Fos expression in neurons of the rat vestibulo-autonomic pathway activated by sinusoidal galvanic vestibular stimulation

Abstract

The vestibular system sends projections to brainstem autonomic nuclei that modulate heart rate and blood pressure in response to changes in head and body position with regard to gravity. Consistent with this, binaural sinusoidally modulated galvanic vestibular stimulation (sGVS) in humans causes vasoconstriction in the legs, while low frequency (0.02-0.04 Hz) sGVS causes a rapid drop in heart rate and blood pressure in anesthetized rats. We have hypothesized that these responses occur through activation of vestibulo-sympathetic pathways. In the present study, c-Fos protein expression was examined in neurons of the vestibular nuclei and rostral ventrolateral medullary region (RVLM) that were activated by low frequency sGVS. We found c-Fos-labeled neurons in the spinal, medial, and superior vestibular nuclei (SpVN, MVN, and SVN, respectively) and the parasolitary nucleus. The highest density of c-Fos-positive vestibular nuclear neurons was observed in MVN, where immunolabeled cells were present throughout the rostro-caudal extent of the nucleus. c-Fos expression was concentrated in the parvocellular region and largely absent from magnocellular MVN. c-Fos-labeled cells were scattered throughout caudal SpVN, and the immunostained neurons in SVN were restricted to a discrete wedge-shaped area immediately lateral to the IVth ventricle. Immunofluorescence localization of c-Fos and glutamate revealed that approximately one third of the c-Fos-labeled vestibular neurons showed intense glutamate-like immunofluorescence, far in excess of the stain reflecting the metabolic pool of cytoplasmic glutamate. In the RVLM, which receives a direct projection from the vestibular nuclei and sends efferents to preganglionic sympathetic neurons in the spinal cord, we observed an approximately threefold increase in c-Fos labeling in the sGVS-activated rats. We conclude that localization of c-Fos protein following sGVS is a reliable marker for sGVS-activated neurons of the vestibulo-sympathetic pathway.

Keywords: blood pressure; heart rate; orthostatic hypotension; otolith organs; rostral ventrolateral medulla; sympathetic nervous system; vasovagal syncope; vestibular nuclei.

Figure 1

Changes in (A) blood pressure (BP) and (B) heart rate (HR) in response to sinusoidal galvanic vestibular stimulation (sGVS) at 0.1 Hz, 2 mA (C). BP fell from 100 to 90 mmHg and HR decreased from 5.5 to 5.1 beats/s. The calibration for BP was taken from an implanted intra-aortic sensor (red trace). Although the changes in BP from the PPG (blue trace) were uncalibrated, the waveforms obtained from PPG and intra-aortic sensors follow similar time courses. Changes in HR, which were calculated from the systolic changes in BP, were the same for the intra-aortic sensor and for PPG. See Section “Materials and Methods” for a further description of processing of the PPG. These results verify that changes in BP and HR can be detected and quantified using PPG data.

Figure 2

Representative vibratome sections through the vestibular nuclei from two sGVS-stimulated (A,B) and two mock (non)stimulated (C,D) rats processed for immunoperoxidase/diaminobenzidine staining of c-Fos protein. c-Fos-immunoreactive neuronal nuclei are apparent in the spinal and medial vestibular nuclei (SpVN, MVN), as well as nucleus tractus solitarius (NTS), of the stimulated animals. Sections from the mock-stimulated animals contained c-Fos-labeled cells in NTS, but rarely in the vestibular nuclei. Scale bar in (D) is for all panels.

Figure 3

A vibratome section through the caudal vestibular nuclei from an sGVS-stimulated rat, stained with anti-c-Fos antibody pre-incubated with a peptide blocker and then further processed for immunoperoxidase/diaminobenzidine staining. Signal was negligible in such control sections, and in those in which primary and/or secondary reagents were omitted from the processing protocol.

Figure 4

A schematic representation of the distribution of c-Fos-positive cells in the vestibular nuclear complex of one rat following sGVS (see Materials and Methods for details) is shown on the left side of the atlas sections (modified from Paxinos and Watson, 1998). The highest overall density of immunostained neurons was present in the medial vestibular nucleus (MVN). These cells were localized almost exclusively in the parvocellular region (MVNpc) and caudal MVN. Immunopositive neurons were present throughout the caudal half of the spinal vestibular nucleus (SpVN), and there was a small dense cluster of immunopositive neurons in the superior vestibular nucleus (SVN). Only labeling in the vestibular nuclei is plotted on this schematic. Approximate Bregma coordinates from the published atlas are indicated to the left. Abbreviations: 6, abducens nucleus; 8vn, vestibular nerve; 8n, vestibulo-cochlear nerve; das, dorsal acoustic stria; DC, dorsal cochlear nucleus; DMSp5, dorsomedial spinal trigeminal nucleus (dorsal D and ventral V subdivisions); DPGi, dorsal paragigantocellular nucleus; ECu, external cuneate nucleus; Gi, nucleus reticularis gigantocellularis; GiA, n. reticularis gigantocellularis, alpha nucleus; icp, inferior cerebellar peduncle; IRt, intermediate reticular nucleus; IS, inferior salivatory nucleus; LVe, lateral vestibular nucleus; lvs, lateral vestibulo-spinal tract; mlf, medial longitudinal fasciculus; MVeMC, medial vestibular nucleus, magnocellular division; MVePC, medial vestibular nucleus, parvocellular division; Pa6, paraabducens nucleus; PCRtA, parvicellular reticular nucleus; pd, predorsal bundle; Pr, prepositus nucleus; py, pyramids; RMg, raphé magnus; RVL, rostral ventrolateral medulla; scp, superior cerebellar peduncle; SGe, supragenual nucleus; sol, solitary tract; Sol, solitary nucleus (ventrolateral VL, rostrolateral RL, and medial M subdivisions); sp5, spinal trigeminal tract (oral O and interstitial I subdivisions); Sp5I, spinal trigeminal nucleus, pars interpolaris; SpVe, spinal (inferior) vestibular nucleus; SuVe, superior vestibular nucleus; ts, tectospinal tract; VCP, ventral cochlear nucleus, posterior division; VeCb, vestibulocerebellar nucleus; Y, Y-group.

Figure 5

Neurons in MVN activated by sGVS, visualized in vibratome sections processed for c-Fos immunoperoxidase/diaminobenzidine staining. The panels illustrate six rostro-caudal levels of the MVN from the same sGVS-stimulated rat. The images were obtained using the same microscopy and imaging conditions, and were subject to the same adjustments of brightness and contrast (see Materials and Methods). In all panels, the midline is to the left. A dense cluster of immunopositive cells is present in the rostral pole of MVNpc (A,B). The few activated neurons in MVNmc (A–D) are small diameter cells; none of the larger diameter neurons of this region were c-Fos-positive. c-Fos-stained cells were scattered throughout the caudal spinal vestibular nucleus (B–F). Approximate Bregma levels are indicated in the upper right of each panel. Abbreviations: MVN, medial vestibular nucleus; MVNmc, medial vestibular nucleus, magnocellular division; MVNpc, medial vestibular nucleus, parvocellular division; NTS, nucleus tractus solitarius; SpVN, spinal vestibular nucleus. Scale bar in (F) represents 100 μm, and is for all panels.

Figure 6

Multiple-label immunofluorescence visualization of c-Fos (green), glutamate (red), and DAPI nuclear stain (blue) in the vestibular nuclei of three different rats stimulated by sGVS. (A) A low magnification overview of rostral medial vestibular nucleus, where there is a discrete cluster of c-Fos immunopositive cells. (B) A cluster of sGVS-activated neurons in SpVN. (C) sGVS-activated neurons in the caudal MVN and SpVN and a dorsoventrally oriented column of labeled cells in the parasolitary nucleus. This panel has an irrelevant primary antibody control overlay, in order to better visualize the anatomical landmarks. Scale bars: 200 μm in (A), 50 μm in (B,C). Abbreviations: MVN, medial vestibular nucleus; MVNpc, medial vestibular nucleus, parvocellular division; NTS, nucleus tractus solitarius; SpVN, spinal vestibular nucleus; SVN, superior vestibular nucleus.

Figure 7

Multiple-label immunofluorescence visualization of c-Fos (green), glutamate (red), and DAPI nuclear stain (blue) in the vestibular nuclei of sGVS-stimulated rats. Three morphological types of vestibular nuclear neurons are activated by sGVS: globular (A,B), multipolar (C), and fusiform (D). The same three morphological cell types send direct projections from the vestibular nuclei to the RVLM (Holstein et al., 2011a). Approximately one third of the c-Fos-positive neurons showed intense glutamate immunofluorescence (C,D). Scale bars in all panels are 20 μm.

Figure 8

Multiple-label immunofluorescence visualization of c-Fos (green), glutamate (red), tyrosine hydroxylase (magenta), and DAPI nuclear stain (blue) in the RVLM of sGVS-stimulated rats. Most, but not all, of the sGVS-activated neurons in RVLM are intensely immunoreactive for tyrosine hydroxylase. The two cells indicated by white arrows in (A) are also intensely glutamate-immunofluorescent, whereas the two c-Fos-positive cells in (B) are not. Scale bars in both panels represent 20 μm.

Figure 9

Schematic diagram of the major cell groups mediating vestibulo-autonomic (red) and baroreflex pathways (green). Although there are vestibular projections to NTS and to CVLM, little convergence of this pathway with baroreflex signals occurs prior to processing in the RVLM. Regions receiving significant convergent baroreflex and vestibulo-sympathetic reflex inputs are indicated in yellow. See text for details. Abbreviations: CVLM, caudal ventrolateral medullary region; IML, intermediolateral cell column; NTS, solitary nucleus; RVLM, rostral ventrolateral medulla; VNC, vestibular nuclear complex.