Vestibular Brainstem Dysmorphology in an Animal Model of Autism Spectrum Disorder

Abstract

Introduction - Prenatal exposure to the antiepileptic drug valproic acid (VPA) is associated with an elevated risk of a diagnosis of autism spectrum disorder (ASD) in humans. Accordingly, in utero exposure to VPA is a clinically relevant and validated animal model of ASD. Previous work from our lab on VPA-exposed animals revealed significant changes in the auditory brainstem and cerebellum. Specifically, we have shown fewer neurons in the auditory brainstem, reduced expression of calcium binding proteins, reduced connectivity and hyperactivation in response to pure tone stimuli. In the cerebellum, we have found smaller Purkinje cells, reduced expression of calbindin (CB) and significant gait ataxia. Based on these findings, we rationalized that brainstem vestibular pathways might similarly be affected in VPA-exposed animals. We therefore hypothesized that in utero VPA exposure will result in fewer neurons in the vestibular nuclei, reduced CB+ immunolabeling, postural instability and poor gait performance after vestibular challenge. Methods - After timed mating, pregnant rats were fed peanut butter meals from embryonic days 7 through 12. Dams in the VPA groups were fed peanut butter with 800mg/kg VPA on E10 and 12. Pups were delivered without interference and were handled by experimenters from postnatal day (P) 15-20. Starting on P21, control and VPA-exposed animals underwent a battery of motor tests and vestibular challenges from P21-28. All morphological studies were done using brain tissue from P28 animals. Results - Compared to control animals, we found significantly fewer neurons in the spinal, lateral and superior vestibular nuclei and larger neuronal cell bodies in the spinal and lateral vestibular nuclei in VPA-exposed animals. In the lateral nucleus we also found significantly more stellate and fusiform neurons but fewer round/oval neurons. Preliminary examination of the number of CB+ terminals in the vestibular nuclei suggests that afferent inputs to these neurons may have reduced CB expression. Behavioral tests suggest that VPA-exposed animals are more severely impacted by vestibular challenge than control animals. Conclusion and Significance - Our results are consistent with both structural and functional changes in animals exposed to VPA in utero. Together, our research provides evidence for significant brainstem dysfunction and we propose these alterations extend to human cases of ASD. This work will clarify the brainstem centers impacted in ASD and may lead to the development of simple, non-invasive tests of vestibular function to screen for ASD.