GABAA receptor-mediated input change on orexin neurons following sleep deprivation in mice
Introduction
Orexins (orexin A and B, also known as hypocretin-1 and hypocretin-2, respectively) are bioactive peptides, which are produced exclusively in the lateral hypothalamus (LH) in mammalian brains and which have been shown to play a critical role in regulating vigilance state transitions (see reviews: (Sakurai, 2007, Sinton, 2011)). The importance of orexin in the maintenance of consolidated bouts of sleep and wakefulness has been convincingly demonstrated by the fact that the sleep disorder narcolepsy with cataplexy is caused by a deficiency in orexin or orexin-producing neurons (orexin neurons) in humans and animals (Chemelli et al., 1999, Nishino et al., 2000, Thannickal et al., 2000). The activity of orexin neurons is increased during wakefulness and decreased during sleep (Lee et al., 2005, Takahashi et al., 2008). Furthermore, acute optogenetic activation of orexin neurons enhances the probability of a transition from sleep to wakefulness (Adamantidis et al., 2007), and chronic stimulation or inhibition of orexin neurons leads to a corresponding alteration in the vigilance state (Sasaki et al., 2011). Orexin neurons receive multiple afferents from several brain regions, including the limbic system, preoptic area, and monoaminergic neurons, and in vitro studies show that the activity of orexin neurons is regulated by several neuropeptides and neurotransmitters (Yoshida et al., 2006, Sakurai and Mieda, 2011). In summary, although the activity of orexin neurons is known to affect the continuity of sleep and wakefulness states, the influence of prolonged wakefulness on the properties of orexin neurons has not been characterized.
γ-Aminobutyric acid (GABA) is the primary and most abundant inhibitory neurotransmitter in the central nervous system (CNS). Sleep-active, GABAergic neurons in the preoptic area/anterior hypothalamus, in addition to local GABAergic interneurons, densely innervate the LH, including direct innervation of orexin neurons (Steininger et al., 2001, Uschakov et al., 2006, Hassani et al., 2010). Endogenous GABA release increases in the LH area during non-rapid eye movement (NREM) sleep (Nitz and Siegel, 1996, Alam et al., 2010). GABA affects sleep regulation via two pharmacologically distinct receptors, the GABAA receptor (GABAAR) and GABAB receptor (GABABR) (Möhler, 2010). In a previous study, we used genetically modified mice to determine the role of the GABABR on orexin neurons in sleep regulation (Matsuki et al., 2009). The specific deletion of GABABRs on orexin neurons led to an instability of orexin neuronal activity and an increment in membrane conductance, resulting in severe fragmentation of vigilance states. However, the role of a GABAAR-mediated inhibitory input on orexin neurons is poorly characterized. GABAARs are pentameric hetero-oligomers and constitute ligand-gated chloride channels (Olsen and Sieghart, 2008). GABAAR subunits are encoded by 19 different genes that have been grouped into eight subclasses based on sequence homology (α1–6, β1–3, γ1–3, δ, ε, θ, π, ρ1–3). The pentameric assembly of these subunits constitutes the functional GABAAR and is dependent on the regional distribution and cell-type-specific expression of each subunit in the CNS (Fritschy and Möhler, 1995, Pirker et al., 2000). Subunits of GABAARs on basal forebrain cholinergic neurons (Modirrousta et al., 2007), and on neurons in the perifornical hypothalamus (Volgin and Kubin, 2007, Volgin et al., 2014) are up-regulated following sleep deprivation (SD), indicating that GABAARs likely play a role in the homeostatic regulation of sleep. However, the molecular function and dynamics of GABAARs on identified orexin neurons under these conditions are unknown.
In this study, we examined the molecular and functional alterations of GABAARs on orexin neurons following SD, i.e., after an increase in homeostatic sleep pressure.
Section snippets
Mice
All experimental procedures involving animals were approved by the Animal Experiment and Use Committee of the University of Tsukuba and were performed strictly in accordance with “Guidelines for proper conduct of animal experiments”, from the Science Council of Japan. Orexin-enhanced green fluorescent protein (eGFP) transgenic mice on a BDF1 background (Yamanaka et al., 2003) were maintained on a 12-h light/dark cycle (lights on from 9:00 A.M. [zeitgeber time: ZT0] to 9:00 P.M. [ZT12]). All
Changes in the expression of GABAARs on orexin neurons after sleep deprivation
To study the alteration in GABAergic input via GABAARs on orexin neurons following SD, we sleep deprived adult orexin-eGFP transgenic mice for 6 h. Control group mice were allowed Ad lib sleep during this period. Immunohistochemically, we studied the expression of several GABAAR subunits on orexin neurons, as well as molecules involved in GABAAR trafficking and inhibitory synapse specialization. Based on previous reports that GABAAR α1, α2, β2, β3, and γ2 subunits are highly expressed in the LH (
Discussion
The orexin system is crucial for transitions between vigilance states and thus for maintaining uninterrupted periods of wakefulness and sleep (Sakurai, 2007). In this study, we have demonstrated, both immunohistochemically and functionally, that prolonged wakefulness affects the GABAergic modulation of orexin neurons through changes in GABAARs.
Conclusion
In this study, we have described a significant change to the input properties of orexin neurons during SD. This change has functional effects and indicates that orexin neurons respond to extended wakefulness with a relatively rapid change to GABAAR subunits that will favor the maintenance of more prolonged sleep bouts once sleep onset occurs. GABAARs on orexin neurons will therefore contribute to at least one of the mechanisms by which orexin modulates vigilance state transitions.
Conflict of interest
The authors declare that no competing interests exist.
Author contributions
T.Ma., M.T., Y.H., T.Mo., and M.Y. designed the research. T. Ma., M.T., and Y.H. performed the experiments and analyzed data. T.Ma., N.M., and M.Y. contributed to analytic tools. T.Ma., C.M.S., T.Mo., and M.Y. analyzed data and wrote the paper.
Acknowledgements
This study is supported by the Cabinet Office, Government of Japan through the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) (M.Y.), the Ministry of Education, Culture, Sports, Science and Technology, World Premier International Research Center Initiative (M.Y.), and the Perot Family Foundation (M.Y.). M.Y. is a former Investigator of the Howard Hughes Medical Institute.
References (43)
- et al.
GABAergic regulation of the perifornical/lateral hypothalamic neurons during non-rapid eye movement sleep in rats
Neuroscience
(2010) - et al.
Circadian and homeostatic regulation of structural synaptic plasticity in hypocretin neurons
Neuron
(2010) - et al.
Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation
Cell
(1999) - et al.
Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2
Neuron
(2007) - et al.
Neurexin–neuroligin signaling in synapse development
Curr Opin Neurobiol
(2007) - et al.
Rapid synaptic scaling induced by changes in postsynaptic firing
Neuron
(2008) - et al.
Huntingtin-associated protein-1 deficiency in orexin-producing neurons impairs neuronal process extension and leads to abnormal behavior in mice
J Biol Chem
(2010) - et al.
Hypocretin (orexin) deficiency in human narcolepsy
Lancet
(2000) - et al.
GABAA receptors: immunocytochemical distribution of 13 subunits in the adult rat brain
Neuroscience
(2000) - et al.
Neuroligin 2 drives postsynaptic assembly at perisomatic inhibitory synapses through gephyrin and collybistin
Neuron
(2009)
Connectomics of orexin-producing neurons: interface of systems of emotion, energy homeostasis and arousal
Trends Pharmacol Sci
Orexin/hypocretin plays a role in the response to physiological disequilibrium
Sleep Med Rev
Neuronal activity of orexin and non-orexin waking-active neurons during wake–sleep states in the mouse
Neuroscience
Reduced number of hypocretin neurons in human narcolepsy
Neuron
Neuroligin 2 is exclusively localized to inhibitory synapses
Eur J Cell Biol
Regionally selective effects of GABA on hypothalamic GABAA receptor mRNA in vitro
Biochem Biophys Res Commun
Synaptic plasticity in sleep: learning, homeostasis and disease
Trends Neurosci
Hypothalamic orexin neurons regulate arousal according to energy balance in mice
Neuron
Short-term sleep deprivation increases intrinsic excitability of prefrontal cortical neurons
Brain Res
Neural substrates of awakening probed with optogenetic control of hypocretin neurons
Nature
Sleep homeostasis modulates hypocretin-mediated sleep-to-wake transitions
J Neurosci
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Present address: Development & Medical Affairs Division, GlaxoSmithKline K.K., Tokyo 151-8566, Japan.