Delayed effects of repeated inescapable severe stress on brain cannabinoid receptor expression and acoustic startle response in adolescent male rats: Relevance to the development of posttraumatic stress disorder and stress-related brain atrophy

Background: Abnormal brain cannabinoid receptor (CBR, CB₁ and CB₂) activities have been implicated in post-traumatic stress disorder (PTSD) and stress-related psychiatric syndromes. We recently reported altered cannabinoid receptor expression in the cerebellum and brain stem of female animals immediately after 3 days repeated severe stress that was absent in the male animals. We hypothesize that this absence of change of CBR in male brain may reflect a delay rather than a lack of stress effect on CBR expression in male animals.Methods: Male adolescent rats were exposed to three-day sessions of 2h repeated restraint/tail shock stress daily before being tested twice for behavioral change in acoustic startle response (ASR): immediately after and seven days after the cessation of the last session of stress. CB₁ and CB₂ mRNA expression levels were quantified in the prefrontal cortex, amygdala, hypothalamus, hippocampus, cerebellum and brain stem of adolescent male rats using real-time PCR. CB₁ and CB₂ protein expression levels in the prefrontal cortex, olfactory, cerebellum and brain stem were determined using Western blot.Key findings: Different from the lack of change in brain CBR expression immediate after the stress in male animals as reported previously, we found significant changes in brain CBR expression7 days after cessation of the stress protocol. Compared to the controls, CB₁ mRNA expression decreased significantly in the prefrontal cortex and brain stem (P<0.01), decreased at a trend level in the hippocampus and hypothalamus (P<0.1), showed no change in the cerebellum, but increased in the amygdala (P<0.05) of stressed male rats. CB₂ mRNA expression decreased significantly in the prefrontal cortex, hypothalamus and brain stem (P<0.05), decreased at a trend level in the amygdala and hippocampus (P<0.1), and increased in the cerebellum (P<0.05) of stressed animals. CB₁ protein expression level and glycosylated CB₁ protein level decreased in the cerebellum but increased in the prefrontal cortex of stressed animals (p<0.05, each). CB₂ protein level, however, decreased significantly in the prefrontal cortex (P<0.01), cerebellum (P<0.05) and brain stem (P<0.05) of the stressed animals. These delayed changes in brain CBR expression levels in male animals paralleled with the transition from a hypo ASR immediately after the stress to a hyper ASR 7 days after the cessation of the stress. Significance: PTSD is a chronic anxiety disorder with a significant period of incubation after trauma exposure and greater prevalence and symptom severity in the females. Different from the previous findings of no change in the cannabinoid system immediately after stress in male animals, significant changes were found in brain cannabinoid receptor expression in male animals seven days after the stress. Importantly, the delayed change in brain CBR appears to parallel with the emergence of enhanced ASR in these animals, suggesting that altered cannabinoid system may contribute significantly to the development of enhanced ASR, a characteristic feature of patients with PTSD. As CB₁ is a potent anxiolytic regulator, the divergent and time-dependent effects of repeated stress on male and female CB₁ expression could contribute to sex dimorphism in PTSD development and symptom severity. In summary, repeated stress caused a delayed effect on brain CBR expression in male animals relative to that in female animals and that could underlie the gender difference in stress-induced anxiety disorders and PTSD.