To investigate the critical maturation time of otolith-related neurons in processing vertical orientations, rats (postnatal day 4 to adults) were studied for functional activation of c-fos expression in brainstem neurons by immuno-/hybridization histochemistry. Conscious rats were subjected to sinusoidal linear acceleration along the vertical plane. Labyrinthectomized and/or stationary controls showed only sporadically scattered Fos-labeled neurons in the vestibular nuclei, confirming an otolithic origin of c-fos expression. Functionally activated Fos expression in neurons of the medial and spinal vestibular nuclei and group x were identifiable by P7 and those in group y by P9. A small number of Fos-labeled neurons characterized by small soma size were found in the ventral part of lateral vestibular nucleus by P9. Other vestibular-related areas such as prepostitus hypoglossal nucleus, gigantocellular reticular nucleus and locus coeruleus of normal experimental rats showed functionally activated c-fos expression at P7. Neurons in dorsal medial cell column and beta subnucleus of the inferior olive only showed functionally activated c-fos expression by the second postnatal week. These findings revealed a unique critical maturation time for each of the vestibular-related brainstem areas in the recognition of gravity-related vertical head orientations. By mapping the three-dimensional distribution of Fos-immunoreactive neurons, we found an even distribution of otolith-related neurons within the spinal vestibular nucleus in groups x and y but a clustered distribution in the middle-lateral-ventral part of the medial vestibular nucleus. Taken together, our findings reveal the developmental profile of neuronal subpopulations within the vertical otolith system, thereby providing an anatomical basis for postnatal coding of gravity-related vertical head movements.

We examined the expression profile of subunits of ionotropic glutamate receptors [N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA)] during postnatal development of connectivity in the rat vestibular nucleus. Vestibular nuclear neurons were functionally activated by constant velocity off-vertical axis rotation, a strategy to stimulate otolith organs in the inner ear. These neurons indicated Fos expression as a result. By immunodetection for Fos, otolith-related neurons that expressed NMDA/AMPA receptor subunits were identified as early as P7, and these neurons were found to increase progressively up to adulthood. Although there was developmental invariance in the percentage of Fos-immunoreactive neurons expressing the NR1, NR2A, GluR1, or GluR2/3 subunits, those expressing the NR2B subunit decreased from P14 onward, and those expressing the GluR4 subunit decreased in adults. These double-immunohistochemical data were corroborated by combined immuno-/hybridization histochemical data obtained from Fos-immunoreactive neurons expressing NR2B mRNA or GluR4 mRNA. The staining of both NR2B and GluR4 in the cytoplasm of these neurons decreased upon maturation. The percentage of Fos-immunoreactive neurons expressing the other ionotropic glutamate receptor subunits (viz. NR1, NR2A, GluR1, and GluR2/3) remained relatively constant throughout postnatal maturation. Triple immunofluorescence further demonstrated coexpression of NR1 and NR2 subunits in Fos-immunoreactive neurons. Coexpression of NR1 subunit with each of the GluR subunits was also observed among the Fos-immunoreactive neurons. Taken together, the different expression profiles of ionotropic glutamate receptor subunits constitute the histological basis for glutamatergic neurotransmission in the maturation of central vestibular connectivity for the coding of gravity-related horizontal head movements.

Summary Autism susceptibility candidate 2 ( AUTS2 ), a risk gene for autism spectrum disorders (ASDs), is implicated in telencephalon development. Because AUTS2 is also expressed in the cerebellum where defects have been linked to ASDs, we investigated AUTS2 functions in the cerebellum. AUTS2 is specifically localized in Purkinje cells (PCs) and Golgi cells during postnatal development. Auts2 conditional knockout (cKO) mice exhibited smaller and deformed cerebella containing immature-shaped PCs with reduced expression of Cacna1a . Auts2 cKO and knock-down experiments implicated AUTS2 participation in elimination and translocation of climbing fiber synapses and restriction of parallel fiber synapse numbers. Auts2 cKO mice exhibited behavioral impairments in motor learning and vocal communications. Because Cacna1a is known to regulate synapse development in PCs, it suggests that AUTS2 is required for PC maturation to elicit normal development of PC synapses and thus the impairment of AUTS2 may cause cerebellar dysfunction related to psychiatric illnesses such as ASDs.

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