Noradrenergic neurons in the rat solitary nucleus participate in the esophageal-gastric relaxation reflex

Abstract

Activation of esophageal mechanosensors excites neurons in and near the central nucleus of the solitary tract (NSTc). In turn, NSTc neurons coordinate the relaxation of the stomach [i.e., the receptive relaxation reflex (RRR)] by modulating the output of vagal efferent neurons of the dorsal motor nucleus of the vagus (DMN). The NSTc area contains neurons with diverse neurochemical phenotypes, including a large population of catecholaminergic and nitrergic neurons. The aim of the present study was to determine whether either one of these prominent neuronal phenotypes was involved in the RRR. Immunohistochemical techniques revealed that repetitive esophageal distension caused 53% of tyrosine hydroxylase-immunoreactive (TH-ir) neurons to colocalize c-Fos in the NSTc. No nitric oxide synthase (NOS)-ir neurons in the NSTc colocalized c-Fos in either distension or control conditions. Local brain stem application (2 ng) of alpha-adrenoreceptor antagonists (i.e., alpha1-prazosin or alpha2-yohimbine) significantly reduced the magnitude of the esophageal distension-induced gastric relaxation to approximately 55% of control conditions. The combination of yohimbine and prazosin reduced the magnitude of the reflex to approximately 27% of control. In contrast, pretreatment with either the NOS-inhibitor NG-nitro-l-arginine methyl ester or the beta-adrenoceptor antagonist propranolol did not interfere with esophageal distension-induced gastric relaxation. Unilateral microinjections of the agonist norepinephrine (0.3 ng) directed at the DMN were sufficient to mimic the transient esophageal-gastric reflex. Our data suggest that noradrenergic, but not nitrergic, neurons of the NSTc play a prominent role in the modulation of the RRR through action on alpha1- and alpha2-adrenoreceptors. The finding that esophageal afferent stimulation alone is not sufficient to activate NOS-positive neurons in the NSTc suggests that these neurons may be strongly gated by other central nervous system inputs, perhaps related to the coordination of swallowing or emesis with respiration.

Fig. 1

Illustration of c-Fos-immunoreactivity (ir) in control vs. esophageal distension cases. Rostral-caudal sections are described in terms of distance (in mm) with respect to calamus scriptorum. Scale bar = 500 μm. Bar graph: overall average number of c-Fos-ir-labeled neurons throughout the nucleus of the solitary tract (NST) in control vs. esophageal distension or acidification cases. ANOVA F_3,33 = 26.0; P < 0.0001; Dunnett’s posttest for comparisons against control: **P < 0.001.

Fig. 2

Distribution of c-Fos-ir neurons in different subregions of the NST. A–C: line drawings of micrographs in Fig. 1 showing the schema used for the identification of different NST divisions for the bar graphs D and E. D: c-Fos distribution (c-Fos-ir cells counted per section) in unstimulated controls. No significant between-group differences. E: c-Fos distribution in rats receiving esophageal distension stimuli. All subregions of the NST are activated by distension, as seen by the increase in c-Fos-positive neurons. Within any given rostral-caudal level, the central subregion expressed more c-Fos (F_8,135 = 11.21, P = 0.044; Bonferroni selected comparisons **P < 0.001).

Fig. 3

Montage of c-Fos, tyrosine hydroxylase (TH), and nitric oxide synthase (NOS) immunohistochemical results. A: illustration of NOS-ir neurons in the core of the central nucleus of the solitary tract (NSTc). Although this area receives esophageal afferent fibers, no NOS-ir neurons here were c-Fos-positive labeled after repeated esophageal distension. Blue-black = NOS-ir; red-brown = c-Fos-ir. B: this section was stained for TH and c-Fos immunoreactivity. Note the high density of TH-ir neurons (arrows) that have been c-Fos activated in response to esophageal distension. These cells surround the NOS-ir core of the NSTc demonstrated in A. Although additional c-Fos-positive cells can be seen as brown nuclei in this core region, they did not react to NOS immunostaining. Blue-black = TH-ir; red-brown = cFos-ir. C: higher magnification image of a cluster of TH-c-Fos-ir neurons in the NSTc. D: A small but nonsignificant number of cells in the medial NST demonstrate NOS-cFos-ir double label after esophageal distension. Blue-black = NOS-ir; red-brown = c-Fos-ir. E: immunohisto-chemical staining shows that dorsal motor nucleus (DMN) neurons (“vacant” regions in this DIC photomicrograph marked by red asterisks) are surrounded by NOS-ir terminal arborizations. However, our physiological studies did not suggest that NOS plays a role in the receptive relaxation reflex (RRR; refer to Fig. 5). Blue-black = NOS-ir fibers. F: TH-ir fibers coursing through the DMN denoted by red arrows. Blue-black = TH-ir fibers. G: NST TH-ir neuron (red) sending an axon (denoted by black arrowheads) to the DMN (methyl green). H: low-power micrograph of the dorsal medulla showing the TH-ir (red-brown) neurons and the “haze” of fine TH-ir fibers in the DMN. Scale bars: A, B = 100 μm; C, D = 20 μm; E, F, G = 10 μm; H = 500 μm.

Fig. 4

Esophageal distension causes a significant increase in the number of TH-c-Fos-double-immunostained neurons in the central division of the NST. Top: plots of the average number of TH or NOS neurons per histological section in control vs. esophageal distension conditions. There was no difference in the number of either phenotype in control vs. distension groups. Bottom: esophageal distension caused a significant increase in the number of TH-containing neurons that also demonstrated c-Fos activation (F_3,40 = 42.0; P < 0.0001; selected Bonferroni posttests **P < 0.001). There was no significant increase in the number of NOS-ir/c-Fos-ir-double-labeled neurons.

Fig. 5

Esophageal distension in food-deprived animals results in RRR of the fundus. Top: sample raw motility records show the effects of receptor blockers on the RRR. Top trace indicates timing and duration of esophageal distension. Amplitude of the control (i.e., pre-drug) RRR is represented in the black traces. Amplitude of the RRR after any specific drug condition (listed at left of motility trace) is represented in gray. Bottom: bar graph summarizing the effects of adrenoceptor antagonists or NOS synthesis inhibition on the strength of the RRR. Data were normalized to a predrug control reflex = to 100%. ANOVA F_5,44 = 13.0, P < 0.0001; Dunnett’s posttest, *P < 0.05, **P < 0.001.

Fig. 6

Unilateral injections of norepinephrine (NE; 40 nl volume; 2 pmol = 0.3 ng) into the left DVC of food-deprived animals equipped with a gastric preload balloon elicits a transient drop in gastric tone not unlike that seen during an RRR induced by mild esophageal distension (refer to Fig. 5). Left: demonstration of drop in fundic tone elicited by microinjection of either PBS or NE. Bar on far left of panel is an illustration of the average magnitude of drop in tone evoked during an RRR (ANOVA F_2,29 = 5.28; P = 0.011; Tukey’s posttest *P < 0.05). Posttests indicate that there is no significant difference between the RRRs evoked in the first part of these studies and the drop in tone elicited by unilateral microinjections of NE. Right: microinjection of 1% Pontamine blue (40 nl volume) verifies location of micropipette in DVC. Scale bar = 200 μm. cc, central canal; ap, area postrema; DMN, dorsal motor nucleus; NST, nucleus of solitary tract.