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

Figures

• 

  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 Chatcre;ChR2 but not in Chatcre 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…

• 

  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 CVNDMV 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 CVNDMV compared to CVNNA in male mice Rhodamine-positive DMV neurons showing pipette (top) and rhodamine (bottom; red) (A). Representative immunofluorescence image of CVNDMV showing biocytin recovered patched cardiac-labeled neurons are cholinergic (B). Representative trace (C) and mean firing rate (D) of CVNDMV neurons show significantly higher spontaneous firing rates compared to CVNNA, with the majority of CVNDMV firing as analyzed by a Mann Whitney test. No statistical differences in resting membrane potential were found between CVNDMV and CVNNA using a two-tailed unpaired Student’s t test (E). Representative traces of membrane responses from CVNNA (top) and CVNDMV (bottom) to stepped current injections (F). Current-voltage (I-V) relationship graph obtained from CVNNA and CVNDMV (G). Rinput was higher in CVNDMV compared to CVNNA as analyzed by a two-tailed unpaired Student’s t test (H). Representative action potential responses in CVNNA (top) and CVNDMV (bottom) in response to 300 pA injection of direct depolarizing current (I). Action potential response curves were higher in CVNDMV compared to CVNNA in response to 50 pA-step…

• 

  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 ChatCre;Phox2bFlp;R26ds−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…