Psilocybin reduces heroin seeking behavior and modulates inflammatory gene expression in the nucleus accumbens and prefrontal cortex of male rats

Abstract

Preclinical and human studies indicate psilocybin may reduce perseverant maladaptive behaviors, including nicotine and alcohol seeking. Such studies in the opioid field are lacking, though opioids are involved in more >50% of overdose deaths. Psilocybin is an agonist at the serotonin 2A receptor (5-HT_2AR), a well-documented target for modulation of drug seeking, and evidence suggests 5-HT_2AR agonists may dampen motivation for opioids. We sought to investigate the therapeutic efficacy of psilocybin in mediating cessation of opioid use and maintenance of long-lasting abstinence from opioid seeking behavior in a rat model of heroin self-administration (SA). Psilocybin or 5-HT_2AR antagonists ketanserin and volinanserin were administered systemically to rats prior to SA of 0.075 mg/kg/infusion of heroin, or relapse following forced abstinence. Psilocybin did not alter heroin taking, but a single exposure to 3.0 mg/kg psilocybin 4-24 hours prior to a relapse test blunted cue-induced heroin seeking. Conversely, 5-HT_2AR antagonists exacerbated heroin relapse. To begin to elucidate mechanisms of psilocybin, drug-naïve rats received psilocybin and/or ketanserin, and tissue was collected from the prefrontal cortex (PFC), a region critical for drug seeking and responsive to psilocybin, 24 hours later for RNA-sequencing. 3.0 mg/kg psilocybin regulated ~2-fold more genes in the PFC than 1.0 mg/kg, including genes involved in the cytoskeleton and cytokine signaling. Ketanserin blocked >90% of psilocybin-regulated genes, including the IL-17a cytokine receptor, Il17ra. Psychedelic compounds have reported anti-inflammatory properties, and therefore we performed a gene expression array to measure chemokine/cytokine molecules in the PFC of animals that displayed psilocybin-mediated inhibition of heroin seeking. Psilocybin regulated 4 genes, including Il17a, and a subset of genes correlated with relapse behavior. Selective inhibition of PFC IL-17a was sufficient to reduce heroin relapse. We conclude that psilocybin reduces heroin relapse and highlight IL-17a signaling as a potential downstream pathway of psilocybin that also reduces heroin seeking.

Keywords: cortex; drug-seeking; heroin; inflammation; nucleus accumbens; opioid; psilocybin; psychedelic; relapse; self-administration; serotonin; transcriptome.

Figure 1:. Psilocybin reduces heroin relapse after forced abstinence but not heroin intake.

(A) Overview of experiment. Animals underwent heroin SA for 8 days. On days 9–11, animals were pretreated with 5-HT_2AR agonist psilocybin 4 hr prior to SA. After 17D abstinence, animals underwent 3 relapse tests on consecutive days and were pretreated with psilocybin 4 hr prior to the test. (B-H) Shown are drug-paired active or inactive lever presses (B-D, G) and heroin infusions during SA (E, F, H). (B-F) SA behavior for each treatment group, prior to psilocybin treatment for days 1–8. Animals were split into treatment groups on day 8, with no significant differences observed in lever responses (D) or heroin infusions (F). (G-H) Heroin SA lever responses (G) and infusions (H) on days 9–11, following pretreatment with psilocybin. (I-K) Active and inactive lever presses during relapse tests on subsequent days (R1, R2, R3) for animals that were pretreated with psilocybin prior to the relapse test. (J-K) Shown are lever presses from panel I combined. Error bars indicate mean +/− standard error of the mean (S.E.M). n=5–12/group. Symbols above line indicate ANOVA: * p<0.05; Post hoc test indicated directly above individual histograms, * p<0.05 vs vehicle; # p<0.05 vs 0.1 mg/kg psilocybin; $ p<0.05 vs 1 mg/kg psilocybin.

Figure 2:. 5-HT_2AR antagonist ketanserin increases heroin self-administration and may exacerbate heroin relapse after forced abstinence at a dose of 3 mg/kg.

(A) Overview of experiment. Animals underwent heroin SA for 8 days. On days 9–11, animals were pretreated with 5-HT_2AR antagonist ketanserin IP 15 min prior to SA. After 17D abstinence, animals underwent 3 relapse tests on consecutive days and were pretreated with ketanserin 15 min prior to the test. (B-H) Shown are drug-paired active or inactive lever presses (B-D, G) and heroin infusions during SA (E, F, H). (B-F) SA behavior for each treatment group, prior to ketanserin treatment for days 1–8. Animals were split into treatment groups on day 8, with no significant differences observed in lever responses (D) or heroin infusions (F). (G-H) Heroin SA lever responses (G) and infusions (H) on days 9–11, following pretreatment with ketanserin. (I-K) Active and inactive lever presses during relapse tests on subsequent days (R1, R2, R3) for animals that were pretreated with ketanserin prior to the relapse test. (J, K) Shown are active lever presses from panel I combined. Error bars indicate mean +/− S.E.M. Post hoc test indicated directly above individual histograms, * p<0.05; n=5–12/group.

Figure 3:. The selective 5-HT_2AR antagonist volinanserin increases heroin relapse.

(A) Overview of experiment. Animals underwent heroin SA for 8 days. On days 9–11, animals were pretreated with 5-HT_2AR antagonist volinanserin IP 15 min prior to SA. After 17D abstinence, animals underwent 3 relapse tests on consecutive days and were pretreated with volinanserin 15 min prior to the test. (B-H) Shown are drug-paired active or inactive lever presses (B-D, G) and heroin infusions during SA (E, F, H). (B-F) SA behavior for each treatment group, prior to volinanserin treatment for days 1–8. Animals were split into treatment groups on day 8, with no significant differences observed in lever responses (D) or heroin infusions (F). (G-H) Heroin SA lever responses (G) and infusions (H) on days 9–11, following pretreatment with volinanserin. (I-K) Active and inactive lever presses during relapse tests on subsequent days (R1, R2, R3) for animals that were pretreated with volinanserin prior to the relapse test. (J, K) Shown are active lever presses from panel I combined. Error bars indicate mean +/− S.E.M. * above solid line denotes p<0.05 for RM-ANOVA between .2 mg/kg volinanserin and vehicle; * directly above histogram indicates unpaired t-test between 0.2 mg/kg volinanserin and vehicle with p<0.05. n=6–10/group.

Figure 4:. Psilocybin induces unique gene expression profiles in the PFC at varying doses that are blocked by ketanserin.

(A) Heatmap of PFC gene expression profiles obtained from tissue collected 24 hours after a single injection of psilocybin and/or ketanserin. (B-E) Volcano plots depicting differential gene expression in the PFC between vehicle treated animals and treatment with 3.0 mg/kg psilocybin (B), 3.0 mg/kg ketanserin (C), 3.0 mg/kg psilocybin and ketanserin (D), or 1.0 mg/kg psilocybin (E). (F) Venn diagram of total genes regulated by psilocybin and/or ketanserin in the PFC and the number of genes regulated by more than one treatment group. (G-H) RRHO plots were generated to compare all gene expression data between animals treated with 3.0 mg/kg psilocybin and 1.0 mg/kg psilocybin (G), 3.0 mg/kg ketanserin and 3.0 mg/kg psilocybin (H), 3.0 mg/kg ketanserin + 3.0 mg/kg psilocybin and psilocybin 3mg/kg alone (I), or 3.0 mg/kg ketanserin + 3.0 mg/kg psilocybin and ketanserin 3mg/kg alone (J). Lower left quadrant represents genes up-regulated in both groups while upper right quadrant represents genes down-regulated in both groups. (K) Gene ontology, KEGG pathway analysis and Uniprot cellular compartments of genes regulated in the PFC following a single exposure of 3.0 mg/kg psilocybin in drug-naïve animals. n=4/group.

Figure 5:. Psilocybin pretreatment blunts heroin relapse and regulates gene expression of inflammatory cytokine and chemokines in the PFC during inhibition of heroin seeking.

(A) Overview of experiment. Animals underwent heroin SA for 10 days, 21D forced abstinence and then a 30-minute relapse test. Animals were pretreated with a single dose of 3.0 mg/kg psilocybin or saline vehicle either 4 or 24 hr prior to the test. Animals were euthanized within 1 hour of completion of the relapse test. (A-F) Shown are drug-paired active or inactive lever presses (B, E) and heroin infusions during SA (C, D, F) for each group, prior to any psilocybin treatment. Animals were balanced for lever responses and heroin infusions (D-F) on day 10 of SA. (G) Active and inactive lever presses during the relapse test for rats that received 3 mg/kg psilocybin 4 or 24 hr prior to testing. Asterisk above solid line denotes p<0.05 for Kruskal-Wallis test; * directly above histogram denotes p<0.05 vs vehicle for Post hoc test. Error bars indicate mean +/− S.E.M. n=8–15/group. (H-I) Volcano plot depicting inflammatory cytokine and chemokine genes, obtained by qPCR array, that were differentially expressed in the PFC of rats that were treated with 3.0 mg/kg psilocybin 4 hr (H) or 24 hr (I) prior to a relapse test, vs vehicle treated animals. Horizontal dotted line denotes p value of 0.05. Vertical dotted line denotes fold change of +/− 30%. Grey circles represent genes that were not significantly altered. Red dots represent genes that meet criteria for significance (J-K) Results of correlation analysis of relapse behavior of rats (from G) with inflammatory cytokine or chemokine gene expression data for the PFC that had psilocybin 4 hr (J) or 24 hr (K) prior to a relapse test. Italicized, bolded values were statistically significant.

Figure 6:. Selective inhibition of IL-17a signaling in the PFC is sufficient to reduce heroin seeking following forced abstinence.

(A) Experimental overview: rats underwent jugular vein catheterization and implantation of PFC cannula. Following recovery, rats underwent heroin SA, forced abstinence and a relapse test. 72 and 24 hours prior to the relapse test, rats received infusion of 50 ng/hemisphere of IL-17a antibody or an IgG control directly into the PFC. (B) Active and inactive lever responses during heroin SA, prior to PFC infusion of IL-17a antibody. (C, D) Heroin infusions made during heroin SA for all rats (C) and depiction of infusions for each treatment group after regrouping into balanced treatment groups (D). (E) Active lever presses during a 30 min relapse test for rats that received IL-17a antibody into the PFC and IgG controls. (F) Fos mRNA in the PFC of rats that received IL-17a antibody prior to a relapse test. (G) Pearson correlation of Fos mRNA expression with active lever responses during the relapse test for rats that received IL-17a antibody or IgG into the PFC prior to the relapse test. Error +/− S.E.M. *p<0.05. n=7–8/group.