Structural and functional connectivity from the dorsomedial hypothalamus to the ventral medulla as a chronological amplifier of sympathetic outflow

Abstract

Psychological stress activates the hypothalamus, augments the sympathetic nervous output, and elevates blood pressure via excitation of the ventral medullary cardiovascular regions. However, anatomical and functional connectivity from the hypothalamus to the ventral medullary cardiovascular regions has not been fully elucidated. We investigated this issue by tract-tracing and functional imaging in rats. Retrograde tracing revealed the rostral ventrolateral medulla was innervated by neurons in the ipsilateral dorsomedial hypothalamus (DMH). Anterograde tracing showed DMH neurons projected to the ventral medullary cardiovascular regions with axon terminals in contiguity with tyrosine hydroxylase-immunoreactive neurons. By voltage-sensitive dye imaging, dynamics of ventral medullary activation evoked by electrical stimulation of the DMH were analyzed in the diencephalon-lower brainstem-spinal cord preparation of rats. Although the activation of the ventral medulla induced by single pulse stimulation of the DMH was brief, tetanic stimulation caused activation of the DMH sustained into the post-stimulus phase, resulting in delayed recovery. We suggest that prolonged excitation of the DMH, which is triggered by tetanic electrical stimulation and could also be triggered by psychological stress in a real life, induces further prolonged excitation of the medullary cardiovascular networks, and could contribute to the pathological elevation of blood pressure. The connectivity from the DMH to the medullary cardiovascular networks serves as a chronological amplifier of stress-induced sympathetic excitation. This notion will be the anatomical and pathophysiological basis to understand the mechanisms of stress-induced sustained augmentation of sympathetic activity.

Figure 1

Scheme of the isolated diencephalon-lower brainstem spinal cord preparation. (A) Lateral view of the preparation before resection of rostral brain structures. The preparation was transected at the level between the hypothalamic infundibulum and the bilateral basal regions of the temporal lobe (both points are shown with red circles, and the cut line is shown with a red dashed line). The cut-surface, with the ventral medulla facing up, was fixed with miniature pins onto a silicon block. A blue arrow shows the direction of microelectrode insertion. (B) Ventral view of the preparation after resection of brain structures rostral to the cut-line indicated in panel A. This preparation included a part of the hypothalamus, midbrain, pons, medulla and cervical spinal cord. The green circle marks the area of recordings. Fluorescence change was quantified in four squared areas. Square 1: rostral ventrolateral medulla (RVLM), Square 2: caudal ventrolateral medulla (CVLM), Square 3: rostral ventromedial medulla (RVMM), Square 4: raphe pallidus (RP). The insertion point of the stimulating microelectrode on the cut surface is shown with a black circle. C4, ventral roots of the fourth cervical spinal cord; cer, cerebellum; CO, optic chiasm; IC, inferior colliculus; SC, superior colliculus; TL, part of the temporal lobe; VI, sixth cranial nerve roots; IX, ninth cranial nerve roots; X, tenth cranial nerve roots; XII, twelfth cranial nerve roots; 3 V, third ventricle.

Figure 2

Distribution of RVLM-projecting neurons in the DMH. A color photomicrograph showing the site of FG injection into the RVLM (A), and retrogradely labeled neurons with FG in the DMH (B). (C, D) The distribution of FG-labeled neurons in two levels of the hypothalamus which contains the DMH (C, D; rostral to caudal). Each red dot indicates one FG-labeled neuron. Note that labeled cell bodies of neurons that projected to the RVLM were found in the DMH and adjacent regions in the hypothalamus. Arc, arcuate nucleus of the hypothalamus; DMH, dorsomedial hypothalamus; f, fornix; VMH, ventromedial hypothalamus; ZI, zona incerta; 3 V, third ventricle.

Figure 3

A photomicrograph showing the site of BDA injection into the DMH (A), and line drawings showing the resulting distribution of BDA-labeled axon fibers (red fine lines) and terminals (red fine dots) in the medulla oblongata (B–D; rostral to caudal). (E) Distribution of anterogradely labeled axon fibers (black-colored) in the ventromedial and ventrolateral medulla. (F–I) Overlapping distribution of BDA-labeled axon fibers and TH-immunoreactive neurons in the RVLM. Note that the BDA-labeled boutons (arrows) are in contiguity with dendrites of the TH-immunoreactive neurons. Such close apposition was frequently observed. Amb, nucleus ambiguus; AP, area postrema; Cu, cuneate nucleus; Gr, gracile nucleus; ic, internal capsule; IO, inferior olivary nucleus; LRt, lateral reticular nucleus; MVe, medial vestibular nucleus; NTS, nucleus of the solitary tract; opt, optic tract; PrH, prepositus hypoglossal nucleus; py, pyramidal tract; SpVe, spinal vestibular nucleus; Sp5, spinal trigeminal nucleus; sp5, spinal trigeminal tract; st, solitary tract; TH, tyrosine hydroxylase; 12, hypoglossal nucleus. For other abbreviations, see Fig. 2 legend.

Figure 4

Representative optical images in a single pulse stimulation experiment. (A) The circle denotes the recording area on the ventral medulla. (B) Pseudocolor images showing fluorescence intensity on the ventral medulla before, during, and after single pulse stimulation. (C) Time course of fluorescence intensity in the red square area of panel B-a. A vertical red bar shows the stimulation period. a: before stimulation, b: 200 ms after the end of stimulation, c: 600 ms after the end of stimulation, d: 2.0 s after the end of stimulation. IX, ninth cranial nerve roots; X, tenth cranial nerve roots; XII, twelfth cranial nerve roots.

Figure 5

Representative optical images in a tetanic stimulation experiment. (A) The circle denotes the recording area. (B) Pseudocolor images showing fluorescence intensity on the ventral medulla before, during, and after tetanic stimulation. (C) Time course of fluorescence intensity in the red square area of panel B-a. A horizontal red bar shows the stimulation period. a: onset of stimulation, b: end of stimulation, c: 5.0 s after the end of stimulation, d: 10.0 s after the end of stimulation, e: 15.0 s after the end of stimulation, f: 20.0 s the end of after stimulation. The excitations of the RVLM, CVLM, RVMM and RP were well detected. RVLM, rostral ventrolateral medulla; CVLM, caudal ventrolateral medulla; RP, raphe pallidus.

Figure 6

Comparison of post-stimulus 75% recovery time in single pulse stimulation and in tetanic stimulation in (A) RVLM, (B) CVLM, (C) RVMM, and (D) RP. RVLM; rostral ventrolateral medulla, CVLM, caudal ventrolateral medulla; RVMM, rostral ventromedial medulla; RP, raphe pallidus. *p < 0.05.