Identification and characterization of a sleep-active cell group in the rostral medullary brainstem

Abstract

Early transection and stimulation studies suggested the existence of sleep-promoting circuitry in the medullary brainstem, yet the location and identity of the neurons comprising this putative hypnogenic circuitry remains unresolved. In the present study, we sought to uncover the location and identity of medullary neurons that might contribute to the regulation of sleep. Here we show the following in rats: (1) a delimited node of medullary neurons located lateral and dorsal to the facial nerve-a region we termed the parafacial zone (PZ)-project to the wake-promoting medial parabrachial nucleus; (2) PZ neurons express c-Fos after sleep but not after wakefulness and hence are sleep active; and (3) cell-body-specific lesions of the PZ result in large and sustained increases (50%) in daily wakefulness at the expense of slow-wave sleep (SWS). Using transgenic reporter mice [vesicular GABA/glycine transporter (Vgat)-GFP], we then show that >50% of PZ sleep-active neurons are inhibitory (GABAergic/glycinergic, VGAT-positive) in nature. Finally, we used a Cre-expressing adeno-associated viral vector and conditional Vgat(lox/lox) mice to selectively and genetically disrupt GABA/glycinergic neurotransmission from PZ neurons. Disruption of PZ GABAergic/glycinergic neurotransmission resulted in sustained increases (40%) in daily wakefulness at the expense of both SWS and rapid eye movement sleep. These results together reveal the location and neurochemical identity of a delimited node of sleep-active neurons within the rostral medullary brainstem.

Figure 1.

PZ neurons: projections to the MPB and sleep activity. Injections of CTB into the MPB (a1). c-Fos and CTB double immunostaining in the PZ of sleeping (n = 3; a2, a4) or awake (n = 3; a3) rats. a4 provides a higher-power view of the box in a2. Note that CTB-immunostained cell bodies are seen in the PZ (a2–a4). Approximately 35% of the CTB-labeled neurons express c-Fos in the sleeping rats (, CTB and c-Fos double-labeled neurons; ▶, CTB single-labeled neurons; a2, a4), whereas only a few single-labeled c-Fos neurons and no double-labeled CTB and c-Fos neurons are seen in the awake rats (a3). c-Fos immunoreactivity is similarly seen in the PZ after sleeping (n = 3; b2) but not wake (n = 3; b1) episodes. Dual immunolabeling of c-Fos and GFP (as a proxy for VGAT-positive neurons) in the PZ after sleep (, GFP and c-Fos double-labeled neurons; ▶, GFP single-labeled neurons; n = 2; c2) and wake (n = 2; c1) episodes in VGAT–GFP mice. Note that ∼45% of GFP-immunoreactive (VGAT+) neurons express c-Fos in the PZ after sleep, but no GFP/c-Fos double-labeled neurons are present in awake mice. 7n, facial nerve.

Figure 2.

PZ lesion in rats. a1 shows anti-OX-SAP-induced bilateral lesions of the PZ in a Nissl-stained section. The extent of the lesion in 10 of the 11 lesioned rats are outlined in a2 and a3. 4V, Fourth ventricle; 7n, facial nerve. b1–b3 represent the hourly percentages (±SEM) of W, SWS, and REM sleep of control (n = 13) and PZ-lesioned rats (n = 11). ^ap < 0.05; ^bp < 0.01; ^cp < 0.001, using a two-way repeated-measures ANOVA. The histograms in c1–c3 correspond to the EEG power spectra (±SEM) in δ (0.5–5 Hz), θ (5–10 Hz), α (or spindle, 10–20 Hz), and β + γ (20–60 Hz) from six PZ-lesioned rats compared with control rats. No significant differences were found using a two-way repeated-measures ANOVA.

Figure 3.

PZ VGAT deletion in Vgat^lox/lox mice. a shows the extent of all AAV–Cre injections of the complete PZ VGAT KO (n = 5) included in the sleep–wake data reported in d1–d3. 4V, Fourth ventricle; 7n, facial nerve; CGPn, central gray pons; Ve/PB, vestibular/parabrachial nuclei area. AAV–Cre injections in the PZ labeled by Cre immunostaining in a Vgat^lox/lox mouse (b1) eliminate the VGAT mRNA signals (in situ hybridization) of the adjacent section (b2) in the same mouse, whereas AAV–GFP (c1) in the PZ of controls does not affect VGAT mRNA signals (c2). d1–d3 represent hourly percentages (±SEM) of W, SWS, and REM sleep of the Vgat^lox/lox mice that received AAV–GFP injections into the PZ (control, n = 6) and littermate Vgat^lox/lox mice that received AAV–Cre injections in the PZ (VGAT deletion, n = 5). ^ap < 0.05; ^bp < 0.01; ^cp < 0.001, a two-way repeated-measures ANOVA. e1–e3 correspond to the main EEG power spectra (±SEM) in δ (0.5–5 Hz), θ (5–10 Hz), α (or spindle, 10–20 Hz), and β + γ (20–60 Hz) from three Vgat^lox/lox mice that received AAV–Cre injections in the PZ (VGAT deletion) and three littermate Vgat^lox/lox control mice that received AAV–GFP injections into the PZ (control). No significant differences were found using a two-way repeated-measures ANOVA.