Pharmacological inhibition of fatty acid amide hydrolase attenuates social behavioural deficits in male rats prenatally exposed to valproic acid

Abstract

Autism spectrum disorders are a group of neurodevelopmental disorders characterised by impaired social interaction, deficits in communication and repetitive stereotyped behaviours. The endocannabinoid system plays an important role in modulating emotionality and social responding, however there have been a paucity of studies investigating this system in autistic animal models. This study investigated the effect of inhibiting fatty acid amide hydrolyase (FAAH), the anandamide catabolic enzyme, on behavioural responding in the valproic acid (VPA) rat model of autism. Male rats prenatally exposed to VPA exhibit an autistic-like behavioural phenotype exemplified as thermal hypoalgesia, reduced social and exploratory behaviour, and enhanced repetitive behaviour. Systemic administration of the FAAH inhibitor PF3845 (10 mg/kg) attenuated the deficit in social behaviour observed in VPA exposed male animals without altering nociceptive, repetitive or exploratory behaviour. In comparison, female VPA exposed rats displayed enhanced repetitive and reduced exploratory behaviour, but no change in social behaviour or thermal nociceptive responding. PF3845 did not alter social, repetitive or thermal nociceptive responding, but reduced exploratory behaviour in a social context in VPA-, but not saline-, exposed females. These data indicate that FAAH inhibition elicits sexual dimorphic effects on behavioural responding in VPA exposed rodents, and support an important role for FAAH in the regulation of social behavioural deficits in autistic males.

Introduction

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterised by impaired social interaction, deficits in communication and restrictive, repetitive stereotyped patterns of behaviours. The aetiology of this spectrum of disorder remains largely unknown, and although several genetic factors have been identified which play a role, known genetic factors are estimated to account for only 5–15% of autism cases. As such, environmental factors and subsequent epigenetic alterations are considered to play particularly important roles in the aetiology of this disorder. Environmental factors identified have included the prenatal exposure to teratogenic agents such as valproic acid (VPA) [1], [2], [3] and knowledge of the association between VPA and ASD has lead to the development of a widely used and validated preclinical model [4]. Exposure of prenatal rats to VPA has been shown to impair neural tube closure resulting in anatomical and neurochemical alterations and consequently behavioural aberrations such as reduced social and exploratory behaviour, enhanced repetitive behaviour and communication deficits, core changes associated with ASD. In addition, VPA exposed animals also exhibit additional behavioural alterations similar to the co-morbid symptoms observed clinically such as altered sensitivity to noxious stimuli (hypo- or hyper-algesia) and increased anxiety [for review see [4]]. Thus, the VPA rodent model provides a useful tool for evaluating the neurobiology underlying ASD and identifying novel therapeutic targets for this neurodevelopmental disorder.

The endocannabinoid system is comprised of the G-protein coupled CB₁ and CB₂ receptors, the endogenous cannabinoid ligands (endocannabinoids) including anandamide (AEA) and 2-archidonylglygerol (2-AG) and the enzymes responsible for the synthesis and catabolism of the endocannabinoids. In addition to CB₁ and CB₂ receptors, endocannabinoids are also known to have affinity for and activity at other receptor targets including the transient receptor potential vanilloid 1 (TRPV1), PPARs, GPR55 and GPR119 [5], [6], [7], [8]. Several lines of evidence have demonstrated that the endocannabinoid system plays an important role in social and emotional processing [9], [10], [11]. For example, gene analysis profiling has demonstrating reduced expression of the gene encoding the CB₁ receptor CNR1 in postmortem brain tissue of people with ASD [12]. Polymorphisms in CNR1, (in particular 2 SNP: rs806377 and rs806380) have been shown to be associated with increased striatal activity [13] and gaze duration [14] to social reward cues happy faces). Given that ASD is associated with atypical eye contact and facial emotion processing, this data suggest that polymorphisms in CNR1 may, in part, underlie the deficits in social reward processing. In accordance, preclinical studies have demonstrated that CB₁ receptors on specific neuronal subtypes (GABA vs Glutamate) modulate social investigatory behaviour of female mice [15]. Social play behaviour has been shown to enhance AEA levels in several brain regions including the amygdala, nucleus accumbens [16], [17] and striatum [11]. Furthermore, enhancing endogenous AEA tone or inhibition of AEA reuptake, results in enhanced social behaviour [17], [18], [19], [20]. However, there is a paucity of studies directly examining alterations or the effects of modulating the endocannabinoid system in preclinical models of ASD. Cortical levels of AEA, but not 2-AG, have been shown to be elevated following social exposure in BTBR T⁺ Itpr3^tf/J (BTBR) mice, [21], an in-bred mouse strain known to exhibit an autistic-like behavioural phenotype. Agonist-induced GTPγS binding of CB₁ receptors is enhanced in the BTBR mouse [21] and pharmacological activation of CB_1/2 receptors (using Δ⁹-THC or WIN55,212-2) has been shown to attenuate the hyperlocomotor activity displayed by these mice [21], [22]. Fragile X syndrome is one of the leading genetic cause of ASD and the FMR1 knockout mouse has provided a useful model in investigating the neurobiology underlying this condition [23]. Recent studies have demonstrated that CB₁ receptor antagonism [24] and FAAH inhibition [25] attenuated cognitive impairments in FMR1-/- mice, in the novel object recognition and passive avoidance test respectively. In comparison, CB₂ receptor antagonism attenuates anxiety-related behaviour, in these animals [24]. However to date, there have been no studies evaluating the impact of endocannabinoid modulation on behavioural responding in non-genetic animal models of ASD. Recent work from our laboratory has demonstrated that on exposure to a social stimulus levels of AEA, and the related N-acylethanolamines, oleoylethalomine and palmitoylethanolamine, are increased in the hippocampus of adolescent rats prenatally exposed to VPA [26]. In addition, while the expression of CB_1/2 receptors are not altered, rats prenatally exposed to VPA exhibited reduced cortical expression of PPARα and GRP55 and reduced hippocampal expression of PPARγ and GPR55 [26], additional receptors targets for endocannabinoids and N-acylethanolamines. Thus, alterations in the endocannabinoid system may underlie the pathophysiology and behaviour alterations observed in this model of ASD. Thus the aim of the present study was to examine the effect of enhancing AEA tone, by pharmacologically inhibiting the enzyme primarily responsible for its catabolism fatty acid amide hydrolyse (FAAH), on social, repetitive and exploratory behaviour in the VPA rat model of autism. In addition, we also evaluated the effect of FAAH inhibition on thermal nociceptive responding, given previous data from our group demonstrating that VPA-exposed animals exhibit thermal hypoalgesia [26], an effect similar to that reported clinically in ASD patients, and the increasing evidence to support a role for the endocannabinoid system in nociceptive processing [27]. Clinical literature indicates a higher prevalence of ASD in males vs female (5:1 ratio) with recent data indicated that ASD females exhibit excessive mutational alterations when compared to males [28], highlighting that females may be somewhat protected against neurodevelopmental disorders such as ASD. Accordingly, VPA exposed males have been shown to exhibit more pronounced behavioural alterations when compared to females; and several studies have now demonstrated sexual dimorphic effects on the cytoarchitecture, neurotransmission and inflammatory mediators in the model [29], [30], [31], [32]. Thus, a further aim of this study was thus to examine if FAAH inhibition elicits sexual dimorphic behavioural changes in rats prenatally exposed to VPA or saline.