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. 2014 Sep;17(9):1217-24.
doi: 10.1038/nn.3789. Epub 2014 Aug 17.

The GABAergic parafacial zone is a medullary slow wave sleep-promoting center

Christelle Anaclet  1 , Loris Ferrari  1 , Elda Arrigoni  1 , Caroline E Bass  2 , Clifford B Saper  1 , Jun Lu  1 , Patrick M Fuller  1
Affiliations

The GABAergic parafacial zone is a medullary slow wave sleep-promoting center

Christelle Anaclet et al. Nat Neurosci. 2014 Sep.

Erratum in

  • Nat Neurosci. 2014 Dec;17(12):1841

Abstract

Work in animals and humans has suggested the existence of a slow wave sleep (SWS)-promoting/electroencephalogram (EEG)-synchronizing center in the mammalian lower brainstem. Although sleep-active GABAergic neurons in the medullary parafacial zone (PZ) are needed for normal SWS, it remains unclear whether these neurons can initiate and maintain SWS or EEG slow-wave activity (SWA) in behaving mice. We used genetically targeted activation and optogenetically based mapping to examine the downstream circuitry engaged by SWS-promoting PZ neurons, and we found that this circuit uniquely and potently initiated SWS and EEG SWA, regardless of the time of day. PZ neurons monosynaptically innervated and released synaptic GABA onto parabrachial neurons, which in turn projected to and released synaptic glutamate onto cortically projecting neurons of the magnocellular basal forebrain; thus, there is a circuit substrate through which GABAergic PZ neurons can potently trigger SWS and modulate the cortical EEG.

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Figures

Figure 1
Figure 1. Cre–dependent expression of the hM3Dq receptor in PZ GABAergic neurons
(a) coronal section outline shows the injection target (delimited node of PZ GABAergic neurons) in Vgat–IRES–cre mice. (b) details of hSyn–DIO–hM3Dq–mCherry–AAV (hM3Dq–AAV) vector injected. (c) GFP immunolabeling in the brain of Vgat–IRES–cre, lox–GFP mice shows the location of GABAergic (VGAT+) PZ neurons (scale bar = 500 μm); (d) higher power photomicrograph of GABAergic PZ neurons targeted for injection (scale bar = 250 μm); (e) morphology of magnocellular PZ GABAergic neurons (scale bar = 65 μm); (f) bilateral expression (brown immunoreactivity in neuropil) of the hM3Dq receptor in PZ GABAergic neurons following AAV–mediated transduction (scale bar = 300 μm). (g) expression of hM3Dq receptors is evident on the cell surface and processes of GABAergic PZ soma (scale bar = 70 μm). (h) high magnification image showing red–brown cytoplasmic and neuropil immunostaining with black nuclear c–Fos immunoreactivity indicates excitation of GABAergic hM3Dq+ PZ neurons by CNO (scale bar = 20 μm). (i) CNO (500 nM bath applied) produced depolarization and firing in hM3Dq–expressing GABAergic PZ neurons in brain slices. 4v: fourth ventricle; 7n: facial nerve; Cre: cre–recombinase; CNO: clozapine–N–oxide; DTg: dorsal tegmental nucleus; PnC: pontine reticular nucleus; PZ: parafacial zone.
Figure 2
Figure 2. Administration of CNO induces all polygraphic manifestations of slow–wave–sleep (SWS) in mice expressing the hM3Dq receptor in GABAergic PZ neurons
Example hypnogram, fast Fourier transform (FFT)–derived delta (0.5–4 Hz) power and EMG activity over 12 hrs following (a) vehicle (b) or CNO (0.3 mg/kg, IP; ZT12) administration in a mouse with bilateral hM3Dq receptor expression in PZ GABAergic neurons. Panel c shows the hypnogram, FFT–derived delta (0.5–4 Hz) power and EMG activity during 12 hr light period following panel b. The raw EEG and EMG traces following vehicle or CNO injection (arrow on the hypnogram) in 2a and b respectively provide unambiguous evidence of CNO–induced SWS/SWA in mice expressing hM3Dq in GABAergic PZ neurons. Note that, as compared to vehicle injection, CNO injection rapidly induced SWS, itself characterized by increased SWA density and amount. Color code: red = wakefulness (W), green = SWS and blue = REM sleep (RS). CNO: clozapine– N–oxide
Figure 3
Figure 3. CNO administration promotes SWS at the expense of both wakefulness and REM sleep
Panels a, b and d show the sleep–wake quantities following vehicle and CNO (0.3 mg/kg, IP; 7 P.M.; n = 13) injections in mice with bilateral expression of the hM3Dq receptor in PZ GABAergic neurons, including the average hourly sleep–wake amounts (% of time ± SEM); the total sleep–wake amounts (± SEM) during (1) the 3 hrs post–injection period (7PM-10PM), (2) the remainder (9 hours) of the dark/active period (10PM-7AM) and (3) the subsequent 12 hour light period (7AM-7PM); and the SWS and REM sleep latencies (± SEM). Panel c shows the SWS power spectrum changes (± SEM) over baseline during the 3 hr post–injection period for vehicle injection as compared with the first hour post–injection period for CNO (0.3 mg/kg, IP; ZT12; n = 8 mice) and the quantitative changes (± SEM) in power for the δ (0.4–4.3 Hz), θ (4.3– 9.8 Hz), α (9.8–19.9 Hz) and β+ γ (19.9–59.8 Hz) frequency bands (± SEM) following vehicle or CNO (0.3 mg/kg, IP; n = 8) administrations. In Panel e time–weighted frequency histograms show the proportion (± SEM) of W or SWS amounts in each bout length to the total amount of W or SWS in the 3 hours post–injection period following vehicle or CNO administration (0.3 mg/kg, IP; n = 13). CNO: clozapine–N–oxide two-way ANOVA followed by a post hoc Bonferroni test or paired T test * p < 0.05.
Figure 4
Figure 4. Activation of PZ GABAergic neurons increases slow–wave–sleep (SWS) during the subjective day
Panels a, b and d show sleep–wake quantities following vehicle and CNO (0.3 mg/kg, IP; 10 A.M.; n = 13) injections in mice with bilateral expression of the hM3Dq receptor in PZ GABAergic neurons, including the average hourly sleep–wake amounts (% of time ± SEM); the total sleep–wake amounts (± SEM) during (1) the 3 hrs post–injection period (10AM-1PM), (2) the remainder (6 hrs) of the light/sleep period (1PM-7PM), (3) the subsequent 12 hr dark period (7PM-7AM) and the next day first 3 hr of the light period (7AM-10AM); and the SWS and REM sleep latencies (± SEM). Panel c shows the SWS power spectrum changes over baseline during the 3 hr post–injection period for vehicle injection as compared with the first, second and third hour post–injection period for CNO (0.3 mg/kg; n = 7 mice) and the quantitative changes (± SEM) in power for the δ (0.4–4.3 Hz), θ (4.3–9.8 Hz), α (9.8–19.9 Hz) and β+ γ (19.9–59.8 Hz) frequency bands (± SEM) following vehicle or CNO (n = 7) administrations. In panel e time–weighted frequency histograms show the proportion (± SEM) of W or SWS amounts in each bout length to the total amount of W or SWS in the 3 hours post–injection period following vehicle or CNO administration (n = 13). CNO: clozapine–N–oxide; two-way ANOVA followed by a post hoc Bonferroni test or paired T test * p < 0.05.
Figure 5
Figure 5. Channelrhodopsin–2–assisted circuit mapping to establish PZVgat→PB→BF and PBVglut2→BFmc→PFC synaptic connectivity
(a–f) To map connectivity of 3rd���order downstream PZVgat targets we injected green–retrograde beads into the BFmc (b–c) and DIO– ChR2–mCherry–AAV into the PZ of Vgat–IRES–cre mice (e–f; mCherry immunoreactivity in brown) and we recorded retrogradelly labeled PB neurons (d). (g) Photostimulation of PZVgat terminals evoked GABAergic IPSCs in BFmc–projecting PB neurons (h) Photo–evoked IPSCs (pIPSCs) and spontaneous IPSCs (sIPSCs) had similar decay kinetics (single exponential fits SD: sIPSC = 0.013 and pIPSC = 0.023; τ: sIPSC = 19.02 ms and pIPSC = 18.70 ms). (i–j) Raster plot and average IPSC probability following photostimulation of PZVgat→PB pathway (50 ms bin; n = 5; ± S.E.M). (k) Photo–evoked GABAergic IPSCs recorded in TTX (1 μM + 4–AP 1 mM), indicating monosynaptic connectivity. (l–n) To map PBVglut2→BFmc→PFC connectivity we injected green–retrograde beads into the PFC and DIO–ChR2–mCherry–AAV into the PB of Vglut2–IRES–cre mice (m, beads; n, mCherry native fluorescence). (o–q) Photostimulation of PBVglut2 terminals produced glutamate release and spike firing in PFC–projecting BFmc neurons (p–q, Vh =−60mV). Photostimulation: 5 ms pulses or 2 ms in k. Bicuculline–methiodide 20 mM and DNQX 30 μM). Scale bars: 500 μm in b and m–n; 30 μm in d–e. Abbreviations: 3V, 3rd ventricle; 7n, facial nerve, ac, anterior commissure; BFmc, magnocellular basal forebrain; HDB, horizontal diagonal band of Broca; LC, locus coerleus; LDT, lateraldorsal tegmental nucleus; MCPO, magnocellular preoptic nucleus; PB, parabrachial nucleus; PFC, prefrontal cortex; PZ: parafacial zone; scp: superior cerebral peduncle; SI: substantia innominate.
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