Organization and transmitter specificity of medullary neurons activated by sustained hypertension: implications for understanding baroreceptor reflex circuitry

Abstract

In situ expression of c-fos observed in response to phenylephrine (PE)-induced hypertension provided a basis for characterizing the organization and neurotransmitter specificity of neurons at nodal points of medullary baroreflex circuitry. Sustained hypertension induced by a moderate dose of PE provoked patterns of c-fos mRNA and protein expression that conformed in the nucleus of the solitary tract (NTS) to the termination patterns of primary baroreceptor afferents and in the caudal ventrolateral medulla (CVLM) to a physiologically defined depressor region. A majority of barosensitive CVLM neurons concurrently displayed markers for the GABAergic phenotype; few were glycinergic. Phenylephrine-sensitive GABAergic neurons that were retrogradely labeled after tracer deposits in pressor sites of the rostral ventrolateral medulla (RVLM) occupied a zone extending approximately 1.4 mm rostrally from the level of the calamus scriptorius, intermingled partly with catecholaminergic neurons of the A1 and C1 cell groups. By contrast, barosensitive neurons of the NTS were found to be phenotypically complex, with very few projecting directly to the RVLM. Extensive colocalization of PE-induced Fos-IR and markers for the nitric oxide phenotype were seen in a circumscribed, rostral, portion of the baroreceptor afferent zone of the NTS, whereas only a small proportion of PE-sensitive neurons in the NTS were found to be GABAergic. PE treatment parameters have been identified that provide a basis for defining and characterizing populations of neurons at the first station in the central processing of primary baroreceptor input and at a key inhibitory relay in the CVLM.

Fig. 1.

Phenylephrine-induced changes in cardiovascular parameters. Time course of changes in the MAP (top) and the baroreflex index (Δ heart rate/Δ MAP) (bottom) induced by sustained intravenous infusion of PE. Data are presented as mean ± SEM; n = 4/dose. PE-induced hypertensive responses differed significantly from controls (***p < 0.005) and from one another (++p < 0.01 compared with the moderate dose). Thesolid bar below the curves represents the 25 min duration of drug or vehicle infusion. The pronounced changes in baroreflex indices are assumed to be indicative of rapid resetting of baroreceptor sensitivity in response to a conditioning pressure ramp.

Fig. 2.

Medullary patterns of c-fos mRNA and Fos protein expression induced by prolonged PE infusion. Dark field (A, B) and bright field (A′, B′, C′) photomicrographs depicting hybridization and avidin–biotin immunoperoxidase localization of c-fos mRNA and Fos-IR in the NTS at the levels of the commissural subnucleus (NTS_c; A,A′) and the area postrema (NTS_ap; B,B′) and in the CVLM (C,C′) at 1 and 2 hr after hypertensive challenge, respectively. C, Combined immuno- and hybridization histochemical detection of choline acetyltransferase-IR cells in the nucleus ambiguus (black cells) and PE-inducedc-fos mRNA (white silver grains), respectively, photographed using polarized epifluorescence illumination. In contrast to the acute infusion model, sustained hypertension resulted in comparable strengths and distributions ofc-fos mRNA and protein expression in each region. This includes robust induction in the dorsal baroreceptor strip of the commissural NTS (NTS_c) that extended into the dorsal and dorsolateral aspects of NTS at the level of area postrema (NTS_ap). Noncatecholaminergic neurons of the CVLM region were also reliably activated.AMB, Nucleus ambiguus; AP, area postrema;cc, central canal; DMX, dorsal motor nucleus of vagus; Gr, gracile nucleus;IO, inferior olive; NTS, nucleus of solitary tract; ts, solitary tract; XII, hypoglossal nucleus. All photomicrographs, 75×.

Fig. 3.

Dose-related patterns of PE-induced Fos-IR expression in medullary neurons. Line drawings of coronal sections through the medulla showing the distribution of Fos-IR nuclei (black dots) at 2 hr after hypertensive challenges induced by moderate (left) or higher (right) doses of PE. In addition to a dose-related increase in Fos-IR neurons in the baroreceptor strip of commissural NTS and the CVLM region, high dose PE also provoked a more-pronounced Fos induction in the medial subnucleus of NTS and in the lateral reticular nucleus and medullary raphe nuclei. A1, A1 noradrenergic cell group; AMB, nucleus ambiguus; AP, area postrema; Cu, cuneate nucleus; dc, dorsal column; dpy, pyramidal decussation;EC, external cuneate nucleus; Gr, gracile nucleus; IO, inferior olive; LRN, lateral reticular nucleus; MV, medial vestibular nucleus;NTS, nucleus of solitary tract; PH, prepositus nucleus; py, pyramidal tract;RVLM, rostral ventrolateral medulla;CVLM, caudal depressor region; SpV, spinal trigeminal nucleus; SV, spinal vestibular nucleus; XII, hypoglossal nucleus.

Fig. 4.

Phenotypic characterization of PE-sensitive neurons in NTS. Top, Schematic drawings of the NTS depicting the subregions in which PE-induced Fos-IR neurons were characterized. These include the dorsal aspect of the commissural division (I) at caudal levels (NTS_c; left), the dorsal and dorsolateral subnuclei (II) at the level of the area postrema (NTS_ap;right), and the medial subnucleus (III) at both levels. Bottom, Comparison of the proportion of Fos-IR neurons within these aspects of the NTS that were colabeled for GAD or GLYT-2 mRNAs, TH-IR, or a retrograde tracer (Fluorogold) after deposits in the RVLM. Data from animals treated with moderate or higher doses of PE are given.AP, Area postrema; cc, central canal;Cu, cuneate nucleus; DMX, dorsal motor nucleus of vagus; Gr, gracile nucleus;NTS, nucleus of solitary tract; ts, solitary tract; XII, hypoglossal nucleus.

Fig. 5.

PE-induced Fos-IR in medullary GABAergic neurons.A–D, Polarized epifluorescence images showing GAD 67 mRNA signal (aqua grains) and peroxidase staining for Fos-IR (brown nuclei; A–C) or TH-IR (brown cytoplasm; D) in the NTS_ap (A), in the commissural NTS (B), and in the CVLM region (C,D). Despite robust expression of GAD in the NTS, only a small subset of neurons in the baroreceptor strip that displayed Fos-IR in response to a moderate PE dose colabeled for GAD mRNA (A, B). By contrast, a substantial fraction of Fos-IR barosensitive neurons in the CVLM region displayed GAD transcripts (C). Very few TH-IR neurons in the CVLM region displayed GAD transcripts (D).Black arrows denote examples of doubly-labeled neurons,open arrows denote cells displaying GAD mRNA only, andarrowheads indicate cells labeled only for Fos-IR (A–C) or TH-IR (D).E, E′, Barosensitive GABAergic neurons projecting to the RVLM; bright-field images of a single field in the CVLM taken at two focal planes to show PE-induced Fos-IR (brown nuclei), silver-enhanced retrograde labeling after WGA-ApoHRP-Gold injection in the RVLM (Retro;black grains; E), and hybridization signals for GAD 67 mRNA (magenta grains;E′). The neuron at the bottom right ofE and E′ displays all three markers. The cell at the left center of E is retrogradely labeled only, whereas the one to the upper right of E′ is positive only for GAD mRNA.dc, Dorsal column; ts, solitary tract. Magnifications: A–D, 150×; E,E′, 375×.

Fig. 6.

Anatomic and phenotypic characterization of PE-sensitive neurons of the VLM. A, B, Three-dimensional histograms showing the number of barosensitive (Fos-IR) neurons seen in response to moderate (A) and higher (B) doses of PE at regular intervals through the medulla (orange columns) and the number colabeled for GAD or GLYT-2 mRNAs, TH-IR, or a retrograde tracer (FG) after deposits in the RVLM at the level indicated. Data are presented as mean ± SEM; n = 6–7/dose. Both PE doses induced c-fos expression concentrated between the level of the calamus scriptorius and that of the caudal aspect of the RVLM. In material from animals treated with a moderate PE dose, Fos-IR neurons were found to be predominantly GABAergic, with a majority projecting to the RVLM. A higher dose of PE gave rise to more robust Fos expression; a diminished proportion of these colabeled for GAD mRNA or the retrograde tracer, and an increased fraction displayed the catecholaminergic phenotype.

Fig. 7.

A subset of barosensitive NTS neurons display markers for the nitric oxide phenotype. Bright-field (right) and polarized epifluorescence (left) renderings showing peroxidase staining for Fos (brown nuclei) and either NADPH diaphorase activity (blue/black cytoplasm; left) or mRNA encoding nitric oxide synthase (aqua silver grains; right) in the dorsolateral part of NTS at the level of the area postrema (NTS_ap). Open arrows, Cells displaying NADPH diaphorase staining or NOS mRNA; filled arrowheads, Fos-IR nuclei; filled arrows, cells displaying both markers. PE-induced Fos-IR was frequently colocalized with markers for NO in the rostral baroreceptive region of the NTS_ap. Cu, Cuneate nucleus. Magnification, 150×.

Fig. 8.

Distribution of GABAergic and RVLM-projecting barosensitive neurons in VLM and their relationship to aminergic cell groups. A–F, Plots of the distribution of cells displaying PE-induced Fos-IR and GAD mRNA (filled circles) or retrogradely labeled after tracer deposits in pressor sites in the RVLM (crosses). The distribution of medullary aminergic (TH-IR) neurons in the same animal (open circles) is shown for comparison. The rostrocaudal distance from the level of the calamus scriptorius is indicated on the lower right-hand cornerof each panel. The results define a barosensitive CVLM region extending ∼1.3–1.4 mm rostrally from the level of the calamus scriptorius in which the bulk of PE-sensitive, RVLM-projecting, and/or GABAergic neurons are found. A1, A1 noradrenergic cell group; Amb, nucleus ambiguus; C1, C1 adrenergic cell group; dpy, pyramidal decussation;IO, inferior olive; LRN, lateral reticular nucleus; py, pyramidal tract;SpV, spinal trigeminal nucleus.

Fig. 9.

Summary of the organization of medullary baroreflex pathways. In the NTS, PE-induced Fos-IR is concentrated in a continuous strip (dark gray) occupying discrete aspects of the commissural and dorsal subnuclei at levels 1 and 2, respectively. We provide evidence here that a rostrally situated subset of barosensitive NTS neurons (level 2) projects directly to pressor sites in the RVLM (level 4) and/or expresses markers for the NO phenotype. Most barosensitive neurons of the NTS, however, do not project directly to the RVLM. The available evidence suggests that they come to influence sympathetic outflow indirectly, via one or more interneurons in the medial division of the NTS (light gray), which in turn projects to the CVLM (dashed lines). Based on criteria of phenotype and connectivity, we define the CVLM as a diffuse, longitudinally organized column of cells, maximally developed atlevel 3, and composed primarily of barosensitive neurons that are GABAergic neurons and project to the RVLM (thick black line). Drawings of sections through the medulla are modified from the atlas of Swanson (1992). AMB, Nucleus ambiguus;AP, area postrema; CVLM, caudal depressor region; NO, nitric oxide; NTS, nucleus of solitary tract; RVLM, rostral ventrolateral medulla;NTS_ap, NTS at the level of area postrema;NTS_c, commissural NTS.