Sex differences in the behavioral and synaptic consequences of a single exposure to cannabinoid at puberty and adulthood

Heavy cannabis consumption among adolescents is associated with significant and lasting neurobiological, psychological and health consequences that depend on the age of first use. Chronic exposure to cannabinoid (CB) agonists during adolescence alters social behavior and prefrontal cortex (PFC) activity in adult rats. However, sex differences on social behavior as well as PFC synaptic plasticity after acute CB activation remain poorly explored. Here, we determined the consequences of a single CB activation differently affects PFC in males and females by assessing social behavior and PFC neuronal and synaptic functions in rats during pubertal or adulthood periods, 24h after a single in-vivo cannabinoid exposure (SCE). During puberty, SCE reduced play behavior in females but not males. In contrast, SCE impaired sociability in both sexes at adulthood. General exploration and memory recognition remained normal at both ages and both sexes. At the synaptic level, SCE ablated endocannabinoid-mediated long-term depression (eCB-LTD) in the PFC of females of both ages and heightened excitability of PFC pyramidal neurons at adulthood, while males were spared. In contrast, SCE was associated to impaired long-term potentiation in adult males. Together, the data indicate behavioral and synaptic sex differences in response to a single in-vivo exposure to cannabinoid at puberty and adulthood.

Introduction 8 comparable to that of the Sham group ( Fig. 1F: U=27, p=0.156, Mann-Whitney U-test), 2 5 9 indicating a specific impairment on social play behavior in pubescent females.

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In contrast to pubescent rats, both male and female adult rats showed reduced social interest 2 6 1 24 h after SCE. Adult male rats administered WIN presented reduced general social 2 6 2 exploration ( Fig. 2A: U=7, p=0.003, Mann-Whitney U-test) as well as reduced sniffing  Similarly, adult cannabinoid-exposed females had less social contact (Fig. 2B: U=14.5, Whitney U-test) with congeners. In addition, SCE did not elicit aggressive behavior in any of 2 6 7 the tested groups (data not shown). Together, these data show that during puberty, SCE is sufficient to alter social behavior in a 2 6 9 sex-specific manner: play behavior was specifically reduced in females while males were 2 7 0 spared. In adults, SCE caused a general impairment in sociability, exhibited by a reduced 2 7 1 number of events related to general exploration and sniffing in both male and female rats. Importantly, we showed that the low socialization observed in pubescent female rats and in 2 7 3 adult rats of both sexes was unlikely due to an impaired exploration since behavioral 2 7 4 parameters unrelated to cognition but linked to general exploration and emotionality, as 2 7 5 rearing and grooming occurrences, were unchanged 24 h after SCE (Table 1).  Whitney U-test; data not shown). The central position of the PFC and eCB system in the regulation of social behavior and the 2 9 6 important role of synaptic plasticity in this structure in mediating experience-dependent 2 9 7 adaptations are well-documented (for review see Araque et al., 2017). At the synaptic level, 2 9 8 activity-dependent plasticity in the PFC -including eCB-mediated long-term depression 2 9 9 (LTD) and NMDAR-mediated long-term potentiation (LTP) -is a common target in animal 3 0 0 models of neuropsychiatric diseases (Scheyer AF et al., 2017). We compared the LTD 3 0 1 9 mediated by the eCB system (eCB-LTD) in the PFC between Sham-and WIN-treated rats of 3 0 2 both sexes at different ages, specifically pubescence and adulthood.

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Low-frequency stimulation of layer V PFC synapses induced comparable LTD in both 3 0 4 control and cannabinoid-exposed pubescent male rats (Fig. 4A: Sham: t (6) =5.596, p=0.001; 3 0 5 WIN: t (4) =3.190,p=0.033;. Similar results were observed in adult male with or hippocampus and accumbens in a previous study (Mato et al., 2004), it appears that in the 3 0 9 male rat PFC, eCB-LTD is not affected 24 h after in-vivo cannabinoid administration. Strikingly, eCB-LTD was ablated in PFC slices obtained from female rats in both age groups.

Age-and sex-dependent ablation of LTP after in-vivo single expsoure to cannabinoid
Considering that the extensive repertoire of synaptic plasticity expressed by medial PFC Mansvelder, 2014) we assessed a second type of plasticity in the PFC which is frequently  p<0.001; WIN, t (6) =2.062, p=0.084; Paired t-test). In contrast, in both age groups, NMDAR-  Sham, t (4) =4.349, p=0.012; WIN, t (7) =3.133, p=0.016; Paired t-test) had normal NMDAR-3 3 0 LTP 24 h following in-vivo cannabinoid exposure.   In adult rats however, sex-specific modifications of the excitability of pyramidal neurons 3 4 7 sampled from females were observed following a single in-vivo cannabinoid exposure. Thus, we observed an alteration of the membrane reaction profile in response to a series of Mann-Whitney U-test). Taken together, these data suggest an overall increase in the 3 6 3 excitability of PFC pyramidal neurons in adult females 24H after SCE. We found that 24 h after a single in-vivo exposure to a cannabinoid, the behavioral, neuronal 3 6 6 and synaptic consequences differ depending on the sex and age of the rat. The current data 3 6 7 indicate a heightened sensitivity of females, especially during pubescence. Specifically, 3 6 8 social behavior and eCB-mediated LTD showed strong deficits in exposed pubescent females 3 6 9 while age-matched male littermates were spared. During adulthood, although reduced social 3 7 0 interactions were observed in both sexes, eCB-mediated synaptic plasticity was ablated 3 7 1 specifically in females and NMDAR-dependent LTP in males.

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Stimulation of CB1R acutely modulates social play in adolescent rats (Trezza and 3 7 3 Vanderschuren, 2008a). We showed that a single exposure to the synthetic cannabinoid WIN (2 mg/kg), at a dose reported to acutely decrease social interactions in male rats (Schneider et specifc effects as long as 24 h after in-vivo exposure. In the pubescent group, cannabinoid-3 7 7 treated females exhibited less social play behavior but normal social investigation, while the 3 7 8 sociability of male littermates exposed to WIN was indistinguishable from that of sham rats. It is important to mention that pubescent female sham rats presented augmented number of considering that in our conditions the play behavior of pubescent females was reduced 24h 3 8 7 after SCE to the same levels of those observed in control males, we may infer that SCE showed that the activation of both CB1 and CB2 receptors (as that observed following cannabinoids during pubescence then males.

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Interactions with age-matched congeners during adolescence are crucial for the development  Gonadal steroids hormones seem to be involved in the sexual differentiation of cannabinoid 4 0 7 sensitivity. Importantly, rat hormonal status (i.e., estrous cycle phase) has been reported to In contrast to the pubescent groups, a unique exposure to WIN triggered a different response 4 1 4 in adults, since both sexes exhibited perturbed social behaviors. Adolescent rodents are more 4 1 5 sensitive to cannabis than adults (Renard et al., 2016a). Surprisingly, here we showed that 24 4 1 6 h after SCE, pubescent males did not display behavioral or synaptic changes, while adult rats 4 1 7 did. As cannabinoid doses, administration route, post-administration intervals and rat strains discrepancy. In addition, we cannot rule out a potential protective effect of gonadal hormones 4 2 0 in pubescent rats, since testosterone protects gonadectomized males against THC dependence 4 2 1 (Marusich et al., 2015b). Thus, considering that this gonadal hormone reaches its peak during 4 2 2 the pubertal period (Pignatelli et al., 2006), we can speculate that testosterone "protected"  Evidence shows that chronic cannabinoid exposure significantly impairs synaptic plasticity deficits resulting from acute cannabinoid exposure largely depend on the brain area. For  Here, we showed that PFC eCB-LTD was ablated in female rats 24 h after SCE regardless of  In rodents, the eCB system sexual differences appear early in development (Craft et al.,   Together, our results reveal behavioral and synaptic sex differences in response to a single in-  The authors declare no conflict of interest.   and female rats differ in brain cannabinoid CB1 receptor density and function and in 5 4 0 behavioural traits predisposing to drug addiction: effect of ovarian hormones. Curr.