The role of GABAB receptors in the vestibular oculomotor system in mice.

Systemic administration of a gamma-amino butyric acid type B (GABAB) receptor agonist, baclofen, affects various physiological and psychological processes. To date, the effects on oculomotor system have been well characterized in primates, however those in mice have not been explored. In this study, we investigated the effects of baclofen focusing on vestibular-related eye movements. Two rotational paradigms, i.e. sinusoidal rotation and counter rotation were employed to stimulate semicircular canals and otolith organs in the inner ear. Experimental conditions (dosage, routes and onset of recording) were determined based on the prior studies exploring the behavioral effects of baclofen in mice. With an increase in dosage, both canal and otolith induced ocular responses were gradually affected. There was a clear distinction in the drug sensitivity showing that eye movements derived from direct vestibulo-ocular reflex pathways were relatively unaltered, while the responses through higher-order neural networks in the vestibular system were substantially decreased. These findings were consistent with those observed in primates suggesting a well-conserved role of GABAB receptors in the oculomotor system across frontal-eyed and lateral-eyed animals. We showed here a previously unrecognized effect of baclofen on the vestibular oculomotor function in mice. When interpreting general animal performance under the drug, the potential contribution of altered balance system should be taken into consideration.

Preserved otolith organ function in caspase-3-deficient mice with impaired horizontal semicircular canal function.

Genetically engineered mice are valuable models for elucidation of auditory and vestibular pathology. Our goal was to establish a comprehensive vestibular function testing system in mice using: (1) horizontal angular vestibulo-ocular reflex (hVOR) to evaluate semicircular canal function and (2) otolith-ocular reflex (OOR) to evaluate otolith organ function and to validate the system by characterizing mice with vestibular dysfunction. We used pseudo off-vertical axis rotation to induce an otolith-only stimulus using a custom-made centrifuge. For the OOR, horizontal slow-phase eye velocity and vertical eye position were evaluated as a function of acceleration. Using this system, we characterized hVOR and OOR in the caspase-3 (Casp3) mutant mice. Casp3 (-/-) mice had severely impaired hVOR gain, while Casp3 (+/-) mice had an intermediate response compared to WT mice. Evaluation of OOR revealed that at low-to-mid frequencies and stimulus intensity, Casp3 mutants and WT mice had similar responses. At higher frequencies and stimulus intensity, the Casp3 mutants displayed mildly reduced otolith organ-related responses. These findings suggest that the Casp3 gene is important for the proper function of the semicircular canals but less important for the otolith organ function.

Inner ear dysfunction in caspase-3 deficient mice.

BACKGROUND: Caspase-3 is one of the most downstream enzymes activated in the apoptotic pathway. In caspase-3 deficient mice, loss of cochlear hair cells and spiral ganglion cells coincide closely with hearing loss. In contrast with the auditory system, details of the vestibular phenotype have not been characterized. Here we report the vestibular phenotype and inner ear anatomy in the caspase-3 deficient (Casp3(-/-)) mouse strain. RESULTS: Average ABR thresholds of Casp3(-/-) mice were significantly elevated (P < 0.05) compared to Casp3(+/-) mice and Casp3(+/+) mice at 3 months of age. In DPOAE testing, distortion product 2F1-F2 was significantly decreased (P < 0.05) in Casp3(-/-) mice, whereas Casp3(+/-) and Casp3(+/+) mice showed normal and comparable values to each other. Casp3(-/-) mice were hyperactive and exhibited circling behavior when excited. In lateral canal VOR testing, Casp3(-/-) mice had minimal response to any of the stimuli tested, whereas Casp3(+/-) mice had an intermediate response compared to Casp3(+/+) mice. Inner ear anatomical and histological analysis revealed gross hypomorphism of the vestibular organs, in which the main site was the anterior semicircular canal. Hair cell numbers in the anterior- and lateral crista, and utricle were significantly smaller in Casp3(-/-) mice whereas the Casp3(+/-) and Casp3(+/+) mice had normal hair cell numbers. CONCLUSIONS: These results indicate that caspase-3 is essential for correct functioning of the cochlea as well as normal development and function of the vestibule.

Polysynaptic inputs to vestibular efferent neurons as revealed by viral transneuronal tracing.

The Bartha strain of the alpha-herpes pseudorabies virus (PrV) was used as a retrograde transneuronal tracer to map synaptic inputs to the vestibular efferent neurons of the Mongolian gerbil, Meriones unguiculatus. Although previous experiments have shown that vestibular efferent neurons respond to visual motion and somatosensory stimuli, the anatomic connections mediating those responses are unknown. PrV was injected unilaterally into the horizontal semicircular canal neuroepithelium of gerbils, where it was taken up by efferent axon terminals. The virus was then retrogradely transported to efferent cell bodies, replicated, and transported into synaptic endings projecting onto the efferent cells. Thirty animals were sacrificed at approximately 5-h increments between 75 and 105 h post-infection after determining that shorter time points had no central infection. Infected cells were visualized immunohistochemically. Temporal progression of neuronal infection was used to determine the nature of primary and higher order projections to the vestibular efferent neurons. Animals sacrificed at 80-94 h post-inoculation exhibited immunostaining in the dorsal and ventral group of vestibular efferent neurons, predominately on the contralateral side. Neurons within the medial, gigantocellular, and lateral reticular formations were among the first cells infected thereafter. At 95 h, additional virus-labeled cell groups included the solitary, area postrema, pontine reticular, prepositus, dorsal raphe, tegmental, the subcoeruleus nuclei, the nucleus of Darkschewitsch, and the inferior olivary beta and ventrolateral subnuclei. Analysis beyond 95 h revealed virus-infected neurons located in the vestibulo-cerebellar and motor cortices. Paraventricular, lateral, and posterior hypothalamic cells, as well as central amygdala cells, were also labeled. Spinal cord tissue exhibited no labeling in the intermediolateral cell column, but scattered cells were found in the central cervical nucleus. The results suggest functional associations among efferent feedback regulation of labyrinthine sensory input and both behavioral and autonomic systems, and support a closed-looped vestibular feedback model with additional open-loop polysynaptic inputs.

Fos responses to short-term adaptation of the horizontal vestibuloocular reflex before and after vestibular compensation in the Mongolian gerbil.

Fos expression in vestibular brainstem and cerebellar regions was evaluated during vestibular adaptation in the Mongolian gerbil. In addition, vestibular adaptation was evaluated in both normal and compensated animals, as vestibular compensation reorganizes the vestibular pathway constraining adaptive processes. Behaviorally, discordant optokinetic and vestibular input induced appropriate high and low gain in horizontal angular vestibuloocular reflex responses. In normal animals, low gain adaptation was more complete than high gain. However, in compensated animals, only low gain adaptation produced adaptive responses both toward and away from the lesion with appropriate gain shifts. High gain adaptation in compensated animals failed to result in gain adaptation for head movements toward the side of the lesion. Fos expression during acute vestibular adaptation in normal animals was found in the flocculus/paraflocculus, the dorsal cap of the inferior olive (IOK), and the prepositus hypoglossi (PrH). Floccular Fos labeling was increased under both high and low gain conditions. IOK and PrH labeling was increased and correlated during low gain conditions, but was reduced and uncorrelated during high gain conditions. The pattern of Fos labeling in compensated animals was asymmetric-favoring the ipsilesional flocculus and contralesional vestibular brainstem. Both compensated high and low gain adaptation groups displayed increased floccular and IOK Fos labeling, but only compensated high gain adaptation produced increased Fos labeling in the medial vestibular nucleus. The behavioral and Fos labeling results are consistent with visual-vestibular adaptation requiring direct vestibular input.

VOR and Fos response during acute vestibular compensation in the Mongolian gerbil in darkness and in light.

We measured binocular horizontal eye movements in the gerbil following unilateral labyrinthectomy during the acute phase (1-24 h) of vestibular compensation. Regardless of whether the animals compensated in the light or the dark, VOR gain progressively reduced following the lesion, and normal oculomotor symmetry was disrupted. Initially, the VOR was comparable at 1 h post-lesion for both visual conditions. However, by 3 h post-lesion the VOR response for head turns away from the lesion continued to drop in animals compensating in the dark. By 24 h, both groups displayed reduced VOR gains, but animals compensating in the light had improved frequency response characteristics. Optokinetic responses became unstable but were generally elevated compared to pre-lesion levels. Animals with vision had reduced optokinetic gains by 24 h, while the OKR response for animals in the dark remained elevated. Brainstem Fos labeling generally increased from 1 to 3 h, then decreased by 24 h. However, at 1 h, Fos labeling in the inferior olivary dorsal cap and prepositus contralateral to the lesion was significantly increased in animals compensating in the light. In both visual conditions, flocculus and paraflocculus Purkinje cell labeling was also observed, and some of the Fos-labeled cells in the medial vestibular nucleus were commissural. Fos in the dorsal cap and prepositus could be attributed to the presence of visual input. While the visually related prepositus Fos labeling preceded improved VOR performance, the dorsal cap appeared to be involved in resolving visual and motor deficits from spontaneous nystagmus.

Visual-vestibular interactions during vestibular compensation: role of the pretectal not in horizontal VOR recovery after hemilabyrinthectomy in rhesus monkey.

Damage to the vestibular labyrinth leads to profound nystagmus and vertigo. Over time, the vestibular-ocular system recovers in a process called vestibular compensation leading to reduced nystagmus and vertigo provided visual signals are available. Our study was directed at identifying sources of visual information that could play a role in vestibular compensation. Specifically, we investigated the role of the pretectal nucleus of the optic tract (NOT) in vestibular compensation after hemilabyrinthectomy (HL) in rhesus monkeys. We chose the NOT because this structure provides critical visual motion information for adaptive modification of the vestibular ocular reflex (VOR). We produced bilateral NOT lesions by injecting the excitotoxin ibotenic acid. We compared vestibular compensation after HL in NOT-lesioned and control animals with intact NOTs. We measured eye movements with an electromagnetic method employing scleral search coils. Measurements included slow-phase eye velocity during spontaneous nystagmus, per- and postrotatory nystagmus and the horizontal VOR (hVOR) gain (eye-velocity/head velocity) associated with per- and postrotatory and sinusoidal (0.2-2.0 Hz; 30-90 degrees/s) whole body oscillation around the earth-vertical axis. VOR gain was low (<0.5) for rotation toward the HL side. Our control animal evinced significant vestibular compensation with VOR gains approaching unity by 100 days post HL. In contrast, monkeys with bilateral lesions of the NOT never obtained this significant recovery with hVOR gains well below unity at 100 days and beyond. Therefore our studies demonstrate that the NOT is an essential source of visual signals for the process of vestibular compensation after HL.

Central distribution of vestibular afferents that innervate the anterior or lateral semicircular canal in the mongolian gerbil.

The central distribution of afferents that innervate the crista ampullaris of the anterior or lateral semicircular canals was determined in gerbils following the direct injection of tracers into one sensory neuroepithelia. Labeled somata were scattered throughout the superior ganglion. The central distribution of fibers demonstrated extensive overlap. The central branch of afferents innervating either canal was located in the rostral part of the nerve. Nerve fibers divided into ascending and descending branches. Ascending branch ramifications terminated in the superior vestibular nucleus, the magnocellular and parvicellular medial vestibular nuclei, and the cerebellum. Cerebellar terminal areas include the flocculus, nodulus and uvula. Descending branch ramifications terminated in the caudal medial, parvicellular medial and descending vestibular nuclei, and the nucleus prepositus hypoglossi. Lateral canal afferents terminated sparsely in nucleus cuneatus. The anterior canal had sparse innervation in the paratrigeminal and gigantocellular reticular formation. This study has shown many similarities in the central distribution of fibers that innervate the anterior and lateral canals and a few areas of segregated input. Projections outside the vestibular nuclei are more extensive than previously determined, including afferents to prepositus hypoglossi, cochlear nuclei, and reticular formation. Projections to the flocculus appear as numerous as those to the vermis.

Central projections of the saccular and utricular nerves in macaques.

The central projections of the utricular and saccular nerve in macaques were examined using transganglionic labeling of vestibular afferent neurons. In these experiments, biotinylated dextran amine was injected directly into the saccular or utricular neuroepithelium of fascicularis (Macaca fascicularis) or rhesus (Macaca mulatta) monkeys. Two to 5 weeks later, the animals were killed and the peripheral vestibular sensory organs, brainstem, and cerebellum were collected for analysis. The principal brainstem areas of saccular nerve termination were lateral, particularly the spinal vestibular nucleus, the lateral portion of the superior vestibular nucleus, ventral nucleus y, the external cuneate nucleus, and cell group l. The principal cerebellar projection was to the uvula with a less dense projection to the nodulus. Principle brainstem areas of termination of the utricular nerve were the lateral/dorsal medial vestibular nucleus, ventral and lateral portions of the superior vestibular nucleus, and rostral portion of the spinal vestibular nucleus. In the cerebellum, a strong projection was observed to the nodulus and weak projections were present in the flocculus, ventral paraflocculus, bilateral fastigial nuclei, and uvula. Although there is extensive overlap of saccular and utricular projections, saccular inputs to the lateral portions of the vestibular nuclear complex suggest that saccular afferents contribute to the vestibulospinal system. In contrast, the utricular nerve projects more rostrally into areas of known concentration of vestibulo-ocular related cells. Although sparse, the projections of the utricle to the flocculus/ventral paraflocculus suggest a potential convergence with floccular projection inputs from the vestibular brainstem that have been implicated in vestibulo-ocular motor learning.

Responses of gerbil utricular afferents to translational motion.

In the present study, we report the sensitivity of utricular afferents to sinusoidal translational motion in the horizontal plane. The head orientation was altered relative to the direction of translational travel in 30 degrees increments to allow determination of the head orientation that elicited the maximal and minimal responses of each afferent neuron. We determined gain and phase relationships at a constant peak linear acceleration of 0.1 g applied at frequencies between 0.20 and 2.0 Hz for multiple head orientations. The response dynamics and vector of maximal sensitivity for the utricular afferents are consistent with those reported for other mammalian species. Irregularly (CV>0.3) and intermediate (0.1

Central projections of the vestibular nerve: a review and single fiber study in the Mongolian gerbil.

The primary purpose of this article is to review the anatomy of central projections of the vestibular nerve in amniotes. We also report primary data regarding the central projections of individual horseradish peroxidase (HRP)-filled afferents innervating the saccular macula, horizontal semicircular canal ampulla, and anterior semicircular canal ampulla of the gerbil. In total, 52 characterized primary vestibular afferent axons were intraaxonally injected with HRP and traced centrally to terminations. Lateral and anterior canal afferents projected most heavily to the medial and superior vestibular nuclei. Saccular afferents projected strongly to the spinal vestibular nucleus, weakly to other vestibular nuclei, to the interstitial nucleus of the eighth nerve, the cochlear nuclei, the external cuneate nucleus, and nucleus y. The current findings reinforce the preponderance of literature. The central distribution of vestibular afferents is not homogeneous. We review the distribution of primary afferent terminations described for a variety of mammalian and avian species. The tremendous overlap of the distributions of terminals from the specific vestibular nerve branches with one another and with other sensory inputs provides a rich environment for sensory integration.

Central projections of the utricular nerve in the gerbil.

The central projections of primary afferent fibers in the utricular nerve, which convey linear head acceleration signals to neurons in the brainstem and cerebellum, are not completely defined. The purpose of this investigation was twofold: 1) to define the central projections of the gerbil utricular afferents by injecting horseradish peroxidase (HRP) and biotinylated dextran amine (BDA) into the utricular macula; and 2) to investigate the projections of individual utricular afferents by injecting HRP intracellularly into functionally identified utricular neurons. We found that utricular afferents in the gerbil projected to all divisions of the vestibular nuclear complex, except the dorsal lateral vestibular nucleus. In addition, terminals were observed in the interstitial nucleus of the eighth nerve, nucleus Y, external cuneate nucleus, and lobules I, IV, V, IX, and X of the cerebellar vermis. No projections appeared in the flocculus or paraflocculus. Fibers traversed the medial and intermediate cerebellar nuclei, but terminals appeared only occasionally. Individual utricular afferents collateralize extensively, projecting to much of the brainstem area innervated by the whole of the utricular nerve. This study did not produce complete filling of individual afferent collateral projections into the cerebellar cortex.