior (SAL-SAL vs. FEN-SAL). Importantly, pretreatment of a separate cohort of fentanyl-treated mice with 5 mg/kg buspirone (FEN-BUS) prior to testing reversed the fentanyl effect upon the latency to first float (D), the time spent floating (E) and the number of floats (F), when compared to their saline-pretreated controls (FEN-SAL). Data represent the mean ± S.E.M. *p < 0.05 vs. SAL or SAL-SAL; +p < 0.05 vs. FEN-SAL.