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Monoclonal L-citrulline immunostaining reveals nitric oxide-producing vestibular neurons.

Nitric oxide is an unstable free radical that serves as a novel messenger molecule in the central nervous system (CNS). In order to understand the interplay between classic and novel chemical communication systems in vestibular pathways, the staining obtained using a monoclonal antibody directed against L-citrulline was compared with the labeling observed using more traditional markers for the presence of nitric oxide. Brainstem tissue from adult rats was processed for immunocytochemistry employing a monoclonal antibody directed against L-citrulline, a polyclonal antiserum against neuronal nitric oxide synthase, and/or NADPH-diaphorase histochemistry. Our findings demonstrate that L-citrulline can be fixed in situ by vascular perfusion, and can be visualized in fixed CNS tissue sections by immunocytochemistry. Further, the same vestibular regions and cell types are labeled by NADPH-diaphorase histochemistry, by the neuronal nitric oxide synthase antiserum, and by our anti-L-citrulline antibody. Clusters of L-citrulline-immunoreactive neurons are present in subregions of the vestibular nuclei, including the caudal portion of the inferior vestibular nucleus, the magnocellular portion of the medial vestibular nucleus, and the large cells in the ventral tier of the lateral vestibular nucleus. NADPH-diaphorase histochemical staining of these neurons clearly demonstrated their multipolar, fusiform and globular somata and long varicose dendritic processes. These results provide support for the suggestion that nitric oxide serves key roles in both vestibulo-autonomic and vestibulo-spinal pathways.

Animals↗

Responses of peripheral vestibular neurons to angular and linear accelerations in the squirrel monkey.

Peripheral neurons innervating semicircular canals can respond to constant linear accelerations. Evidence is presented that, in our preparation, the response is artifactual and arises from thermal gradients introduced by the surgical exposure. Otolith neurons do not respond to even intense angular accelerations. Canal plugging abolishes the response of the corresponding afferents to angular acceleration, without obviously affecting resting activity. The procedure does not prevent the canals from responding to linear accelerations. The latter response, unlike that seen in intact canals, is not due to thermal gradients and may be related to the mechanisms underlying the persistent component of barbecue nystagmus.

Acceleration↗

Central compensation of vestibular deficits. II. Influences of roll tilt on different-size lateral vestibular neurons after ipsilateral labyrinth deafferentation.

The activity of 168 Deiters' neurons projecting to lumbosacral segments of the spinal cord has been recorded in precollicular decerebrate cats after ipsilateral acute (aVN) or chronic vestibular neurectomy (cVN), and their response characteristics to sinusoidal stimulation of contralateral labyrinth receptors at the standard parameters (roll tilt at 0.026 Hz, 10 degrees peak displacement) have been related to cell size inferred from the conduction velocity of the corresponding axons. These findings were compared with those elicited in decerebrate cats with both vestibular nerves intact. In all experimental conditions, the higher the coefficient of variation (CV) of the vestibulospinal neurons, reflecting a more irregular unit discharge, the lower was the mean discharge rate at rest. However, the proportion of regularly discharging units (with the lowest CV) decreased after aVN but increased after cVN. The relation found in control experiments, i.e., the faster the conduction velocity of vestibulospinal axon the lower was the unit discharge at rest, was lost after aVN due to a decrease in resting discharge rate of the slow neurons. The mean discharge rate of these units, however, recovered after cVN, so that the negative correlation between resting discharge rate and axonal conduction velocity was reestablished. After aVN, the decrease in resting discharge rate of the slow vestibulospinal neurons was not associated with significant changes in gain (impulses per second per degree) of the unit responses to standard parameters of tilt, so that the sensitivity of these units (percentage change of the mean discharge rate per degree) increased; on the other hand, the resting discharge rate of the fast neurons, which remained almost unchanged after aVN, was associated with a significant increase in gain, thus leading to an average increase in response sensitivity of these units.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Autoradiographic measurement of tritiated agmatine as an indicator of physiologic activity in Hermissenda visual and vestibular neurons.

[3H]Agmatine (amino-4-guanidobutane) has been shown to be potentially useful for identifying and assessing the ACh sensitivity of specific neurons. Small cationic amines are able to permeate ACh-activated ion channels in sympathetic neurons and vertebrate endplates. Sensory neurons of the photic pathway in the nudibranch mollusc Hermissenda crassicornis are cholinergic and the synaptic interactions between the photic and vestibular systems have been well characterized electrophysiologically. We have therefore tested the feasibility of using autoradiography with [3H]agmatine, (a) to identify known ACh-responsive postsynaptic cells and (b) to examine its ability to serve as an indicator of physiologic activity within the photic and vestibular pathways under conditions of darkness and light stimulation. Scintillation counting revealed that approximately 70% of the radioactivity was associated with the CNS while approximately 30% was found in the processing fluids, indicating that routine glutaraldehyde-osmium fixation and subsequent processing for epoxy embedding allows retention of substantial amounts of the radiolabel. The autoradiographic results consistently demonstrated that the uptake patterns for [3H]agmatine did reflect some of the known neuronal interactions under the experimental conditions of light and dark. The accuracy extended to the second order cells of the optic ganglion and to putative interneurons along the photic tract in the cerebropleural ganglion. Since all the neurons in these pathways are unipolar with their synaptic interactions occurring only at the terminal endings, the radiolabel accumulated in the somata resulted from retrograde axonal transport. In the photic-vestibular pathways, the highest silver grain densities were found over structures (cell bodies or axon tracts) with increased synaptic activity coupled with higher levels of cellular activity (i.e. increased excitatory postsynaptic potentials or increased spontaneous impulse activity). Slightly less label was found in cells which received increased numbers of inhibitory postsynaptic potentials that produced hyperpolarization and a transient cessation of impulse activity under conditions of illumination. Therefore, the uptake levels of [3H]agmatine as revealed by autoradiography appear to reflect not only changes in sensitivity or density of ACh-activated channels but also changes in cellular activity as indicated by increased amounts of retrograde transport. These results represent the first example of the effective use of this radiolabel as an indicator of synaptic activity in invertebrates and in sensory systems.

Agmatine↗