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

Anaclet C, Ferrari L, Arrigoni E, Bass CE, Saper CB, Lu J, Fuller PM - Nat. Neurosci. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Division of Sleep Medicine, Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

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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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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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.

Mentions: In a previous study we showed that PZ sleep–active neurons project to the wake– promoting parabrachial nucleus (PB)13. Given that projections from glutamatergic PB neurons to the magnocellular basal forebrain (BFmc), but not the thalamus, are indispensable for maintaining cortical activation and wakefulness18, 19, we hypothesized that GABAergic PZ neurons might promote SWS and SWA by inhibiting the wake–promoting PB–BFmc–cortex circuit. In other words, we predict that PZ GABAergic neurons monosynaptically project to and produce inhibitory postsynaptic events in PB neurons that specifically and monosynaptically innervate neurons of the BFmc. To test this hypothesis, we injected a cre–dependant vector containing channelrhodopsin–2 (DIO–ChR2(H134R)–mCherry–AAV; ChR2–mCherry) into the PZ and retrograde fluorescent microspheres were injected into the ipsilateral BFmc of Vgat– ires–cre mice (Fig. 5a). Histological assessment confirmed accurate bead placement in the BF (Fig. 5b–c) and mCherry–positive somas restricted to the GABAergic PZ (Fig. 5e–f). Photo– stimulation of PZVgat cell bodies expressing ChR2–mCherry elicited robust photocurrents and trains of brief blue–light flashes entrained the firing of PZVgat neurons up to 5 Hz (Supplementary Fig. 7). To determine whether activation of PZ axons evoked GABA release in the PB, we photostimulated PB slices containing ChR2–mCherry expressing axons that originated from PZVgat neurons (Fig. 5d). The flashes of blue light evoked fast inhibitory postsynaptic currents (IPSCs) in PB neurons that were retrogradely labeled from the BFmc (n = 7/18 neurons). The light stimuli induced synaptic events of amplitude comparable to spontaneous IPSCs (Fig. 5h), indicating that the responses were in the physiological range. Moreover, based upon the rapid kinetics, both the spontaneous and photo–evoked responses occurred in the soma and proximal dendrites of the cell. The photo–evoked IPSCs were also completely abolished by bicuculline (20 μM, Fig. 5g) indicating that these responses were mediated by the release of GABA and by the activation of GABAA postsynaptic receptors. In BFmc–projecting PB neurons that exhibited synaptic responses (PZVgat→PB) the probability of any given light pulse evoking a synchronous IPSC was 58.0 ± 5.6% (n = 7, Fig. 5i–j). Three stimuli were used to show that 1) the response was consistent across stimulation, and 2) no depression occurred following repeated stimulation, i.e., individual synaptic response had equivalent amplitudes. The photo–evoked IPSCs in PB neurons projecting to BFmc had an average onset delay of 6.2 ± 0.3 ms, a peak amplitude of 23.1 ± 3.2 pA, and a charge transfer of 1.02 ± 0.21 pC (single light pulses; n = 7). Photostimulation evoked IPSC’s in PB neurons even in the presence of tetrodotoxin (TTX; n = 4; Fig. 5k) supporting a direct synaptic connectivity from PZ to PB (PZVgat→PB→BFmc).


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

Anaclet C, Ferrari L, Arrigoni E, Bass CE, Saper CB, Lu J, Fuller PM - Nat. Neurosci. (2014)

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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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.
Mentions: In a previous study we showed that PZ sleep–active neurons project to the wake– promoting parabrachial nucleus (PB)13. Given that projections from glutamatergic PB neurons to the magnocellular basal forebrain (BFmc), but not the thalamus, are indispensable for maintaining cortical activation and wakefulness18, 19, we hypothesized that GABAergic PZ neurons might promote SWS and SWA by inhibiting the wake–promoting PB–BFmc–cortex circuit. In other words, we predict that PZ GABAergic neurons monosynaptically project to and produce inhibitory postsynaptic events in PB neurons that specifically and monosynaptically innervate neurons of the BFmc. To test this hypothesis, we injected a cre–dependant vector containing channelrhodopsin–2 (DIO–ChR2(H134R)–mCherry–AAV; ChR2–mCherry) into the PZ and retrograde fluorescent microspheres were injected into the ipsilateral BFmc of Vgat– ires–cre mice (Fig. 5a). Histological assessment confirmed accurate bead placement in the BF (Fig. 5b–c) and mCherry–positive somas restricted to the GABAergic PZ (Fig. 5e–f). Photo– stimulation of PZVgat cell bodies expressing ChR2–mCherry elicited robust photocurrents and trains of brief blue–light flashes entrained the firing of PZVgat neurons up to 5 Hz (Supplementary Fig. 7). To determine whether activation of PZ axons evoked GABA release in the PB, we photostimulated PB slices containing ChR2–mCherry expressing axons that originated from PZVgat neurons (Fig. 5d). The flashes of blue light evoked fast inhibitory postsynaptic currents (IPSCs) in PB neurons that were retrogradely labeled from the BFmc (n = 7/18 neurons). The light stimuli induced synaptic events of amplitude comparable to spontaneous IPSCs (Fig. 5h), indicating that the responses were in the physiological range. Moreover, based upon the rapid kinetics, both the spontaneous and photo–evoked responses occurred in the soma and proximal dendrites of the cell. The photo–evoked IPSCs were also completely abolished by bicuculline (20 μM, Fig. 5g) indicating that these responses were mediated by the release of GABA and by the activation of GABAA postsynaptic receptors. In BFmc–projecting PB neurons that exhibited synaptic responses (PZVgat→PB) the probability of any given light pulse evoking a synchronous IPSC was 58.0 ± 5.6% (n = 7, Fig. 5i–j). Three stimuli were used to show that 1) the response was consistent across stimulation, and 2) no depression occurred following repeated stimulation, i.e., individual synaptic response had equivalent amplitudes. The photo–evoked IPSCs in PB neurons projecting to BFmc had an average onset delay of 6.2 ± 0.3 ms, a peak amplitude of 23.1 ± 3.2 pA, and a charge transfer of 1.02 ± 0.21 pC (single light pulses; n = 7). Photostimulation evoked IPSC’s in PB neurons even in the presence of tetrodotoxin (TTX; n = 4; Fig. 5k) supporting a direct synaptic connectivity from PZ to PB (PZVgat→PB→BFmc).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Division of Sleep Medicine, Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

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.

Show MeSH
Related in: MedlinePlus