Endogenous opioid system modulates conditioned cocaine reward in a sex-dependent manner

Abstract

Cocaine predictive cues and contexts exert powerful control over behaviour and can incite cocaine seeking and taking. This type of conditioned behaviour is encoded within striatal circuits, and these circuits and behaviours are, in part, regulated by opioid peptides and receptors expressed in striatal medium spiny neurons. We previously showed that augmenting levels of the opioid peptide enkephalin in the striatum facilitates acquisition of cocaine conditioned place preference (CPP), while opioid receptor antagonists attenuate expression of cocaine CPP. However, whether striatal enkephalin is necessary for acquisition of cocaine CPP and maintenance during extinction remains unknown. To address this, we generated mice with a targeted deletion of enkephalin from dopamine D2-receptor expressing medium spiny neurons and tested them in a cocaine CPP paradigm. Low striatal enkephalin levels did not attenuate acquisition of CPP. However, expression of preference, assessed after acute administration of the opioid receptor antagonist naloxone, was blocked in females, regardless of genotype. When saline was paired with the cocaine context during extinction sessions, females, regardless of genotype, extinguished preference faster than males, and this was prevented by naloxone when paired with the cocaine context. We conclude that while striatal enkephalin is not necessary for acquisition, expression, or extinction of cocaine CPP, expression and extinction of cocaine preference in females is mediated by an opioid peptide other than striatal enkephalin. The unique sensitivity of females to opioid antagonists suggests sex should be a consideration when using these compounds in the treatment of cocaine use disorder.

Keywords: cocaine reward; enkephalin; striatum.

Figure 1.. Validation of proenkephalin gene knockout from D2 receptor expressing striatal medium spiny neurons.

(A-D) Significant reduction of proenekphalin (Penk: 6 – 14 / genotype), but no change in μ opioid receptor (Oprm: 3 / genotype), δ opioid receptor (Oprd: 3 / genotype), or prodynorphin (Pdyn: 3 / genotype) in the ventral (VS) and dorsal (DS) striatum of D2-PenkKO, compared to Penk^f/f. (E) Representative images of in situ hybridization of Penk mRNA (orange), dopamine D2 receptor mRNA (Drd2, green), and DAPI (cyan) in the VS (top) and cortex (CTX, bottom) in Penk^f/f (left) and D2-PenkKO (right). Arrows indicate colocalization of DAPI and Penk. Triangles indicate colocalization of DAPI and Drd2. (F) Significant reduction in total number of Penk positive cells (left), total number of Penk puncta (middle), and average number of Penk puncta per cell (right) in the VS (top), but not the CTX (bottom) of D2-PenkKO compared to Penk^f/f controls (8 / genotype). (G) Representative immunohistochemistry images of VS and CTX in Penk^f/f and D2-PenkKO showing met-enkephalin (Enk, red), Cre recombinase (Cre, green), and DAPI (blue). Arrows indicate colocalization of DAPI and Enk. Triangles indicate colocalization of DAPI and Cre. (H) Proportions of Enk+ (blue), Cre+ (beige), co-labeled (black), and Enk- Cre- cells (white) in the VS (top), DS (middle), and CTX (bottom) of Penk^f/f (left) and D2-PenkKO (right). (I) D2-PenkKO show fewer percent of Enk positive cells in the VS and DS, but not the CTX, compared to Penk^f/f (6 – 9 / genotype). (J) Mass spectrometry showed D2-PenkKO have less met- (Met) and leu- enkephalin (Leu) in the striatum compared to Penk^f/f (3 samples, 9 mice / genotype). * p < 0.05; ** p < 0.01; **** p < 0.0001 D2-PenkKO vs. Penk^f/f

Figure 2.. Non-selective opioid antagonist naloxone blocks the expression of cocaine preference selectively in female mice.

(A) Experimental timeline of cocaine CPP. Mice received cocaine (15 mg/kg; COC) paired with a distinct floor (conditioned stimulus positive, CS+) and saline (SAL) on a different floor (CS-). (B) Percent time on the cocaine-paired floor during Pretest, Post Test 1, Post Test 2, and Post Test 3 for male (filled) and female (open) control Penk^f/f (blue) and D2-PenkKO (brown) mice (Males = 21–23 / genotype; Females = 20–22 / genotype). All mice showed a significant increase in percent time on the cocaine floor after 4 and 8 conditioning days compared to Pretest (****p < 0.0001). Naloxone pretreatment (10 mg/kg; NAL) blocked expression of cocaine preference in females compared to males at Post Test 3 (** p < 0.01) and compared to females at Post Test 2 (**** p < 0.0001).

Figure 3.. Females show greater extinction of cocaine preference than males and naloxone prevents extinction in D2-PenkKOs.

(A) Timeline of extinction training. After naloxone-primed Post-Test 3, Penk^f/f and D2-PenkKO mice were divided into subsets to receive naloxone (10 mg/kg; NAL) or saline (SAL) 20 min prior to saline injection and were placed on the cocaine-paired (CS+) or saline-paired (CS-) floor. Preference was tested after 4 and 8 days of extinction (EXT Test 1 and 2). (B,D) Male (filled) and female (open) Saline-Extinction mice, regardless of genotype, extinguished preference after 8 extinction trials (*p < 0.05; Males = 7–8 / genotype; Females = 7 / genotype). Females showed a trend to extinguish after 4 trials (p = 0.08). (C,E) Naloxone-Extinction mice, regardless of sex or genotype, did not extinguish cocaine preference (Males = 8–9 / genotype; Females = 8–9 / genotype). (F,G) When post-extinction preference was normalized to the naloxone-primed Post Test, 3 Saline-Extinction D2-PenkKOs showed greater extinciton than Penk^f/f (*p < 0.05), and this was prevented in the Naloxone-Extinction group (*p < 0.05). (H) Females show greater extinction than males at Extinction Test 2 (***p < 0.001), and naloxone prevented extinction selectively in D2-PenkKO mice (**p < 0.01).

Figure 4.. Greater extinction in females following saline-challenge at Post-conditioning Test 3.

(A) Timeline of extinction training. After saline-primed Post-Test 3, Penk^f/f and D2-PenkKO mice were divided into subsets to receive naloxone (10 mg/kg; NAL) or saline (SAL) 20 min prior to saline injection and were placed on the cocaine-paired (CS+) or saline-paired (CS-) floor. Preference was tested after 4 and 8 days of extinction (EXT Test 1 and 2). (B,D) Female (open), but not male (filled) Saline-Extinction mice, regardless of genotype, extinguished preference after 8 extinction trials (*p < 0.05; Males = 4–5 / genotype; Females = 4–6 / genotype). Females showed a trend to extinguish after 4 trials (p = 0.06). (C,E) Naloxone-primed treatment during extinction prevented extinction in all mice, regardless of sex or genotype (Males = 5–6 / genotype; Females = 5–6 / genotype). (F) Comparison between the sexes in the Naloxone-Extinction group, though, revealed that naloxone prevented extinction selectively in female mice (*p < 0.05). Additionally, Saline-Extinction females show greater extinction than Saline-Extinction males at Extinction Test 2 (*p < 0.05).