Bmal1 knockdown suppresses wake and increases immobility without altering orexin A, corticotrophin-releasing hormone, or glutamate decarboxylase.

OBJECTIVE: To determine the effect of Bmal1 knockdown (KD) on sleep, activity, immobility, hypothalamic levels of orexin, corticotrophin-releasing hormone (CRH), and GABAergic glutamate decarboxylase (GAD). METHODS: We used Bmal1 siRNA, or control siRNA intracerebroventricular (ICV) injection to knock down Bmal1 in C57BL/6 mice. Sleep polysomnography, wheel-running activity, and tail suspension test were performed. Polysomnographic (PSG) recordings in both groups were preceded by ICV injection made during both the light phase and the dark phase. We also measured brain orexin A and CRH using an ELISA and measured GAD using immunoblotting. RESULTS: Compared with control group, Bmal1 KD group had reduced wheel activity and increased immobility. Compared with control, the Bmal1 KD group had reduced wheel activity and increased immobility. During the first 24 hours after treatment, we observed that control siRNA induced a much greater increase in sleep during the dark phase, which was associated with lower orexin levels. However, beginning 24 hours after treatment, we observed an increase in sleep and a decrease in time spent awake during the dark phase in the Bmal1 KD group. These changes were not associated with changes in brain levels of orexin A, CRH, or GAD. CONCLUSION: Bmal1 KD led to reduced activity, increased immobility, and dramatic reduction in time spent awake as well as an increase in sleep during the dark phase. Early after injection, there was a slight change in sleep but brain levels of orexin, CRH, and GAD remain unchanged. Control siRNA also affected sleep associated with changes in orexin levels.

Proteomic profiling of the hypothalamus in two mouse models of narcolepsy.

Narcolepsy is a disabling neurological disorder of sleepiness linked to the loss of neurons producing orexin neuropeptides in the hypothalamus. Two well-characterized phenotypic mouse models of narcolepsy, loss-of-function (orexin-knockout), and progressive loss of orexin (orexin/ataxin-3) exist. The open question is whether the proteomics signatures of the hypothalamus would be different between the two models. To address this gap, we utilized a label-free proteomics approach and conducted a hypothalamic proteome analysis by comparing each disease model to that of wild type. Following data processing and statistical analysis, 14 484 peptides mapping to 2282 nonredundant proteins were identified, of which 39 proteins showed significant differences in protein expression across groups. Altered proteins in both models showed commonalties in pathways for mitochondrial dysfunction and neuronal degeneration, as well as altered proteins related to inflammatory demyelination, insulin resistance, metabolic responses, and the dopaminergic and monoaminergic systems. Model-specific alterations in insulin degraded enzyme (IDE) and synaptosomal-associated protein-25 were unique to orexin-KO and orexin/ataxin-3, respectively. For both models, proteomics not only identified clinically suspected consequences of orexin loss on energy homeostasis and neurotransmitter systems, but also identified commonalities in inflammation and degeneration despite the entirely different genetic basis of the two mouse models.

Hippocampal and motor fronto-cortical neuroligin1 is increased in an animal model of depression.

Neuroligins (NLGNs) regulate synaptic excitability, neuronal signaling and sleep. We hypothesize that alteration of NLGNs is involved in the pathology of depression and tested the hypothesis in a model of depression using Wistar Kyoto (WKy) rat and its control, the Wistar (Wis) rat. We first evaluated behavioral deficits using the forced swim test and then characterized alterations of NLGN1 and NLGN2 with RT-PCR and Western Blotting in the prefrontal cortex, motor frontal cortex and hippocampus. Compared with controls of Wis rats, (1) the WKy rats had significantly shorter swim time and longer immobile time; (2) NLGN1 mRNA levels was higher in the motor frontal cortex and hippocampus in the WKy model; (3) NLGN1 protein was significantly higher in the motor frontal cortex, the prefrontal cortex and the hippocampus in the WKy model; (4) NLGN2 mRNA was significantly higher in the motor frontal cortex but significantly lower in the hippocampus in the WKy model. We concluded that NLGN1 gene and protein expression is higher in the motor frontal cortex, hippocampus and in the prefrontal cortex in the WKy rats suggesting that alterations of NLGN1 is involved in the pathology of depression but need to be further evaluated in human.

7,8-Dihydroxyflavone reduces sleep during dark phase and suppresses orexin A but not orexin B in mice.

Brain-derived neurotrophic factor (BDNF) binds to Tropomyosin-receptor-kinase B (TrkB) receptors that regulate synaptic strength and plasticity in the mammalian nervous system. 7,8-Dihydroxyflavone (DHF) is a recently identified small molecule Trk B agonist that has been reported to ameliorate depression, attenuate the fear response, improve memory consolidation, and exert neuroprotective effects. Poor and disturbed sleep remains a symptom of major depressive disorder and most current antidepressants affect sleep. Therefore, we conducted sleep/wake recordings and concomitant measurement of brain orexins, endogenous peptides that suppress sleep, in mice for this study. Baseline polysomnograph recording was performed for 24 h followed by treatment with either 5 mg/kg of DHF or vehicle at the beginning of the dark phase. Animals were sacrificed the following day, one hour after the final treatment with DHF. Orexin A and B were quantified using ELISA and radioimmunoassay, respectively. Total sleep was significantly decreased in the DHF group, 4 h after drug administration in the dark phase, when compared with vehicle-treated animals. This difference was due to a significant decrease of non-rapid eye movement sleep, but not rapid eye movement sleep. DHF increased power of alpha and sigma bands but suppressed power of gamma band during sleep in dark phase. Interestingly, hypothalamic levels of orexin A were also significantly decreased in the DHF group (97 pg/mg) when compared with the vehicle-treated group (132 pg/mg). However, no significant differences of orexin B were observed between groups. Additionally, no change was found in immobility tests.

Chromosome 1 replacement increases brain orexins and antidepressive measures without increasing locomotor activity.

Decreased orexin level has been well demonstrated in patients suffering from narcolepsy, depression accompanied with suicide attempt; obstructive sleep apnea and comorbidity were also demonstrated in these diseases. As C57BL/6J (B6) mice are more "depressed" and have lower brain orexins than A/J mice, B6 mice having chromosome 1 replacement (B6A1 mice) might have restored orexin levels and less depressive behavior. We studied the behavior of 4-6 month old B6, A/J and B6A1 mice with forced swim, tail suspension, and locomotor activity tests. The animals were then sacrificed and hypothalamus and medullas dissected from brain tissue. Orexins-A and -B were determined by radioimmunoassay. Compared with A/J mice, B6 mice displayed several signs of depression, including increased immobility, increased locomotors activity, and decreased orexin A and -B levels in both the hypothalamus and medulla. Compared to B6 mice, B6A1 mice exhibited significantly higher levels of orexins-A and -B in both brain regions. B6A1 mice also exhibited antidepressive features in most of measured variables, including decreased locomotor activity, decreased immobility and increased swim in tail suspension test; compared with B6 mice, however. B6A1 mice also reversed immobility in the early phase of the swim test. In summary, B6 mice exhibited depressive attributes compared with A/J mice, including increased locomotor activity, greater immobility, and decreased brain orexins, these were largely reversed in B6A1 mice. We conclude that orexin levels modulate these B6 behaviors, likely due to expression of A/J alleles on Chromosome 1.

Effects of orexin 2 receptor activation on apnea in the C57BL/6J mouse.

BACKGROUND: The hypothesis was that an orexin 2 receptor (OX2R) agonist would prevent sleep-related disordered breathing. METHODS: In C57BL/6J (B6) mice, body plethysmography was performed with and without EEG monitoring of state (wakefulness, NREM and REM sleep). Outcome was apnea rate/h during sleep-wake states at baseline and with an intracerebroventricular administration of vehicle, 4 nMol of agonist OB(DL), and 4 nMol of an antagonist, TCS OX2 29. RESULTS: A significant reduction (p=0.035, f=2.99) in apneas/hour occurred, especially with the agonist. Expressed as a function of the change from baseline, there was a significant difference among groups in Wake (p=0.03, f=3.8), NREM (p=0.003, f=6.98) and REM (p=0.03, f=3.92) with the agonist reducing the rate of apneas during sleep from 29.7±4.7 (M±SEM) to 7.3±2.4 during sleep (p=0.001). There was also a reduction in apneas during wakefulness. Administration of the antagonist did not increase event rate over baseline levels. CONCLUSIONS: The B6 mouse is a preclinical model of wake-and sleep-disordered breathing, and the orexin receptor agonist at a dose of 4 nMol given intracerebroventricularly will reduce events in sleep and also wakefulness.