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

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A Corrigendum to this article was published on 21 November 2014

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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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Figure 1: Cre-dependent expression of the hM3Dq receptor in PZ GABAergic neurons.
Figure 2: Administration of CNO induces all polygraphic manifestations of SWS in mice expressing the hM3Dq receptor in GABAergic PZ neurons.
Figure 3: CNO administration promotes SWS at the expense of both wakefulness and REM sleep.
Figure 4: Activation of PZ GABAergic neurons increases SWS during the subjective day.
Figure 5: ChR2-assisted circuit mapping to establish PZVgat→PB→BF and PBVglut2→BFmc→PFC synaptic connectivity.

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  • 28 August 2014

    In the version of this article initially published, the colors of the data points were reversed relative to the key in Figure 3d, left panel. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We are grateful to Q.H. Ha, M. Ha and S. Keating for superb technical assistance and M. Debruyne for helpful comments. We are also indebted to B. Lowell and L. Vong (Beth Israel Deaconess Medical Center) for supplying us with the breeder pairs for developing our Vgat-IRES-cre and Vglut2-IRES-cre mouse lines. This work was supported by US National Institutes of Health grants NS073613, NS061863, NS062727, NS061841, NS085477, DA024763, MH103399 and HL095491 and by the G. Harold and Leila Y. Mathers Foundation.

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Authors and Affiliations

Authors

Contributions

C.A., E.A., C.B.S., J.L. and P.M.F. designed the experiments. C.A. and L.F. carried out the experiments. C.A., C.E.B., E.A. and P.M.F. provided reagents and analytic tools. C.A., L.F., E.A. and P.M.F. analyzed the data. C.A., E.A., C.B.S. and P.M.F. wrote the paper.

Corresponding authors

Correspondence to Jun Lu or Patrick M Fuller.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The extent of transduced neurons (that is, mCherry-positive somas) is shown for individual Vgat-IRES-cre mice that received bilateral injections of hM3Dq-AAV.

Rostral medullary regions containing hM3Dq transfected neurons are outlined for n = 13 mice included in the study, across 4 panels. hM3Dq-expressing neurons were located in the rostral medulla, near the medullo-pontine junction, lateral to the facial nerve, ventral to the locus coeruleus and vestibular complex and medial to the principal sensory and spinal trigeminal nuclei.

Supplementary Figure 2 hM3Dq receptor expression in PZ GABAergic neurons does not alter baseline (that is, in the absence of ligand (CNO)) sleep-wake.

Panels a, b & c show hourly sleep-wake amounts (± SEM) in Vgat-IRES-cre mice (n = 13) expressing hM3Dq receptor in GABAergic PZ neurons as compared with non-cre-expressing littermate mice (n = 12). Panels d, e & f show sleep-wake power bands (± SEM) (δ 0.4-4.3 Hz, θ 4.3-9.8, α 9.8-19.9 Hz, β + γ 19.9-59.8 Hz) from the first half of the dark (active) period in both groups of mice (n = 8 hM3Dq+ and 9 controls), and expressed in % of total power (0.4-59.8 Hz). Neither sleep-wake amounts nor the state-specific power bands differed between Vgat-ires-cre mice expressing hM3Dq in GABAergic PZ neurons and non-hM3Dq-expressing wild type littermate mice, indicating that expression of the (inactive) hM3Dq receptor has no impact on baseline SWS, wake, REM sleep or the respective state EEG spectral content.

Source data

Supplementary Figure 3 Administration of the hM3Dq receptor ligand, CNO, has no effect on the sleep-wake cycle of control mice, that is, non–Cre-expressing littermates.

Panels a, b & c show hourly sleep-wake amounts (± SEM) following injections of CNO (0.3 mg/kg, IP; 7PM) as compared with vehicle injection in control mice (n = 12). Panel d, e & f show sleep-wake power bands (± SEM) (δ 0.4-4.3 Hz, θ 4.3-9.8, α 9.8-19.9 Hz, β + γ 19.9-59.8 Hz) from the first three hours of the dark period in these mice (n = 7) following vehicle or CNO injections and expressed in percentage of baseline power band at the same time of the day (7PM-10PM). Neither sleep-wake amounts nor the state-specific power spectra differed following CNO and vehicle injections in control mice, indicating that CNO at the dosage used in this study is pharmacologically inert on sleep, wake and REM and associated EEG spectra. CNO: clozapine-N-oxide.

Source data

Supplementary Figure 4 Activation of PZ GABAergic neurons induces normal slow-wave sleep (SWS) during the subjective day (inactive period, a time of high sleep drive in mice).

SWS power spectra and frequency band expressed in % of total power. Panel a shows the SWS power spectrum during the 3 hrs post-injection period for vehicle injection as compared with the first hour post-injection period for CNO (0.3 mg/kg, IP; 7PM; n = 8 mice). Panel b shows 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 following CNO (0.3 mg/kg, IP; ZT12; n = 8) administration. CNO: clozapine-N-oxide.

Source data

Supplementary Figure 5 c-Fos expression in the cortex of PZ M3-expressing mice and littermate controls following acute CNO administration.

Mice expressing hM3Dq in PZ GABAergic neurons and non-cre-expressing littermates were sacrificed 2 hours after acute administration of CNO (0.3mg/kg, IP, 7PM, which is the beginning of the active period) and their brains were processed for c-Fos, a marker of cellular activation. (a-b) Mice expressing hM3Dq in PZ GABAergic neurons show dense PZ c-fos expression (b) following administration of the ligand, CNO, whereas (c-d) non-cre-expressing littermate mice show sparse c-fos PZ expression (d) following administration of CNO. (a) Consistent with the induction of behavioral sleep and electrographic SWS following CNO administration, very little c-Fos was observed in cortical regions of mice expressing hM3Dq in PZ GABAergic neurons, i.e., cellular cortex was quiescent. (c) By contrast, non-cre-expressing mice exhibited high c-Fos expression levels in cortical regions, i.e., neuronal activity. These cortical c-Fos findings are consistent with the behavioral and EEG changes observed following CNO administration in hM3Dq-expressing mice (high SWS, low wake) and non-cre-expressing littermates (low SWS, high wake). Scale bar = 100 μm. CNO: clozapine-N-oxide.

Supplementary Figure 6 Administration of CNO significantly increases waking and decreases SWS in mice expressing the hM4Di receptor in GABAergic PZ neurons.

Panels a, b & c show hourly sleep-wake amounts (± SEM) following injections of CNO (0.3 mg/kg, IP; 10AM) as compared with vehicle injection in mice with confirmed bilateral PZ transduction of hM4Di-AAV. Panel d, e & f show sleep-wake power bands (± SEM) (δ 0.4-4.3 Hz, θ 4.3-9.8, α 9.8-19.9 Hz, β + γ 19.9-59.8 Hz) from the first three hours after injection in these mice following vehicle or CNO injections and expressed in percentage of baseline power band at the same time of the day (10AM-1PM; a time of high ‘sleep drive’). Wake amounts are significantly increased and both SWS and REM sleep amounts were significantly decreased during 2 hr post CNO injection as compared with control injection. No statistically significant changes were seen in the EEG frequency bands between control and CNO condition. CNO: clozapine-N-oxide. * p < 0.05.

Source data

Supplementary Figure 7 PZ Vgat neurons expressing ChR2-mCherry respond to direct stimulation with blue-light pulses.

(a) After an injection of AAV-DIO-ChR2(H134R)-mCherry virus in Vgat-IRES-cre mice, PZVgat neurons express ChR2-mCherry (scale bar = 20 µm) (b) ChR2-mediated current (Vh = -60mV, in TTX 1 µM) evoked by a 300 ms blue-light pulse (blue bar). (c) Current-clamp (top traces) and voltage-clamp (bottom traces; Vh = -60mV) recordings of a ChR2-mCherry expressing PZVgat neuron following photostimulation at different frequencies (trains of ten 2 ms blue-light flashes, blue bars). The response to the last light flash of each stimulation paradigm is represented in the insert on the right. All recordings were conducted using a K-gluconate-based pipette solution.

Supplementary Figure 8 Schematic model of the principal findings described in this study.

When activated in vivo GABAergic PZ neurons potently and rapidly induce SWS and cortical SWA. Activation of GABAergic PZ neurons also results in the release of synaptic GABA (red arrow) onto BFmc-projecting PB neurons (green arrow). Moreover, activation of glutamatergic PB neurons results in the release of synaptic glutamate (blue arrow) onto cortically-projecting BF neurons. The net result is a dramatic reduction in ascending activating influences normally provided by the PB and BFmc to the cortex. This is reflected in the cortical EEG as a decrease in the fast frequencies that are characteristic of cortical activation/desynchronization and wakefulness and the appearance of slow waves and delta waves that are characteristic of cortical synchronization and SWS. BFmc: magnocellular basal forebrain; PB: parabrachial nucleus; PFC: prefrontal cortex; PZ: parafacial zone; SWS: slow-wave-sleep; SWA: slow-wave activity.

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Anaclet, C., Ferrari, L., Arrigoni, E. et al. The GABAergic parafacial zone is a medullary slow wave sleep–promoting center. Nat Neurosci 17, 1217–1224 (2014). https://doi.org/10.1038/nn.3789

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