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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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Related in: MedlinePlus

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.
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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.

Mentions: To test the ability of GABAergic PZ neurons to initiate SWS in behaving animals, we placed bilateral injections of an adeno–associated viral (AAV) vector containing an excitatory modified muscarinic G protein–coupled receptor (DIO–hM3Dq–mCherry–AAV10; Fig. 1b) expressed in a cre–dependent manner into the PZ (Fig. 1a–b,f–g and Supplementary Fig. 1) of Vgat–IRES–cre mice14 (Fig. 1c–e) and non–cre expressing littermates. Robust cell–surface expression of the hM3Dq receptors was observed on GABAergic PZ neurons of Vgat–IRES–cre mice (Fig. 1f–g) but was absent in non–cre–expressing littermates, confirming the requirement for cre activity to enable hM3Dq expression within PZ GABAergic neurons. Injections of the hM3Dq agonist clozapine–N–oxide [CNO 0.3mg/kg, intraperitoneal (IP)], which is otherwise pharmacologically inert, also drove c–Fos expression in hM3Dq–expressing GABAergic neurons in the PZ (Fig. 1h), confirming ligand–induced activation of hM3Dq–expressing GABAergic PZ neurons in vivo. CNO–driven depolarization and firing of hM3Dq–expressing GABAergic PZ neurons was also confirmed in whole cell, current clamp recordings (Fig. 1i). Importantly, in baseline condition, we observed no differences between Vgat–IRES–cre mice expressing hM3Dq in GABAergic PZ neurons and non–hM3Dq–expressing littermate mice in hourly sleep–wake or the EEG power spectra (Supplementary Fig. 2 and Supplementary Table 1), demonstrating that, in the absence of the ligand (CNO), hM3Dq receptors are without effect on sleep–wake parameters. Similarly, IP injections of CNO were without significant effect on these same parameters in non–hM3Dq–expressing mice (Supplementary Fig. 3 and Supplementary Table 2), indicating that electrographic or physiologic changes observed in hM3Dq–expressing mice following CNO administration is directly linked to the activation of PZ GABAergic neurons.


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: To test the ability of GABAergic PZ neurons to initiate SWS in behaving animals, we placed bilateral injections of an adeno–associated viral (AAV) vector containing an excitatory modified muscarinic G protein–coupled receptor (DIO–hM3Dq–mCherry–AAV10; Fig. 1b) expressed in a cre–dependent manner into the PZ (Fig. 1a–b,f–g and Supplementary Fig. 1) of Vgat–IRES–cre mice14 (Fig. 1c–e) and non–cre expressing littermates. Robust cell–surface expression of the hM3Dq receptors was observed on GABAergic PZ neurons of Vgat–IRES–cre mice (Fig. 1f–g) but was absent in non–cre–expressing littermates, confirming the requirement for cre activity to enable hM3Dq expression within PZ GABAergic neurons. Injections of the hM3Dq agonist clozapine–N–oxide [CNO 0.3mg/kg, intraperitoneal (IP)], which is otherwise pharmacologically inert, also drove c–Fos expression in hM3Dq–expressing GABAergic neurons in the PZ (Fig. 1h), confirming ligand–induced activation of hM3Dq–expressing GABAergic PZ neurons in vivo. CNO–driven depolarization and firing of hM3Dq–expressing GABAergic PZ neurons was also confirmed in whole cell, current clamp recordings (Fig. 1i). Importantly, in baseline condition, we observed no differences between Vgat–IRES–cre mice expressing hM3Dq in GABAergic PZ neurons and non–hM3Dq–expressing littermate mice in hourly sleep–wake or the EEG power spectra (Supplementary Fig. 2 and Supplementary Table 1), demonstrating that, in the absence of the ligand (CNO), hM3Dq receptors are without effect on sleep–wake parameters. Similarly, IP injections of CNO were without significant effect on these same parameters in non–hM3Dq–expressing mice (Supplementary Fig. 3 and Supplementary Table 2), indicating that electrographic or physiologic changes observed in hM3Dq–expressing mice following CNO administration is directly linked to the activation of PZ GABAergic neurons.

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