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Dissociating orexin-dependent and -independent functions of orexin neurons using novel Orexin-Flp knock-in mice
Srikanta Chowdhury,
Chi Jung Hung,
Shuntaro Izawa,
Ayumu Inutsuka,
Meiko Kawamura,
Takashi Kawashima,
Haruhiko Bito,
Itaru Imayoshi,
Manabu Abe,
Kenji Sakimura,
Akihiro Yamanaka ,
Nagoya University, Japan;
CREST, JST, Honcho Kawaguchi, Japan;
Niigata University, Japan;
The University of Tokyo, Japan;
Kyoto University, Japan;
Srikanta Chowdhury
Chi Jung Hung
Shuntaro Izawa
Ayumu Inutsuka
Meiko Kawamura
Takashi Kawashima
Haruhiko Bito
Itaru Imayoshi
Manabu Abe
Kenji Sakimura
Akihiro Yamanaka
Uninterrupted arousal is important for survival during threatening situations. Activation of orexin/hypocretin neurons is implicated in sustained arousal. However, orexin neurons produce and release orexin as well as several co-transmitters including dynorphin and glutamate. To disambiguate orexin-dependent and -independent physiological functions of orexin neurons, we generated a novel Orexin-flippase (Flp) knock-in mouse line. Crossing with Flp-reporter or Cre-expressing mice showed gene expression exclusively in orexin neurons. Histological studies confirmed that orexin was knock-out in homozygous mice. Orexin neurons without orexin showed altered electrophysiological properties, as well as received decreased glutamatergic inputs. Selective chemogenetic activation revealed that both orexin and co-transmitters functioned to increase wakefulness, however, orexin was indispensable to promote sustained arousal. Surprisingly, such activation increased the total time spent in cataplexy. Taken together, orexin is essential to maintain basic membrane properties and input-output computation of orexin neurons, as well as to exert awake-sustaining aptitude of orexin neurons.
Orexin A (hypocretin-1) and orexin B (hypocretin-2) (de Lecea et al., 1998; Sakurai et al., 1998), generated from the same precursor protein called prepro-hypocretin (encoded by hypocretin gene), are endogenous ligands for two closely related G-protein-coupled receptors termed orexin-1 receptor (OX1R) and orexin-2 receptor (OX2R) (Sakurai et al., 1998). Although orexin was named for its effect on inducing feeding behavior, it gained immense interest in sleep research as the knockout (KO) of prepro-orexin or dysfunction of OX2R in canines or mice reportedly mimics the human sleep disorder narcolepsy (Chemelli et al., 1999; Lin et al., 1999). Narcolepsy is a neurological disorder characterized by fragmented sleep/wakefulness, persistent daytime sleepiness and brief episodes of muscle weakness called cataplexy, which is often triggered by positive emotions. Narcolepsy patients showed a loss of overall hypocretin mRNA, confirming the association between orexin neuronal loss and the pathogenesis of narcolepsy (Peyron et al., 2000; Thannickal et al., 2000).
A small number of orexin-producing neurons (orexin neurons) are exclusively distributed in the lateral hypothalamic area (LHA) and perifornical area, but send projections widely throughout the major brain areas (Nambu et al., 1999; Peyron et al., 1998). Thus, it is no surprise that orexin neurons have diverse physiological roles, including the regulation of sleep/wakefulness (Chemelli et al., 1999; Lin et al., 1999), energy homeostasis (Yamanaka et al., 2003), thermoregulation (Tupone et al., 2011), as well as regulation of heart rate and blood pressure (Zhang et al., 2006). Using optogenetic and/or chemogenetic techniques, we and others showed that orexin neurons modulate sleep/wake cycles in rodents (Adamantidis et al., 2007; Sasaki et al., 2011; Tsunematsu et al., 2011). In addition to these, employing transgenic mice, we reported that chemogenetic activation of orexin neurons increased locomotion, feeding behavior and metabolism (Inutsuka et al., 2014). Thus, orexin neurons are thought to interact with the neuroregulatory, autonomic and neuroendocrine systems, and perform critical roles in the regulation of sleep/wakefulness and energy homeostasis.
The physiological activity of orexin neurons is modulated by multiple neural inputs and humoral factors. These inputs include GABAergic neurons in the preoptic area, serotonergic neurons in the dorsal and median raphe nuclei, central amygdala, basal forebrain cholinergic neurons, the bed nucleus of the stria terminalis, supraventricular zone, and the dorsomedial, lateral and posterior hypothalamus (Sakurai et al., 2005; Yoshida et al., 2006). Recently, we reported that serotonergic neurons in the raphe nucleus inhibit orexin neurons both directly and indirectly (Chowdhury and Yamanaka, 2016). Orexin neurons are also found to respond to multiple humoral factors and neuropeptides (Inutsuka and Yamanaka, 2013; Sakurai, 2014). Most interestingly, orexin neurons form a positive-feedback circuitry among themselves in the LHA by activating other orexin neurons through OX2R to maintain the wake-active network at optimum level and/or for a sustained period (Yamanaka et al., 2010).
However, orexin neurons contain other neurotransmitters including glutamate (Rosin et al., 2003), dynorphin (Chou et al., 2001) and galanin (Håkansson et al., 1999). Therefore, to better understand the physiology and pathophysiology of orexin/hypocretin system, it is essential to disambiguate the roles of orexin from other co-transmitters. To address this, we generated novel Orexin-flippase (Flp) knock-in (KI) mice, which express Flp recombinase under control of hypocretin gene in mice. Employing this novel mouse line, we found that orexin performs critical roles in maintaining basic electrophysiological properties of orexin neurons as well as to initiate feed-forward activation of orexin neurons by facilitating excitatory glutamatergic inputs. Focusing on sleep/wakefulness, we also manipulated orexin neuronal activity and evaluated their physiological effects in freely-moving mice. To achieve this, we employed a chemogenetic technique, Designer Receptors Exclusively Activated by Designer Drugs (DREADD) (Armbruster et al., 2007). Our results showed orexin plays a major role in the awake-sustaining aptitude of orexin neurons. Although the phenotypic consequences of orexin neurons activation in increased wakefulness were reinforced for by co-transmitters, these co-transmitters rather deteriorated cataplexy. Together, these data clearly identified the importance of orexin neuropeptides for the physiological function of orexin neurons and their postsynaptic partners.
Generation of novel Orexin-Flp (OF) mice
We previously generated Orexin-Cre (Inutsuka et al., 2014) or orexin-tetracycline-controlled trans-activator (Orexin-tTA) (Tabuchi et al., 2013) transgenic mice in which Cre recombinase or tTA was expressed in orexin neurons to allow regulation of transgene expression in the orexin neurons. However, expression of Cre or tTA in those transgenic mice was driven by a 3.2 kb short gene fragment upstream of the human hypocretin gene. Thus, we generated a new mouse by targeting the hypocretin gene of the mouse genome. Employing a homologous recombination system, here we generated Orexin-Flp (OF) mice by knocking in the EGFP-2A-Flp transgene just downstream of the translation initiation site of the hypocretin gene in-frame (Figure 1A). To visualize the function of Flp recombinase, we bred these mice with Flp-reporter (FSF-mTFP1) mice (Imayoshi et al., 2012) in which fluorescent protein mTFP1 is expressed in a Flp-dependent manner (Figure 1B). Immunohistochemical analysis showed mTFP expression in 80.9 ± 4.4% of orexin-immunoreactive neurons. We observed no ectopic expression of EGFP and/or mTFP1 protein in melanin-concentrating hormone (MCH)-immunoreactive (n = 4 mice; Figure 1C and D) or in any other non-orexin-immunoreactive neurons in these bigenic mice. In other words, all reporter gene-expressing neurons were orexin-immunoreactive (IR). Together, these data confirmed that functional Flp was expressed exclusively in the orexin neurons of newly generated OF mice.
Generation of OF mice.
(A) Schematic representations of the hypocretin gene, targeting vector, and targeted gene. To achieve orexin neuron-specific expression of Flp recombinase, we inserted EGFP-2A-Flp just behind the translation initiation site of the hypocretin gene in-frame. Viral T2A peptide is cleaved just after translation, and EGFP and Flp recombinase localize independently. DT, diphtheria toxin; Neo, neomycin-resistant gene expression cassette. (B) Structure of the reporter gene in the presence of Flp. Orexin-Flp; FSF-mTFP1 bigenic mice were generated to express mTFP1 in orexin neurons. (C) Representative pictures from coronal brain sections of Orexin-Flp; FSF-mTFP1 bigenic mice. Arrow indicates mTFP1 and/or EGFP expressing orexin neurons and the arrowhead indicates the position of the MCH neuron. Orexin-IR, orexin-immunoreactive; MCH-IR, MCH-immunoreactive. Inset scale bar: 20 µm. (D) Summary of the co-expression analysis (n = 4 mice). The p values were determined using one-way ANOVA test followed by a post-hoc Tukey analysis. Data represent the mean ± SEM.
Source data for Figure 1D.
OF mice enable the dual targeting of adjacent cell types
By taking advantage of the vast repertoire of cell type-specific Cre- or tTA-expressing mice, the new OF mice may enable the expression of various genes in different subsets of neurons (Figure 2A). To confirm this, we next generated two different bigenic mice by crossing OF mice with either MCH-Cre (encoded by Pmch gene) or Gad67-Cre (encoded by Gad1 gene) mice (Figure 2B and E). MCH-Cre or Gad67-Cre mice exclusively express Cre recombinase in MCH (Higo et al., 2009) or GABA neurons (Kong et al., 2010), respectively. To test whether we could target dual cell types simultaneously inside the LHA, we performed injection of an AAV cocktail composed of an equal volume of AAV(DJ)-CMV-FLEX-tdTomato and AAV(DJ)-CMV-dFRT-hrGFP in both Orexin-Flp; MCH-Cre and Orexin-Flp; Gad67-Cre bigenic mice (Figure 2C and F). Given that MCH and GABA neurons are distinct neuronal subsets from orexin neurons in the LHA (Broberger et al., 1998; Rosin et al., 2003), we expected a discrete expression of tdTomato and hrGFP in both bigenic mice. Indeed, we observed that Flp and Cre recombinase-driven fluorescent protein expression did occur in distinct populations (Figure 2D and G) in coronal brain sections (n = 3 mice for each). These data demonstrated that a broader range of opportunities for cell type-specific manipulation and/or activity recording is possible with OF mice.
Gene expression control using OF mice to target different cell types within the same brain region.
(A) Schematic showing Cre (left) and Flp (right) recombinase-dependent gene expression control. (B and E) The breeding scheme for Orexin-Flp; MCH-Cre and Orexin-Flp; Gad67-Cre bigenic mice, respectively. (C and F) Schematic drawings showing micro-injection of the AAV cocktail into the LHA of bigenic mice. (D and G) Representative coronal brain sections showing the segregated expression of tdTomato and hrGFP in a Cre and Flp recombinase-dependent manner, respectively.
OF (KI/KI) homozygote knockout orexin peptides
Since the EGFP-2A-Flp sequence was inserted in-frame at the start codon of the hypocretin gene using the KI method, we reasoned that KI/KI homozygous mice are essentially orexin KO mice. To confirm, immunohistochemical studies were conducted to compare the expression of EGFP, orexin and dynorphin in the LHA of heterozygous OF (KI/-) and homozygous OF (KI/KI) mice (Figure 3A and B). As expected, we found that EGFP expressing neurons were distributed in the LHA, however, orexin-immunoreactivity was absent in the LHA in OF (KI/KI) homozygous mice (Figure 3B). In heterozygous OF (KI/-) mice, 41.1 ± 5.2% of orexin-positive neurons were colocalized with EGFP (Figure 3—figure supplement 1B). This value was lower than results obtained from reporter mice (80.9 ± 4.4%), and is likely due to detection limitations of EGFP fluorescence due to low levels of expression. We stained every 4th slice of mouse brain containing LHA and counted 567 ± 25 orexin-positive and 673 ± 55 dynorphin-positive cells/animal in heterozygous mice (n = 3 mice). Among the counted neurons, 84.9 ± 4.4% of dynorphin-positive neurons co-expressed orexin (Figure 3—figure supplement 1A). However, while we counted 661 ± 15 dynorphin-positive cells/animal, we did not find any orexin-positive neurons in the homozygous mice (n = 3 mice, Hetero vs Homo, p=0.847 (dynorphin), p=2.4e-5 (orexin), unpaired t-test), showing that the number of dynorphin-positive cells were comparable to that in heterozygous animals. These immunohistochemical studies confirmed that orexin neurons in homozygous mice did not express orexin, however, expression of dynorphin was unaffected.
Immunohistochemical confirmation of OF mice.
(A and B) Representative coronal brain sections showing the expression of EGFP (green), orexin (red) and dynorphin (blue). GFP-IR, GFP-immunoreactive; Dynorphin-IR, Dynorphin-immunoreactive; Orexin-IR, orexin-immunoreactive.
Electrophysiological properties of orexin neurons lacking orexin
The expression of EGFP in the OF mice allowed us to visualize orexin neurons in the acute brain slice preparations. To this end, we sought to evaluate the importance of orexin neuropeptides in conserving the electrophysiological properties of orexin neurons. Therefore, we recorded and compared resting membrane potentials (Vrest), firing frequency, input resistance and capacitance in OF (KI/-), OF (KI/KI), and Orexin-EGFP (Tg/Tg) (OE) (Yamanaka et al., 2003) mice (Figure 4A–4E). Orexin neurons in the OF (KI/KI) mice were found to have significantly hyperpolarized membrane potential (−58.8 ± 1.2 mV; n = 21 cells) compared to OF (KI/-) mice (−51.6 ± 0.9 mV; n = 21 cells, p=1.0e-5) and OE mice (−50.4 ± 0.9 mV; n = 22 cells, p=3.5e-7; Figure 4A–i–4A-iv). Spontaneous firing frequency measured by cell-attached recordings were also found to be significantly lower in orexin neurons in OF (KI/KI) mice (1.5 ± 0.2 Hz; n = 25 cells) compared to OF (KI/-) mice (2.5 ± 0.3 Hz; n = 21 cells, p=0.015) and OE mice (2.6 ± 0.2 Hz; n = 25, p=0.004; Figure 4B–i–4B-iv). This lower discharge rate could be attributed to the hyperpolarized membrane potential of orexin neurons in homozygous mice. We next measured the input resistance of identified neurons by measuring the voltage deviation generated by current injection from −100 to +100 pA in the current clamp protocol. In mice lacking orexin peptide (OF (KI/KI) mice), orexin neurons were found to have significantly lower input resistance (485.9 ± 35.8 MΩ; n = 23 cells) compared to OF (KI/-) mice (639.9 ± 48.2 MΩ; n = 21 cells, p=0.037) and OE mice (638.2 ± 43.4 MΩ; n = 24 cells, p=0.032; Figure 4C–i–4C-iv). The lower input resistance in neurons lacking orexin peptides suggests that greater synaptic inputs (current injection) are necessary to generate changes in membrane potential. We also compared the membrane capacitance measured during whole-cell recordings. Interestingly, we observed that orexin neurons in OF (KI/KI) mice had significantly higher membrane capacitance (35.4 ± 1.3 pF; n = 25 cells) than OF (KI/-) mice (29.9 ± 1.5 pF; n = 25 cells, p=0.012) and OE mice (30.0 ± 1.3 pF; n = 25 cells, p=0.014, one-way ANOVA post-hoc Tukey; Figure 4E). This higher capacitance in orexin neurons that lack orexin peptides reflects the increased surface area of the plasma membrane. Taken together, these data clearly suggest that orexin neuropeptide is essential for maintaining the active and passive electrophysiological properties of orexin neurons.
Electrophysiological properties of orexin neurons with/without orexin.
(A-C) show representative traces recorded from OF (KI/-) (i), OF (KI/KI) (ii) and OE (iii) mice. (A) Membrane potential in whole-cell current clamp recordings. (B) Spontaneous firing in cell-attached recordings. (C) Step current injection-induced membrane potential changes. Panel iv is a summary of the data in panel i to iii. (D) Representative images showing EGFP expression in acute coronal brain slices during electrophysiological recording. (E) Cell capacitance from whole-cell current clamp recording. The p values were determined using one-way ANOVA test followed by a post-hoc Tukey analysis. Data represent the mean ± SEM.
Source data for Figure 4Aiv, 4Biv, 4Civ and 4E.
Orexin mediates feed-forward activation of orexin neurons via facilitation of glutamatergic inputs
To characterize whether the excitatory and inhibitory synaptic inputs onto orexin neurons are regulated by the presence or absence of orexin peptides, we recorded glutamatergic and GABAergic inputs using the voltage clamp method. We recorded spontaneous excitatory post-synaptic currents (sEPSCs) from EGFP-expressing orexin neurons in the presence of picrotoxin (400 μM), a GABA-A receptor antagonist. We found that orexin neurons lacking orexin protein had significantly lower sEPSC frequency while the amplitudes were unaffected (Figure 5A–5E). The average inter-event interval for all recorded sEPSCs of orexin neurons in OF (KI/KI) mice was 247.2 ± 48.6 ms (n = 29 cells) while that of OF (KI/-) mice was 116.7 ± 7.6 ms (n = 25 cells, p=0.01 vs OF (KI/KI)) and OE mice was 77.9 ± 7.4 ms (n = 26 cells, p=5.8e-4 vs OF (KI/KI), one-way ANOVA pot-hoc Tukey; Figure 5D). The sEPSC amplitude of orexin neurons in OF (KI/KI) mice was 22.6 ± 1.3 pA (n = 29 cells) while that of OF (KI/-) mice was 22.6 ± 1.3 pA (n = 25 cells, p=1.0 OF (KI/KI)) and in OE mice was 20.5 ± 1.1 pA (n = 26 cells, p=0.46 vs OF (KI/KI), one-way ANOVA post-hoc Tukey; Figure 5E).
Orexin neurons receive fewer glutamatergic inputs in the absence of orexin.
(A) Representative sEPSC traces recorded from EGFP-expressing neurons at a holding potential of −60 mV. (B-C) Cumulative probability plot for the representative traces shown in A. Bar diagrams in D and E summarize the sEPSC data. (D) Inter-event interval. (E) amplitude (n = 25–29 cells). F, Representative sIPSC traces recorded from EGFP-expressing neurons at a holding potential of −60 mV. (G-H) Cumulative probability plot for the representative traces shown in F. Bar diagrams in (I and J) summarize the sIPSC data. (G) Inter-event interval. (H) amplitude (n = 17–22 cells). The p values were calculated by one-way ANOVA followed by a post-hoc Tukey test. Data represent the mean ± SEM.
Source data for Figure 5D, E and I, J.
Next, we recorded spontaneous inhibitory post-synaptic currents (sIPSCs) from orexin neurons in the presence of CNQX (20 μM) and AP-5 (50 μM) to block glutamatergic inputs. Although sIPSCs in neurons lacking orexin peptides showed a tendency toward decreasing frequency and amplitude, these changes were not statistically significant. The inter-event interval for sIPSCs in orexin neurons in OF (KI/KI) mice was 835.0 ± 124.6 ms (n = 17 cells) while that of OF (KI/-) mice was 642.4 ± 70.9 ms (n = 22 cells, p=0.35 vs OF (KI/KI)) and in OE mice was 595.0 ± 96.7 ms (n = 21 cells, p=0.21 vs OF (KI/KI), one-way ANOVA post-hoc Tukey; Figure 5H). The sIPSC amplitude in orexin neurons in OF (KI/KI) mice was 48.4 ± 6.1 pA (n = 17 cells) while that of OF (KI/-) mice was 52.5 ± 3.9 pA (n = 22 cells, p=0.86 vs OF (KI/KI)) and in OE mice was 57.7 ± 6.5 pA (n = 21 cells, p=0.48 vs OF (KI/KI), one-way ANOVA post-hoc Tukey; Figure 5I). These, along with previous, results indicate that orexin plays a role in maintaining the physiological input-output functions in orexin neurons.
OF (KI/KI) mice showed symptoms in narcolepsy
We reasoned that if orexin is successfully knocked out from OF (KI/KI) mice, it must show the sign of behavioral arrest, cataplexy, which is defined as the sudden and reversible episodes of the drop of voluntary muscle tone while remains fully conscious during the episodes (Tabuchi et al., 2014). Thus, we recorded and analyzed the baseline sleep/wakefulness cycle in OF (KI/KI) mice. Behavioral states shown by OF (KI/KI) mice were classified in four states which includes either wakefulness, REM, NREM or cataplexy (see Materials and methods). All recorded OF (KI/KI) mice showed cataplexy attack, especially during the start of the dark period. We also compared the vigilance state parameters of the OF (KI/KI) mice with those of wild type (WT) control mice and the values are presented in Table 1. As expected, OF (KI/KI) mice showed less wakefulness and higher NREM and REM sleep compared to the WT control animals (Table 1). Along with these changes, orexin KO mice showed an increased number of bouts and decreased average duration in all vigilance states (Table 1), suggesting fragmentation of the sleep-wakefulness and an inability to maintain sustained arousal in the absence of orexin peptides. However, in comparison to the previously generated orexin KO (Chemelli et al., 1999) as well as to the orexin neuron-ablated mice (Hara et al., 2001), newly generated OF (KI/KI) mice showed higher REM sleep and wakefulness and a lower amount of NREM sleep. This is presumably due to changes in the recording environment, different criteria of the definition of vigilance state and/or changes in the genetic background as the WT control mice show similar changes.
Vigilance state parameters recorded from OF (KI/KI) and WT animals.
The table shows total time spent in individual states in minutes, duration of respective states in seconds and number of episodes (bouts) observed in either 24 hr, or only in light or dark period in OF (KI/KI, n = 7 mice; WT, n = 8 mice). *, p<0.05. The p values were determined using two-tailed unpaired student's t-test. Values are represented as the mean ± SEM.
NREM
Orexin-Flp (KI/KI)
24 hr Total time (min) 96.0 ± 5.7 92.2 ± 4.6 18.7 ± 6.3 584.6 ± 19.1 555.7 ± 14.8 740.7 ± 18.3 792.1 ± 19.1
Duration (sec) 55.7 ± 4.2 57.3 ± 2.4 61.5 ± 12.1 81.4 ± 7.0 94.8 ± 5.8 102.1 ± 8.4* 189.9 ± 16.8
Bouts 110.7 ± 8.5 96.8 ± 7.5 17.4 ± 5.2 442.3 ± 37.4* 359.3 ± 20.8 453.0 ± 36.4* 358.6 ± 20.8
Light period Total time (min) 57.3 ± 3.8* 68.4 ± 2.9 2.1 ± 0.8 355.1 ± 13.5* 390.8 ± 4.9 305.5 ± 14.6* 260.8 ± 7.2
Duration (sec) 48.4 ± 3.9* 58.9 ± 1.8 48.9 ± 13.0 93.4 ± 7.5 94.6 ± 5.0 80.1 ± 4.6* 63.1 ± 4.2
Bouts 73.4 ± 7.6 70.0 ± 4.4 2.0 ± 0.7 234.6 ± 17.3 251.8 ± 14.1 231.0 ± 17.6 251.1 ± 13.9
Dark period Total time (min) 38.7 ± 4.8* 23.8 ± 2.5 16.6 ± 5.6 229.5 ± 12.4* 164.9 ± 10.5 435.2 ± 11.3* 531.3 ± 12.7
Duration (sec) 63.0 ± 6.2 55.6 ± 4.0 74.1 ± 19.8 69.3 ± 8.2* 94.9 ± 7.6 124.0 ± 12.7* 316.6 ± 30.9
Bouts 37.3 ± 4.3* 26.8 ± 3.5 15.4 ± 4.7 207.7 ± 21.1* 107.5 ± 10.5 222.0 ± 20.6* 107.5 ± 10.6
Chemogenetic activation of orexin neurons lacking orexin neuropeptides
Next, we sought to isolate the physiological effects of orexin from those of all other neurotransmitters co-released by orexin neurons. To achieve this, we employed chemogenetics, DREADD. Using Flp recombinase-dependent gene expression, hM3Dq was exclusively expressed in orexin neurons. Here, hM3Dq was fused with mCherry to detect expression and localization. AAV(9)-CMV-dFRT-hM3Dq-mCherry was injected bilaterally into the LHA of both homozygous OF (KI/KI) and heterozygous OF (KI/-) mice (Figure 6A). We quantified the number of orexin-positive cells in every 4th brain slices. The number of orexin-immunoreactive cells was 439 ± 23 cells/mouse in the brain of heterozygous mice and, among these, 92.1 ± 0.6% expressed mCherry (n = 3 mice; Figure 6B). Moreover, a very similar number of LHA neurons expressed mCherry in both heterozygous and homozygous mice: 422 ± 80 cells/animal (n = 6 mice) in OF (KI/-) and 455 ± 74 cells/animal (n = 6 mice) in OF (KI/KI) mice expressed mCherry (p=0.76, unpaired t-test). To confirm the selective activation of hM3Dq-expressing neurons in vivo, we measured the expression of an immediate early gene product, c-Fos, which is a surrogate molecular marker of neuronal activity. Following the behavioral studies, six randomly selected mice from both the heterozygous and homozygous groups received i.p. administration of either saline or clozapine N-Oxide (CNO) (1.0 mg/kg). Animals were perfused, and tissues were collected 90 min after the injection. c-Fos staining of brain slices confirmed that CNO selectively activated hM3Dq-expressing neurons in both heterozygous and homozygous mice. In heterozygous mice, the ratio of c-Fos expression in mCherry-positive cells of saline or CNO injected mice was 11.6 ± 1.9% or 89.2 ± 1.4%, respectively (n = 3 mice/group, p=9.2e-5, Figure 6C–6D). In homozygous mice, the ratio of c-Fos expression in mCherry-positive cells of saline or CNO injected mice was 10.6 ± 1.8% or 90.3 ± 1.1%, respectively (n = 3 mice/group, p=9.6e-4, paired t-test, Figure 6C–6D). Thus, we concluded that the DREADD system successfully enabled selective activation of orexin neurons in both heterozygous and homozygous mice.
Selective chemogenetic activation of orexin neurons.
(A) Intracranial injection of AAV in OF mice to achieve Flp-dependent expression of hM3Dq-mCherry fusion protein in orexin neurons. (B) Representative images showing the expression of mCherry in orexin-immunoreactive neurons in heterozygous OF (KI/-) mice. (C) c-Fos expression after i.p. administration of either saline or CNO in both OF (KI/-) and OF (KI/KI) mice. (D) Summary of the immunostaining data shown in C. CNO administration can significantly increase neuronal activity in both heterozygous and homozygous mice (n = 3 mice per group). (E) Schematic showing the schedule of i.p. administration during sleep recording. The p values were determined by a two-tailed student's t-test; data represent the mean ± SEM.
Orexin, and not other co-transmitters, was critical to promote sustained wakefulness and preventing cataplexy
Next, we compared the effect of chemogenetic activation of orexin neurons on sleep/wakefulness. Mice were injected with either saline or CNO during the light (L) period (at 10:00 AM) and the dark (D) period (at 10:00 PM; Figure 6E). Expectedly in OF (KI/-) mice, activation of orexin neurons increased total time spent in wakefulness (n = 9 mice, Figures 7B–I and 8D–I) and decreased time in REM sleep (Figure 7B–ii and D-ii) and in NREM sleep (Figure 7B–iii and D-iii) after CNO administration during both the light and dark periods (Figure 7—figure supplement 1). However, while the effects of orexin neuronal activation in OF (KI/KI) mice were comparable to the OF (KI/-) control during the dark period, it was dampened during the light period (n = 8 mice, Wakefulness: Figure 7B–i and D–I; REM sleep: Figure 7B–ii and D-ii; and NREM sleep: Figure 7B–iii and B-iii, and Figure 7—figure supplement 1).
Chemogenetic activation of orexin neurons resulted in altered sleep/wakefulness based on the availability of orexin.
(A) Line graph with symbols showing the time spent in wakefulness (i), REM sleep (ii), NREM sleep (iii), and cataplexy (iv) during each hour for the 6 hr following CNO or saline administration during light period. B, Bar graph showing the 2 hr average of the total time spent in wakefulness (i), REM sleep (ii), NREM sleep (iii), and cataplexy (iv) following CNO or saline injection during the light period. C, Scatter plot showing the averaged bout and duration in wakefulness (i), REM sleep (ii), NREM sleep (iii), and cataplexy (iv). The data in D-F are shown similar to the representation in A-C, respectively, recorded during the dark period (OF (KI/-): n = 9 mice and OF (KI/KI): n = 8 mice). Data represent the mean ± SEM in both the line and bar graph. The p values were determined by either two-way ANOVA followed by a post-hoc Bonferroni test or paired student's t-test (cataplexy).
Source data for Figure 7A, B, C, E and F.
CNO-induced cataplexy was not different from naïve cataplexy.
(A) hypnogram of 2 hr after saline or CNO administration. (B) typical traces showing EEG signal, EEG power spectrum and EMG of cataplexy episode. (C) line graph (left) and bar graph (right) showing relative power spectrum of EEG during cataplexy episode in the light period (n = 8 mice). Data represent the mean ± SEM in both the line and bar graph.
Source data for Figure 8C.
This clear difference could be explained by the ability of co-transmitters to partially compensate for the increased wakefulness. Therefore, we hypothesized that the co-transmitters could eventually rescue OF (KI/KI) mice from cataplexy as well. Surprisingly, however, activation of orexin neurons that lacked orexin peptide rather deteriorate the cataplexy during the light periods while it showed similar propensity during the dark period (saline (L): 0.2 ± 0.1 min/hr; CNO (L): 0.9 ± 0.3 min/hr, n = 8 mice, p=0.04; saline (D): 3.0 ± 1.2 min/hr; CNO (D): 5.2 ± 1.4 min/hr, n = 8 mice, p=0.14; Figure 7B-iv and 7D-iv). CNO itself is reported to have no effect on the initiation or maintenance of cataplectic attack in the Prepro-orexin KO (OXKO) mice (Mahoney et al., 2017). These results clearly suggested that neurotransmitters other than orexin could partially compensate for abnormalities in the regulation of sleep/wakefulness, excluding cataplexy.
We also compared the effect of orexin neuronal activation in the number of episodes (bouts) and average time spent in each vigilance state. Whereas, in OF (KI/-) mice, total observed bouts decrease in all three vigilance states for 2 hr after CNO injection (Wakefulness: Figure 7C–i and E–i; REM: Figure 7C–ii and E-ii, NREM: Figure 7C–iii and 7E-iii), they remain unaffected in case of OF (KI/KI) mice (Wakefulness: Figure 7C–i and E–i; REM: Figure 7C–ii and 7E-ii, NREM: Figure 7C–iii and 7E-iii; Cataplexy: Figure 7C-iv and 7E-iv). Conversely, orexin neuronal activation increased average wake duration in OF (KI/-) mice (Figure 7C–i and E–i); and decreased REM sleep (Figure 7C–ii and 7E-ii) and NREM sleep duration (Figure 7C–iii and 7E-iii). However, in case of OF (KI/KI) mice, while the total wake time was increased after chemogenetic activation (Figure 7B and 7E i-iv), duration of wake was not extended (Wakefulness: Figure 7C–i and E–i; REM: Figure 7C–ii and 7E-ii; NREM: Figure 7C–iii and 7E-iii; Cataplexy: Figure 7C-iv and 7E-iv). Together, all these experimental analyses indicated that orexin neuropeptide was indispensable to promote sustained arousal, where the co-transmitters could not compensate.
CNO-induced cataplexy was not different from naïve cataplexy
Finally, we sought to answer the question of whether CNO-induced increase in cataplexy attack showed similar properties to naïve cataplexy. We analyzed and compared the relative power spectrum EEG during the cataplexy episode in the daytime after saline or CNO administration as chemogenetic activation of orexin KO neurons resulted in increased cataplexy in the daytime (Figure 8). Relative power analysis showed no difference in either power of the delta, theta, alpha or beta wave (n = 8 mice, Figure 8C), suggesting that CNO-induced cataplexy showed similar electrocortical activity as the naïve cataplexy attack observed in OF (KI/-) mice.
It has been shown that orexin neurons possess several neurotransmitters and release them together with orexin. However, orexin- or OX2R-KO mice closely phenocopy the symptoms observed in human narcolepsy, which is caused by the specific degeneration of orexin neurons. This might suggest that other neurotransmitters have either no or insignificant role in the regulation of sleep/wakefulness. The physiological importance of orexin can be explored in several ways including central administration, KO, functional manipulation or ablation of source neurons, among others. Here, we dissociated the effect of orexin by applying electrophysiological analyses and neural activity manipulation to orexin neurons that lack orexin peptide using novel KI mice. Previously, we generated Orexin-Cre or Orexin-tTA transgenic mice in which a short 3.2 kb fragment from the 5′-upstream region of the human hypocretin gene was used as a promoter (Inutsuka et al., 2014; Sakurai et al., 1999; Tabuchi et al., 2013; Yamanaka et al., 2003). However, the random integration of the Orexin-Cre or Orexin-tTA transgene into the genome could result in ectopic gene expression in non-targeted cells as well as unexpected expression during development, as the timing and regulation of gene expression might be affected by genes located near the integrated locus. This could be problematic if these mouse lines were bred with reporter mice. In contrast, KI permits accurate temporo-spatial gene expression control. However, the knock-in method has the disadvantage that orexin content in OF (KI/-) mice could be slightly decreased since Flp was knocked into the hypocretin locus. Chemelli et al. reported that orexin A content was decreased to 75% in heterozygous mice. (Chemelli et al., 1999). OF (KI/-) mice showed a shorter duration of wakefulness compared with WT control mice, and is likely due to the decrease in orexin. The Flp function was restricted in orexin neurons in the OF mice as confirmed by reporter mice. To our knowledge, OF mice represent the first line which enabled exclusive gene expression in orexin neurons by crossing with reporter mice. Moreover, combining Flp and Cre-driver mice enable us to express different genes in different neuronal subtypes to apply neural manipulations and/or activity readout simultaneously. By crossing OF mice with either MCH-Cre or Gad67-Cre, we generated Orexin-Flp; MCH-Cre and Orexin-Flp; Gad67-Cre bigenic mice, and argued that OF mice could be an essential tool for studying the functional connectome between orexin neurons and any other neurons in the hypothalamus. Besides, OF mice can also be useful for analyzing long-range neuronal connections. By using the Orexin-Flp; Gad67-Cre bigenic mice, we recently found that GABAergic neurons in the ventral tegmental area inhibited orexin neuronal activity by making monosynaptic inhibitory projections and regulate NERM sleep in mice (Chowdhury et al., 2019).
Previously, we reported that orexin neuropeptide depolarized orexin neurons via OX2R and mediates a positive-feedback loop both directly and indirectly (Yamanaka et al., 2010). In agreement with this, we also found that orexin facilitates glutamate release onto orexin neurons. However, several basic electrophysiological properties of orexin neurons were also found to be altered in the absence of orexin peptides, including hyperpolarized membrane potential, lower discharge rate and input resistance. Surprisingly, the capacitance of orexin neurons that lack orexin peptide was significantly larger than that of orexin neurons possessing orexin. This indicates that orexin neurons lacking orexin have a larger surface of the cell membrane since capacitance reflects the surface area of the cytoplasmic membrane. We did not directly measure the difference in diameter of cell size since an approximately 3 pF increase is estimated to be associated with a 1–2 µm increase in diameter as a spherical body. This might suggest that orexin signaling through either OX2R or glutamatergic excitatory inputs is involved in the regulation of cell size. However, this study cannot conclude whether the observed altered electrophysiological properties in the orexin KO neurons is directly caused by the absence of orexin, or if the lack of orexin signaling results in developmental compensation that contributes to these differences. Several studies have shown the role of orexin in the developing brain. Satoyanova et al. showed that orexin A and B are expressed at 1 week of age during early postnatal brain development (Stoyanova et al., 2010). In addition to this, van den Pol et al. showed that orexin increased the temporal synchrony of action potentials in developing locus coeruleus cells (van den Pol et al., 2002). These reports suggest that orexin might have a role in brain development. To fully characterize some of these unexpected effects of orexin in maintaining the biophysical properties of orexin neurons, in vivo genome editing using the CRISPR/Cas9 system or hypocretin gene knockout after maturation using an inducible Cre recombinase such as CreERT2, will likely be required.
Orexin neurons are known to play an essential role in maintaining uninterrupted wakefulness as human narcolepsy patients show chronic daytime sleepiness (Crocker et al., 2005; Thannickal et al., 2000). Disruption of orexin signaling in mice, rats and dogs produce a very similar phenotype which includes short bouts of wakefulness and increased transitions of vigilance states (Chemelli et al., 1999; Mochizuki et al., 2004). These suggest that orexin might be the key component released from the orexin neurons in performing the wake-maintaining role of orexin neurons. Our study showed further evidence in support of this hypothesis in the mouse model as a putative increase in the probability of co-transmitters release from orexin neurons could not rescue from the short bout and frequent state-transition phenomenon. Chemogenetic activation of orexin neurons prolonged the duration of wakefulness only in the presence of orexin, suggesting that orexin is crucial to achieve long-duration wakefulness. Moreover, orexin administration can also make wakefulness with high cognitive ability in non-human primates (Deadwyler et al., 2007). Such effect of orexin is also expected in human society, and our results indicate the irreplaceability of orexin.
On the contrary, we found that other behavioral outcomes of activating orexin neurons, including increased wakefulness and decreased REM and NREM sleep, can be compensated for by co-transmitters. This effect was obvious especially during the dark (active) period when baseline activity of orexin neurons is presumably higher (Estabrooke et al., 2001; Lee et al., 2005; Mileykovskiy et al., 2005). Interestingly, some other behavioral effects, like the prevention of cataplexy, cannot be compensated for by co-transmitters. Rather, it deteriorates the condition. This might suggest that the role of orexin in the prevention of cataplexy is not simply to activate post-synaptic neurons since glutamate (co-transmitter) also activates post-synaptic neurons. Rather, neurons that are not directly innervated by orexin neurons might also be involved in the regulation of cataplexy. It is also possible that the released orexin diffuses in the CSF since intracerebroventricular injection of orexin inhibited cataplexy (Mieda et al., 2004). Moreover, orexin co-transmitter dynorphin, which inhibits orexin neurons both directly and indirectly by depressing glutamatergic afferent inputs to orexin neurons, might also play a key role in such deterioration of cataplexy (Li and van den Pol, 2006).
The release of orexin follows a circadian rhythm that is also strongly related to locomotion (Zhang et al., 2004). In rodents, the level of orexin peptide in the CSF is low during the light period and high during the active-wake period (Zeitzer et al., 2003). Moreover, orexin neurons supposedly show higher activity during active wakefulness and become less active or inactive during REM or NREM sleep (Lee et al., 2005; Mileykovskiy et al., 2005). Our data also suggest that the activity of orexin neurons is differentially regulated during the light and dark periods. It is possible that the higher endogenous activity of orexin neurons during the dark period may facilitate CNO-induced transmitter release resulting in improved wakefulness in OF(KI/KI) mice.
In summary, here we dissociated the role of orexin at the cellular and behavioral level. We suggest that the primary function of orexin is to maintain the electrophysiological balance, the input-output functions in orexin neurons and most importantly, to exert the function of orexin neurons in maintaining sustained wakefulness.
(species) or
Source or
Strain, strain background AAV(9)-CMV-dFRT-hM3Dq-mCherry This paper NA Titer: 1.0 × 1012particles/ml
Strain, strain background AAV(DJ)-CMV-dFRT-hrGFP This paper NA Titer: 3.7 × 1012particles/ml
Strain, strain background AAV(DJ)-CMV-FLEX-tdTomato This paper NA Titer: 2.0 × 1012particles/ml
Genetic reagent (Mus musculus) Orexin-Flippase This paper Orexin-Flp Knock-in mice; EGFP-2A-Flp was placed just behind the translational initiation site of the hypocretin gene in-frame
Genetic reagent (Mus musculus) Glutamic acid decarboxylase 67-Cre Higo et al., 2009 Gad67-Cre
Genetic reagent (Mus musculus) R26-CAG-FRT-STOP-FRT-mTFP1 Imayoshi et al., 2012 FSF-mTFP1
Genetic reagent (Mus musculus) Melanin-concentrating hormone-Cre Kong et al., 2010 MCH-Cre
Genetic reagent (Mus musculus) Orexin-enhanced green fluorescence protein Yamanaka et al., 2003 Orexin-EGFP
Antibody goat polyclonal anti-orexin Santa Cruz Biotechnology sc-8070 (1/1000)
Antibody rabbit polyclonal anti-MCH Sigma-Aldrich M8440 (1/2000)
Antibody rabbit polyclonal anti-prodynorphin Merck Millipore AB5519 (1/100)
Antibody mouse monoclonal anti-c-Fos Santa Cruz Biotechnology E-8 (1/500)
Antibody mouse monoclonal anti-DsRED Santa Cruz Biotechnology sc-390909 (1/1000)
Antibody mouse monoclonal anti-GFP Fujifilm Wako Pure Chemical Corporation mFX75 (1/1000)
Sequence-based reagent (primer) forward primer: 5'-CTCATTAGTACTCGGAAACTGCCC-3' This paper NA Primer for PCR genotyping of orexin-Flp mouse tail DNA
Sequence-based reagent (primer) reverse primer: 5'-AAGCACTATCATGGCCTCAGTAGT-3' This paper NA Primer for PCR genotyping of orexin-Flp mouse tail DNA
Chemical compound, drug clozapine-N-oxide (CNO) Enzo Life Sciences BML-NS105-0025
Chemical compound, drug Picrotoxin Sigma-Aldrich P1675
Chemical compound, drug tetrodotoxin (TTX) Alomone Labs Cat# T-550
Chemical compound, drug D-2-Amino-5-phosphopentanoic acid (AP5) Alomone Labs Cat# D-145
Chemical compound, drug 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) Sigma-Aldrich Cat# C127
Chemical compound, drug Lidocaine N-ethyl bromide (QX-314 bromide) Alomone Labs Cat# Q-100
Software, algorithm Origin 2017 Lightstone Origin 2018
Software, algorithm SleepSign Kissei Comtec Version 3
Software, algorithm pClamp 10.5Software and Algorithms Molecular Devices RRID:SCR_011323
Software, algorithm ImageJ https://imagej.nih.gov/ij/ RRID:SCR_003070
Other DAPI stain Thermo Fisher Scientific Cat# D1306
All experimental protocols in this study involving the use of mice were approved and were performed in accordance with the approved guidelines of the Institutional Animal Care and Use Committees of the Research Institute of Environmental Medicine, Nagoya University, Japan (Approval number #18232, #18239). Mice were group housed unless stated otherwise, on a 12 hr light-dark cycle (lights were turned on at 8:00 AM), with free access to food and water. All efforts were made to reduce the number of animals used and to minimize the suffering and pain of animals.
Generation of OF knock-in mice
To generate the OF knock-in mice, we designed a targeting vector in which Flp recombinase cDNA was fused to enhanced green fluorescent protein (EGFP) with the 2A peptide gene (EGFP-2A-Flp) and was placed just behind the translational initiation site of the hypocretin gene in-frame. The knock-in vector was constructed with the MC1 promoter-driven diphtheria toxin gene, a 5.44 kb fragment at the 5' site. Flp recombinase, including a nuclear localization signal cDNA, was fused to EGFP with the T2A peptide sequence, a Pgk-1 promoter-driven neomycin phosphotransferase gene (neo) flanked by two Dre recognition target (rox) sites, and a 5.16 kb fragment at the 3' site (Figure 1A). The sequence of EGFP-2A-Flp was codon-optimized for expression in mammalian cells. Linearized targeting vector was electroporated into embryonic stem cells from the C57BL/6 mouse line (RENKA), and corrected targeted clones were isolated by southern blotting. Two founders were obtained, and the B line was used in this study. PCR genotyping of mouse tail DNA was performed with the following primers: knock-in forward, 5'-CTCATTAGTACTCGGAAACTGCCC-3'; knock-in reverse, 5'-AAGCACTATCATGGCCTCAGTAGT-3'.
Generation of Orexin-Flp; FSF-mTFP1, Orexin-Flp; MCH-Cre and Orexin-Flp; Gad67-Cre bigenic mice
After several generations of breeding, OF mice were separately bred with either R26-CAG-FRT-STOP-FRT-mTFP1 (FSF-mTFP1) (Imayoshi et al., 2012), MCH-Cre (Kong et al., 2010), or glutamic acid decarboxylase at 67 K-dalton (Gad67)-Cre (Gad67-Cre) (Higo et al., 2009) mice to generate Orexin-Flp; FSF-mTFP1, Orexin-Flp; MCH-Cre or Orexin-Flp; Gad67-Cre bigenic mice, respectively.
Generation and microinjection of viral vectors
Adeno-associated viral (AAV) vectors were produced using the AAV Helper-Free System (Agilent Technologies, Inc, Santa Clara, CA, USA). The virus purification method was modified from a previously published protocol (Inutsuka et al., 2016). Briefly, HEK293 cells were transfected with a pAAV vector, pHelper and pAAV-RC (serotype nine or DJ; purchased from Cell Biolabs Inc, San Diego, CA, USA) plasmid using a standard calcium phosphate method. Three days after transfection, cells were collected and suspended in artificial CSF (aCSF) solution (in mM: 124 NaCl, 3 KCl, 26 NaHCO3, 2 CaCl2, 1 MgSO4, 1.25 KH2PO4 and 10 glucose). Following multiple freeze-thaw cycles, the cell lysates were treated with benzonase nuclease (Merck, Darmstadt, Germany) at 37°C for 30 min, and were centrifuged 2 times at 16,000 g for 10 min at 4°C. The supernatant was used as the virus-containing solution. Quantitative PCR was performed to measure the titer of purified virus. Virus aliquots were then stored at −80°C until use.
A dFRT cassette was used for Flp-dependent gene expression control. A dFRT cassette is composed of two different FRT sequences (FRT and F3) located in a cis position. In the presence of Flp, sequence between dFRT is reversed and fixed. To express hM3Dq exclusively in Flp-expressing neurons, we stereotactically injected 600 nl of AAV(9)-CMV-dFRT-hM3Dq-mCherry (viral titer: 1.0 × 1012 particles/ml) virus into both brain hemispheres of OF mice using the following coordinates: −1.4 mm posterior to the bregma, 0.8 mm lateral to the midline, −5.0 mm ventral to the brain surface. Mice were anesthetized with 1.5–2.0% isoflurane (Wako Pure Chemical Industries, Osaka, Japan) using a Univentor 400 Anaesthesia Unit (Univentor Ltd., Malta) throughout the entire surgery. These mice were used in the behavioral experiments beginning at least 3 weeks post-injection. For Flp-dependent expression of humanized Renilla reniformis green fluorescent protein (hrGFP) and Cre-dependent expression of tdTomato, we stereotactically injected 600 nl of virus cocktail containing equal volume of AAV(DJ)-CMV-dFRT-hrGFP (viral titer: 3.7 × 1012 particles/ml) and AAV(DJ)-CMV-FLEX-tdTomato (viral titer: 2.0 × 1012 particles/ml) into one brain hemisphere of OF mice using the same coordinates described above.
Mice were anesthetized with 10% somnopentyl (1.0 mg/kg, Kyoritsu Seiyaku Corporation, Tokyo, Japan) and were perfused transcardially with 20 ml of ice-cold saline. This perfusion was immediately followed by another 20 ml of 10% ice-cold formalin (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Brains were then isolated and postfixed in 10% formalin solution at 4°C overnight. Subsequently, brains were immersed in 30% sucrose in PBS at 4°C for at least 2 days. Coronal brain slices of 40 μm thickness were generated using a cryostat (Leica CM3050 S; Leica Microsystems, Wetzlar, Germany). For staining, coronal brain sections were immersed in blocking buffer (1% BSA and 0.25% Triton-X in PBS), then incubated with primary antibodies at 4°C overnight. The sections were then washed with blocking buffer and incubated with secondary antibodies for 1 hr at room temperature (RT). After washing, brain sections were mounted and examined using a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan or IX71, Olympus, Tokyo, Japan).
Antibodies and stains
Primary antibodies were diluted in the blocking buffer as follows: anti-orexin-A goat antibody (Santa Cruz, Dallas, TX) at 1:1000, anti-MCH rabbit antibody (Sigma-Aldrich) at 1:2000, anti-prodynorphin guinea pig antibody (Merck Millipore, Billerica, MA) at 1:100, anti-c-Fos rabbit antibody (Santa Cruz) at 1:500 and anti-GFP mouse antibody (Wako, Japan) at 1:1000. Secondary antibodies included: CF 488- or CF 594-conjugated anti-goat antibody (Biotium Inc, Hayward, CA), CF 647-conjugated anti-rabbit antibody (Biotium), CF 680-conjugated anti-guinea pig antibody (Biotium) and CF 488-conjugated anti-mouse antibody (Biotium); all were diluted at 1:1000 in blocking buffer.
Acute brain slice preparations and electrophysiological recording
OF and OE (Yamanaka et al., 2003) mice of both sexes, aged 2–5 months, were used for electrophysiological recordings. Brain slice preparations and subsequent electrophysiological recording were modified from a previously published protocol (Chowdhury and Yamanaka, 2016). Briefly, mice were deeply anesthetized using isoflurane (Wako) and decapitated at around 11:00 AM. Brains were quickly isolated and chilled in ice-cold oxygenated (95% O2 and 5% CO2) cutting solution (in mM: 110 K-gluconate, 15 KCl, 0.05 EGTA, 5 HEPES, 26.2 NaHCO3, 25 Glucose, 3.3 MgCl2 and 0.0015 (±)−3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid). After trimming the brain, coronal brain slices of 300 µm thickness that contained the LHA were generated using a vibratome (VT-1200S; Leica, Wetzlar, Germany) and were temporarily placed in an incubation chamber containing oxygenated bath solution (in mM: 124 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 1.23 NaH2PO4, 26 NaHCO3 and 25 Glucose) in a 35°C water bath for 60 min. Slices were then incubated at RT in the same incubation chamber for another 30–60 min for recovery.
Acute brain slices were transferred from the incubation chamber to a recording chamber (RC-26G; Warner Instruments, Hamden, CT, USA) equipped with an upright fluorescence microscope (BX51WI; Olympus, Tokyo, Japan), and were superfused with oxygenated bath solution at the rate of 1.5 ml/min using a peristaltic pump (Dynamax; Rainin, Oakland, CA, USA). An infrared camera (C3077-78; Hamamatsu Photonics, Hamamatsu, Japan) was installed in the fluorescence microscope along with an electron multiplying charge-coupled device camera (EMCCD, Evolve 512 delta; Photometrics, Tucson, AZ, USA) and both images were separately displayed on monitors. Micropipettes of 4–6 MΩ resistance were prepared from borosilicate glass capillaries (GC150-10; Harvard Apparatus, Cambridge, MA, USA) using a horizontal puller (P-1000; Sutter Instrument, Novato, CA, USA). Patch pipettes were filled with KCl-based internal solution (in mM: 145 KCl, 1 MgCl2, 10 HEPES, 1.1 EGTA, 2 MgATP, 0.5 Na2GTP; pH 7.3 with KOH) with osmolality between 280–290 mOsm. Positive pressure was introduced in the patch pipette as it approached the cell. For whole-cell current clamp or voltage clamp recordings, a giga-seal of resistance >1 GΩ was made between the patch pipette and the cell membrane by releasing the positive pressure upon contacting the cell. The patch membrane was then ruptured by gentle suction to form a whole-cell configuration. Electrophysiological properties of the cells were monitored using the Axopatch 200B amplifier (Axon Instrument, Molecular Devices, Sunnyvale, CA). Output signals were low-pass filtered at 5 kHz and digitized at a 10 kHz sampling rate. Patch clamp data were recorded through an analog-to-digital (AD) converter (Digidata 1550A; Molecular Devices) using pClamp 10.2 software (Molecular Devices). Blue light with a wavelength of 475 ± 17.5 nm was generated by a light source that used a light-emitting diode (Spectra light engine; Lumencor, Beaverton, OR, USA) and was guided to the microscope stage with a 1.0 cm diameter fiber. Brain slices were illuminated through the objective lens of the fluorescence microscope. EGFP-expressing orexin neurons were identified by its fluorescence. For cell-attached recording, a seal of resistance <1 GΩ was made and spontaneous firing was recorded. The resting membrane potentials (Vrest) were measured from offline analysis of current clamp recordings using the predefined fitting function provided by Clampfit. We performed standard exponential fitting with zero shift for the initial 20 s of data to measure the Vrest of recorded neurons. Firing frequency was also calculated from offline analysis of the initial 60 s of the cell-attached recording data. sEPSCs were recorded with picrotoxin (400 μM) and sIPSCs were recorded with AP-5 (50 μM) and CNQX (20 μM) in bath solutions. Both sEPSCs and sIPSCs were recorded in the presence of KCl-based pipette solutions that included 1 mM of QX-314.
EEG-EMG surgery, data acquisition, and vigilance state determination
Virus injected age-matched male mice were implanted with EEG and EMG electrodes for polysomnographic recording under isoflurane anesthesia following the protocol published elsewhere (Tabuchi et al., 2014). Immediately after surgery, each mouse received an i.p. injection of 10 ml/kg of analgesic solution containing 0.5 mg/ml of Carprofen (Zoetis Inc, Japan). The same analgesic at the same dose was administered again 1 day after surgery. Mice were singly housed for 7 days during recovery. Mice were then connected to a cable with a slip ring in order to move freely in the cage and were habituated with the cable for another 7 days. The first 3 days were treated as the adaptation period for the animals to acclimate to the new environment and to intraperitoneal (i.p.) administration (10 ml/kg) of saline. On days 4 and 5, the mice were injected with saline (day 4) and CNO (Enzo Life Sciences, Farmingdale, NY, USA) (day 5) at 10:00 AM during the light period. On days 7 and 8, they were injected with saline (day 7) and CNO (day 8) at 10:00 PM during the dark period (See Figure 6). CNO was dissolved in water to make a stock solution (10 mg/ml) and was diluted with saline to a final concentration of 100 µg/ml just prior to i.p. administration.
EEG and EMG signals were amplified (AB-610J, Nihon Koden, Japan), filtered (EEG 1.5–30 Hz and EMG 15–300 Hz), digitized (at a sampling rate of 128 Hz), recorded (Vital Recorder, Kissei Comtec Co., Ltd, Japan) and finally analyzed (SleepSign, Kissei Comtec). Animal behavior was monitored through a CCD video camera (Amaki Electric Co., Ltd., Japan) during both the light and dark periods. The dark period video recording was assisted by infrared photography (Amaki Electric Co., Ltd., Japan) and an infrared sensor (Kissei Comtec). EEG and EMG data were automatically scored in 4 s epochs and classified as wake, rapid eye movement sleep, and non-rapid eye movement sleep. The EEG analysis yielded power spectra profiles over a 0 ~ 20 Hz window with 1 Hz resolution for delta (1–5 Hz), theta (6–10 Hz), alpha (11–15 Hz), and beta (16–20 Hz) bandwidths. All auto-screened data were examined visually and corrected. The criteria for vigilance states were the same as described previously (Tabuchi et al., 2014). Briefly, (i) wake (low EEG amplitude with high EMG or locomotion score), (ii) non-rapid eye movement (NREM) sleep (low EMG and high EEG delta amplitude), and (iii) rapid eye movement (REM) sleep (low EMG, low EEG amplitude with high theta activity, and should be followed by NREM). Cataplexy was tracked using a combination of multiple criteria: muscle atonia lasting ≥10 s, predominance of theta activity and more than 40 s of wakefulness before the cataplectic attack.
Data analysis and presentation
Immunostaining data were analyzed and processed with ImageJ (US National Institute of Health) and BZ-X Analyzer (Keyence BZ-X710 microscope). Electrophysiological analysis was performed with either Clampfit10 (Molecular Devices, Sunnyvale, CA) or Minianalysis software (Synaptosoft Inc, Decatur, GA). Electrophysiological data were saved as American Standard Code for Information Interchange (ASCII) files and further data calculations were performed in Microsoft Excel. Graphs were generated in Origin 2017 (OriginLab, Northampton, MA) using data from Excel. Statistical analysis was also performed with Origin 2017. Graphs were generated using Canvas 15 (ACD Systems, Seattle, WA).
Experimental design and statistical analysis
The electrophysiological effects of knocking out a single neuropeptide in its source neurons were analyzed by slice electrophysiology. Individual sample sizes for slice patch-clamp recordings (n number of neurons) are reported separately for each experiment. The physiological effects of activating orexin neurons that lack orexin neuropeptide were also analyzed. In all cases, five or more animals were used for each parameter tested. All statistical tests, including the exact p values, are described when used. No statistical analyses were used to predetermine sample sizes. All data are presented as the mean ± standard error of the mean (SEM). For all statistical tests *p<0.05, **p<0.01, ***p<0.001 were considered significant and p>0.05 was considered not significant (ns).
All data generated or analysed during this study are included in the manuscript and supporting files.
Adamantidis AR
Aravanis AM
Deisseroth K
de Lecea L
(2007) Neural substrates of awakening probed with optogenetic control of hypocretin neurons
Armbruster BN
Li X
Pausch MH
Herlitze S
Roth BL
(2007) Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand
Broberger C
Sutcliffe JG
Hökfelt T
(1998) Hypocretin/orexin- and melanin-concentrating hormone-expressing cells form distinct populations in the rodent lateral hypothalamus: relationship to the neuropeptide Y and agouti gene-related protein systems
https://doi.org/10.1002/(SICI)1096-9861(19981228)402:4<460::AID-CNE3>3.0.CO;2-S
Chemelli RM
Willie JT
Sinton CM
Elmquist JK
Scammell T
Lee C
Williams SC
Xiong Y
Kisanuki Y
Fitch TE
Nakazato M
Saper CB
Yanagisawa M
(1999) Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation
Chou TC
Lee CE
Lu J
Hara J
Beuckmann CT
Sakurai T
Scammell TE
(2001) Orexin (hypocretin) neurons contain dynorphin
The Journal of Neuroscience 21:RC168.
https://doi.org/10.1523/JNEUROSCI.21-19-j0003.2001
Chowdhury S
Matsubara T
Miyazaki T
Ono D
Fukatsu N
Sudo Y
Yamanaka A
(2019) GABA neurons in the ventral tegmentalarea regulate non-rapid eye movementsleep in mice
(2016) Optogenetic activation of serotonergic terminals facilitates GABAergic inhibitory input to orexin/hypocretin neurons
Crocker A
España RA
Papadopoulou M
Faraco J
Honda M
Mignot E
(2005) Concomitant loss of dynorphin, NARP, and orexin in narcolepsy
Neurology 65:1184–1188.
https://doi.org/10.1212/01.WNL.0000168173.71940.ab
Kilduff TS
Peyron C
Gao X
Foye PE
Danielson PE
Fukuhara C
Battenberg EL
Gautvik VT
Bartlett FS
Frankel WN
van den Pol AN
Bloom FE
Gautvik KM
(1998) The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity
PNAS 95:322–327.
https://doi.org/10.1073/pnas.95.1.322
Deadwyler SA
Porrino L
Siegel JM
Hampson RE
(2007) Systemic and nasal delivery of Orexin-A (Hypocretin-1) Reduces the effects of sleep deprivation on cognitive performance in nonhuman primates
Estabrooke IV
McCarthy MT
Ko E
(2001) Fos expression in orexin neurons varies with behavioral state
Håkansson M
Meister B
(1999) Leptin receptor- and STAT3-immunoreactivities in hypocretin/orexin neurones of the lateral hypothalamus
Journal of Neuroendocrinology 11:653–663.
Nambu T
Sugiyama F
Yagami K
Goto K
(2001) Genetic ablation of orexin neurons in mice results in narcolepsy, Hypophagia, and obesity
Higo S
Akashi K
Tamamaki N
(2009) Subtypes of GABAergic neurons project axons in the neocortex
Frontiers in Neuroanatomy 3:25.
https://doi.org/10.3389/neuro.05.025.2009
Imayoshi I
Hirano K
Sakamoto M
Miyoshi G
Imura T
Kitano S
Miyachi H
Kageyama R
(2012) A multifunctional teal-fluorescent Rosa26 reporter mouse line for cre- and Flp-mediated recombination
Neuroscience Research 73:85–91.
https://doi.org/10.1016/j.neures.2012.02.003
Inutsuka A
Inui A
Tabuchi S
Tsunematsu T
Lazarus M
(2014) Concurrent and robust regulation of feeding behaviors and metabolism by orexin neurons
Yamashita A
Nakai J
Ohkura M
Taguchi T
(2016) The integrative role of orexin/hypocretin neurons in nociceptive perception and analgesic regulation
(2013) The physiological role of orexin/hypocretin neurons in the regulation of sleep/wakefulness and neuroendocrine functions
Frontiers in Endocrinology 4:.
https://doi.org/10.3389/fendo.2013.00018
Kong D
Vong L
Parton LE
Ye C
Tong Q
Hu X
Choi B
Lowell BB
(2010) Glucose stimulation of hypothalamic MCH neurons involves K(ATP) channels, is modulated by UCP2, and regulates peripheral glucose homeostasis
Cell Metabolism 12:545–552.
https://doi.org/10.1016/j.cmet.2010.09.013
Lee MG
Hassani OK
Jones BE
(2005) Discharge of identified orexin/hypocretin neurons across the sleep-waking cycle
(2006) Differential target-dependent actions of coexpressed inhibitory dynorphin and excitatory hypocretin/orexin neuropeptides
Lin L
Li R
Kadotani H
Rogers W
Lin X
Qiu X
de Jong PJ
Nishino S
(1999) The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene
Mahoney CE
Agostinelli LJ
Brooks JN
(2017) GABAergic neurons of the central amygdala promote cataplexy
Mieda M
(2004) Orexin peptides prevent cataplexy and improve wakefulness in an orexin neuron-ablated model of narcolepsy in mice
Mileykovskiy BY
Kiyashchenko LI
(2005) Behavioral correlates of activity in identified hypocretin/orexin neurons
Mochizuki T
McCormack S
(2004) Behavioral state instability in orexin knock-out mice
Mizukami K
Hosoya Y
(1999) Distribution of orexin neurons in the adult rat brain
Tighe DK
Heller HC
(1998) Neurons containing hypocretin (orexin) project to multiple neuronal systems
The Journal of Neuroscience 18:9996–10015.
Ripley B
Overeem S
Charnay Y
Nevsimalova S
Aldrich M
Reynolds D
Albin R
Hungs M
Pedrazzoli M
Padigaru M
Kucherlapati M
Fan J
Maki R
Lammers GJ
Bouras C
Kucherlapati R
(2000) A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains
Nature Medicine 6:991–997.
Rosin DL
Weston MC
Sevigny CP
Stornetta RL
Guyenet PG
(2003) Hypothalamic orexin (hypocretin) neurons express vesicular glutamate transporters VGLUT1 or VGLUT2
Ishii M
Matsuzaki I
Tanaka H
Kozlowski GP
Wilson S
Arch JR
Buckingham RE
Haynes AC
Carr SA
Annan RS
McNulty DE
Liu WS
Terrett JA
Elshourbagy NA
Bergsma DJ
(1998) Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior
Moriguchi T
Furuya K
Kajiwara N
Nakamura T
(1999) Structure and function of human prepro-orexin gene
Nagata R
Kawamura H
Tsujino N
Muraki Y
Kageyama H
Kunita S
Takahashi S
Koyama Y
Shioda S
(2005) Input of orexin/hypocretin neurons revealed by a genetically encoded tracer in mice
(2014) The role of orexin in motivated behaviours
Sasaki K
Suzuki M
Roth B
(2011) Pharmacogenetic modulation of orexin neurons alters sleep/wakefulness states in mice
Stoyanova II
Rutten WL
le Feber J
(2010) Orexin-A and orexin-B during the postnatal development of the rat brain
Cellular and Molecular Neurobiology 30:81–89.
Sugio S
Xu M
Tanaka KF
Tominaga M
(2013) Influence of inhibitory serotonergic inputs to orexin/hypocretin neurons on the diurnal rhythm of sleep and wakefulness
Sleep 36:1391–1404.
https://doi.org/10.5665/sleep.2972
Black SW
Maruyama M
Takagi K
Minokoshi Y
(2014) Conditional ablation of orexin/hypocretin neurons: a new mouse model for the study of narcolepsy and orexin system function
Thannickal TC
Moore RY
Nienhuis R
Ramanathan L
Gulyani S
Cornford M
(2000) Reduced number of hypocretin neurons in human narcolepsy
Boyden ES
(2011) Acute optogenetic silencing of orexin/hypocretin neurons induces slow-wave sleep in mice
Tupone D
Madden CJ
Cano G
Morrison SF
(2011) An orexinergic projection from perifornical hypothalamus to raphe pallidus increases rat Brown adipose tissue thermogenesis
Ghosh PK
Liu RJ
Aghajanian GK
Gao XB
(2002) Hypocretin (orexin) enhances neuron activity and cell synchrony in developing mouse GFP-expressing locus coeruleus
The Journal of Physiology 541:169–185.
(2003) Hypothalamic orexin neurons regulate arousal according to energy balance in mice
Fukazawa Y
(2010) Orexin directly excites orexin neurons through orexin 2 receptor
Yoshida K
(2006) Afferents to the orexin neurons of the rat brain
Zeitzer JM
Buckmaster CL
Parker KJ
Hauck CM
Lyons DM
(2003) Circadian and homeostatic regulation of hypocretin in a primate model: implications for the consolidation of wakefulness
Zhang S
Yoshida Y
Wisor JP
Edgar DM
(2004) Lesions of the suprachiasmatic nucleus eliminate the daily rhythm of hypocretin-1 release
Sleep 27:619–627.
https://doi.org/10.1093/sleep/27.4.619
Fukuda Y
Kuwaki T
(2006) Orexin neuron-mediated skeletal muscle vasodilation and shift of baroreflex during defense response in mice
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 290:R1654–R1663.
Yang Dan
Reviewing Editor; University of California, Berkeley, United States
Catherine Dulac
Senior Editor; Harvard University, United States
Reviewer; University of California, Berkeley, United States
Thank you for submitting your article "Dissociating orexin-dependent and -independent functions of orexin neurons using novel orexin-Flpknock-in mice" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Yang Dan as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Catherine Dulac as the Senior Editor.
Orexin neurons are known to be important for sustained arousal, and they release glutamate and dynorphin in addition to orexin. This paper addresses the effect of knocking out orexin on the property of orexinergic neurons and on the outcome of activating orexinergic neurons. They found that orexin knock out altered the electrophysiological properties of the neurons and reduced their glutamatergic inputs. While all transmitters contribute to arousal, orexin is crucial for sustained arousal and prevention of cataplexy. Interestingly, activating orexinergic neurons in an orexin deficient background causes increase in cataplexy. This novel finding is expected to lead to future discoveries on the mechanism of cataplexy. The new mouse line they generated is of great interest to the field, given the highly specific expression of Flp in orexin neurons.
Discussion section: "The Flp function was restricted in orexin neurons in the OF mice as confirmed by reporter mice."
To conclude this, it is essential to describe how many% of reporter positive cells are also orexin positive, which seems not provided. (The authors only state how many% of orexin positive cells are also reporter positive.)
Subsection "OF (KI/KI) mice showed symptoms in narcolepsy": "Sleep state parameters for orexin knockout mice are presented in Table 1. These values were comparable with our previously generated orexin neuron-ablated mice (Tabuchi et al., 2014a), as well as with the previously generated orexin-knockout mice (Chemelli et al., 1999)."
This part is not very convincing. First, in case of Tabuchi et al., 2014a, the amount of each sleep stage changes dynamically following DTA expression so it is difficult to compare sleep amount to the current KI mice. According to Table 1, the KI mice show high REM sleep amount (96 min.), whereas NREM sleep amount is quite low (584 min.). According to Chemelli et al., 1999, Orexin KO mice exhibit normal amount of REM sleep (88 min.) and high amount of NREM sleep (761 min.). Thus, contrary to the manuscript, the values do not seem "comparable" to both papers that were cited. In addition, according to Hara et al., 2001, mice in which orexinergic neurons were ablated by ataxin-expression exhibited a similar trend to Chemelli et al., 1999, i.e. normal amount of REM sleep (88 min.) and high amount of NREM sleep (731 min.). The characterization of the sleep architecture of the KI mice generated here is important because unexpected effects of expressing Flp in these neurons are possible. The recording environment might have affected the sleep differently from Chemelli et al., 1999 or Hara et al., 2001. To truly characterize the KI mice, please also provide sleep data of wt control mice.
The authors find that chemogenetic activation of orexin neurons in the KI/- mice strongly increases the average duration of wake bouts, while activation of orexin neurons w/o orexin in the KI/KI mice has no effect on wake duration (Figure 7C, F). The control recordings (with saline injections) show no significant differences between the two mouse lines. However, also during spontaneous sleep/wake behavior mice show sustained periods of wakefulness, especially during the dark period (Lo et al., 2004, Stephenson et al., 2013), implying that the control recordings with saline in Figure 7 should contain long wake periods. Consequently, if the release of orexin by orexin neurons was indispensable for sustained wake periods, KI/KI mice (w/o orexin in orexin neurons) should show shortened wake periods under control (saline) conditions. As this is not the case, the reported data in their present form demonstrate that orexin is sufficient, but not indispensable for sustained wake periods. This point should be clarified.
As the duration of wake durations typically follows a bimodal distribution with short and long wake bouts (Lo et al., 2004, Stephenson et al., 2013), potential effects on long wake periods in the control recordings might be occluded by averaging. By separating the distribution of wake periods into short and long periods, the authors could test whether the absence of orexin specifically affects either of these two distributions. A second reason why the authors could not detect differences under control conditions might be that the analyses in Figure 7C, F only include the first two hours after saline injection (which might not contain enough wake periods to detect differences). By extending their analysis to the complete control recordings, can the authors find shortened wake periods in KI/KI mice supporting a necessary role of orexin in sustaining wakefulness?
In the previous version of the manuscript, we stated that "We observed no ectopic expression of EGFP and/or mTFP1 protein in melanin-concentrating hormone (MCH)-immunoreactive (n = 4 mice; Figure 1C and 1D) or in any other non-orexin-immunoreactive neurons in these bigenic mice". However, to further clarify this description, we added the following sentence:
"In other words, all reporter gene-expressing neurons were orexin-immunoreactive (IR)."
In addition, we added a bar graph to show the percentage of reporter gene-positive cells that were also orexin-positive in Figure 1D.
As requested by the reviewer, we performed sleep recordings and analysis in wild type (WT) control mice (n = 8) and compared these results with OF (KI/KI) mice (n = 7). These results have been added to Table 1. OF (KI/KI) mice showed a significant increase in the number of bouts and a decreased average duration in all vigilance states compared with WT mice. These results suggest fragmentation of sleep/wakefulness and an inability to maintain sustained arousal in the absence of orexin. However, we observed a higher amount of REM sleep even in the WT control mice (92 min) compared with values reported for WT mice by Chemelli et al., 1999 (73 min). Although our REM sleep results for WT mice were similar to that reported by Hara et al., 2001 (92 min), our WT mice showed a higher level of wakefulness (792 min) and less NREM sleep (556 min) than observed by either Chemelli et al. (awake: 661 min, NREM: 703 min) or Hara et al. (awake: 654 min, NREM: 692 min). These differences might be caused by either i) the position of the EEG electrodes, or ii) the criteria used for the definition of vigilance state and the recording environment.
i) Position of EEG electrodes:
Our position: AP -1.5, ML -1.0 and AP -5.4, ML -1.0, mm.
Chemelli et al., 1999: AP1.1, ML ± 1.45 and AP-3.5, ML ± 1.45, mm.
Duran et al. (Sleep 2018) reported that differences in electrode position resulted in different REM sleep times. Our electrode position enabled detection of theta activity from the hippocampus with high sensitivity since the electrode was positioned close to the hippocampus. Thus, the observed increase in REM sleep time under our recording conditions compared with other reports might be caused by such differences in electrode position.
ii) Criteria for the definition of vigilance state: Our definition of drowsiness differed from that used by others. While Chemelli et al. defined drowsiness as NREM sleep, we defined it as wakefulness since we identified NREM sleep as having a low EMG and high delta amplitude by EEG. This might be caused by less NREM sleep and longer wakefulness compared with previous reports.
Therefore, we hypothesize that one potential reason for the differences in the amount of NREM and REM sleep in orexin-Flp mice may be the differences in recording conditions, environment and criteria used for sleep/wakefulness analysis, and not due to the expression of Flp. Moreover, patch clamp recording from orexin neurons in orexin-Flp (KI/-) heterozygous mice showed no abnormality in electrophysiological parameters compare with orexin-EGFP mice, confirming that Flp expression did not induce unexpected effect (Figure 4). Nevertheless, to avoid any confusion, we have removed the citation of Tabuchi et al. and added Hara et al. We added the following sentences to the Results:
"We also compared the vigilance state parameters of the OF (KI/KI) mice with those of wild type (WT) control mice and the values are presented in Table 1. As expected, OF (KI/KI) mice showed less wakefulness and higher NREM and REM sleep compared to the WT control animals (Table 1). Along with these changes, orexin KO mice showed an increased number of bouts and decreased average duration in all vigilance states (Table 1), suggesting fragmentation of the sleep-wakefulness and an inability to maintain sustained arousal in the absence of orexin peptides. However, in comparison to the previously generated orexin KO (Chemelli et al., 1999) as well as to the orexin neuron-ablated mice (Hara et al., 2001), newly generated OF (KI/KI) mice showed higher REM sleep and wakefulness and a lower amount of NREM sleep. This is presumably due to changes in the recording environment, different criteria of the definition of vigilance state and/or changes in the genetic background as the WT control mice show similar changes."
As the duration of wake durations typically follows a bimodal distribution with short and long wake bouts (Lo et al., 2004, Stephenson et al., 2013), potential effects on long wake periods in the control recordings might be occluded by averaging. By separating the distribution of wake periods into short and long periods, the authors could test whether the absence of orexin specifically affects either of these two distributions. A second reason why the authors could not detect differences under control conditions might be that the analyses in Figure 7C,F only include the first two hours after saline injection (which might not contain enough wake periods to detect differences). By extending their analysis to the complete control recordings, can the authors find shortened wake periods in KI/KI mice supporting a necessary role of orexin in sustaining wakefulness?
To address whether KI/KI mice (that lack orexin expression in orexin neurons) exhibit shortened periods of wakefulness under control (saline) conditions, we extended our analysis for 12 hours after the injection of saline. However, the duration and number of bouts of wakefulness were not significantly different. The duration of wakefulness in heterozygous (n = 4) and homozygous (n = 6) mice were 106.3 ± 16.8 sec and 99.5 ± 11.9 sec (Author response image 1; p = 0.7, unpaired t-test), respectively. The number of bouts of wakefulness in heterozygous and homozygous mice was 228.0 ± 23.1 and 252.8 ± 20.8 (Author response image 1, p = 0.5, unpaired t-test), respectively.
Bouts and duration of wakefulness in OF (KI/-) (n = 4) and OF (KI/KI) (n = 7) mice after saline injection for 12 hr).
Data represent mean ± S.E.M.
It is, however, notable that both the duration and bouts of wakefulness in heterozygous mice was not comparable to those of WT mice (Table 1; duration: 316.6 ± 30.9 sec, bouts: 107.5 ± 10.6, without saline injection during the dark period). This is presumably because of decrease in orexin in heterozygous mice since the Flp gene was knocked-in to the prepro-orexin gene locus. We could not compare sleep/wakefulness values from heterozygous mice with previous reports including that of Chemelli et al., since they did not provide such values. However, Chemelli et al. did describe a decrease in the content of orexin peptides in the brain of heterozygous mice, orexin-A was 75% and orexin-B was 83% of wild type mice. While we did not measure orexin content in orexin-Flp mice, the orexin content in heterozygous mice should be decreased. Thus, the decreased duration in both heterozygous and homozygous mice compared with WT mice demonstrates that orexin is indispensable for sustaining the period of wakefulness. We added this limitation in our results to the Discussion as follows:
"However, the knock-in method has the disadvantage that orexin content in OF (KI/-) mice could be slightly decreased since Flp was knocked into the prepro-orexin locus. Chemelli et al. reported that orexin A content was decreased to 75% in heterozygous mice. (Chemelli et al., 1999). OF (KI/-) mice showed a shorter duration of wakefulness compared with WT control mice, and is likely due to the decrease in orexin."
Given the reasons described above (that heterozygous mice were not normal due to a slight decrease in orexin), we compared the number of short, middle and long bouts of wakefulness between OF (KI/KI) homozygous mice (n = 7) and wild type mice (n = 8) during the dark period (Author response image 2). The wakefulness duration was divided into three categories, less than 5 min (short), 5 min to 1 hr (middle) and more than 1 hr (long) according to Stephenson et al., 2013. During the dark period (12 hr active period), OF (KI/KI) homozygous mice showed a significantly larger number of wakefulness episodes of short and middle durations compared with those of WT mice (short p = 0.0007 vs WT; middle p = 0.0001 vs WT; unpaired t-test). On the contrary, OF (KI/KI) homozygous mice exhibited no wakefulness episodes longer that 1 hr (p = 0.000001 vs WT, unpaired t-test). Taken together, these results strongly suggest that orexin is indispensable for sustained wakefulness.
Number of wakefulness bouts of short duration (~5 min), middle duration (5 min-1 hr) and long duration (1 hr~) in WT mice and OF (KI/KI) mice during the dark period.
Department of Neuroscience II, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan
Department of Neural Regulation, Graduate School of Medicine, Nagoya University, Nagoya, Japan
CREST, JST, Honcho Kawaguchi, Saitama, Japan
Present address
Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong, Bangladesh
Conceptualization, Data curation, Formal analysis, Writing—original draft, Writing—review and editing
Data curation, Formal analysis, Investigation
Data curation
Conceptualization, Resources
Department of Animal Model development, Brain Research Institute, Niigata University, Niigata, Japan
Department of Neurochemistry, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
Research Center for Dynamic Living Systems, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
Conceptualization, Supervision, Funding acquisition, Writing—original draft, Writing—review and editing
yamank@riem.nagoya-u.ac.jp
Japan Science and Technology Agency (JPMJCR1656)
Ministry of Education, Culture, Sports, Science, and Technology (26293046)
Ministry of Education, Culture, Sports, Science and Technology (26640041)
Ministry of Education, Culture, Sports, Science and Technology (16H01271)
Ministry of Education, Culture, Sports, Science and Technology (18KK0223)
We thank S Tsukamoto, Y Miyoshi, A Inui, and G Wang for technical assistance.
Animal experimentation: All experimental protocols in this study involving the use of mice were approved and were performed in accordance with the approved guidelines of the Institutional Animal Care and Use Committees of the Research Institute of Environmental Medicine, Nagoya University, Japan (Approval number #18232, #18239).
Catherine Dulac, Harvard University, United States
Yang Dan, University of California, Berkeley, United States
Accepted: May 9, 2019
Version of Record published: June 4, 2019 (version 1)
© 2019, Chowdhury et al.
orexin/hypocretin
flippase recombinase
chemogenetics
sleep/wakefulness
GABA neurons in the ventral tegmental area regulate non-rapid eye movement sleep in mice
Srikanta Chowdhury, Takanori Matsubara ... Akihiro Yamanaka
Research Article Jun 4, 2019
Sleep/wakefulness cycle is regulated by coordinated interactions between sleep- and wakefulness-regulating neural circuitry. However, the detailed mechanism is far from understood. Here, we found that glutamic acid decarboxylase 67-positive GABAergic neurons in the ventral tegmental area (VTAGad67+) are a key regulator of non-rapid eye movement (NREM) sleep in mice. VTAGad67+ project to multiple brain areas implicated in sleep/wakefulness regulation such as the lateral hypothalamus (LH). Chemogenetic activation of VTAGad67+ promoted NREM sleep with higher delta power whereas optogenetic inhibition of these induced prompt arousal from NREM sleep, even under highly somnolescent conditions, but not from REM sleep. VTAGad67+ showed the highest activity in NREM sleep and the lowest activity in REM sleep. Moreover, VTAGad67+ directly innervated and inhibited wake-promoting orexin/hypocretin neurons by releasing GABA. As such, optogenetic activation of VTAGad67+ terminals in the LH promoted NREM sleep. Taken together, we revealed that VTAGad67+ play an important role in the regulation of NREM sleep.


Palm Beach is about the sea, the waves, the blue skies, the constant passage of clouds, the pristine avenues of palms trees, the idyllic villas and sublime services, and above all the people. I hope you will enjoy this Flagler Museum wedding in Palm Beach!
Palm Beach was established as a resort by Henry Morrison Flagler, who made the Atlantic coast barrier island accessible via his Florida East Coast Railway. He was also the creator and owner of the worldwide famous resort The Breakers and The Flagler Museum.
One of the iconic establishments in Palm Beach is a luxurious hotel "The Breakers". The night before the wedding the rehearsal dinner is held at The Breakers. It is a delicious insight into the wedding festivities we will enjoy the following day. Here are some images from this party with a tropical theme. In the morning the gentlemen enjoy a round of golf before they start to get ready. As the guys dress a fun atmosphere and plenty of excitement is in the room. Once the groom is ready he will come to the bride's home where they will see each other for the first time. Following that, we will then go to the Flagler Museum where a charming ceremony will be held after sunset.
The bride is getting ready at her family home in Palm Beach with her 10 bridesmaids. As they have hair and makeup done I go around the house, both inside and outside to take some photos and prepare a few ideas for the "First Look". Finishing my coverage of their home I then move to capturing some details. I love details and have fun with her accessories. She has a stunning Oscar de la Renta dress, it is totally unique. A gentle Tiffany Blue hue, beads and floral details are discretely suggestive and appropriate for our Palm Beach location. The dress really comes alive when the light shines through the fabric.
As well as photographing the wedding dress I photograph the ten bridesmaids dresses too, it takes some time to create the shot but how many times do I get to photograph 10 bridesmaids dresses!
I also take pictures of the rings, invites, flowers, bracelet, earrings, veil, and perfume. Once these are complete it is time for the star of the show to have some photos… Yoshi the dog! Well, I'm joking of course, he is not the star but he is very important, and as he will not be able to come to the wedding venue, the Flagler Museum, I need to make sure we involve him in the day now. After some fun photos with Yoshi and the girls, they all start to put their dresses on.
The bride's mother and sister help her into the dress and she puts on her shoes and accessories. Next up we have the "First look" between her and the groom and then some more pictures together with Yoshi before we head to seal this Palm Beach wedding at the Flagler Museum.
The Henry Morrison Flagler Museum is a stunning venue in Palm Beach. It was built by Henry Flagler for his wife as a wedding present…. how romantic! On arrival I get to work with the family photos, we finish these and I take our happy couple for some portraits at sunset. Around about 6pm it is dark and I have to start to prepare myself for the wedding ceremony which will be in the Pavilion at the Flagler Museum.
6.30pm, it is now dark outside. The guests enter the pavilion at the Flagler Museum and find their seats. The processional is perfect and an exuberant bride enters with her father. The ceremony is emotional, a few tears and some good laughs ensue, they exchange vows and rings, and a kiss concludes the union. With the wedding ceremony over we move to the Grand Hall in the Flagler Museum to kick off the celebrations.
Palm Beach Flagler Museum wedding reception and party!
The dinner reception starts with their first dance and then the father-daughter and mother-son dances take place simultaneously. There are also a couple of fun speeches during the evening and constant live music from an excellent band. During the reception, a team is quickly flipping the location where we had the ceremony. Outside the pavilion string lights, lounge furniture, a bar and photo corner have all been setup. Everyone transitions to the party area for cake cutting and dancing, an amazing high energy band drives the party along…. everyone has a brilliant time!
At midnight there is a send-off at the front of the Flagler Museum and our newlyweds get into their Rolls Royce to be whisked away into the night.
This Palm Beach Flagler Museum wedding was a blast! I loved the location and the technical challenges of shooting in a low light venue at night time. I always find it inspiring to work in a new location, being unfamiliar with a wedding venue is stimulating as it forces me to think on my feet and create on the spur of the moment…. that's fun!
If you liked this event have a look at this wedding in Texas.


text:
Flags flying at half-staff across Iowa
Updated: 9:24 AM CDT Sep 11, 2013
Gov. Terry Branstad is calling for all flags in the state to fly at half-staff to honor the victims of the Sept. 11, 2001, terrorist attacks.Branstad ordered all the flags in Iowa to be lowered Wednesday from 8 a.m. to 5 p.m.Flags flown at half-staff include those at the state Capitol, the Capitol complex and other public buildings throughout the state.Branstad is encouraging businesses and schools to do the same as a sign of respect for the thousands of people killed in the 2001 attacks.
Gov. Terry Branstad is calling for all flags in the state to fly at half-staff to honor the victims of the Sept. 11, 2001, terrorist attacks.
Branstad ordered all the flags in Iowa to be lowered Wednesday from 8 a.m. to 5 p.m.
Flags flown at half-staff include those at the state Capitol, the Capitol complex and other public buildings throughout the state.
Branstad is encouraging businesses and schools to do the same as a sign of respect for the thousands of people killed in the 2001 attacks.

redpajama_set_name: RedPajamaCommonCrawl


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As an avid reader, I love finding new books that I can recommend. I've put together the 2019 best books guide, so you can find new books for yourself and to give as gifts.
If you are looking for an addictive thriller, The Secretary by Renee Knight is the perfect choice. Warning: it is hard to put down once you start reading.


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Brenda Thompson, the owner/operator of Whispering Hearts Horse Rescue, with some of her horses.
Hamilton Region
Rescuing the horse rescuer
By Mark McNeilContributing Columnist
Tue., June 11, 2013timer3 min. read
updateArticle was updated Feb. 29, 2020
HAGERSVILLE It's not exactly a case of the rescuer needing to be rescued. But it's close.
Whispering Hearts Horse Rescue, a 20-hectare equine rehabilitation farm for abused and neglected animals, is facing its toughest year yet.
Fallout from the racehorse industry decline means a deluge of discarded horses needing help. That same explosion of castaway animals has flooded the marketplace and it's harder than ever to find adoptive homes for their rehabilitated horses. And on top of that, there's a severe hay shortage and prices have skyrocketed.
"We would normally spend an average of $8,000 in hay per year, but this year we're looking $45,000," says Brenda Thompson, the owner/operator of the rescue, located 6 kilometres east of Hagersville.
Last year the hay crop in this part of the province was ravaged by a caterpillarlike pest called armyworm. The insect gets its name from its reputation of lining up and chomping through fields in a militarylike fashion.
Next month the rescue will host its annual Open House fundraising event. They're hoping to raise $20,000 to help offset escalating expenses. Last year they raised $14,000.
"Without the event we wouldn't survive," she said "It takes us over the edge."
Normally the money raised would be used for capital projects around the farm, expanding the barn, buying equipment, etc. But this year they need to put the money into vaccinating the herd, other vet bills and hay.
The farm costs $90,000 a year to run with most of the funds coming out of Thompson and her husband's day jobs. They charge a $500 adoption fee to people wanting to buy one of their rescue horses. But that covers only a small portion of the cost of rehabilitating and caring for the horse — usually for a year — to have it ready for adoption.
Thompson started the horse rescue because she used to work for the Ontario SPCA, and "when I did cruelty investigations, I would realize there was nowhere to put the horses."
So in 2007 the Thompsons bought the farm and started taking in horses in crisis.
"I never imagined it would get as big as it has," she says.
They were thinking maybe 40 to 50 horses at a time. But this year they have 79, and that's after having to turn down 80 more that were offered to Whispering Hearts.
Over the winter they took in 36 critical horses. As well as being sadly underfed, "most had lice and were full of worms with skin infections.
"We took in more this winter than we ever had," Thompson said. "And the horses that came in were at death's door. But we managed to save every one of them."
She thinks it's going to get worse before it gets better.
"There are free horses everywhere. Look on Kijiji ... a lot of people are taking in horses that probably shouldn't be."
Vivian LaFlamme, an inspector at the Hamilton/Burlington SPCA, says often well-meaning people take in horses on their hobby farm but just aren't up to the task of caring for them.
"Just because you have a pasture that isn't enough," she said. "The problem is some of these horses end up with people who lack the experience for proper standards of care."
And when the people finally realize they can't handle the costs and responsibilities of horse ownership, or the SPCA comes knocking at their door, she said, the horses often end up at Whispering Hearts for critical care.
LaFlamme says, "Whenever we've used Whispering Hearts the animals received the best of care. The horses always improved so much there. They are given really good care."
Whispering Hearts Open House Tack and Garage Sale
When: Saturday, July 13 from 8 a.m. to 5 p.m.
Where: 1652 Concession 10, Hagersville
Information: 905-768-9951 and www.whhrescue.com


Each 12-hr period thereafter - $18.
Payment Methods: Cash (U.S. currency), check (in U.S. currency), American Express, MasterCard, Visa, Diners Club, Carte Blance and Discover cards are accepted for payment of parking fees.
E-Z Pass - is a faster way to pay for your airport parking and is accepted in all lots.
All customers who plan to park at Newark Liberty International Airport for more than 30 days should contact Five Star Parking the day you arrive at 973-961-6421 or 973-961-6422. Please provide them with your name, license plate number and the estimated number of days you plan to park at Newark. Any vehicle parked over 30 days will be towed and the owner will be subject to retrieval costs.
Phone Numbers to use: Parking - 1-888-397-4636, then press 22 to receive up-to-date information.
Off Airport Parking: In addition to the on-airport parking, there are a number of privately operated parking lots located near the airport.
where is the cell phone lot located?
Do you need to reserve a space in advance at Parking Lot C?


Lectures and workshops from the Science in Society archive on the New Science of the Organism. For articles in other categories, please see the SiS archive menu.
Video Clip of Jim Oschman lecturing on "Energy Medicine & Quantum Jazz". From the Celebrating ISIS - Quantum Jazz Biology event, March 2011.
Video clip of Dr. Mae-Wan Ho on "Why Beauty is Truth and Truth Beauty". From the Celebrating ISIS - Quantum Jazz Biology event, March 2011.
The organism is quantum coherent, with water playing the lead role in creating and maintaining the quantum coherence and in energizing the organism. Mae-Wan Ho Ph. D.
What Can Development Tell us about Evolution?
The Ufaina Indians in the Colombian Amazon believe in a vital force called fufaka which is present in all living things. The source of this vital force is the sun. From the sun, it reaches earth and is constantly recycled among plants, animals and human beings.
Organisms are so enigmatic from the thermodynamic point of view that Lord Kelvin, coinventor of the Second Law of thermodynamics, specifically excluded them from its dominion Ehrenberg, 1967 , while Schrödinger 1944 suggested they feed upon negative entropy to free themselves from all the entropy they cannot help producing.