Sex- and Brain Region-specific Changes in Gene Expression in Male and Female Rats as Consequences of Methamphetamine Self-administration and Abstinence

Abstract

Sex differences in METH use exist among human METH users and in animal models of METH addiction. Herein, we tried to identify potential differences in gene expression between female and male rats after Methamphetamine self-administration (METH SA). Rats were trained to self-administer METH using two 3-hours daily sessions for 20 days. Cue-induced drug seeking was measured on withdrawal days 3 (WD3) and 30 (WD30). Rats were euthanized twenty-four hours after WD30. Prefrontal cortex (PFC) and hippocampus (HIP) were dissected to measure mRNA expression. Both female and male rats increased their METH intake and showed increased METH seeking during withdrawal. Female had higher basal level expression of hypocretin receptor 1 (Hcrtr1) and prodynorphin (Pdyn) mRNAs in the PFC and HIP. Basal corticotropin releasing hormone receptor 1 (Crhr1), Crh receptor 2 (Crhr2), hypocretin receptor 2 (Hcrtr2) and opioid receptor kappa 1 (Oprk1) mRNA levels were higher in the PFC of females. Male rats had higher basal levels of Crh and Crhr1 in HIP. METH SA was associated with increased Crh and Crhr1 in the HIP of both sexes and Crhr2 only in female HIP. Importantly, increased Crh and Crhr1 mRNA levels correlated positively with incubation of METH craving in both sexes, supporting their potential involvement, in part, in the regulation of this behavioral phenomenon. When taken together, our results identified sexual dimorphic baseline differences in rats. We also detected dimorphic responses in animals that had self-administered METH. These observations highlight the importance of understanding the molecular neurobiology of sex differences when therapeutic interventions are planned against METH addiction.

Keywords: Methamphetamine self-administration; addiction; hippocampus; prefrontal cortex; sexual dimorphism.

Fig. 1.. Female and male rats escalate their METH intake during METH SA.

(A) Patterns of weekly METH taking behaviors by female and male rats. (B) Drug intake by low [(female MLT, n = 9) and (male MLT, n = 4)] and high [(female MHT, n = 8) and (male MHT, n = 11)] METH taker rats. (C) METH-seeking behaviors by female and male rats at WD3 and WD30. Key to statistics: *P < 0.05, **P < 0.01, ***P < 0.001, female and male METH groups compared with respective saline groups; #P < 0.05, ##P <0.01, ###P < 0.001, comparison of male METH group with female METH group; $P <0.05, $ $P < 0.01, $ $ $P < 0.001, comparison of weekly METH intake of female and male rats with respect to METH intake on the first week of training; !P < 0.05, !!P < 0.01, !!!P < 0.001, comparison between female vs male METH takers. ^P < 0.05 within sex comparison of lever pressing at WD3 and WD30. All values represent means + SEM of number of animals indicated in the figure. Ct, Control Saline Rats; MLT, METH Low Takers; MHT, METH High Takers.

Fig. 2.. Expression of Prodynorphin (Pdyn) and Proenkephalin (Penk) after METH SA and withdrawal.

Relative Pdyn mRNA levels in female and male METH rats compared to controls in the (A) prefrontal cortex (PFC) and (B) hippocampus (HIP) after 30 days of withdrawal from METH SA. Relative Penk mRNA levels in female and male METH rats compared with controls are shown in (C) PFC and (D) HIP. Key to statistics: Key to statistics: *P < 0.05, **P < 0.01, ***P < 0.001, comparison control vs low and high METH taker groups; #P < 0.05, ##P < 0.01, ###P < 0.001, comparison between low and high METH taker groups; !P < 0.05, !!P < 0.01, !!!P < 0.001, comparison between female and male. Ct, Control Saline Rats, MLT, METH Low Takers; MHT, METH High Takers.

Fig. 3.. Correlation of Prodynorphin (Pdyn) and Proenkephalin (Penk) mRNA with METH seeking behavior.

(A) Pdyn mRNA levels are negatively correlated with active levels presses in both female and male METH SA rats at WD30 in PFC but only (B) in male rats in the HIP. (C) Penk mRNA levels are negatively correlated with active levels presses in male METH SA rats at WD30 in PFC. (D) No correlation was found between Penk mRNA levels and active levels presses in male and female METH SA rats in HIP.

Fig. 4.. Expression of Opioid receptors (Oprk1, Oprm1 and Oprd1) after METH SA and withdrawal.

Oprd1 mRNA levels in the (A) PFC and (B) HIP of female and male rats. Oprk1 mRNA levels in the (C) PFC and (D) of female and male rats. Oprm1 mRNA levels in the (E) PFC and (F) of female and male rats. Keys to statistics are as in Fig. 2.

Fig. 5.. Correlation between active lever presses on WD30 and expression of opioid receptors.

(A) Oprd1 mRNA levels are negatively correlated with active levels presses in female METH SA rats at WD30 in PFC. (B) No correlation was found between Oprd1 mRNA levels and active levels presses in male and female METH SA rats in HIP. Oprk1 mRNA levels are negatively correlated with active levels presses in (C) female METH SA rats at WD30 in the PFC and (D) both female and male METH SA rats in HIP

Fig. 6.. Expression of Crh system in response to METH SA and withdrawal.

Crh mRNA levels in the (A) PFC and (B) HIP of female and male rats. Crhr1 mRNA levels in the (C) PFC and (D) HIP of female and male rats. Crhr2 mRNA levels in the (E) PFC and (F) HIP of female and male rats. Key to statistics is as in Fig. 2.

Fig. 7.. Correlation between Crh and its receptor with active lever presses on WD30.

(A) Changes in PFC Crh mRNA levels are negatively correlated with active levels presses only in male METH SA rats at WD30. (B) Changes in HIP Crh mRNA levels showed positive correlation with active levels presses in female and male METH SA rats at WD30. (C) PFC Crhr1 mRNA levels are negatively correlated with active levels presses in female METH SA rats at WD30. (D) Changes in HIP Crhr1 mRNA levels are positively correlated with active levels presses in female and male METH SA rats at WD30. (E) Crhr2 mRNA levels are negatively correlated with active levels presses in female METH SA rats at WD30 in PFC. (F) Changes in HIP Crhr2 mRNA levels are positively correlated with active levels presses in female METH SA rats at WD30.

Fig. 8.. Expression of Hypocretin receptors after METH SA and withdrawal.

Relative Hcrtr1 mRNA levels in the (A) PFC and (B) HIP of female and male rats. Hcrtr2 mRNA levels the (C) PFC and (D) HIP of female and male rats. Key to statistics is as in Fig. 2.

Fig. 9.. Correlation between active lever presses on WD30 and expression level of hypocretin receptors.

(A) PFC Hcrtr1 mRNA levels are positively correlated with active levels presses in male METH SA rats at WD30. (B) HIP Hcrtr1 mRNA levels are negatively correlated active levels presses in female and male METH SA rats. (C) PFC Hcrtr2 mRNA levels are negatively correlated with active levels presses in female and male METH SA rats at WD30. (D) No correlation was found between Hcrtr2 mRNA levels and active levels presses in male and female METH SA rats in the HIP.