Regular articlePharmacological inhibition of fatty acid amide hydrolase attenuates social behavioural deficits in male rats prenatally exposed to valproic acid
Graphical abstract
Inhibition of FAAH, the enzyme that preferentially metabolises the endocannabinoid anandamide (AEA), using PF3845, attenuates social deficits in male rats prenatally exposed to valproic acid (VPA), an environmental animal model of autism.
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 5ā15% 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+ Itpr3tf/J (BTBR) mice, [21], an in-bred mouse strain known to exhibit an autistic-like behavioural phenotype. Agonist-induced GTPĪ³S binding of CB1 receptors is enhanced in the BTBR mouse [21] and pharmacological activation of CB1/2 receptors (using Ī9-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 CB1/2 receptors are not altered, rats prenatally exposed to VPA exhibited reduced cortical expression of PPARĪ± and GRP55 and reduced hippocampal expression of PPARĪ³ 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.
Section snippets
Animals
Male and female Sprague-Dawley rats (200ā300Ā g; Charles River Laboratories UK) were mated following determination of the oestrus phase of the reproductive cycle. The presence of spermatozoa in vaginal smears indicated the first day of gestation (G0.5). Following copulation, female rats were housed singly and maintained at constant temperature (21Ā Ā±Ā 2Ā Ā°C) and humidity (30%-35%) under reverse lighting conditions (12:12Ā h darkālight, lights on from 1900 to 0700Ā h). Food and water were available ad
Male, but not female, rats prenatally exposed to VPA exhibit thermal hypoalgesia, an effect not modulated by FAAH inhibition
In the hotplate test, VPA-exposed male, but not female, rats exhibited a significant increase in latency to respond to the noxious heat stimulus (F1,26Ā =Ā 10.233 PĀ =Ā 0.004) when compared to control animals (Fig. 1), indicating heat hypoalgesia. Systemic administration of the FAAH inhibitor PF3845 did not alter the latency to respond in the hotplate test of male or female, saline- or VPA-exposed animals.
Prenatal VPA exposure or FAAH inhibition does not alter locomotor activity
Analysis of behaviour during the 5Ā min acclimatization period in the sociability arena revealed
Discussion
The endocannabinoid system has been shown to play an important modulatory role in social and emotional processing, however there have been a paucity of studies investigating the effect of modulation of this system in preclinical models of impaired social processing or ASD. The present study demonstrated that the FAAH inhibitor PF3845 attenuates impaired social responding, without modulating hypoalgesia, enhanced repetitive or reduce exploratory behaviour, in male rats prenatally exposed to VPA.
Conflicts of interest
The authors have no conflicts of interest to declare.
Acknowledgements
The authors would like to gratefully acknowledge the technical assistance provided by Rebecca Henry, Lisa Flannery and Nikita Burke. This work was supported by the Wellcome Trust Biomedical Vacation Scholarship and funding from the discipline of Physiology, National University of Ireland Galway, Ireland.
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