Sex differences between CRF1 receptor deficient mice following naloxone-precipitated morphine withdrawal in a conditioned place aversion paradigm: implication of HPA axis

Abstract

Background: Extinction period of positive affective memory of drug taking and negative affective memory of drug withdrawal, as well as the different response of men and women might be important for the clinical treatment of drug addiction. We investigate the role of corticotropin releasing factor receptor type one (CRF1R) and the different response of male and female mice in the expression and extinction of the aversive memory.

Methodology/principal finding: We used genetically engineered male and female mice lacking functional CRF1R. The animals were rendered dependent on morphine by intraperitoneally injection of increasing doses of morphine (10-60 mg/kg). Negative state associated with naloxone (1 mg/kg s.c.)-precipitated morphine withdrawal was examined by using conditioned place aversion (CPA) paradigm. No sex differences for CPA expression were found in wild-type (n = 29) or CRF1R knockout (KO) mice (n = 29). However, CRF1R KO mice presented less aversion score than wild-type mice, suggesting that CRF1R KO mice were less responsive than wild-type to continuous associations between drug administration and environmental stimuli. In addition, CPA extinction was delayed in wild-type and CRF1R KO male mice compared with females of both genotypes. The genetic disruption of the CRF1R pathway decreased the period of extinction in males and females suggesting that CRF/CRF1R is implicated in the duration of aversive memory. Our results also showed that the increase in adrenocorticotropic hormone (ACTH) levels observed in wild-type (n = 11) mice after CPA expression, were attenuated in CRF1R KO mice (n = 10). In addition, ACTH returned to the baseline levels in males and females once CPA extinction was finished.

Conclusion/significance: These results suggest that, at least, CPA expression is partially due to an increase in plasma ACTH levels, through activation of CRF1R, which can return when CPA extinction is finished.

Fig 1. Experimental schedule for the conditioned place aversion training.

Mice were treated with a chronic escalating-dose morphine regimen for 4 days in the conditioning period. On day 4, 1 h later last morphine injection, naloxone (1mg/kg, s.c.) was administered. We examined the extinction of naloxone CPA testing for aversion from day 6 to 13.

Fig 2. Changes in body weight.

(A) Effect of saline or morphine injection on body weight in wild-type (WT) or knockout (CRF1R KO) mice. The animals received increasing doses of morphine (10–60 mg/kg, i.p.) or saline every 12 hours during four days. (B) Effect of naloxone (nx, 1mg/kg, s.c.) injection on body weight loss in WT and KO mice treated with morphine or saline. Data are the mean ± SEM. ***p<0.001 versus WT mice treated with saline, or saline+nx; +p<0.05 versus KO mice treated with saline or saline+nx; &&&p<0.001 versus male of WT mice treated with morphine+nx; ###p<0.001 versus female of KO mice treated with morphine+nx.

Fig 3. Conditioned place aversion (CPA) of morphine withdrawal.

A) Expression of CPA induced by naloxone (nx, 1 mg/kg s.c.) in wild-type (WT) or knockout (CRF1R KO) mice treated with morphine or saline. The score was calculated for each mouse as the difference between the postconditioning and the preconditioning time spent in the drug-paired compartment. B) Extinction of CPA training. Aversion scores from day 5 to 13 for male (m) and female (f) from WT and CRF1R KO are shown. Data are expressed as the mean ± SEM. ***p<0.001 versus WT mice treated with saline+naloxone; +p<0.05, ++p<0.01&p<0.05 versus KO mice treated with saline+nx, &p<0.05, &&&p<0.001 versus WT mice; #p<0.05, ##p<0.01 versus male of CRF1R KO mice; $$p<0.01 versus male of WT mice.

Fig 4. Behaviour effects by naloxone (nx) precipitated morphine withdrawal.

We have evaluated the somatic signs: A) jumping, B) rearing; C) rubbing, D) grooming, E) Diarrhoea, and F) Freezing behavior induced after naloxone (1 mg/kg, s.c.)-injection to morphine or saline- treated mice during 18 min. We have also evaluated the time to first immobilization (G). Data are expressed as the mean ± SEM. *p<0.05,***p<0.001 versus WT mice treated with saline+nx; +p<0.05, ++p<0.01, +++p<0.001 versus KO mice treated with saline+nx; &p<0.05, &&p<0.01, &&&p<0.001 versus WT mice treated with morphine+nx; ##p<0.01, ###p<0.001 versus WT mice treated with saline+nx; $p<0.05, $$p<0.01 versus male of WT mice treated with saline+nx; çp<0.05 versus female of KO mice treated with morphine+naloxone.

Fig 5. ACTH and corticosterone plasma levels after the expression and extinction of CPA training.

ACTH (A,B) and corticosterone (C,D) plasma levels in wild-type (WT) and knockout (CRF1R KO) mice after CPA expression and extinction training. Data are expressed as the mean ± SEM. ***p<0.001 versus WT mice treated with saline+naloxone (nx); &&&p<0.001 versus WT mice treated with morphine+nx; +p<0.05, ++p<0.01 versus male of WT and CRF1R KO mice.