Dorsal motor vagal neurons can elicit bradycardia and reduce anxiety-like behavior

Abstract

Cardiovagal neurons (CVNs) innervate cardiac ganglia through the vagus nerve to control cardiac function. Although the cardioinhibitory role of CVNs in nucleus ambiguus (CVN^NA) is well established, the nature and functionality of CVNs in dorsal motor nucleus of the vagus (CVN^DMV) is less clear. We therefore aimed to characterize CVN^DMV anatomically, physiologically, and functionally. Optogenetically activating cholinergic DMV neurons resulted in robust bradycardia through peripheral muscarinic (parasympathetic) and nicotinic (ganglionic) acetylcholine receptors, but not beta-1-adrenergic (sympathetic) receptors. Retrograde tracing from the cardiac fat pad labeled CVN^NA and CVN^DMV through the vagus nerve. Using whole-cell patch-clamp, CVN^DMV demonstrated greater hyperexcitability and spontaneous action potential firing ex vivo despite similar resting membrane potentials, compared to CVN^NA. Chemogenetically activating DMV also caused significant bradycardia with a correlated reduction in anxiety-like behavior. Thus, DMV contains uniquely hyperexcitable CVNs and is capable of cardioinhibition and robust anxiolysis.

Keywords: Behavioral neuroscience; Biological sciences; Natural sciences; Neuroscience; Physiology; Systems neuroscience.

Graphical abstract

Figure 1

Optogenetic stimulation of DMV elicits bradycardia in male and female mice Genetic crossbreeding paradigm used to generate transgenic mice harboring loxP-flanked ChR2 in Chat-positive motor neurons (A). Photostimulation-evoked action potentials from DMV motor neurons to 20 Hz stimulation (B). Representative diagram illustrating confirmed locations of optogenetic probes (C). Representative images of DMV (top) and NA (bottom) stained for the neuronal activation marker, c-Fos (middle panel; red) and GFP (left panel; green) immunoreactivity confirming c-Fos activation in DMV, but not NA, after DMV photostimulation (D). Schematic illustrating time course of optogenetic studies in awake mice (E). Representative trace (F) and mean HR (G) showing optogenetic stimulation of DMV produced a bradycardia in mice expressing Chat^cre;ChR2 but not in Chat^cre mice. Representative trace (H) and mean HR (I) showing i.p. muscarinic parasympathetic blocker, methyl-scopolamine, eliminated the photostimulation-induced bradycardia, while sympathetic blockage with β-1 receptor blocker atenolol mildly reduced this bradycardia. Representative images of probe site (top) and immunohistochemical staining of NA showing c-Fos activation after stimulation of NA (J). Representative trace (K) and mean HR (L) showing the nicotinic antagonist, hexamethonium (i.p.) abolished photostimulation-induced bradycardia in both DMV and NA. Bars represent mean and SEM. Blue bars/shading indicates light stimulation. Data analyzed by a repeated measure one-way ANOVA with Tukey’s post hoc (B and I) or repeated measure two-way ANOVA with Šídák’s post hoc when appropriate (G and L). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Figure 2

DMV innervates cardiac tissue through the vagus nerve in male mice Schematic showing injection into the epicardial fat pad and labeling in both CVN brain regions (A). Representative images of DMV and NA after cardiac injection of retrograde tracers, rhodamine (left in red) and cholera toxin subunit B (CT-B; right in green) (B). Both rhodamine and CT-B significantly labeled cardiac projecting neurons in NA and DMV as analyzed by a repeated measure two-way ANOVA with Šídák’s post hoc (C). Representative images of DMV after a right cervical vagotomy (C). Representative image (D) and mean cell count (E) showing a right cervical vagotomy significantly attenuated CVN^DMV numbers ipsilateral to vagotomy as analyzed by a two-tailed paired Student’s t test. Bars represent mean and SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Figure 3

Differential electrophysiological properties of CVN^DMV compared to CVN^NA in male mice Rhodamine-positive DMV neurons showing pipette (top) and rhodamine (bottom; red) (A). Representative immunofluorescence image of CVN^DMV showing biocytin recovered patched cardiac-labeled neurons are cholinergic (B). Representative trace (C) and mean firing rate (D) of CVN^DMV neurons show significantly higher spontaneous firing rates compared to CVN^NA, with the majority of CVN^DMV firing as analyzed by a Mann Whitney test. No statistical differences in resting membrane potential were found between CVN^DMV and CVN^NA using a two-tailed unpaired Student’s t test (E). Representative traces of membrane responses from CVN^NA (top) and CVN^DMV (bottom) to stepped current injections (F). Current-voltage (I-V) relationship graph obtained from CVN^NA and CVN^DMV (G). R_input was higher in CVN^DMV compared to CVN^NA as analyzed by a two-tailed unpaired Student’s t test (H). Representative action potential responses in CVN^NA (top) and CVN^DMV (bottom) in response to 300 pA injection of direct depolarizing current (I). Action potential response curves were higher in CVN^DMV compared to CVN^NA in response to 50 pA-step injections of direct depolarizing current as analyzed by a repeated measure two-way ANOVA with Šídák’s post hoc (J). Data represent mean and SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Figure 4

Chemogenetic stimulation of DMV produces bradycardia and reduces anxiety in both male and female mice Illustration of coronal hindbrain section showing injection site and Chat and Phox2b expression (A, top left); Schematic of AAV1-CreON/FlpON-hM3Dq-HA viral construct (A, bottom); Venn diagram showing expected expression of hM3Dq-HA and H2b-GFP (A, top right). Representative images of rostral, intermediate, caudal DMV and NA stained for HA (magenta) and H2b-GFP (green) immunoreactivity in Chat^Cre;Phox2b^Flp;R26^ds−HTB mice after DMV injection of AAV1-CreON/FlpON-hM3Dq-HA (B). Percentage of H2b-GFP+ cells immunoreactive for hM3Dq-HA in the rostral, intermediate and caudal DMV and NA (C). Effect of CNO on HR in DMV-hM3Dq mice over 24-h period. CNO (1 mg/kg, i.p.) injected at time = 0 min. Data analyzed using repeated measure ANOVA with Tukey’s post hoc (D). Effect of i.p. saline, CNO and CNO+MA on HR in DMV-hM3Dq (magenta) and control mice (black); comparisons are to baseline using a repeated measure ANOVA with Tukey’s post hoc (E). Schematic of open field experiment (F). Effects of i.p. saline vehicle and CNO on time in the center (G) and time spent moving (H) for the open field, in seconds. Data analyzed using two tailed paired Student’s t test (G,H). Correlation between changes in HR and center time between saline and CNO conditions in DMV-hM3Dq mice (I). Schematic of elevated plus maze (EPM) experiment (J). Effects of i.p. saline vehicle, CNO and CNO+MA administration on open-arm time (K) and time spent moving (L) in the elevated plus maze, in seconds. Data analyzed using repeated measure ANOVA with Tukey’s post hoc (K, L). Pearson correlation between changes in HR and open-arm time between saline and CNO conditions in DMV-hM3Dq mice (M). Effects of i.p. MA and AQ-RA-741 on open-arm time in elevated plus maze, compared to saline; same saline and CNO+MA data as in Figure 4K. Data analyzed using repeated measure ANOVA with Tukey’s post hoc (N). Data represent mean and SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.