Sex-dependent vulnerability to a neuroendocrine disruptor in a mouse model of autism spectrum disorders.

Autism is a neurodevelopmental disorder defined by core symptoms that include impaired social and communicative skills and repetitive behaviors, and it is strongly biased towards males, with a male-female ratio of 4:1. The etiological bases of autism are still largely unexplained, but there is general agreement that autism results from complex interaction between multiple genes conferring vulnerability and diverse environmental factors, including early prenatal exposure to environmental contaminants. A role for steroid hormones has been also hypothesized in autism etiology, as fetal exposure to higher levels of testosterone could contribute to the hypermasculinization of sex dimorphic brain areas reported in some autistic individuals (the Extreme Male Brain theory of autism, see [2]). Notably perinatal exposure to widespread environmental pollutants with known endocrine disrupting activity (i.e. heavy metals, organophosphate pesticides) appears to increase risk of developing autism spectrum disorders [8]. BTBR T+tf/J (BTBR) is an inbred strain of mice that displays several behavioural traits relevant to autism, including social deficits and repetitive behaviours in adults, analogous to the first and third diagnostic symptoms of autism, as well as an unusual pattern of ultrasonic vocalizations in pups, resembling the atypical crying seen in some autistic infants. Furthermore, BTBR pups show faster acquisition of some developmental milestones [4]. Alterations in brain levels of serotonin and oxytocin have been reported in this strain of mice, but surprisingly few information is available on steroid hormones status of BTBR. Chlorpyrifos (CPF) is a non-persistent organophosphate (OP) largely used as insecticide in both agricultural and urban communities. It acts as a developmental neurotoxicant by targeting immature central nervous system at doses below the threshold that elicits overt cholinergic toxicity [1]. A recent study [6] indicates that environmentally relevant prenatal exposure to CPF in children can alter the morphology of some brain areas involved in cognitive and behavioural processes, inducing loss or reversal of expected sex differences. Numerous animal studies have confirmed long-term effects of developmental exposure to CPF both on behaviour and neural systems in absence of significant brain acetylcholinesterase (AChE) inhibition. In particular, several evidences indicate that CPF acts as a neuroendocrine disrupter: developmental exposure to this OP in rodent models induces changes in social and anxiety responses and influences neuroendocrine markers (oxytocin, vasopressin, estrogen receptors) in hypothalamic and amygdaloidal regions [7]. In the present study we analyzed the effects of developmental exposure to CPF in BTBR mice. We aimed at evaluating in this mouse model of autism whether prenatal exposure to low CPF doses i) interfered with early neurobehavioral development of the offspring, ii) modulated the characteristic autistic-like behavioral profile of these mice at adulthood, iii) affected the expression of oxytocin, vasopressin and their related receptors, and estrogen receptor ERα and β in brain areas involved in the control of social responses. Pregnant BTBR mice were administered from gestational day 14 to gestational day 17 with either vehicle or CPF at a daily dose of 6 mg/kg by oral gavages. Offspring of both sexes underwent assessment of sensorimotor milestones and ultrasound emission on postnatal days 4, 6, 8 and 12. At adulthood, the social responses of females were assessed in a free social interaction test with a same-sex companion, whereas the courtship behavior of adult males (including ultrasonic calls) was analyzed upon presentation of an estrous untreated female. Our finding indicated that CPF did not grossly alter the neurodevelopmental profile of BTBR mice, but significantly reduced spontaneous motor activity and head movements while increasing immature motor patterns such as pivoting. CPF exposure also increased both frequency and duration of ultrasonic calls emitted by pups in isolation. Of note all these effects were more marked in the male sex. At adulthood, CPF associated alterations were found predominantly in males, while in social interactions between females we did not found any significant deficit in social investigation. Specifically, CPF-exposed males presented a particular shift in social investigation when presented with a receptive female, as they increased sniffing of the head and body areas and reduced sniffing of the anogenital area of their female partners. Such abnormalities in social investigation were associated with marked increase in the rate of ultrasonic vocalizations emitted during the courtship. Still in progress analyses of mRNA expression as for several neuroendocrine markers in hypothalamus and amygdala evidenced in males a significant CPF-induced decrease of vasopressin receptor 1A in the hypothalamus and ERα in the amygdala, and a general trend towards a diminished expression of ERβ and oxytocin precursor in the hypothalamus. Such findings, though still preliminary, suggest that in BTBR male mice prenatally exposed to CPF long-term changes in limbic/hypothalamic circuits might contribute to the unusual courtship profile. Altogether, we showed here that BTBR male mice are specifically vulnerable to a widely-diffused neurotoxicant acting as a neuroendocrine disruptor in both rodents and humans. These findings open the way for future experimental studies on the interaction among vulnerable gene backgrounds, hormonal homeostasis and environmental neurotoxicants in the etiology of sex-biased neurodevelopmental disorders.