Projection neurons of the vestibulo-sympathetic reflex pathway

Abstract

Changes in head position and posture are detected by the vestibular system and are normally followed by rapid modifications in blood pressure. These compensatory adjustments, which allow humans to stand up without fainting, are mediated by integration of vestibular system pathways with blood pressure control centers in the ventrolateral medulla. Orthostatic hypotension can reflect altered activity of this neural circuitry. Vestibular sensory input to the vestibulo-sympathetic pathway terminates on cells in the vestibular nuclear complex, which in turn project to brainstem sites involved in the regulation of cardiovascular activity, including the rostral and caudal ventrolateral medullary regions (RVLM and CVLM, respectively). In the present study, sinusoidal galvanic vestibular stimulation was used to activate this pathway, and activated neurons were identified through detection of c-Fos protein. The retrograde tracer Fluoro-Gold was injected into the RVLM or CVLM of these animals, and immunofluorescence studies of vestibular neurons were conducted to visualize c-Fos protein and Fluoro-Gold concomitantly. We observed activated projection neurons of the vestibulo-sympathetic reflex pathway in the caudal half of the spinal, medial, and parvocellular medial vestibular nuclei. Approximately two-thirds of the cells were ipsilateral to Fluoro-Gold injection sites in both the RVLM and CVLM, and the remainder were contralateral. As a group, cells projecting to the RVLM were located slightly rostral to those with terminals in the CVLM. Individual activated projection neurons were multipolar, globular, or fusiform in shape. This study provides the first direct demonstration of the central vestibular neurons that mediate the vestibulo-sympathetic reflex.

Keywords: RVLM; blood pressure; orthostatic hypotension; vestibular.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflicts of interest.

Copyright © 2013 Wiley Periodicals, Inc.

Figures

Figure 1

Schematic diagram illustrating the main cell groups and pathways mediating the interaction between the vestibulo-sympathetic reflex (light gray) and the baroreflex pathway (dark gray). Convergence of these two pathways (black) appears to occur in the RVLM. The large arrow from the VNCc to the RVLM illustrates the activated vestibular projection neurons.

Figure 2

C-Fos protein (red) visualized by immunofluorescence staining of vestibular neurons activated using low frequency sinusoidal galvanic vestibular stimulation (sGVS). Activated vestibular neurons are illustrated at two levels of the vestibular nuclear complex (VNC), and in both the spinal and medial vestibular nuclei (SpVN and MVN, respectively). DAPI (blue) was used as a marker for neuronal nuclei. As previously reported (Holstein et al., 2012), c-Fos-positive vestibular neurons were observed in the caudal half of SpVN and throughout the non-magnocellular MVN after sGVS stimulation. Atlas templates were obtained from (Paxinos and Watson, 2005). Scale bars: 20 μm.

Figure 3

Vibratome sections from an sGVS-stimulated (A) and a non-stimulated (B) rat, processed identically and contemporaneously for immunoperoxidase/diaminobenzidine staining of c-Fos protein. A: The sGVS stimulation resulted in substantial accumulation of c-Fos protein in neurons in MVN but not the prepositus nucleus (NPH). B. In the non-stimulated rat, c-Fos-immunoreactive neuronal nuclei were apparent in the solitary nucleus (NTS), but rarely in SpVN or MVN. In both panels, the midline is to the right. Scale bar in A is for both panels: 100 μm.

Figure 4

Photomicrographs of immunoperoxidase/diaminobenzidine-stained Vibratome sections from two rats, one with a FluoroGold tracer injection into RVLM (A) and the other with a similar tracer injection into CVLM (B). Estimated Bregma levels are based on matching anatomical boundaries with atlas drawing from (Paxinos and Watson, 2005). Scale bar in B: 1 mm for both panels.

Figure 5

Examples of injection site maps. The location of the injection site and extent of the local diffusion of tracer was identified in each animal using immunoperoxidase/diaminobenzidine-stained sections through the medulla. This site was then plotted on drawings of rat brain stereotaxic atlas sections (Paxinos and Watson, 2005) by matching the anatomical structures present on the ventral aspect of stained sections to the atlas images. In the two rats depicted in the figure, the injection site in the rostral ventrolateral medullary region (RVLM) extended from Bregma −12.00 to −12.66 and is illustrated by filled shapes; the injection site in the caudal ventrolateral medulla (CVLM) extended from Bregma −12.9 to −13.68 and is illustrated with hatchmark-filled shapes. For both injection sites, the shading opacity reflects the density of the labeling observed in the tissue sections.

Figure 6

Immunoperoxidase/diaminobenzidine-stained Vibratome sections through the caudal vestibular nuclei illustrating the three morphological types of vestibular neurons that were retrogradely-filled following a FluoroGold tracer injection into CVLM. Multipolar (A, B-right, D), globular (B-left, C and G) and fusiform (E, F and H) cells (arrows) were observed throughout the caudal half of SpVN and through the caudal and parvocellular MVN. Estimated Bregma levels are based on matching anatomical boundaries with atlas drawing from (Paxinos and Watson, 2005). Scale bar in H: 50 μm for all panels.

Figure 7

Location of vestibular neurons that were activated by sGVS and had direct projections to the ipsilateral (filled circles on left side of each drawing) or contralateral (Xs on right side of each drawing) RVLM. The highest density of activated vestibular neurons with direct projections to RVLM was observed at Bregma −11.64, and the majority of these activated projection neurons were observed at or rostral to this level.

Figure 8

Location of vestibular neurons that were activated by sGVS and had direct projections to the ipsilateral (filled circles on left side of each drawing) or contralateral (Xs on right side of each drawing) CVLM. The highest density of cells was observed at Bregma −11.88, with most of these activated projection neurons observed at or caudal to this level.

Figure 9

Immunofluorescence images of c-Fos-positive (magenta) SpVN neurons with direct projections to RVLM. The FluoroGold tracer (green) has a punctate appearance in the somata and dendrites of retrogradely-filled neurons. Multipolar (A, B, F), globular (C, E) and fusiform (D) neurons were observed, and there were no differences in the cytology of neurons projecting ipsilaterally (A–D) vs contralaterally (E–F). Scale bar in each panel: 10 μm.

Figure 10

Examples of c-Fos (magenta) and FluoroGold (green) labeled vestibular neurons in MVN neurons with direct projections to RVLM. Fusiform (A), multipolar (B, D, right), and globular (C, D left) neurons were observed, as were activated projection neurons with ipsilateral (A, B) and contralateral (C, D) projections. Scale bar in each panel: 10 μm

Figure 11

Immunofluorescence images of c-Fos-positive (magenta) SpVN neurons (A–D) and MVN neurons (E–H) with direct projections to the CVLM (FluoroGold; green). Cells with ipsilateral (A, B, E, F) and contralateral (C, D, G, H) terminal fields were observed. Scale bar in each panel: 10 μm.