Regulatory role of orexin on postnatal maturation of the central vestibular system in rats

Abstract

Orexin is a hypothalamic neuropeptide which is associated with motor function in the vestibular nucleus (VN) complex. Deficiency of orexin in adult rats results in a sudden loss of muscle strength as in cataplexy and deterioration in the performance of vestibular-mediated behaviors.

To evaluate whether early postnatal perturbation of orexinergic circuits in the VN exerts any effects on the vestibular-related behaviors, we perturbed the activities of orexin receptors in the VN of postnatal day (P) 1 rat by implanting polymer (Elvax) slices loaded with orexin, orexin receptor antagonist (SB334867) or agonist ([Ala11, D-Leu15]-orexin-B) onto the dorsal surface of VN for slow release of the drug to the underlying VN. Specific behavioral tests including negative geotaxis, surface righting, air righting, balanced beam, rotarod, tail suspension, and dead reckoning were performed on these rats at different postnatal ages. Improvement of vestibular-related behavior was observed in rats pretreated with orexin receptor antagonist, while derangement of vestibular-related behaviors were observed in rats pretreated with orexin or its agonist. Interestingly, perturbation delivered to the VN at P14 did not impose any effect on animals’ behavior, indicating the presence of a critical period of orexinergic perturbation. To ascertain the source of orexinergic projection, we conducted behavioral experiments in rats which had received stereotaxic injection of a designed shOrexin adeno-associated virus (AAV) into the lateral hypothalamus (LH) of rat pups at P4. The virus expresses designed shRNA cocktails which reduces orexin mRNA and protein expression by more than 2 folds as indicated in results of western blot and quantitative polymerase chain reaction. Similar alternation in vestibular behavior were also observed in the experiments of air righting, tail suspension and dead reckoning, thereby providing further evidence with regard to the role of orexin on functional maturation of the VN complex during postnatal development.

Given that glutamatergic receptors at the VN constitutes to the coordination of motor function, we hypothesized that behavioral alternation upon early orexinergic perturbation may be associated with glutamatergic circuits in the VN. Significant decrease in protein levels of AMPA receptor subunit GluA1 and GluR2 as well as NMDA receptor GluR1 were observed in the medial vestibular nucleus (MVN) of P6 rats pretreated at P1 with orexin and its agonist. In addition, significant increase in spine density and maturation were observed in the MVN of P14 rats pretreated at P1 with orexin and its agonist. Such change in morphology of dendritic spine among MVN neurons may account for the delay in behavioral emergence of vestibular-related responses.

Altogether, our findings indicate that orexin modulates the glutamatergic circuits within the VN, and thereby regulate the expression of vestibular behavior and dendritic spine morphology of the MVN neuron. These provide us insights for development of novel therapeutic approach in children patients suffered from cataplexy and vestibular disorder.