The finding of orexin/hypocretin deficiency in narcolepsy patients suggests that this hypothalamic neuropeptide plays a crucial role in regulating sleep/wakefulness states. However, very little is known about the synaptic input of orexin/hypocretin-producing neurons (orexin neurons). We applied a transgenic method to map upstream neuronal populations that have synaptic connections to orexin neurons and revealed that orexin neurons receive input from several brain areas. These include the amygdala, basal forebrain cholinergic neurons, GABAergic neurons in the preoptic area, and serotonergic neurons in the median/paramedian raphe nuclei. Monoamine-containing groups that are innervated by orexin neurons do not receive reciprocal connections, while cholinergic neurons in the basal forebrain have reciprocal connections, which might be important for consolidating wakefulness. Electrophysiological study showed that carbachol excites almost one-third of orexin neurons and inhibits a small population of orexin neurons. These neuroanatomical findings provide important insights into the neural pathways that regulate sleep/wakefulness states.

In this paper, there was an error in the description of the fusion protein used in the transgenic construct. We described the construct as TTC::GFP in the text and in Figure 1A. The order of the components of the fusion protein was wrong. The fusion protein used in this study should be described as GFP::TTC (the tetanus toxin C-terminal fragment is fused to the C terminus of GFP). Although this correction does not affect the data or the conclusions of the paper, the authors would like to apologize to readers who have been misled by these mistakes. The authors also wish to correct the affiliations of Natsuko Tsujino and Yoshimasa Koyama as listed above.

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Orexins, also called hypocretins, are a recently described pair of neuropeptides orexin-A and -B. Orexins are implicated in energy homeostasis and arousal. Orexin-containing neurons are specifically located in the perifornical, dorsomedial, lateral, and posterior hypothalamus. Orexin neurons have been shown to regulate directly or indirectly the activity of aminergic nuclei. However, little is known about how the orexin neuronal activity is regulated in the brain. Despite the importance to study orexin neurons, it has been difficult to identify and directly examine their electrophysiological properties, because orexin neurons of the LHA are scarce, diffusely distributed, and lack distinct morphological features. To facilitate finding orexin neurons, we made transgenic mice in which orexin neurons specifically express enhanced green fluorescent protein (EGFP). Here we characterize the electrophysiological properties of orexin neurons using slice preparations from transgenic mice in which orexin neurons specifically express green fluorescent protein. Orexin neurons showed high frequency firing with little adaptation by injecting a positive current. The hyperpolarizationactivated current was observed in orexin neurons by a negative current injection. The neurotransmitters, which were implicated in sleep/wake regulation, affected the activity of orexin neurons; norepinephrine, dopamine, and serotonin hyperpolarized, while carbachol depolarized orexin neurons in either the presence or absence of tetrodotoxin. It has been reported that orexins directly or indirectly activate the nuclei that are the origin of the neurons containing these neurotransmitters. Our data suggest that orexin neurons have reciprocal neural circuits between these nuclei for an either positive or negative feedback loop and orchestrate the activity of these neurons to regulate the vigilance states.

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Orexin A and B are neuropeptides implicated in the regulation of sleep/wakefulness and energy homeostasis. The regulatory mechanism of the activity of orexin neurons is not precisely understood. Using transgenic mice in which orexin neurons specifically express yellow cameleon 2.1, we screened for factors that affect the activity of orexin neurons (a total of 21 peptides and six other factors were examined) and found that a sulfated octapeptide form of cholecystokinin (CCK-8S), neurotensin, oxytocin, and vasopressin activate orexin neurons. The mechanisms that underlie CCK-8S-induced activation of orexin neurons were studied by both calcium imaging and slice patch-clamp recording. CCK-8S induced inward current in the orexin neurons. The CCK A receptor antagonist lorglumide inhibited CCK-8S-induced activation of orexin neurons, whereas the CCK B receptor agonists CCK-4 (a tetrapeptide form of cholecystokinin) and nonsulfated CCK-8 had little effect. The CCK-8S-induced increase in intracellular calcium concentration was eliminated by removing extracellular calcium but not by an addition of thapsigargin. Nifedipine, -conotoxin, -agatoxin, 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride, and SNX-482 had little effect, but La 3ϩ , Gd 3ϩ , and 2-aminoethoxydiphenylborate inhibited CCK-8S-induced calcium influx. Additionally, the CCK-8S-induced inward current was dramatically enhanced in the calcium-free solution and was inhibited by the cation channel blocker SKF96365, suggesting an involvement of extracellular calcium-sensitive cation channels. CCK-8S did not induce an increase in intracellular calcium concentration when membrane potential was clamped at Ϫ60 mV, suggesting that the calcium increase is induced by depolarization. The evidence presented here expands our understanding of the regulation of orexin neurons and the physiological role of CCK in the CNS.

Orexin neurons are directly and indirectly regulated by catecholamines in a complex manner. J Neurophysiol 96: 284 -298, 2006. First published April 12, 2006 doi:10.1152/jn.01361.2005. We reported elsewhere that orexin neurons are directly hyperpolarized by noradrenaline (NA) and dopamine. In the present study, we show that NA, dopamine, and adrenaline all directly hyperpolarized orexin neurons. This response was inhibited by the ␣ 2 adrenergic receptor (␣ 2 -AR) antagonist, idazoxan or BRL44408, and was mimicked by the ␣ 2 -AR-selective agonist, UK14304. A low concentration of Ba 2ϩ inhibited NA-induced hyperpolarization, which suggests that activation of G protein coupled inward rectifier potassium channels is involved in the response. In the presence of a high concentration of idazoxan, NA induced depolarization or inward current. This response was inhibited by ␣ 1 -AR antagonist, prazosin, which suggests the existence of ␣ 1 -ARs on the orexin neurons along with ␣ 2 -AR. We also examined the effects of NA on glutamatergic and GABAergic synaptic transmission. NA application dramatically increased the frequency and amplitude of spontaneous inhibitory synaptic currents (sIPSCs) and inhibited excitatory synaptic currents (sEPSCs) in orexin neurons; however, NA decreased the frequency of miniature EPSCs (mEPSCs) and IPSCs and the amplitude of evoked EPSCs and IPSCs through the ␣ 2 -AR, because the NA response on mPSCs was inhibited by idazoxan. These results suggest that the NA-induced increase in sIPSC frequency and amplitude is mediated via ␣ 1 -ARs on the somata of GABAergic neurons that innervate the orexin neurons. Calcium imaging using orexin/YC2.1 transgenic mouse brain revealed that NA-induced inhibition of orexin neurons is not altered by sleep deprivation or circadian time in mice. The evidence presented here revealed that orexin neurons are regulated by catecholamines in a complex manner.

Fibrosis of the lung constitutes a major clinical challenge and novel therapies are required to alleviate the associated morbidity and mortality. Investigating the antifibrotic efficacy of drugs that are already in clinical practice offers an efficient strategy to identify new therapies. The phosphodiesterase 4 (PDE4) inhibitors, approved for the treatment of chronic obstructive pulmonary disease, harbor therapeutic potential for pulmonary fibrosis by augmenting the activity of endogenous antifibrotic mediators that signal through cyclic AMP. In this study, we tested the efficacy of several PDE4 inhibitors including a novel compound (Compound 1) in a murine model of lung fibrosis that results from a targeted type II alveolar epithelial cell injury. We also compared the antifibrotic activity of PDE4 inhibition to the two therapies that are FDA‐approved for idiopathic pulmonary fibrosis (pirfenidone and nintedanib). We found that both preventative (day 0–21) and therapeutic (day 11–21) dosing regimens of the PDE4 inhibitors significantly ameliorated the weight loss and lung collagen accumulation that are the sequelae of targeted epithelial cell damage. In a therapeutic protocol, the reduction in lung fibrosis with PDE4 inhibitor administration was equivalent to pirfenidone and nintedanib. Treatment with this class of drugs also resulted in a decrease in plasma surfactant protein D concentration, a reduction in the plasma levels of several chemokines implicated in lung fibrosis, and an in vitro inhibition of fibroblast profibrotic gene expression. These results motivate further investigation of PDE4 inhibition as a treatment for patients with fibrotic lung disease.