Ectopic expression of melanopsin in orexin/hypocretin neurons enables control of wakefulness of mice in vivo by blue light
Highlights
► hOPN4 was expressed in hypothalamic orexin/hypocretin neurons in the double-transgenic mice. ► hOPN4-expressing orexin neurons showed long-lasting activation by blue light. ► Blue light stimulation in the hypothalamus initiated wakefulness in the mice in vivo.
Introduction
Growing optogenetic approaches to neuroscience, channelrhodopsin and other optogenetic tools are emerging (Berndt et al., 2011, Boyden et al., 2005, Chow et al., 2010, Zhang et al., 2006). Channelrhodopsin was originally discovered in a non-mammalian model organism, Chlamydomonas reinhardtii, and it has been shown to directly form a channel that carries cationic ions (Nagel et al., 2002, Nagel et al., 2003, Wang et al., 2009). In contrast, melanopsin (OPN4) is a photosensitive G-protein-coupled photopigment (Hatori and Panda, 2010, Sexton et al., 2012) that was originally found in a subtype of mammalian retinal ganglion cells (Hattar et al., 2002). OPN4 can be activated by blue light, and it is coupled with Gq-type G protein to open subtypes of transient receptor potential (TRP) channels and regulate cell excitability for several tens of seconds (Sexton et al., 2012). By this machinery, OPN4 shows several characteristic features: high photosensitivity, regulation of intracellular Ca2+ dynamics, and long activation kinetics (Koizumi et al., 2013). In fact, it was shown that ectopic expression of OPN4 in non-melanopsin retinal ganglion cells successfully restored vision with higher photosensitivity and longer activation kinetics (Lin et al., 2008). The aim of the present study was to express OPN4 ectopically in hypothalamic orexin/hypocretin neurons to control its excitability for a long duration.
Orexins/hypocretins are neuropeptides expressed in a specific population of neurons in the lateral hypothalamic area (LHA) (de Lecea et al., 1998, Sakurai et al., 1998). Animals that lack these peptides, orexin neurons, or functional orexin-2 receptors show phenotypes similar to that of the sleep disorder narcolepsy (Chemelli et al., 1999, Hara et al., 2001, Lin et al., 1999, Willie et al., 2003), that is, sleep/wake fragmentation and cataplexy. It has been shown that orexin neurons fire phasically during active wakefulness and are silent during slow wave sleep (SWS) (Lee et al., 2005, Mileykovskiy et al., 2005). These findings indicate that the orexin system has a critical role in the regulation of sleep/wakefulness, especially in the maintenance of arousal. Optogenetical approaches to regulate sleep/wakefulness cycles have already been conducted by expressing channelrhodopsin-2 (ChR2) to activate orexin neurons (Adamantidis et al., 2007, Carter et al., 2009) and by expressing halorhodopsin to suppress activity of these neurons (Tsunematsu et al., 2011). In the present study, we successfully demonstrated that blue light photostimulation activated orexin neural activity in vitro and initiated the electroencephalography (EEG) pattern that reflects long-lasting wakefulness in vivo for several tens of seconds by ectopic expression of OPN4 in orexin neurons. In contrast to ChR2, OPN4 can be activated for a long period by just a short pulse of blue light. Once OPN4 has been activated, the cells maintain their activity for several tens of seconds even after the blue light has been switched off.
Section snippets
Animal usage
All experimental procedures involving the transgenic animals described in the results section were approved by the National Institute for Physiological Sciences Animal Care and Use Committee and were in accordance with NIH guidelines. All efforts were made to minimize animal suffering or discomfort and to reduce the number of animals used.
Generation of the double-transgenic mice line (orexin-tTA; Bitet-O hOPN4/mCherry mice)
As described in the results section, we crossed orexin-tTA mice (line G5, Tabuchi et al., manuscript in submission) with Bitet-O hOPN4/mCherry mice to obtain
OPN4 expression in orexin neurons of the Orexin-tTA; Bitet-O hOPN4/mCherry double-transgenic mice
First, we established a double-transgenic mice line, orexin-tTA; Bitet-O hOPN4/mCherry mice, derived by crossing orexin-tTA mice with Bitet-O hOPN4/mCherry mice to obtain the expression of human OPN4 (hOPN4) specifically in orexin neurons. As shown in Fig. 1A and B, we used a tetracycline-controlled gene expression system. Briefly, the tTA protein, which is driven by the human prepro-orexin promoter, binds on the tet-O sequence (3.2 kb). Then tTA bi-directionally induces gene expression (mCherry
Discussion
In the present study, we successfully expressed hOPN4 in orexin neurons of orexin-tTA; Bitet-O hOPN4/mCherry double-transgenic mice. A short blue light pulse successfully excited orexin neurons in vitro and initiated the EEG pattern that reflects long-lasting wakefulness from SWS in vivo. We confirmed that activation of orexin neurons using OPN4 induced wakefulness in mice. This result shows good agreement with the previous reports that optogenetic photostimulation of orexin neurons via a ChR2
Acknowledgements
We thank Dr. Satoru Moritoh for his technical support. This study was financially supported by a Grant-in-Aid for challenging Exploratory Research (AY, AK) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This study was also supported by the Takeda Foundation (AY, KFT, AK) and by the Yamada Foundation (AK, KFT).
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