NEW PHARMACOLOGICAL TOOLS FOR AUTISM RESEARCH: OXYTOCIN RECEPTOR MUTANT MICE AND ZEBRAFISH AS NEUROBEHAVIOURAL MODELS

Autism is a neurodevelopmental disorder which is characterized by severe and pervasive impairment in reciprocal socialization, qualitative impairment in communication and repetitive or stereotyped behaviour associated to resistance to change. Oxytocin (OT) is a peptidic hormone best known for its role in lactation and parturition. Recently, it has been also shown by several studies involving different lines of knock-out mice to play an important role in the central nervous system (CNS) by acting on the regulation of social, emotional, aggressive behaviour and on learning and memory. Furthermore, prosocial effects following OT administration in humans have been shown. The link between OT and autism has already been traced in preliminary clinical studies as autism affected patients received a beneficial outcome from treatment with OT. Pharmacokinetic (very short half-life), pharmacodynamic (unspecific binding to vasopressin (AVP) receptors) properties and the presence of peripheral side effects of OT, though, make this peptide an unsuitable target for a future clinical use. It is important, in this perspective, to characterize specific animal models in order to validate the use of OT analogs with more suitable characteristics for preclinical research. To this end, a characterization of the behavioural phenotype of OTR knock-out mice (OTR-/-) and heterozygous littermates (OTR+/-) in comparison with their wild type counterparts (OTR+/+) has been carried out. General home cage behaviour, sensory, motor abilities and emotional behaviour were not affected by the altered genotype. Interestingly, both and OTR+/- and OTR-/- mice exhibited a significant social impairment as quantified in both the sociability and social novelty tests. Furthermore, OTR-/- mice displayed much higher levels of aggression when facing a stranger mouse as a higher number of attacks and tail rattlings has been registered in the neutral cage paradigm. Moreover, when tested in the reversal phase of a T-maze task for their cognitive flexibility, OTR-/- showed a profound impairment in responding to the changes applied in their established routine. All in all, the OTR mutant mouse model provides full range autism-related aberrant behaviours, displaying social impairments, altered aggressive behaviour, a strong resistance to change and stereotyped behaviour. Mechanisms underlying the aberrant phenotype revealed by mutant mice were investigated through autoradiographic binding experiments for both OTR and vasopressin 1A receptor (V1aR) distribution. In addition, pharmacological treatments with OT, AVP and V1aR antagonist SR49059 were done. Binding experiments were carried out in specific brain areas known to exert a key role in integrating the processing of olfactory information that is crucial to regulate social and emotional behaviour in rodents. OTR-/- animals displayed an almost undetectable OTR binding in all tested areas. Furthermore, a slight compensatory up-regulation of V1aR in the hippocampus and a significant down-regulation of the V1aR expression in the anterior olfactory nucleus, amygdala, ventral pallidum and lateral septum were found. As for heterozygous mutants their phenotype appeared as halfway between the wild type and knock out counterparts for OTR binding sites and V1aR binding has been subjected to a slight reduction in ventral pallidum and anterior olfactory nucleus only. Hence, the previously mentioned aberrant behavioural phenotype displayed by OTR mutant mice could be due to an altered OT/AVP receptors concentration in crucial brain areas. Interestingly, intracerebroventicular treatment with both OT and AVP (0.5ng/mouse) in mutant mice was able to rescue the impairment shown in all behavioural tasks. Furthermore, pre-treatment with V1aR antagonist SR49059 (0.5 ng/mouse), which per se did not exert any effect, in association with OT, blocked the social, aggressive and cognitive enhancing effects of the neuropeptide. Our results suggest a strong involvement of AVP, alongside OT, and in particular the subtype 1A of the AVP receptor, in the modulation of social abilities and cognitive flexibility of OTR mutant mice. Finally, as an increasing interest for the use of zebrafish for social behavioural analyses to study the genetic basis of behaviour is rising, we also evaluated zebrafish potential as a screening tool for neuropsychiatric diseases involving deficits in social behaviour. To this end, we analyzed the effect of OT, AVP but most importantly isotocin (ISO) and vasotocin (AVT) (zebrafish homologues of OT and AVP, respectively) using the shoaling preference test as social paradigm. Dose-response parabolic curves were obtained and all neuropeptides showed significant efficacy in enhancing social interaction, suggesting the involvement of the oxytocin/vasopressin systems and their analogs in the modulation of zebrafish social behaviour. In conclusion, our findings indicate that OTR-/- and in part OTR+/- mice display autistic-like symptoms rescued by administration of AVP and OT to young adult animals. The OTR mutant mouse is thus instrumental to investigate the neurochemical and synaptic abnormalities underlying autistic-like disturbances and to test new strategies of pharmacological intervention. We also suggest the use of zebrafish as an alternative animal model for the study of social behaviour, especially as a screening tool: future studies involving new molecules acting on OT and AVP systems will be carried out, taking advantage of this new promising model.