1. Volume 46, Issue 2, Pages 297-308 (April 2005)
Input of Orexin/Hypocretin Neurons Revealed by a Genetically Encoded Tracer in Mice 
Takeshi Sakurai, Ruby Nagata, Akihiro Yamanaka, Hiroko Kawamura, Natsuko Tsujino, Yo Muraki, Haruaki Kageyama, Satoshi Kunita, Satoru Takahashi, Katsutoshi Goto, Yoshimasa Koyama, Seiji Shioda, Masashi Yanagisawa 
Neuron 
Volume 46, Issue 2, Pages (April 2005)
DOI: /j.neuron
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2. Figure 1
Transgenic Mice Expressing TTC::GFP Fusion Protein in Orexin Neurons
(A) Structure of orexin/TTC::GFP transgene. SP, human ICAM signal sequence; TTC, tetanus toxin C-terminal fragment; mPrmI, murine protamine-1 gene fragment containing 3′ half of exon 1, intron 1, and exon 2.
(B) Expression of TTC::GFP fusion protein by orexin-containing neurons in LHA of orexin/TTC::GFP transgenic mice, shown by double-label immunofluorescence. (Left panel) GFP-like immunoreactivity (GFP-ir) (green). (Middle panel) Orexin-like immunoreactivity (orexin-ir) (red). (Right panel) Merged image. Lower panels show high-power views of region shown in yellow rectangles in corresponding upper panels. Arrows show local interneurons, which are positive for GFP-ir and negative for orexin-ir. Scale bar, 50 μm.
(C) Colocalization of β-galactosidase and orexin-ir in the LHA of orexin/TTC::GFP transgenic mice shown by double labeling by β-galactosidase staining (blue) and orexin-ir (brown). Because these two transgenes were integrated in the same locus and driven by the same promoter, we could monitor the site of transgene expression by β-galactosidase staining. The β-galactosidase staining of brains of orexin/TTC::GFP lines showed that the transgene was eutopically expressed by orexin neurons. The right panel shows a high-power view of the region shown in the yellow rectangle in the left panel. Scale bar, 50 μm.
(D) In situ hybridization histochemistry showing that the transgene mRNA is exclusively localized in the LHA. Coronal sections of the transgenic mouse brains hybridized with digoxigenin-labeled antisense probe for GFP. (Left panel) Low-power field. (Right panel) Higher-power field of the regions shown by yellow rectangle in the left panel. No signal beyond background was generated by sense probe (data not shown). We did not detect any signals outside the LHA in the all transgenic lines.
Neuron  , DOI: ( /j.neuron )
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3. Figure 2
Retrograde Transfer of TTC::GFP Fusion Protein from Cell Bodies to Synaptic Terminals Revealed by Immunoelectron Microscopy Using Anti-GFP Antiserum
(A) Electron micrograph showing a GFP-ir cell body receiving a synapse from a GFP-ir axon terminal. Perinuclear vesicle-like structures are intensely immunostained in the cell body (expression origin). The cytoplasm in a synaptic terminal making a synapse on the GFP-ir soma also contains strongly immunoreactive vesicles. Pre- and postsynaptic membranes are both strongly stained, showing that the fusion protein is transferred trans-synaptically.
(B) Schematic drawing of (A) Mt, mitochondria. Scale bar, 1 μm.
(C and D) High-power views of regions shown by rectangles in (A). Arrowhead shows the synapse structure. Arrows show immunostained vesicle-like structures.
Neuron  , DOI: ( /j.neuron )
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4. Figure 3 Distribution of Afferent Cells to Orexin Neurons
Schematic drawings of localization of TTC::GFP fusion protein from rostral (A) to caudal (O) in transgenic mouse brain rostrocaudal coronal sections revealed by immunohistochemical staining using anti-GFP antiserum. Blue stars, orexin neurons. Red dots, weakly or moderately stained cells. Red squares, strongly stained cell bodies. 7N, facial nucleus; 8vn, vestibular root of vestibulocochlear nerve; 10N, dorsal motor nucleus of vagus; aci, anterior commissure, intrabulbar; aca, anterior commissure, anterior part; AcbSh, accumbens nucleus, shell region; Aco, anterior cortical amygdaloid nucleus; AHP, anterior hypothalamic area; Arc, arcuate hypothalamic nucleus; BLA, basolateral amygdaloid nucleus; BMA, basomedial amygdaloid nucleus, anterior part; BMP, basomedial amygdaloid nucleus, posterior part; BST; bed nucleus of stria terminalis; C1, C1 adrenaline cells; Ce, central amygdaloid nucleus; cp, cerebral peduncle; D3, dorsal third ventricle; DG, dentate gyrus; DM, dorsomedial hypothalamic nucleus; DR, dorsal raphe nucleus; f, fornix; HDB, nucleus of horizontal limb of diagonal band; IL, infralimbic cortex; IS, inferior salivatory nucleus; LA, lateroanterior hypothalamic nucleus; LC, locus coeruleus; LDT, laterodorsal tegmental nucleus; LH, lateral hypothalamus; LHb, lateral habenular nucleus; lo, lateral olfactory tract; LRt, lateral reticular field; LV, lateral ventricle; m5, motor root of trigeminal nerve; MCPO, magnocellular preoptic nucleus; ml, medial lemniscus; MiTg, microcellular tegmental nucleus; mlf, medial longitudinal fasciculus; MnR, median raphe nucleus; Mo5, motor tegmental nucleus; MPO, medial preoptic area; MS, medial septal nucleus; mt, mammillothalamic tract; MTu, medial tuberal nucleus; NTS, nucleus of solitary tract; PAG, periaqueductal gray PH, posterior hypothalamus; Pir, piriform cortex; PMCo, posteromedial cortical amygdaloid nucleus; PPT, pedunculopontine tegmental nucleus; PrL, prelimbic cortex; py, pyramidal tract; RMg, raphe magnus nucleus; Rpa, raphe pallidus nucleus; Sch, suprachiasmatic nucleus; SNC, substantia nigra, compact part; SNR, substantia nigra, reticular part; SO, supraoptic nucleus; sol, solitary tract; sp5, spinal trigeminal tract; st, stria terminalis; SuM, supramammillary nucleus; TMN, tuberomammillary nucleus; Tu, olfactory tubercle; VDB, nucleus of vertical limb of diagonal band; VLPO, ventrolateral preoptic area; VMH, ventromedial hypothalamic nucleus; VTA, ventral tegmental area.
Neuron  , DOI: ( /j.neuron )
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5. Figure 4 Cholinergic Neurons Send Input to Orexin Neurons
GFP-positive neurons in cholinergic cells in orexin/TTC::GFP transgenic mouse brains are demonstrated by immunohistochemical staining using anti-GFP antiserum. Scale bars, 50 μm. (A) Coronal brain section at approximately bregma +1.1 mm stained with anti-GFP antibody. (B) High-power view of region shown in yellow rectangle in (A). Several strongly stained cells are observed in the vertical and horizontal limb of the diagonal band nucleus (VDB and HDB). (C) Coronal brain section at approximately bregma −0.7 mm stained with anti-GFP antibody. (D) High-power view of region shown in yellow rectangle in (C). The basal nucleus of Meynert contains many strongly stained cells. BLA, basolateral amygdaloid nucleus; BMA, basomedial amygdaloid nucleus; opt, optic tract. (E) Colocalization of GFP-ir and choline acetyl transferase (ChAT)-ir in transgenic mouse. (Left panels) GFP-positive neurons are stained with green fluorescence (Alexa 488). (Middle panels) ChAT-positive cells are stained with red fluorescence (Alexa 594). (Right panels) Merged image. Colocalization of GFP-ir and ChAT-ir in VBD/HBD region, basal nucleus of Meynert, LDT, and DMH is shown. The number of GFP-ir cells in the LDT/PPT was very small, but these cells showed ChAT-ir (shown by arrow).
Neuron  , DOI: ( /j.neuron )
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6. Figure 5 Cholinergic Effects on Orexin Neurons
Effects of a muscarinic agonist, carbachol, on GFP-expressing orexin neurons in hypothalamic slice preparations from orexin/EGFP transgenic mice (Yamanaka et al., 2003a; Yamanaka et al., 2003b). (A and B) The effect of carbachol was analyzed in current-clamp mode in the absence (upper panels) or presence (lower panels) of tetrodotoxin (1 μM). Orexin neurons showed a variety of responses to carbachol: 27% showed depolarization (A), 6% showed hyperpolarization (B), and 67% showed no effect (data not shown). (C) Relationship between peak inward current (pA) (mean ± SD) and concentration of carbachol (n = 10) in orexin neurons in which carbachol evoked an excitatory effect. Holding potential (Vh) was clamped at −60 mV. (D) Relationship between peak outward current (pA) (mean ± SD) and dose of carbachol (n = 10) in orexin neurons in which carbachol evoked an inhibitory effect. Holding potential (Vh) was −60 mV. (E) Distribution of orexin neurons showing depolarization (excitatory; red circles), hyperpolarization (inhibitory; green circles), or no effect (blue circles) of carbachol application during current clamp, analyzed in several successive slice preparations from the rostral, middle, or caudal region of the hypothalamus.
Neuron  , DOI: ( /j.neuron )
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7. Figure 6 Neurons in POA Send Input to Orexin Neurons
(A) Preoptic area of transgenic mouse brain section at approximately bregma mm stained with anti-GFP antibody. MPO, medial preoptic area; LPO, lateral preoptic area.
(B) Ventrolateral preoptic area (VLPO) contains several cells densely labeled with anti-GFP antiserum. Scale bar, 50 μm.
(C) Double-immunofluorescence analysis showing that most GFP-positive neurons in the VLPO region contain GABA-ir. (Left panel) GFP-ir neurons in VLPO region in transgenic mouse brain. (Middle panel) GABA-ir observed in same brain section. Scale bar, 50 μm. (Right panel) Merged image.
Neuron  , DOI: ( /j.neuron )
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8. Figure 7
Immunohistochemical Study Showing that Serotonergic Cells in MnR Send Input to Orexin Neurons
(A) Median raphe nucleus of coronal section of transgenic mouse brain at approximately bregma −4.4 mm stained with anti-GFP antibody.
(B) High-power view of region shown in yellow rectangle in (A). Scale bar, 50 μm.
(C) (Upper panels) Colocalization of GFP-ir and 5-HT-ir in the median raphe (MnR). (Left panels) GFP-positive neurons are stained with green fluorescence (Alexa 488). (Middle panels) 5-HT-positive cells are stained with red fluorescence (Alexa 594). (Right panels) Merged image. Arrows show examples of 5-HT-positive cells that also contain GFP-ir. (Lower panels) GFP-ir cells were not observed in DR, although amorphous GFP-ir staining was observed in this region. Scale bars, 50 μm. Aq, aqueduct.
Neuron  , DOI: ( /j.neuron )
Copyright © 2005 Elsevier Inc. Terms and Conditions

9. Figure 8
Schematic Drawing Showing Major Input and Output Systems of Orexin Neurons in the Network Implicated in the Regulation of Sleep/Wakefulness States
Acb, nucleus accumbens; Amyg, amygdala; Arc, arcuate nucleus; BF, basal forebrain; BST, bed nucleus of the stria terminalis; PH, posterior hypothalamus; POA, preoptic area; TMN, tuberomammillary nucleus; LC, locus coeruleus; DR, dorsal raphe; MnR, median raphe; LDT, laterodorsal tegmental nucleus. Red lines show efferents, while blue lines show afferents of orexin neurons. Purple lines show other relevant neuronal pathways between important brain regions for sleep/wakefulness regulation. During wakefulness, innervation by cholinergic cells in the basal forebrain excites orexin neurons. Orexin neurons activate these cholinergic neurons, resulting in a positive feedback loop that reinforces their activity. Orexin neurons might be also activated by the influences from the amygdala and the BST during wakefulness. Orexin neurons send excitatory innervation to monoaminergic cells in the brainstem, including LC, DR, and TMN, and thereby elicit cortical arousal. Serotonergic cells in the DR and noradrenergic cells in the LC send inhibitory projections to POA sleep-active cells to further maintain wakefulness. Serotonergic neurons in the MnR send inhibitory projections to orexin neurons; this innervation may act as a negative feedback system that limits activity of orexin neurons appropriately during wakefulness. During the sleep period, POA sleep-active cells send inhibitory projections to cholinergic cells and orexin neurons to silence these cells, which results in decreased activity of monoaminergic neurons in the brainstem.
Neuron  , DOI: ( /j.neuron )
Copyright © 2005 Elsevier Inc. Terms and Conditions