Orexin (hypocretin) gene transfer diminishes narcoleptic sleep behavior in mice

Gene transfer has proven to be an effective neurobiological tool in a number of neurodegenerative diseases, but it is not known if it can correct a sleep disorder. Narcolepsy is a neurodegenerative sleep disorder linked to the loss of neurons containing the neuropeptide orexin, also known as hypocretin. Here, a replication-defective herpes simplex virus-1 amplicon-based vector was constructed to transfer the gene for mouse prepro-orexin into mice with a genetic deletion of the orexin gene. After in vitro tests confirmed successful gene transfer into cells, the gene vector was delivered to the lateral hypothalamus of orexin knockout (KO) mice where the orexin peptide was robustly expressed in the somata and processes of numerous neurons, and the peptide product was detected in the cerebrospinal fluid. During the 4-day life-span of the vector the incidence of cataplexy declined by 60%, and the levels of rapid eye movement sleep during the second half of the night were similar to levels in wild-type mice, indicating that narcoleptic sleep-wake behavior in orexin KO mice can be improved by targeted gene transfer.     

Narcoleptic orexin receptor knockout mice express enhanced cholinergic properties in laterodorsal tegmental neurons

Pharmacological studies of narcoleptic canines indicate that exaggerated pontine cholinergic transmission promotes cataplexy. As disruption of orexin (hypocretin) signaling is a primary defect in narcolepsy with cataplexy, we investigated whether markers of cholinergic synaptic transmission might be altered in mice constitutively lacking orexin receptors (double receptor knockout; DKO). mRNA for Choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the high‐affinity choline transporter (CHT1) but not acetylcholinesterase (AChE) was significantly higher in samples from DKO than wild‐type (WT) mice. This was region‐specific; levels were elevated in samples from the laterodorsal tegmental nucleus (LDT) and the fifth motor nucleus (Mo5) but not in whole brainstem samples. Consistent with region‐specific changes, we were unable to detect significant differences in Western blots for ChAT and CHT1 in isolates from brainstem, thalamus and cortex or in ChAT enzymatic activity in the pons. However, using ChAT immunocytochemistry, we found that while the number of cholinergic neurons in the LDT and Mo5 were not different, the intensity of somatic ChAT immunostaining was significantly greater in the LDT, but not Mo5, from DKO than from WT mice. We also found that ChAT activity was significantly reduced in cortical samples from DKO compared with WT mice. Collectively, these findings suggest that the orexins can regulate neurotransmitter expression and that the constitutive absence of orexin signaling results in an up‐regulation of the machinery necessary for cholinergic neurotransmission in a mesopontine population of neurons that have been associated with both normal rapid eye movement sleep and cataplexy.     

Orexin in sleep,addiction and more: Is the perfect insomnia drug at hand?

Orexins A and B (hypocretins 1 and 2) and their two receptors (OX₁R and OX₂R) were discovered in 1998 by two different groups. Orexin A and B are derived from the differential processing of a common precursor, the prepro-orexin peptide. The neuropeptides are expressed in a few thousand cells located in the lateral hypothalamus (LH), but their projections and receptor distribution are widespread throughout the brain. Remarkably, prepro peptide and double (OX₁R/OX₂R) receptor knock out (KO) mice reproduce a sleep phenotype known in humans and dogs as narcolepsy/cataplexy. In humans, this disease is characterized by the absence of orexin producing cells in the LH, and severely depleted levels of orexin the cerebrospinal fluid. Null mutation of the individual OX₁R or OX₂R in mice substantially ameliorates the narcolepsy/cataplexy phenotype compared to the OX₁R/OX₂R KO, and highlights specific roles of the individual receptors in sleep architecture, the OX₁R KO demonstrating an a attenuated sleep phenotype relative to the OX₂R KO. It has therefore been suggested that orexin is a master regulator of the sleep-wake cycle, with high activity of the LH orexin cells during wake and almost none during sleep. Less than 10 years later, the first orexin antagonist, almorexant, a dual orexin receptor antagonist (DORA), was reported to be effective in inducing sleep in volunteers and insomnia patients. Although development was stopped for almorexant and for Glaxo’s DORA SB-649868, no less than 4 orexin receptor antagonists have reached phase II for insomnia, including Filorexant (MK-6096) and Suvorexant (MK-4305) from Merck. Suvorexant has since progressed to Phase III and dossier submission to the FDA. These four compounds are reported as DORAs, however, they equilibrate very slowly at one and/or the other orexin receptor, and thus at equilibrium may show more or less selectivity for OX₁R or OX₂R. The appropriate balance of antagonism of the two receptors for sleep is a point of debate, although in rodent models OX₂R antagonism alone appears sufficient to induce sleep, whereas OX₁R antagonism is largely devoid of this effect. Orexin is involved in a number of other functions including reward and feeding, where OX₁R (possibly OX₂R) antagonists display anti-addictive properties in rodent models of alcohol, smoking, and drug self-administration. However, despite early findings in feeding and appetite control, orexin receptor antagonists have not produced the anticipated effects in models of increased food intake or obesity in rodents, nor have they shown marked effects on weight in the existing clinical trials. The role of orexin in a number of other domains such as pain, mood, anxiety, migraine and neurodegenerative diseases is an active area of research. The progress of the orexin field is thus extraordinary, and the community awaits the clinical testing of more receptor selective antagonists in sleep and other disorders, as well as that of orexin agonists, with the latter expected to produce positive outcomes in narcolepsy/cataplexy and other conditions.     

Optogenetic activation of melanin‐concentrating hormone neurons increases non‐rapid eye movement and rapid eye movement sleep during the night in rats

Neurons containing melanin‐concentrating hormone (MCH) are located in the hypothalamus. In mice, optogenetic activation of the MCH neurons induces both non‐rapid eye movement (NREM) and rapid eye movement (REM) sleep at night, the normal wake‐active period for nocturnal rodents [R. R. Konadhode et al. (2013) J. Neurosci., 33, 10257–10263]. Here we selectively activate these neurons in rats to test the validity of the sleep network hypothesis in another species. Channelrhodopsin‐2 (ChR2) driven by the MCH promoter was selectively expressed by MCH neurons after injection of rAAV‐MCHp‐ChR2‐EYFP into the hypothalamus of Long–Evans rats. An in vitro study confirmed that the optogenetic activation of MCH neurons faithfully triggered action potentials. In the second study, in Long–Evans rats, rAAV‐MCH‐ChR2, or the control vector, rAAV‐MCH‐EYFP, were delivered into the hypothalamus. Three weeks later, baseline sleep was recorded for 48 h without optogenetic stimulation (0 Hz). Subsequently, at the start of the lights‐off cycle, the MCH neurons were stimulated at 5, 10, or 30 Hz (1 mW at tip; 1 min on – 4 min off) for 24 h. Sleep was recorded during the 24‐h stimulation period. Optogenetic activation of MCH neurons increased both REM and NREM sleep at night, whereas during the day cycle, only REM sleep was increased. Delta power, an indicator of sleep intensity, was also increased. In control rats without ChR2, optogenetic stimulation did not increase sleep or delta power. These results lend further support to the view that sleep‐active MCH neurons contribute to drive sleep in mammals.     

Narcoleptic episodes in orexin-deficient mice are increased by both attractive and aversive odors

Orexin-deficient mice are an established animal model for narcolepsy. In human patients, narcoleptic events are mainly triggered by emotional events. However, the role of emotional stimuli in murine narcolepsy is not well understood. The present study investigated the effects of attractive and aversive odor stimuli, i.e. urine samples of coyote and female mice, on narcoleptic episodes (cataplexy, sleep attacks) in orexin-deficient mice. Here, we first demonstrate that exposure to both attractive and aversive odors significantly increase the number of narcoleptic episodes in orexin-deficient mice. This behavioral paradigm may be of high interest for studies focused on the question how emotions can trigger narcoleptic episodes.     

Deep brain stimulation of hypothalamus for narcolepsy-cataplexy in mice

BackgroundNarcolepsy type 1 (NT1, narcolepsy with cataplexy) is a disabling neurological disorder caused by loss of excitatory orexin neurons from the hypothalamus and is characterized by decreased motivation, sleep-wake fragmentation, intrusion of rapid-eye-movement sleep (REMS) during wake, and abrupt loss of muscle tone, called cataplexy, in response to sudden emotions.ObjectiveWe investigated whether subcortical stimulation, analogous to clinical deep brain stimulation (DBS), would ameliorate NT1 using a validated transgenic mouse model with postnatal orexin neuron degeneration.MethodsUsing implanted electrodes in freely behaving mice, the immediate and prolonged effects of DBS were determined upon behavior using continuous video-electroencephalogram-electromyogram (video/EEG/EMG) and locomotor activity, and neural activation in brain sections, using immunohistochemical labeling of the immediate early gene product c-Fos.ResultsBrief 10-s stimulation to the region of the lateral hypothalamus and zona incerta (LH/ZI) dose-responsively reversed established sleep and cataplexy episodes without negative sequelae. Continuous 3-h stimulation increased ambulation, improved sleep-wake consolidation, and ameliorated cataplexy. Brain c-Fos from mice sacrificed after 90 min of DBS revealed dose-responsive neural activation within wake-active nuclei of the basal forebrain, hypothalamus, thalamus, and ventral midbrain.ConclusionAcute and continuous LH/ZI DBS enhanced behavioral state control in a mouse model of NT1, supporting the feasibility of clinical DBS for NT1 and other sleep-wake disorders.     

Development of Cataplexy in Genetically Narcoleptic Dobermans

Forty-two genetically narcoleptic Doberman puppies [20 pure narcoleptic (N) puppies (from four narcoleptic × narcoleptic crosses) and 22 backcross narcoleptic (BN) puppies (from six narcoleptic × heterozygous crosses)] were systematically observed during the developmental period (4–24 weeks) to assess the age at onset and severity of cataplexy, a pathological manifestation of REM sleep atonia seen in narcolepsy. The mean age of onset of cataplexy was 9.69 ± 1.15 weeks, with a median age of 7 weeks. The severity of cataplexy increased with age and reached a plateau at around 16–24 weeks. The effects of cross type (N vs BN) and sex on the development of cataplexy were analyzed. There was no difference in severity between N and BN puppies (P = 0.51). However, females had more severe cataplexy than males (P = 0.01), and this trend was preserved in five of the six litters that had both male and female puppies. These results suggest that the pathophysiological process in genetic canine narcolepsy emerges during the early developmental period and that it may involve a differential development in males and females. Furthermore, our results revealed that cataplexy onset corresponds to the emergence of adult-like REM sleep and to previously reported neuroanatomical and neurochemical abnormalities in canine narcolepsy.     

Developmental divergence of sleep-wake patterns in orexin knockout and wild-type mice

Narcolepsy, a disorder characterized by fragmented bouts of sleep and wakefulness during the day and night as well as cataplexy, has been linked in humans and nonhuman animals to the functional integrity of the orexinergic system. Adult orexin knockout mice and dogs with a mutation of the orexin receptor exhibit symptoms that mirror those seen in narcoleptic humans. As with narcolepsy, infant sleep-wake cycles in humans and rats are highly fragmented, with consolidated bouts of sleep and wakefulness developing gradually. Based on these common features of narcoleptics and infants, we hypothesized that the development of sleep-wake fragmentation in orexin knockout mice would be expressed as a developmental divergence between knockouts and wild-types, with the knockouts lagging behind the wild-types. We tested this hypothesis by recording the sleep-wake patterns of infant orexin knockout and wild-type mice across the first three postnatal weeks. Both knockouts and wild-types exhibited age-dependent, and therefore orexin-independent, quantitative and qualitative changes in sleep-wake patterning. At 3 weeks of age, however, by which time the sleep and wake bouts of the wild-types had consolidated further, the knockouts lagged behind the wild-types and exhibited significantly more bout fragmentation. These findings suggest the possibility that the fragmentation of behavioural states that characterizes narcolepsy in adults reflects reversion back toward the more fragmented sleep-wake patterns that characterize infancy.     

Changes in orexinergic immunoreactivity of the piglet hypothalamus and pons after exposure to chronic postnatal nicotine and intermittent hypercapnic hypoxia

We recently showed that orexin expression in sudden infant death syndrome (SIDS) infants was reduced by 21% in the hypothalamus and by 40–50% in the pons as compared with controls. Orexin maintains wakefulness/sleeping states, arousal, and rapid eye movement sleep, abnormalities of which have been reported in SIDS. This study examined the effects of two prominent risk factors for SIDS, intermittent hypercapnic hypoxia (IHH) (prone‐sleeping) and chronic nicotine exposure (cigarette‐smoking), on orexin A (OxA) and orexin B (OxB) expression in piglets. Piglets were randomly assigned to five groups: saline control (n = 7), air control (n = 7), nicotine [2 mg/kg per day (14 days)] (n = 7), IHH (6 min of 7% O₂/8% CO₂ alternating with 6‐min periods of breathing air, for four cycles) (n = 7), and the combination of nicotine and IHH (N + IHH) (n = 7). OxA/OxB expression was quantified in the central tuberal hypothalamus [dorsal medial hypothalamus (DMH), perifornical area (PeF), and lateral hypothalamus], and the dorsal raphe, locus coeruleus of the pons. Nicotine and N + IHH exposures significantly increased: (i) orexin expression in the hypothalamus and pons; and (ii) the total number of neurons in the DMH and PeF. IHH decreased orexin expression in the hypothalamus and pons without changing neuronal numbers. Linear relationships existed between the percentage of orexin‐positive neurons and the area of pontine orexin immunoreactivity of control and exposure piglets. These results demonstrate that postnatal nicotine exposure increases the proportion of orexin‐positive neurons in the hypothalamus and fibre expression in the pons, and that IHH exposure does not prevent the nicotine‐induced increase. Thus, although both nicotine and IHH are risk factors for SIDS, it appears they have opposing effects on OxA and OxB expression, with the IHH exposure closely mimicking what we recently found in SIDS.     

Essential role of endogenous calcitonin gene‐related peptide in pain‐associated plasticity in the central amygdala

The role of the neuropeptide calcitonin gene‐related peptide (CGRP) is well established in nociceptive behaviors. CGRP is highly expressed in the projection pathway from the parabrachial nucleus to the laterocapsular region of the central amygdala (CeC), which plays a critical role in relaying nociceptive information. The CeC is a key structure in pain behavior because it integrates and modulates nociceptive information along with other sensory signals. Previous studies have demonstrated that blockade of the amygdalar CGRP‐signaling cascade attenuates nociceptive behaviors in pain models, while CGRP application facilitates amygdalar synaptic transmission and induces pain behaviors. Despite these lines of evidence, it remains unclear whether endogenous CGRP is involved in the development of nociceptive behaviors accompanied with amygdalar plasticity in a peripheral inflammation model in vivo. To directly address this, we utilized a previously generated CGRP knockout (KO) mouse to longitudinally study formalin‐induced plasticity and nociceptive behavior. We found that synaptic potentiation in the right PB‐CeC pathway that was observed in wild‐type mice was drastically attenuated in the CGRP KO mice 6 h post‐inflammation, when acute nociceptive behavior was no longer observed. Furthermore, the bilateral tactile allodynia 6 h post‐inflammation was significantly decreased in the CGRP KO mice. In contrast, the acute nociceptive behavior immediately after the formalin injection was reduced only at 20–25 min post‐injection in the CGRP KO mice. These results suggest that endogenous CGRP contributes to peripheral inflammation‐induced synaptic plasticity in the amygdala, and this plasticity may underlie the exaggerated nociception–emotion linkage in pain chronification.     

Orexin‐A increases the activity of globus pallidus neurons in both normal and parkinsonian rats

Orexin is a member of neuropeptides which was first identified in the hypothalamus. The globus pallidus is a key structure in the basal ganglia, which is involved in both normal motor function and movement disorders. Morphological studies have shown the expression of both OX₁ and OX₂ receptors in the globus pallidus. Employing single unit extracellular recordings and behavioural tests, the direct in vivo electrophysiological and behavioural effects of orexin‐A in the globus pallidus were studied. Micro‐pressure administration of orexin‐A significantly increased the spontaneous firing rate of pallidal neurons. Correlation analysis revealed a negative correlation between orexin‐A induced excitation and the basal firing rate. Furthermore, application of the specific OX₁ receptor antagonist, SB‐334867, decreased the firing rate of pallidal neurons, suggesting that endogenous orexinergic systems modulate the firing activity of pallidal neurons. Orexin‐A increased the excitability of pallidal neurons through both OX₁ and OX₂ receptors. In 6‐hydroxydopamine parkinsonian rats, orexin‐A‐induced increase in firing rate of pallidal neurons was stronger than that in normal rats. Immunostaining revealed positive OX₁ receptor expression in the globus pallidus of both normal and parkinsonian rats. Finally, postural test showed that unilateral microinjection of orexin‐A led to contralateral deflection in the presence of systemic haloperidol administration. Further elevated body swing test revealed that pallidal orexin‐A and SB‐334867 induced contralateral‐biased swing and ipsilateral‐biased swing respectively. Based on the electrophysiological and behavioural findings of orexin‐A in the globus pallidus, the present findings may provide a rationale for the pathogenesis and treatment of Parkinson's disease.     

Orexins Excite Neurons of the Rat Cerebellar Nucleus Interpositus Via Orexin 2 Receptors In Vitro

Orexins are newfound hypothalamic neuropeptides implicated in the regulation of feeding behavior, sleep–wakefulness cycle, nociception, addiction, emotions, as well as narcolepsy. However, little is known about roles of orexins in motor control. Therefore, the present study was designed to investigate the effect of orexins on neuronal activity in the cerebellum, an important subcortical center for motor control. In this study, perfusing slices with orexin A (100 nM–1 μM) or orexin B (100 nM–1 μM) both produced neurons in the rat cerebellar interpositus nucleus (IN) a concentration-dependent excitatory response (96/143, 67.1%). Furthermore, both of the excitations induced by orexin A and B were not blocked by the low-Ca²⁺/high-Mg²⁺ medium (n = 8), supporting a direct postsynaptic action of the peptides. Highly selective orexin 1 receptor antagonist SB-334867 did not block the excitatory response of cerebellar IN neurons to orexins (n = 22), but [Ala¹¹, D-Leu¹⁵] orexin B, a highly selective orexin 2 receptor (OX₂R) agonist, mimicked the excitatory effect of orexins on the cerebellar neurons (n = 18). These results demonstrate that orexins excite the cerebellar IN neurons through OX₂R and suggest that the central orexinergic nervous system may actively participate in motor control through its modulation on one of the final outputs of the spinocerebellum.     

Impaired fear extinction in mice lacking protease nexin‐1

The serine protease inhibitor protease‐nexin‐1 (PN‐1) has been shown to modulate N‐methyl‐d ‐aspartate receptor (NMDAR)‐mediated synaptic currents and NMDAR‐dependent long‐term potentiation of synaptic transmission. Here, we analysed the role of PN‐1 in the acquisition and extinction of classical auditory fear conditioning, two distinct forms of learning that both depend on NMDAR activity in the amygdala. Immunostaining revealed that PN‐1 is expressed throughout the amygdala, primarily in γ‐aminobutyric acid containing neurons of the central amygdala and intercalated cell masses (ITCs) and in glia. Fear extinction was severely impaired in mice lacking PN‐1 (PN‐1 KO). Consistent with a role for the basal nucleus of the amygdala in fear extinction, we found that, compared with wild‐type (WT) littermate controls, PN‐1 KO mice exhibited decreased numbers of Fos‐positive neurons in the basal nucleus after extinction. Moreover, immunoblot analysis of laser‐microdissected amygdala sub‐nuclei revealed specific extinction‐induced increases in the level of phosphorylated alpha‐calcium/calmodulin protein kinase II in the medial ITCs and in the lateral subdivision of the central amygdala in WT mice. These responses were altered in PN‐1 KO mice. Together, these data indicate that lack of extinction in PN‐1 KO mice is associated with distinct changes in neuronal activity across the circuitry of the basal and central nuclei and the ITCs, supporting a differential impact on fear extinction of these amygdala substructures. They also suggest a new role for serine protease inhibitors such as PN‐1 in modulating fear conditioning and extinction.     

Treatment with immunosuppressive and anti-inflammatory agents delays onset of canine genetic narcolepsy and reduces symptom severity

All Doberman pinschers and Labrador retrievers homozygous for a mutation of the hypocretin (orexin) receptor-2 (hcrtr2) gene develop narcolepsy under normal conditions. Degenerative changes and increased display of major histocompatibility complex class II antigens have been linked to symptom onset in genetically narcoleptic Doberman pinschers. This suggests that the immune system may contribute to neurodegenerative changes and narcoleptic symptomatology in these dogs. We therefore attempted to alter the course of canine genetic narcolepsy, as an initial test of principle, by administering a combination of three immunosuppressive and anti-inflammatory drugs chosen to suppress the immune response globally. Experimental dogs were treated with a combination of methylprednisolone, methotrexate and azathioprine orally starting within 3 weeks after birth, and raised in an environment that minimized pathogen exposure. Symptoms in treated and untreated animals were quantified using the food elicited cataplexy test (FECT), modified FECT and actigraphy. With drug treatment, time to cataplexy onset more than doubled, time spent in cataplexy during tests was reduced by more than 90% and nighttime sleep periods were consolidated. Short-term drug administration to control dogs did not reduce cataplexy symptoms, demonstrating that the drug regimen did not directly affect symptoms. Treatment was stopped at 6 months, after which experimental animals remained less symptomatic than controls until at least 2 years of age. This treatment is the first shown to affect symptom development in animal or human genetic narcolepsy. Our findings show that hcrtr2 mutation is not sufficient for the full symptomatic development of canine genetic narcolepsy and suggest that the immune system may play a role in the development of this disorder.     

Ectopic Overexpression of Orexin Alters Sleep/Wakefulness States and Muscle Tone Regulation during REM Sleep in Mice

Orexins (also called hypocretins), which are neuropeptides exclusively expressed by a population of neurons specifically localized in the lateral hypothalamic area, are critically implicated in the regulation of sleep/wake states. Orexin deficiency results in narcoleptic phenotype in rodents, dogs, and humans, suggesting that orexins are important for maintaining consolidated wakefulness states. However, the physiological effect of constitutive increased orexinergic transmission tone, which might be important for understanding the effects of orexin agonists that are promising candidates for therapeutic agents of narcolepsy, has not been fully characterized. We report here the sleep/wakefulness abnormalities in transgenic mice that exhibit widespread overexpression of a rat prepro-orexin transgene driven by a β-actin/cytomegalovirus hybrid promoter (CAG/orexin transgenic mice). CAG/orexin mice exhibit sleep abnormalities with fragmentation of non-rapid eye movement (REM) sleep episode and a reduction in REM sleep. Non-REM sleep was frequently disturbed by short episodes of wakefulness. EEG/EMG studies also reveal incomplete REM sleep atonia with abnormal myoclonic activity during this sleep stage. These results suggest that endogenous orexinergic activity should be appropriately regulated for normal maintenance of sleep states. Orexinergic transmission should be activated during wakefulness, while it should be inactivated or decreased during sleep state to maintain appropriate vigilance states.     

New Approaches for the Study of Orexin Function

Orexin is a neuropeptide produced by a specific subset of neurones located in the lateral hypothalamic area. Mice lacking either prepro‐orexin or orexin receptor 2, as well as those in which orexin‐producing neurones (orexin neurones) are deleted, share a common phenotype: altered sleep–wake regulation and the sudden onset of muscle atonia. These symptoms are similar to the human sleep disorder narcolepsy. In this review, we describe recent advances in the study of orexin function with a particular emphasis on microscopic techniques that better characterise the neuronal networks involving orexin neurones, as well as recent optogenetic approaches that allow for the activation or inhibition of specific neurones by expressing different light‐activated proteins. In particular, the use of orexin/halorhodopsin and orexin/channelrhodopsin‐2 transgenic mice has demonstrated an important role for orexin neurones in regulating the sleep–wake cycle and state of arousal in vivo. Further refinement of these in vitro and in vivo techniques will allow for a more detailed understanding of the interaction of orexin with other neurotransmitter pathways in the brain.     

Role of the posterodorsal medial amygdala in predator odour stress‐induced puberty delay in female rats

Puberty onset is influenced by various factors, including psychosocial stress. The present study investigated cat‐odour stress on puberty onset and oestrous cyclicity in rats. Female weanling rats were exposed to either soiled cat litter or fresh unused litter for 10 consecutive days. Following vaginal opening (VO), rats were smeared for 14 days to determine oestrous cyclicity. Anxiety‐like behaviour was assessed using standard anxiety tests. Brains were collected to determine corticotrophin‐releasing factor (CRF), CRF receptor 1 (CRF‐R1) and CRF receptor 2 (CRF‐R2) mRNA in the paraventricular nucleus (PVN), as well as the central nucleus of the amygdala (CEA) and the medial nucleus of the amygdala (MEA). Cat odour delayed VO and first oestrus, disrupted oestrous cycles and caused anxiogenic responses. Cat odour elicited increased CRF mRNA expression in the PVN but not in the CeA. CRF‐R1 and CRF‐R2 mRNA levels in the PVN and CeA were unaffected by cat odour; however, CRF‐R1 mRNA levels were decreased in the MeA. The role of CRF signalling in the MeA, particularly its posterodorsal subnucleus (MePD), with respect to pubertal timing was directly examined by unilateral intra‐MePD administration of CRF (0.2 nmol day^‐1 for 14 days) via an osmotic mini‐pump from postnatal day 24 and was shown to delay VO and first oestrus. These data suggest that CRF signalling in the MePD may be associated with predator odour‐induced puberty delay.     

Serotonergic‐postsynaptic receptors modulate gripping‐induced immobility episodes in male taiep rats

The Taiep rat is a myelin mutant with a motor syndrome characterized by tremor, ataxia, immobility, epilepsy, and paralysis. The rat shows a hypomyelination followed by a progressive demyelination. During immobilities taiep rats show a REM‐like sleep pattern and a disorganized sleep‐wake pattern suggesting taiep rats as a model of narcolepsy‐cataplexy. Our study analyzed the role of postsynaptic serotonin receptors in the expression of gripping‐induced immobility episodes (IEs) in 8‐month‐old male taiep rats. The specific postsynaptic serotonin agonist ±1‐(2,5‐dimethoxy‐4‐iodoamphetamine hydrochloride (±DOI) decreased the frequency of gripping‐induced IEs, but that was not the case with α‐methyl‐serotonin maleate (α‐methyl‐5HT), a nonspecific postsynaptic agonist. Although the serotonin antagonists, ketanserine and metergoline, produced a biphasic effect, first a decrease followed by an increase with higher doses, similar effects were obtained with a mean duration of gripping‐induced IEs. These findings correlate with the pharmacological observations in narcoleptic dogs and humans in which serotonin‐reuptake inhibitors improve cataplexy, particularly in long‐term treatment that could change the serotonin receptor levels. Polysomnographic recordings showed an increase in the awakening time and a decrease in the slow wave and rapid eye movement sleep concomitant with a decrease in immobilities after use of ±DOI, this being stronger with the highest dose. Taken together, our results show that postsynaptic serotonin receptors are involved in the modulation in gripping‐induced IEs caused by the changes in the organization of the sleep‐wake cycle in taiep rats. It is possible that specific agonists, without side effects, could be a useful treatment in human narcoleptic patients. Synapse 63:737–744, 2009. © 2009 Wiley‐Liss, Inc.     

Narcolepsy and the hypocretin system--where motion meets emotion

Narcolepsy is a neurological disorder that is characterized by excessive daytime sleepiness and cataplexy--a loss of muscle tone generally triggered by certain strong emotions with sudden onset. The underlying cause of most cases of human narcolepsy is a loss of neurons that produce hypocretin (Hcrt, also known as orexin). These cells normally serve to drive and synchronize the activity of monoaminergic and cholinergic cells. Sleepiness results from the reduced activity of monoaminergic, cholinergic and other cells that are normally activated by Hcrt neurons, as well as from the loss of Hcrt itself. Cataplexy is caused by an episodic loss of activity in noradrenergic cells that support muscle tone, and a linked activation of a medial medullary cell population that suppresses muscle tone. Current treatments for narcolepsy include stimulants to combat sleepiness and antidepressants to reduce cataplexy. Sodium oxybate produces both reductions in cataplexy and improved waking alertness. Future treatments are likely to include Hcrt or Hcrt agonists to reverse the underlying neurochemical deficit.     

Muscarinic‐2 and orexin‐2 receptors on GABAergic and other neurons in the rat mesopontine tegmentum and their potential role in sleep–wake state control

Acetylcholine (ACh) plays an important role in the promotion of paradoxical sleep (PS) with muscle atonia through the muscarinic‐2 receptor (M2R) in the mesopontine tegmentum. Conversely, orexin (Orx or hypocretin) appears to be critical for the maintenance of waking with muscle tone through the orexin‐2 (or hypocretin‐B) receptor (Orx2R), which is lacking in dogs having narcolepsy with cataplexy. In dual‐immunostained material viewed under fluorescence microscopy, we examined the presence and distribution of M2R or Orx2R labeling on all neuronal nuclei (NeuN)‐stained neurons or on glutamic acid decarboxylase (GAD)‐stained neurons through the mesopontine tegmentum. Applying stereological analysis, we determined that many neurons bear M2Rs on their membrane (≈6,300), including relatively large, non‐GABAergic cells, which predominate (>75%) in the oral and caudal pontine (PnO and PnC) reticular fields, and small, GABAergic cells (≈2,800), which predominate (>80%) in the mesencephalic (Mes) reticular formation. Many neurons bear Orx2Rs on their membrane (≈6,800), including relatively large, non‐GABAergic cells, which predominate (>70%) through all reticular fields, and comparatively few GABAergic cells (≈700). In triple‐immunostained material viewed by confocal microscopy, many large neurons in PnO and PnC appear to bear both M2Rs and Orx2Rs on their membrane, indicating that ACh and Orx could exert opposing influences of inhibition vs. excitation on putative reticulo‐spinal neurons and thus attenuate vs. facilitate activity and muscle tone. A few GABAergic cells bear both receptors and could as PS inhibitor neurons serve under these different influences to control PS effector neurons and accordingly gate PS and muscle atonia appropriately across sleep–wake states. J. Comp. Neurol. 510:607–630, 2008. © 2008 Wiley‐Liss, Inc.