α1-adrenergic receptors facilitate inhibitory neurotransmission to cardiac vagal neurons in the nucleus ambiguus

Abstract

The cholinergic cardiac vagal neurons (CVNs), located in the nucleus ambiguus, are the origin of cardioinhibitory parasympathetic activity. Catecholaminergic neurons in nearby regions of the brainstem, including the C1 and C2 cell groups, are thought to play a key role in both arousing from sleep and maintaining wakefulness. Because norepinephrine (NE) could play an important role in influencing the activity of CVNs, particularly in response to sleeping/waking and arousal states, the present study investigated the contribution of α(1)-adrenergic receptor activation to augment inhibitory and/or blunt excitatory neurotransmission to CVNs. To test the effects of α(1)-adrenergic receptor activation, CVNs were labeled in rats by retrograde tracing and synaptic events were recorded by whole cell voltage clamp techniques in vitro. Prazosin, an inverse agonist of α(1)-adrenergic receptor, significantly decreased the frequency of both GABAergic and glycinergic neurotransmission to CVNs. Activation of α(1)-adrenergic receptors by the α(1)-adrenergic receptor agonists NE or phenylephrine (PE) both significantly increased GABAergic and glycinergic inhibitory event frequency. This effect was prevented by the sodium channel blocker tetrodotoxin (TTX). Activation of α(1)-adrenergic receptors did not alter glutamatergic neurotransmission to CVNs. This study indicates that α(1)-adrenergic receptor activation in the brainstem can facilitate inhibitory GABAergic and glycinergic neurotransmission so as to reduce CVN activity; this synaptic modulation may play a role in the tachycardia seen during NE-dependent behavioral arousal.

Figure 1. Representative images of tyrosine-hydoxylase (TH) positive neurons (green) in the C1 and C2 regions, with fluorescent localization of CVNs in the NA (red, by the presence of the fluorescent tracer rhodamine (X491))

Schematic figure was adapted from Paxinos and Watson. (CVN: cardiac vagal neuron; NTS: nucleus of the solitary tract; pr: pyramidal complex; PrBo: pre-Bötzinger complex)

Figure 2. The α₁-adrenergic receptor inverse agonist prazosin (3μM) significantly decreased the spontaneous inhibitory neurotransmission to cardiac vagal neurons (CVNs)

A. The mean frequency and amplitude averages for GABAergic IPSCs (n=7). B. The mean frequency and amplitude averages for glycinergic IPSCs (n=11).* indicates a significant difference (p<0.05) between groups.

Figure 3. Norepinephrine (NE) was applied in the presence of the α₂-adrenergic antagonist atipamezole (1 μM) and β-adrenergic receptor antagonist propanolol (10 μM) to examine the effect of α₁-adrenergic receptor activation on inhibitory pathways to CVNs

A. NE significantly augmented both GABAergic and glycinergic neurotransmission to CVNs. The mean frequency and amplitude averages for GABAergic IPSCs (n=7). B. The mean frequency and amplitude averages for glycinergic IPSCs (n=9).* indicates a significant (p<0.05) difference between groups.

Figure 4. The selective α₁-adrenergic receptor agonist phenylephrine (PE; 50 μM) was applied to examine the effect of α₁-adrenergic receptor activation on spontaneous inhibitory events in cardiac vagal neurons (CVNs)

PE increased both GABAergic and glycinergic IPSC frequency in CVNs. A. The mean frequency and amplitude averages for GABAergic IPSCs (n=7). B. The mean frequency and amplitude averages for glycinergic IPSCs (n=7).* indicates a significant difference (p<0.05) between groups.

Figure 5. The selective α₁-adrenergic receptor agonist phenylephrine (PE; 50 μM) was applied in the presence of the sodium channel blocker TTX (1 μM) to examine the potential mechanisms responsible for the increase in inhibitory neurotransmission to cardiac vagal neurons (CVNs) after α₁-adrenergic receptors activation

Control bath condition in A and B includes TTX which prevented the inhibition that occurred with α₁-adrenergic receptor activation. A. The mean frequency and amplitude averages for GABAergic IPSCs (n=7). B. The mean frequency and amplitude averages for glycinergic IPSCs (n=8). C. The mean frequency and amplitude averages for glutamatergic EPSC after PE (n=9).