Effects of central hypocretin-1 administration on hemodynamic responses in young-adult and middle-aged rats

The prevalence of hypertension in middle age correlates with impaired autonomic regulation and as norepinephrinergic neurons decline with increasing age, and this reduction may contribute to this impairment. Central hypocretin-activated norepinephrinergic neurons contribute to sympathetic regulation. In the present study we compared sympathoadrenal effects of intracerebroventricular (i.c.v.) hypocretin-1(5 nmol) between young-adult (12-14 weeks) and middle-aged (12-14 months) rats. Arterial blood pressure, heart rate and plasma catecholamines were assessed under pentobarbital anesthesia. In addition, we compared hypocretin-1 and K(+)-evoked norepinephrine release from the cerebrocortical slices prepared from young-adult and middle-aged rats. We also examined whether the novel hypocretin receptor-1 antagonist (SB-334867) could reverse these hypocretin-1 effects both in vivo and in vitro. I.c.v. hypocretin-1 significantly increased blood pressure by some 7%, heart rate by 9% and plasma norepinephrine concentrations by 100% in young-adult rats. In middle-aged rats these parameters did not change. Plasma epinephrine did not increase in either group. There was a significant correlation between changes in mean arterial pressure and plasma norepinephrine. Similarly, hypocretin-1 evoked norepinephrine release from cerebrocortical slices prepared from young-adult rats was significantly higher than that of middle-aged rats whilst K(+)-evoked release did not differ between the groups. SB-334867 significantly attenuated hypocretin-1-increased blood pressure and both in vivo and in vitro norepinephrine release. The present data suggest that hypocretinergic neurons may contribute to the regulation of central but not adrenal sympathetic activity. Moreover, sympathetic regulation by hypocretinergic neurones may disappear in middle-age in rats.     

Effects of Medroxyprogesterone Acetate on Sleep of Healthy Young Male Adults

Abstract: The effects of a single 100 mg dose of medroxyprogesterone acetate (MPA) on human sleep were investigated using healthy young male adults. After a baseline night recording, two consecutive MFA nights and the following two withdrawal nights were recorded. The total sleep time was reduced after the 2nd MPA night and the decrease was significant on the 1st withdrawal night. The time of intrasleep awakening increased after the 2nd MPA night. Stage 1 sleep increased significantly on the 2nd MPA and 1st withdrawal nights. Stage 4 sleep decreased and the sleep latency gradually increased after the MPAadministration. REM sleep reduced slightly on the 1st withdrawal night. These results indicate that MPA has a mild arousal action on human sleep and that the action may be pronounced a few days after administration of MPA.     

The novel brain neuropeptide, orexin-A, modulates the sleep-wake cycle of rats

Orexin-A is a novel neuropeptide initially isolated from hypothalamic extracts but now known to be present in fibres distributed throughout the rat CNS including many regions associated with sleep-wake regulation. The recognition of a particularly dense innervation of orexinergic nerves in the locus coeruleus, together with the observed increase in firing rate of locus coeruleus neurons following application of orexin-A in vitro, further highlighted a potential involvement of the peptide in modulating the arousal state. The present study was undertaken to determine the effects of intracerebroventricularly (ICV) administered orexin-A on the sleep-wake cycle of conscious rats using electroencephalographic and electromyographic recordings. When administered at the onset of the normal sleep period, orexin-A (1, 10 or 30 microg/rat ICV) produced a dose-dependent increase in the time rats spent awake during the second and third hours after dosing. The enhancement of arousal was accompanied by a marked reduction in paradoxical sleep and deep slow wave sleep at the highest dose. The latency to the first occurrence of paradoxical sleep was also prolonged. This overall profile of increased arousal and decreased paradoxical sleep is consistent with a high rate of firing of locus coeruleus neurons as would be expected to occur following ICV administration of orexin-A. It is concluded that orexin-A may play an important physiological role in regulating the sleep-wake state, a hypothesis considerably strengthened by the recently reported narcoleptic phenotype of prepro-orexin (the precursor for orexin-A) knockout mice.     

Effects on sleep and wakefulness of the injection of hypocretin-1 (orexin-A) into the laterodorsal tegmental nucleus of the cat

Anatomical data demonstrate a dense projection, in the cat, from hypocretin (orexin) neurons in the hypothalamus to the laterodorsal tegmental nucleus (LDT), which is a critical pontine site that is involved in the regulation of the behavioral states of sleep and wakefulness. The present study was therefore undertaken to explore the hypocretinergic control of neurons in the LDT vis-à-vis these behavioral states. Accordingly, hypocretin-1 was microinjected into the LDT of chronic, unanesthetized cats and its effects on the percentage, latency, frequency and duration of wakefulness, quiet (non-REM) sleep and active (REM) sleep were determined. There was a significant increase in the time spent in wakefulness following the microinjection of hypocretin-1 into the LDT and a significant decrease in the time spent in active sleep. The increase in the percentage of wakefulness was due to an increase in the duration of episodes of wakefulness; the reduction in active sleep was due to a decrease in the frequency of active sleep episodes, but not in their duration. These data indicate that hypocretinergic processes in the LDT play an important role in both of the promotion of wakefulness and the suppression of active sleep.     

Diurnal variation of cerebrospinal fluid hypocretin-1 (Orexin-A) levels in control and depressed subjects.

BACKGROUND: Hypocretins, excitatory neuropeptides at monoaminergic synapses, appear to regulate human sleep-wake cycles. Undetectable cerebrospinal fluid hypocretin-1 levels are seen in narcolepsy, which is frequently associated with secondary depression. Shortened rapid eye movement latency is observed in both narcolepsy and depression. Cerebrospinal fluid hypocretin-1 levels have not been reported in mood disorders. METHODS: We examined hypocretin-1 levels in 14 control and 15 depressed subjects. Cerebrospinal fluid was drawn continuously in supine subjects for 24 hours with an indwelling intrathecal catheter under entrained light-dark conditions. Depressed subjects were studied before and after 5 weeks of sertraline (n=10, three nonresponders) or bupropion (n=5, two nonresponders). RESULTS: Hypocretin-1 levels varied slightly (amplitude 10%) but significantly across the diurnal cycle in control subjects, with amplitude significantly reduced in depression (3%). Levels were lowest at midday, surprising for a hypothetically wake-promoting peptide. Mean hypocretin levels trended higher in depressive than in control subjects. Hypocretin-1 levels decreased modestly but significantly after sertraline (-14%) but not bupropion. CONCLUSIONS: Our results are consistent with previous physiologic findings in depression indicating dampened diurnal variations in hypocretin-1. The finding that sertraline but not bupropion slightly decreased cerebrospinal fluid hypocretin-1 indicates a serotoninergic influence on hypocretin tone.     

Hypothalamo-preoptic Histaminergic Projections in Sleep-Wake Control in the Cat

Cats were chronically implanted with electrodes for polygraphic recordings and cannulae for intracerebral microinjections in order to study the functional role of histaminergic innervation of the preoptic-anterior hypothalamus in sleep-wake control. α-Fluoromethylhistidine (αFMH, 50 μg in 1 μl), a specific inhibitor of the histamine-synthesizing enzyme, when injected bilaterally into the preoptic area, where numerous histaminergic fibres and terminal-like structures are present, caused a significant increase in deep slow wave sleep (S2) and paradoxical sleep (PS) and a decrease in wakefulness. In contrast, microinjections of histamine (5 or 30 μg in 1 μl) in the same area dose-relatedly increased wakefulness and decreased both slow wave sleep and paradoxical sleep. The effects of histamine were reduced by pretreatment with mepyramine (1 mg/kg i. p.), a well known histamine H₁ receptor antagonist, and were mimicked by a local injection of impromidine (1 μg in 1 μl), a potent histamine H₂ receptor agonist. Microinjections of mepyramine alone (120 μg in 1 μl) caused an increase in slow wave sleep. These results suggest that preoptic histaminergic innervation is involved in sleep-wake control and that the action might be mediated via both H₁ and H₂ receptors.     

Early neonatal 192 IgG saporin induces learning impairments and disrupts cortical morphogenesis in rats

We have shown previously that neonatal intraventricular injections of the selective cholinergic immunotoxin 192 IgG saporin on postnatal day 7 (pnd 7) induce marked cholinergic loss in hippocampus and neocortex and a learning impairment on pnd 15. In the present study, we analysed the behavioural, morphological and neurochemical effects of earlier intraventricular injection of the immunotoxin 192 IgG saporin (pnd 1 and 3). We hypothesised that these earlier lesions would interrupt a critical stage in neocortical maturation, and impair behavior more profoundly than the later lesions. Passive avoidance (PA) learning and locomotor activity during the PA test were assessed on pnd 15. Retention of the PA task was assessed on pnd 16. Reactivity to spatial and object novelty was assessed on pnd 180 in a spatial open field test with five objects. Choline acetyltransferase (ChAT) activity was measured in basal forebrain targets on pnd 20 and pnd 180. Neonatal administration of 192 IgG saporin resulted in a slower acquisition of the PA task in females; retention and locomotor activity were not affected. On pnd 180, reaction to spatial novelty was mildly impaired in lesioned rats of both sexes. There was a marked reduction of ChAT in the hippocampus and neocortex of lesioned rats of both sexes, at both ages. Morphological analysis of the somatosensory cortex of lesioned rats revealed alterations in cortical development with sex specific variations in total cortical thickness. These results suggest that interrupting cholinergic basal forebrain innervation of neocortex and hippocampus during the first postnatal days affects the development of cognitive behaviour, neurochemistry and cortical organisation in a sex specific manner. Furthermore, the alterations in cortical organization are more profound than those noted after a lesion later in postnatal development. These behavioural and morphological abnormalities could be considered a model for several neurodevelopmental disorders associated with mental retardation.     

Effects of intravenous caffeine administered to healthy males during sleep

Pharmacological challenge paradigms have been useful for elucidating the phenomenology and neurobiology of panic attacks. A drawback of the pharmacological challenge method is that individual differences in baseline arousal and outcome expectancy can lead to different subjective and physiological drug responses. One method for eliminating differences in baseline arousal and expectancy is to perform pharmacological challenges during non-rapid eye movement (non-REM) sleep. In the present study, fourteen healthy male volunteers received caffeine (5 mg/kg) and placebo (normal saline) during non-REM sleep on two successive nights, in a single-blind manner. Caffeine, compared to placebo, was associated with increased arousal, sleep disruption, and elevations in adrenocorticotropic hormone (ACTH) and cortisol. In one subject, caffeine infusion during sleep induced a panic attack. These findings indicate that caffeine leads to increased arousal and hypothalamic-pituitary-adrenal axis (HPA) axis activation in the absence of high baseline anxiety and expectancy bias. Further, they suggest that similar techniques can be employed in patient populations to elucidate the neurobiology of sleep panic attacks. Depression and Anxiety 5:21–28, 1997. © 1997 Wiley-Liss, Inc.     

Postsynaptic excitation of prefrontal cortical pyramidal neurons by hypocretin-1/orexin A through the inhibition of potassium currents

Hypocretins are crucial for the regulation of wakefulness by the excitatory actions on multiple subcortical arousal systems. To date, there is little information about the direct postsynaptic excitatory effects of hypocretins on the neurons in prefrontal cortex (PFC), which is important for higher cognitive functions and is correlated with level of wakefulness. In this study, we tested the excitatory effects of hypocretin-1 on acutely isolated PFC pyramidal neurons of rats and studied the possible ionic mechanisms by using whole-cell patch-clamp techniques. Puff application of hypocretin-1 caused a dose-dependent excitation. Further observations that perfusion of Ca2+-free artificial cerebrospinal fluid did not influence the depolarizing effects of hypocretin-1, in conjunction with the findings that hypocretin-1 could decrease net whole-cell K+ currents, demonstrate that the excitatory effects of hypocretin-1 on PFC neurons are mediated by the inhibition of K+ currents but not Ca2+ influx. Finally, the decrease in K+ currents induced by hypocretin-1 was abolished by a protein kinase C (PKC) inhibitor (BIS II) or a phospholipase C (PLC) inhibitor (D609), suggesting that PKC and PLC appear to be involved in mediating the inhibitory effects of hypocretin-1 on K+ currents. These results indicate that hypocretin-1 exerts a postsynaptic excitatory action on PFC neurons through the inhibition of K+ currents, which probably results from activation of PKC and PLC signaling pathways.     

CSF hypocretin-1 levels in restless legs syndrome

CSF hypocretin-1 levels at 6 PM did not significantly differ between patients with restless legs syndrome (RLS) and control subjects as measured by direct radioimmunoassay and after acid extraction. The authors did not observe significant differences between early onset and late onset RLS. Hypocretin-1 levels did not correlate with RLS severity or polysomnographic measures. These results contrast with previous findings reporting significantly increased CSF hypocretin-1 in the late evening and mostly in early onset RLS.     

The orexinergic neurons receive synaptic input from C1 cells in rats

The C1 cells, located in the rostral ventrolateral medulla (RVLM), are activated by pain, hypoxia, hypoglycemia, infection, and hypotension and elicit cardiorespiratory stimulation, adrenaline and adrenocorticotropic hormone (ACTH) release, and arousal. The orexin neurons contribute to the autonomic responses to acute psychological stress. Here, using an anatomical approach, we consider whether the orexin neurons could also be contributing to the autonomic effects elicited by C1 neuron activation. Phenylethanolamine N‐methyl transferase‐immunoreactive (PNMT‐ir) axons were detected among orexin‐ir somata, and close appositions between PNMT‐ir axonal varicosities and orexin‐ir profiles were observed. The existence of synapses between PNMT‐ir boutons labeled with diaminobenzidine and orexinergic neurons labeled with immunogold was confirmed by electron microscopy. We labeled RVLM neurons with a lentiviral vector that expresses the fusion protein ChR2‐mCherry under the control of the catecholaminergic neuron‐selective promoter PRSx8 and obtained light and ultrastructural evidence that these neurons innervate the orexin cells. By using a Cre‐dependent adeno‐associated vector and TH‐Cre rats, we confirmed that the projection from RVLM catecholaminergic neurons to the orexinergic neurons originates predominantly from PNMT‐ir catecholaminergic (i.e., C1 cells). The C1 neurons were found to establish predominantly asymmetric synapses with orexin‐ir cell bodies or dendrites. These synapses were packed with small clear vesicles and also contained dense‐core vesicles. In summary, the orexin neurons are among the hypothalamic neurons contacted and presumably excited by the C1 cells. The C1–orexin neuronal connection is probably one of several suprabulbar pathways through which the C1 neurons activate breathing and the circulation, raise blood glucose, and facilitate arousal from sleep. J. Comp. Neurol. 522:3834–3846, 2014. © 2014 Wiley Periodicals, Inc.     

Effects of orexins on the hypothalamic-pituitary-adrenal system

The effects of the recently identified neuropeptides orexin-A and orexin-B on the hypothalamic-pituitary-adrenal (HPA) system were investigated. An in vivo system was used to assess the central effects of both orexin-A and orexin-B. Different doses of the orexins (2.8-560 pmol) were administered intracerebroventricularly (i.c.v.) to adult male rats, and plasma corticosterone was used as an index of the degree of the activation of the HPA system. Both peptides exhibited a clear dose-response action, although orexin-B proved to be less effective than orexin-A. Pretreatment with the corticotropin-releasing hormone (CRH) antagonist alpha-helical CRH9-41 completely prevented the action of the orexins. Orexin-A, orexin-B or adrenocorticotropic hormone (ACTH) was further administered intraperitoneally (i.p.). While ACTH evoked a significant adrenal response, the orexins did not influence the basal secretion. Adrenal slices, oxygenized and perifused with Krebs' solution, were also treated with orexin-A, orexin-B or ACTH. Both orexins failed to modify the release of corticosterone, but ACTH induced a marked adrenal response. This study suggests that these appetite-regulating peptides might activate the HPA system at a central level but neither orexin-A nor orexin-B appears to modulate directly the adrenal corticosterone release.     

Differences in arousal response between aged and middle-aged patients with obstructive sleep apnea syndrome

Abstract The aim of this study is to show the clinical significance of the differences in arousal response at a termination of apnea/hypopnea between aged and middle-aged patients with obstructive sleep apnea syndrome (OSAS). We polygraphically assessed electrocardiographic (ECG) and electroencephalographic (EEG) arousal. Electrocardiographic arousal was defined as an abrupt increase in heart rate at a termination of apnea/hypopnea. Our findings showed that EEG and ECG arousal at a termination of apnea/hypopnea were significantly suppressed in aged patients with OSAS, which might provide useful information on the pathophysiology of OSAS.     

Fluctuation of extracellular hypocretin-1 (orexin A) levels in the rat in relation to the light–dark cycle and sleep–wake activities

Hypocretins/orexins are neuropeptides implicated in sleep regulation and the sleep disorder narcolepsy. In order to examine how hypocretin activity fluctuates across 24 h with respect to the sleep-wake cycle, we measured changes in extracellular hypocretin-1 levels in the lateral hypothalamus and medial thalamus of freely moving rats with simultaneous sleep recordings. Hypocretin levels exhibited a robust diurnal fluctuation; levels slowly increased during the dark period (active phase), and decreased during the light period (rest phase). Levels were not correlated with the amount of wake or sleep in each period. Although an acute 4-h light-shift did not alter hypocretin levels, 6-h sleep deprivation significantly increased hypocretin release during the forced-wake period. Hypocretin activity is, thus, likely to build up during wakefulness and decline with the occurrence of sleep. These findings, together with the fact that a difficulty in maintaining wakefulness during the daytime is one of the primary symptoms of hypocretin-deficient narcolepsy, suggest that hypocretin activity may be critical in opposing sleep propensity during periods of prolonged wakefulness.     

Effects of 192 IgG-saporin on acetylcholinesterase histochemistry in male and female rats

Sex hormones may exert neuroprotective effects in various models of brain lesions. Male and female Long-Evans rats were subjected to intracerebroventricular injections of 2 microg 192 IgG-saporin or vehicle. Starting 2 days before surgery, half the male rats were treated with estradiol for 7 days. Three weeks after surgery, they were sacrificed for histochemical staining of acetylcholinesterase (AChE) and densitometric evaluations. The lesion induced a substantial to dramatic decrease of the AChE-positive fiber density in the cingulate, somatosensory, piriform, retrosplenial and perirhinal cortices, and in the hippocampus. Weak effects were found in the striatum. There was no significant decrease in the dorsal thalamus. Sex had no significant effect on AChE-positive staining in any brain area. In males, estradiol treatment did not alter the effects of 192 IgG-saporin. These results show that sex or estradiol treatment in male rats does not interfere with the immunotoxic effects of intracerebroventricular injections of 192 IgG-saporin on cholinergic projections from the basal forebrain.     

Effects of midbrain raphe stimulation and lesion on EEG activity in rats

In order to clarify whether the dorsal raphe (d-R) and medial raphe nuclei (m-R) of the midbrain play a different role in EEG activity, the effects of either electrical stimulation or lesion of each raphe nucleus were examined in rats. After m-R stimulation, EEG activity changed to a marked drowsy pattern. D-R stimulation, however, failed to produce such effects. M-R lesion elicited a significant arousal in EEG. The change in hippocampal EEG was the most characteristic, i.e., synchronized waves with an extreme increase in voltage. D-R lesion did not produce such a change. The hippocampal EEG arousal was easily abolished by L-5-hydroxytryptophan without any change in the cortical EEG or behavior. These results suggest that the m-R plays a more important role than the d-R in regulating the hippocampal EEG activity, especially in inhibiting the appearance of high voltage synchronized waves in the hippocampus.     

The behavioral functions of the cholinergic basalforebrain : lessons from 192 IgG-SAPORIN

Until recently, our understanding of the functional neuroanatomy of the cholinergicbasal forebrain (CBF) has been hindered by the lack of a lesioning technique that is truly selective.The development of the immunotoxin 192 IgG-saporin (192-sap) has greatly improved our abilityto create specific lesions of the CBF. Rats with such lesions have been studied in a wide variety ofbehavioral paradigms of learning, memory, and attention. Complete or near-complete destructionof the CBF results in deficits in a variety of behavior paradigms including passive avoidance,spatial tasks (water and radial mazes), delayed matching to position/sample, and attentional tasks.However, interpretation of many experiments is hampered by incomplete lesions and/orconcomitant damage to cerebellar Purkinje neurons. Future studies will need to address theseissues. Recent development of a similar immunotoxin that is effective in primates should permitmore sophisticated behavioral analysis of CBF function. Additionally, immunotoxins selective forother types of neurons, such as the noradrenergic selective anti-DBH-saporin, will permit analysisof the behavioral functions of other diffusely projecting systems and how these other systems mayinteract with the CBF.