Adolescent Δ9-tetrahydrocannabinol exposure differently affects histone modifications in the brain of female and male rats

Abstract

Despite the increasing evidence of a possible interaction between adolescent Cannabis abuse and the subsequent development of psychiatric disorders, Cannabis remains the illicit drug most abused by adolescents. We have previously demonstrated that female rats chronically treated during adolescence with increasing doses of delta-9-tetrahydrocannabinol (THC), the main psychoactive ingredient of cannabis, develop a depressive/psychotic-like phenotype in adulthood. Interestingly, only chronic adolescent exposure to THC, but not adult exposure, led to this complex phenotype, suggesting that adolescence may represent a more vulnerable period for the adverse effect of THC. However, the neurobiology of this vulnerability is not still clear. Considering the important role assumed by epigenetics in the etiopathogenesis of psychiatric disorders, the main goal of this thesis is to extend our knowledge on the impact of adolescent THC exposure on histone modifications occurring in other brain areas involved in the different aspects of the depressive/psychotic-like phenotype described in our animals. Specifically, we considered the Hippocampus for its involvement in cognition, the Nucleus Accumbens for its role in the reward circuit, and the Amygdala for its relevance in the emotional behaviour. To investigate the existence of age-specificity of THC effects, we performed the same analysis also after adult THC treatment. To investigate sex-dependency of THC response, we also checked THC response in adolescent male animals. First of all, adolescent (PND 35-45) and adult (PND 75-85) female Sprague-Dawley rats were treated twice a day with increasing intraperitoneal (ip) doses of THC: 2.5 mg/kg, 5 mg/kg, and 10 mg/kg or with its vehicle. Two, 24 and 48 hours after the end of the treatment, the brain areas of interest were collected and. Histone modifications associated with both transcriptional repression (H3K9 di- and tri-methylation, H3K27 trimethylation) and activation (H3K9 and H3K14 acetylation) were evaluated. Chronic THC exposure affected histone modifications in the brain of female rats in a region- and age-specific manner. Indeed, THC acted on different targets depending on the considered brain areas and, remarkably, the adolescent brain was generally more sensitive to THC exposure compared to the adult one. Specifically, in the Hippocampus of adolescent rats, THC induced a reduction of H3K14ac levels 2 hours after the end of the treatment. This was followed by a significant increase in di- and tri-methylation of H3K9 at 24 hours. Regarding the Nucleus Accumbens, H3K9me3 was significantly increased 2 hours after the end of the treatment. This enhancement was maintained 24 hours later, and it was paralleled by a significant increase in H3K9me2 and H3K14ac levels. On the contrary, at 48h, H3K9me3 levels, as well as H3K9me2 and H3K14ac levels were significantly reduced. In the Amygdala, THC administration induced a significant increase in H3K9me2 levels 2 hours after the end of the treatment. Twenty-four hours later, while this alteration returned to control values, H3K9me3 levels were significantly enhanced. Adult female rats exposed to chronic THC showed a different pattern of histone alterations. In the Hippocampus and Nucleus Accumbens, H3K14 acetylation levels were significantly increased, respectively, 2 and 24 hours after the end of the treatment. Intriguingly, a more complex picture is present in the adult Amygdala, in which a significant decrease in H3K9me2 and H3K27me3 were induced immediately after the cease of the treatment. Twenty-four hours later H3K9ac was significantly reduced, and at 48 hours, H3K14ac levels were significantly decreased. As a whole, the investigation performed in female rats suggests that in the adolescent brain THC induced a primary effect represented by changes leading to transcriptional repression, whereas the primary effect induced by adult THC exposure led to transcriptional activation. Interestingly, only in the adolescent brain, the primary effect was followed by a homeostatic response to counterbalance the THC-induced repressive effect, except in the amygdala. The presence of a more complex response in the adolescent brain may be part of the mechanisms that make the adolescent brain vulnerable to THC adverse effects. The second aim of this thesis was to extend our knowledge on the impact of adolescent THC exposure on histone modifications occurring in different brain areas of male rats. To this aim, adolescent (PND 35-45) male Sprague-Dawley rats were treated with the same protocol previously described for females and we conducted the same analysis in the Prefrontal Cortex, Hippocampus and Nucleus Accumbens. Chronic THC exposure affected histone modifications in the brain of male rats in a region- specific manner. Surprisingly, in the Prefrontal cortex and Hippocampus, we did not found any histone alterations at any intervals of time, and only in the Nucleus Accumbens we found significant alterations in H3K9me3 levels. Specifically, H3K9me3 was decreased immediately after the end of the treatment and then increased 24h later. Further studies are needed to clarify the epigenetic landscape in the brain of male rats and how it could account for the development of the psychotic-like phenotype described in these animals. However, it is possible to conclude that Cannabis abuse during adolescence could impair the brain network functionality acting through a mechanism involving histone modifications that is characterized by sex-specificity