Article Figures & Data
Figures
- Figure 1.
Selective expression ChR2 in LC neurons. In TH-Cre-ChR2-EYFP transgenic mice, strong expression of ChR2-EYFP defines the LC. The LC noradrenergic neurons express the highest levels of transgene in pontomedullary brainstem tissue. A, Transverse coronal section showing that ChR2-EYFP (green) is strongly expressed within the LC located near the dorsal edge, whereas the CVNs (labeled with the retrograde fluorescent tracer XRITC, red) in the NA are localized to the ventrolateral medulla, Scale bar, 80 μm. B, Magnified representative confocal image taken from the callout region in A, from another slice of tissue, showing the ChR2-EYFP (green) expression in the cell membrane and processes of LC neurons. Scale bar, 80 μm. C–E, Confocal images showing individual neurons, with ChR2-EYFP expression (yellow) localized to the cell membrane (C) and TH immunopositivity in the cytoplasm, shown in blue (D). Merged image: ChR2 expression is limited to TH immunopositive neurons, Scale bar, 9 μm. F, Representative action potential trains recorded under current-clamp configuration from a LC neuron expressing ChR2-EYFP indicating that ChR2 activation increase firing from 2.3 ± 0.6 Hz to 4.4 ± 0.8 Hz (n = 13, p < 0.001). G, Voltage-clamp recording of a LC neuron expressing ChR2-EYFP responding with a rapid excitatory inward current in response to photoactivation of ChR2. In this and all subsequent figures, *p < 0.05, **p < 0.01, and ***p < 0.001.
- Figure 2.
Optogenetic stimulation of LC ChR2-expressing neurons facilitates inhibitory neurotransmission to CVNs in the NA. Optogenetic stimulation of LC neurons did not change the frequency or amplitude of EPSCs in CVNs (Fig. 2A, n = 9); however, photostimulation of LC neurons increased the frequency of IPSCs in CVNs by 21 ± 5% (n = 20, p = 0.02; Fig. 2B). Optogenetic activation of LC neurons increased the frequency of isolated glycinergic IPSCs in CVNs by 27 ± 8% (Fig. 2C, p = 0.003, n = 26) and augmented isolated GABAergic IPSCs in CVNs by 21 ± 5% (Fig. 2D, p = 0.001, n = 26), respectively. A typical action potential pattern of LC firing with optogenetic stimulation inhibited the spontaneous firing in a CVN, recorded in current-clamp configuration (Fig. 2E).
- Figure 3.
The α1-adrenergic receptor inverse agonist prazosin (3 μm) blocked the evoked increase of inhibitory glycinergic and GABAergic postsynaptic inputs in CVNs. Photostimulation of LC neurons increased glycinergic IPSC frequency by 18 ± 6% (n = 7, p = 0.03), which was prevented (1.8 ± 5.5%) in the presence of prazosin (n = 7, p = 0.8). Representative traces are shown on the top left and summary data are shown on the bottom left graph. In control conditions, photostimulation of LC neurons also increased GABAergic neurotransmission to CVNs by 24.4 ± 3% (n = 9, p = 0.006), but in the presence of prazosin, the increase in GABAergic IPSCs was abolished (−7.8 ± 9%, n = 9, p = 0.8, right traces and graph).
- Figure 4.
The α2-adrenergic receptor antagonist yohimbine (2 μm) did not significantly alter the increase in inhibitory GABAergic or glycinergic neurotransmission to CVNs upon LC photoactivation. The photostimulation of LC neurons increased glycinergic IPSC frequency by 19 ± 5% (n = 10, p = 0.03) in control and by 19 ± 4% in the presence of yohimbine (n = 10, p = 0.04, left traces and graph). In control conditions, photostimulation of LC neurons also increased GABAergic neurotransmission to CVNs by 30 ± 7% (n = 9, p = 0.03). In the presence of yohimbine (2 μm), the increase in GABAergic IPSCs was maintained (27.6 ± 8%, n = 9, p = 0.02, right traces and graph).
- Figure 5.
The selective β1-adrenergic receptor antagonist atenolol (2 μm) prevented the increase in glycinergic, but not GABAergic, IPSCs in CVNs. The photostimulation of LC neurons increased glycinergic neurotransmission to CVNs by 21.7 ± 7% (n = 10, p = 0.016), but this increase was prevented by atenolol (−3.4 ± 4%, p = 0.4, n = 10). Representative traces are shown on the top left and summary data on the bottom left graph. However, atenolol did not alter the increase in inhibitory GABAergic neurotransmission to CVNs. The photostimulation of LC neurons increased GABAergic neurotransmission to CVNs by 18.6 ± 5% (n = 9, p = 0.02; in control, top right trace and right graph). In the presence of atenolol, GABAergic neurotransmission to CVNs increased by 21 ± 8% (n = 9, p = 0.03, bottom right trace and right graph) upon photoactivation of LC neurons.
