Presentations

Abstract

Introduction:

The dorsal motor nucleus of the vagus (DMV) is a functionally heterogeneous vagal motor region which controls a variety of digestive and metabolic functions but also innervates the heart, where its role in controlling heart rate is less clear. We therefore chemogenetically activated DMV neurons in awake behaving mice while monitoring heart rate. The results from these studies also prompted our investigation of the relationship between the DMV's role in controlling heart rate and behavior.

Methods:

To selectively activate DMV neurons, we injected an adeno-associated virus (AAV) which expresses the excitatory chemogenetic receptor, hM3Dq (8 injections, 40nl each), only after recombination by both Cre and Flp recombinases, into the DMV of Chat-Cre::Phox2b-Flp mice. Several months later, we injected the hM3Dq ligand clozapine N-oxide (CNO; 1mg/kg) via intraperitoneal injection (n=4 females, 5 males; mean age ± S.D., 32 ± 2 weeks) to activate hM3Dq+ DMV neurons while measuring heart rate via a non-invasive ECG system (ECGenie).

Results:

We measured heart rate 20 minutes prior to CNO and 0, 20, 40 minutes and 1, 2, 6, 8, 24 hours later. CNO administration significantly but reversibly decreased heart rate (mean ± standard deviation, S.D.: 20min before CNO, 705 ± 15 bpm; 40min after CNO, 536 ± 86 bpm, p=0.0271; 24hr after CNO, 727 ± 25 bpm, p=0.7347; one-way ANOVA, all timepoints, F1.660, 6.638=10.31; Dunnet's post-hoc test, p=0.0107). Interestingly, when we intraperitoneally injected the peripheral muscarinic blocker, methyl-atropine (MA; 0.1mg/kg), in the same mice 20 minutes post CNO administration, heart rate values returned to baseline (baseline, 747 ± 5 bpm; after CNO, 640 ± 19 bpm, p=0.0078 vs. baseline; after methyl-atropine, 801 ± 4 bpm, p=0.0004 vs. baseline). In order to identify the specific neurons responsible, we repeated these studies, without the injection of MA, in Calb2-Cre::Chat-Flp mice (n=4 females, 5 males; mean age ± S.D., 29 ± 2 weeks) to target Calb2+ DMV neurons, a recently identified DMV molecular subtype. However, in striking contrast to what we observed when activating DMV neurons generally, administering CNO to activate Calb2+ DMV neurons specifically did not significantly affect heart rate (mean ± S.D.: 20min before CNO, 691 ± 69 bpm; 40min after CNO, 718 ± 31 bpm, p=0.6787; 24hr after CNO, 710 ± 54 bpm, p=0.4701; one-way ANOVA, all timepoints, F2.911, 11.65=0.6277; Dunnet's, p=0.6069). A higher dose of CNO (3.5mg/kg) also had no effect on heart rate in Calb2-Cre::Chat-Flp mice (data not shown). Together, these studies indicate that DMV neurons are capable of suppressing heart rate through peripheral muscarinic signaling and that the underlying neurons likely do not express Calb2.

After observing fewer anxious behaviors in the hM3Dq+ Chat-Cre::Phox2b-Flp mice given CNO, we also decided to investigate the role of DMV neurons in reducing anxiety-like behaviors in both hM3Dq+ mice (n=4 females, 4 males; mean age ± S.D., 32 ± 2 weeks) and control mice (n=4 females, 4 males; mean age ± S.D., 29 ± 2 weeks) using an elevated plus maze. We injected CNO 20 minutes prior to placing the mice in an elevated plus maze where their movements were recorded for 10 minutes. hM3Dq+ mice given CNO spent significantly more time in the open arms of the maze, an indicator of reduced anxiety-like behavior, than the same mice given saline vehicle (CNO, 16.9 ± 4.83 s; vehicle, 0.23 ± 0.23 s; p=0.023). Like its effect on the induced bradycardia discussed above, injection of MA with CNO in hM3Dq+ mice served to negate the effects of CNO on behavior, again indicating peripheral muscarinic signaling by the DMV. When control mice were given CNO or CNO and MA, however, there was no significant change in the time spent in the open arms of the maze, indicating that neither CNO nor MA reduce anxiety-like behavior on their own.

Conclusion:

Uncovering the DMV's role in heart rate will expand our understanding of how this clinically relevant metric, associated with a host of cardiovascular disorders, is controlled. Furthermore, elucidating the moderate correlation between the CNO-induced bradycardia and increased time in the open arms of the maze (r = 0.6394) could prove vital for novel therapies for individuals suffering from anxiety disorders.