Substance P Immunoreactivity Exhibits Frequent Colocalization with Kisspeptin and Neurokinin B in the Human Infundibular Region

Neurons synthesizing neurokinin B (NKB) and kisspeptin (KP) in the hypothalamic arcuate nucleus represent important upstream regulators of pulsatile gonadotropin-releasing hormone (GnRH) neurosecretion. In search of neuropeptides co-expressed in analogous neurons of the human infundibular nucleus (Inf), we have carried out immunohistochemical studies of the tachykinin peptide Substance P (SP) in autopsy samples from men (21-78 years) and postmenopausal (53-83 years) women. Significantly higher numbers of SP-immunoreactive (IR) neurons and darker labeling were observed in the Inf of postmenopausal women than in age-matched men. Triple-immunofluorescent studies localized SP immunoreactivity to considerable subsets of KP-IR and NKB-IR axons and perikarya in the infundibular region. In postmenopausal women, 25.1% of NKB-IR and 30.6% of KP-IR perikarya contained SP and 16.5% of all immunolabeled cell bodies were triple-labeled. Triple-, double- and single-labeled SP-IR axons innervated densely the portal capillaries of the infundibular stalk. In quadruple-labeled sections, these axons formed occasional contacts with GnRH-IR axons. Presence of SP in NKB and KP neurons increases the functional complexity of the putative pulse generator network. First, it is possible that SP modulates the effects of KP and NKB in axo-somatic and axo-dendritic afferents to GnRH neurons. Intrinsic SP may also affect the activity and/or neuropeptide release of NKB and KP neurons via autocrine/paracrine actions. In the infundibular stalk, SP may influence the KP and NKB secretory output via additional autocrine/paracrine mechanisms or regulate GnRH neurosecretion directly. Finally, possible co-release of SP with KP and NKB into the portal circulation could underlie further actions on adenohypophysial gonadotrophs.     

The neuroendocrine pathways and mechanisms for the control of the reproduction in female pigs

Within the hypothalamic-pituitary-gonad (HPG) axis, the major hierarchical component is gonadotropin-releasing hormone (GnRH) neurons, which directly or indirectly receive regulatory inputs from a wide array of regulatory signals and pathways, involving numerous circulating hormones, neuropeptides, and neurotransmitters, and which operate as a final output for the brain control of reproduction. In recent years, there has been an increasing interest in neuropeptides that have the potential to stimulate or inhibit GnRH in the hypothalamus of pigs. Among them, Kisspeptin is a key component in the precise regulation of GnRH neuron secretion activity. Besides, other neuropeptides, including neurokinin B (NKB), neuromedin B (NMB), neuromedin S (NMS), α-melanocyte-stimulating hormone (α-MSH), Phoenixin (PNX), show potential for having a stimulating effect on GnRH neurons. On the contrary, RFamide-related peptide-3 (RFRP-3), endogenous opioid peptides (EOP), neuropeptide Y (NPY), and Galanin (GAL) may play an inhibitory role in the regulation of porcine reproductive nerves and may directly or indirectly regulate GnRH neurons. By combining data from suitable model species and pigs, we aim to provide a comprehensive summary of our current understanding of the neuropeptides acting on GnRH neurons, with a particular focus on their central regulatory pathways and underlying molecular basis.     

Nicotinic α4 Receptor-Mediated Cholinergic Influences on Food Intake and Activity Patterns in Hypothalamic Circuits

Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating appetite and have been shown to do so by influencing neural activity in the hypothalamus. To shed light on the hypothalamic circuits governing acetylcholine’s (ACh) regulation of appetite this study investigated the influence of hypothalamic nAChRs expressing the α4 subunit. We found that antagonizing the α4β2 nAChR locally in the lateral hypothalamus with di-hydro-ß-erythroidine (DHβE), an α4 nAChR antagonist with moderate affinity, caused an increase in food intake following free access to food after a 12 hour fast, compared to saline-infused animals. Immunocytochemical analysis revealed that orexin/hypocretin (HO), oxytocin, and tyrosine hydroxylase (TH)-containing neurons in the A13 and A12 of the hypothalamus expressed the nAChR α4 subunit in varying amounts (34%, 42%, 50%, and 51%, respectively) whereas melanin concentrating hormone (MCH) neurons did not, suggesting that DHβE-mediated increases in food intake may be due to a direct activation of specific hypothalamic circuits. Systemic DHβE (2 mg/kg) administration similarly increased food intake following a 12 hour fast. In these animals a subpopulation of orexin/hypocretin neurons showed elevated activity compared to control animals and MCH neuronal activity was overall lower as measured by expression of the immediate early gene marker for neuronal activity cFos. However, oxytocin neurons in the paraventricular hypothalamus and TH-containing neurons in the A13 and A12 did not show differential activity patterns. These results indicate that various neurochemically distinct hypothalamic populations are under the influence of α4β2 nAChRs and that cholinergic inputs to the lateral hypothalamus can affect satiety signals through activation of local α4β2 nAChR-mediated transmission.     

Tyrosine Hydroxylase Is Short-Term Regulated by the Ubiquitin-Proteasome System in PC12 Cells and Hypothalamic and Brainstem Neurons from Spontaneously Hypertensive Rats: Possible Implications in Hypertension

Aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR) and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86±15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2±8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92±22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7±0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasme activity. Proteasome activity was significantly reduced by 67±4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.     

Phenotypic alterations of neuropeptide Y and calcitonin gene-related peptide-containing neurons innervating the rat temporomandibular joint during carrageenan-induced arthritis

The aim of this study was to identify immunoreactive neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) neurons in the autonomic and sensory ganglia, specifically neurons that innervate the rat temporomandibular joint (TMJ). A possible variation between the percentages of these neurons in acute and chronic phases of carrageenan-induced arthritis was examined. Retrograde neuronal tracing was combined with indirect immunofluorescence to identify NPY-immunoreactive (NPY-IR) and CGRP- immunoreactive (CGRP-IR) neurons that send nerve fibers to the normal and arthritic temporomandibular joint. In normal joints, NPY-IR neurons constitute 78±3%, 77±6% and 10±4% of double-labeled nucleated neuronal profile originated from the superior cervical, stellate and otic ganglia, respectively. These percentages in the sympathetic ganglia were significantly decreased in acute (58±2% for superior cervical ganglion and 58±8% for stellate ganglion) and chronic (60±2% for superior cervical ganglion and 59±15% for stellate ganglion) phases of arthritis, while in the otic ganglion these percentages were significantly increased to 19±5% and 13±3%, respectively. In the trigeminal ganglion, CGRP-IR neurons innervating the joint significantly increased from 31±3% in normal animals to 54±2% and 49±3% in the acute and chronic phases of arthritis, respectively. It can be concluded that NPY neurons that send nerve fibers to the rat temporomandibular joint are located mainly in the superior cervical, stellate and otic ganglia. Acute and chronic phases of carrageenan-induced arthritis lead to an increase in the percentage of NPY-IR parasympathetic and CGRP-IR sensory neurons and to a decrease in the percentage of NPY-IR sympathetic neurons related to TMJ innervation.Key words: trigeminal ganglion, otic ganglion, superior cervical ganglion, arthritis, temporomandibular joint.     

Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry

Summary A method was developed that allows the analysis of neuropeptides and monoamines in a single tissue section by the application of the unlabeled antibody method for peptide staining to tissue sections freeze-dried for formaldehyde-induced monoamine histofluorescence. The hypothalamic magnocellular system of male albino rats served as a model for this study; neurons were stained with anti-neurophysin sera, which mark the vasopressin- and oxytocin-associated proteins. Neurophysin-containing perikarya appeared to be surrounded by catecholamine-containing varicosities. This phenomenon was seen to varying degrees within the supraoptic and paraventricular nuclei. The juxtaposition of varicosities and peptidergic neurons suggests an afferent fiber-target neuron relationship that might favor a functional interaction between monoamines and neuropeptides.Supported by USPHS Grant and postdoctoral NS15816 (JRS) fellowship AG01575 (THM)     

Ultrasound-Assisted Extraction of Arabinogalactan and Dihydroquercetin Simultaneously from Larix gmelinii as a Pretreatment for Pulping and Papermaking

An ultrasound-assisted extraction (UAE) method using ethanol was applied for extracting arabinogalactan (AG) and dihydroquercetin (DHQ) simultaneously from larch wood, as a pretreatment for pulping and papermaking. The extraction parameters were optimized by a Box-Behnken experimental design with the yields of AG and DHQ as the response values. Under optimum conditions (three extractions, each using 40% ethanol, for 50 min, 200 W ultrasound power and 1∶18 solid-liquid ratio), the yields of AG and DHQ were 183.4 and 36.76 mg/g, respectively. After UAE pretreated, the wood chips were used for Kraft pulping (KP) and high boiling solvent pulping (HBSP). The pulping yield after pretreatment was higher than that of untreated (the pulping yields of untreated HBSP and KP were 42.37% and 39.60%, and the pulping yields of HBSP and KP after UAE-pretreated were 44.23% and 41.50% respectively), as indicated by a lower kappa number (77.91 and 27.30 for untreated HBSP and KP; 77.01 and 26.83 for UAE-pretreated HBSP and KP). Furthermore, the characteristics of paper produced from pretreated wood chips were superior to those from the untreated chips: the basis weight was lower (85.67 and 82.48 g·cm⁻² for paper from untreated KP and HBSP; 79.94 and 80.25 g·cm⁻² for paper from UAE-pretreated KP and HBSP), and the tensile strengths, tearing strengths, bursting strengths, and folding strengths were higher than these of paper after UAE-pretreated, respectively.     

MCT2 Expression and Lactate Influx in Anorexigenic and Orexigenic Neurons of the Arcuate Nucleus

Hypothalamic neurons of the arcuate nucleus control food intake, releasing orexigenic and anorexigenic neuropeptides in response to changes in glucose concentration. Several studies have suggested that the glucosensing mechanism is governed by a metabolic interaction between neurons and glial cells via lactate flux through monocarboxylate transporters (MCTs). Hypothalamic glial cells (tanycytes) release lactate through MCT1 and MCT4; however, similar analyses in neuroendocrine neurons have yet to be undertaken. Using primary rat hypothalamic cell cultures and fluorimetric assays, lactate incorporation was detected. Furthermore, the expression and function of MCT2 was demonstrated in the hypothalamic neuronal cell line, GT1-7, using kinetic and inhibition assays. Moreover, MCT2 expression and localization in the Sprague Dawley rat hypothalamus was analyzed using RT-PCR, in situ hybridization and Western blot analyses. Confocal immunohistochemistry analyses revealed MCT2 localization in neuronal but not glial cells. Moreover, MCT2 was localized to ∼90% of orexigenic and ∼60% of anorexigenic neurons as determined by immunolocalization analysis of AgRP and POMC with MCT2-positives neurons. Thus, MCT2 distribution coupled with lactate uptake by hypothalamic neurons suggests that hypothalamic neurons control food intake using lactate to reflect changes in glucose levels.     

β-Arrestin Regulates Estradiol Membrane-Initiated Signaling in Hypothalamic Neurons

Estradiol (E2) action in the nervous system is the result of both direct nuclear and membrane-initiated signaling (EMS). E2 regulates membrane estrogen receptor-α (ERα) levels through opposing mechanisms of EMS-mediated trafficking and internalization. While ß-arrestin-mediated mERα internalization has been described in the cortex, a role of ß-arrestin in EMS, which underlies multiple physiological processes, remains undefined. In the arcuate nucleus of the hypothalamus (ARH), membrane-initiated E2 signaling modulates lordosis behavior, a measure of female sexually receptivity. To better understand EMS and regulation of ERα membrane levels, we examined the role of ß-arrestin, a molecule associated with internalization following agonist stimulation. In the present study, we used an immortalized neuronal cell line derived from embryonic hypothalamic neurons, the N-38 line, to examine whether ß-arrestins mediate internalization of mERα. β-arrestin-1 (Arrb1) was found in the ARH and in N-38 neurons. In vitro, E2 increased trafficking and internalization of full-length ERα and ERαΔ4, an alternatively spliced isoform of ERα, which predominates in the membrane. Treatment with E2 also increased phosphorylation of extracellular-signal regulated kinases 1/2 (ERK1/2) in N-38 neurons. Arrb1 siRNA knockdown prevented E2-induced ERαΔ4 internalization and ERK1/2 phosphorylation. In vivo, microinfusions of Arrb1 antisense oligodeoxynucleotides (ODN) into female rat ARH knocked down Arrb1 and prevented estradiol benzoate-induced lordosis behavior compared with nonsense scrambled ODN (lordosis quotient: 3 ± 2.1 vs. 85.0 ± 6.0; p < 0.0001). These results indicate a role for Arrb1 in both EMS and internalization of mERα, which are required for the E2-induction of female sexual receptivity.     

Menopause and the human hypothalamus: evidence for the role of kisspeptin/neurokinin B neurons in the regulation of estrogen negative feedback

Menopause is characterized by depletion of ovarian follicles, a reduction of ovarian hormones to castrate levels and elevated levels of serum gonadotropins. Rather than degenerating, the reproductive neuroendocrine axis in postmenopausal women is intact and responds robustly to the removal of ovarian hormones. Studies in both human and non-human primates provide evidence that the gonadotropin hypersecretion in postmenopausal women is secondary to increased gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. In addition, menopause is accompanied by hypertrophy of neurons in the infundibular (arcuate) nucleus expressing KiSS-1, neurokinin B (NKB), substance P, dynorphin and estrogen receptor alpha (ERalpha) mRNA. Ovariectomy in experimental animals induces nearly identical findings, providing evidence that these changes are a compensatory response to ovarian failure. The anatomical site of the hypertrophied neurons, as well as the extensive data implicating kisspeptin, NKB and dynorphin in the regulation of GnRH secretion, provide compelling evidence that these neurons are part of the neural network responsible for the increased levels of serum gonadotropins in postmenopausal women. We propose that neurons expressing KiSS-1, NKB, substance P, dynorphin and ERalpha mRNA in the infundibular nucleus play an important role in sex-steroid feedback on gonadotropin secretion in the human.     

Exogenous Melatonin Alleviates Skeletal Muscle Wasting by Regulating Hypothalamic Neuropeptides Expression in Endotoxemia Rats

To investigate whether exogenous melatonin (MLT) could alleviate skeletal muscle wasting by regulating hypothalamic neuropeptides expression. Adult male Sprague Dawley rats were intraperitoneally injected with lipopolysaccharide (LPS) (10 mg/kg), followed by MLT (30 mg/kg/day) or saline for 3 days. Hypothalamic tissues and skeletal muscle were obtained on day 3. Skeletal muscle wasting was measured by the mRNA expression of two E3 ubiquitin ligases, muscle atrophy F-box and muscle ring finger 1 as well as 3-methylhistidine (3-MH) and tyrosine release. Three hypothalamic neuropeptides (POMC, AgRP, CART) expression were detected in all groups. POMC expression knockdown was achieved by ARC injection of lentiviruses containing shRNA against POMC. Two weeks after ARC viruses injection, rats were i.p. injected with LPS (10 mg/kg) followed by MLT (30 mg/kg/day) or saline for 3 days. Brain tissues were harvested for immunostaining. In septic rats, 3-MH, tyrosine release and muscle atrophic gene expression were significantly decreased in MLT treated group. POMC and CART expression were lower while AgRP expression was higher in MLT treated group. Furthermore, in septic rats treated with MLT, muscle wasting in those with lower expression of neuropeptide POMC did not differ from those with normal POMC expression. Exogenous MLT could alleviate skeletal muscle wasting in septic rats by regulating hypothalamic neuropeptides.

     

A Self-Report Risk Index to Predict Occurrence of Dementia in Three Independent Cohorts of Older Adults: The ANU-ADRI

Background and Aims

The Australian National University AD Risk Index (ANU-ADRI, http://anuadri.anu.edu.au) is a self-report risk index developed using an evidence-based medicine approach to measure risk of Alzheimer''s disease (AD). We aimed to evaluate the extent to which the ANU-ADRI can predict the risk of AD in older adults and to compare the ANU-ADRI to the dementia risk index developed from the Cardiovascular Risk Factors, Aging and Dementia (CAIDE) study for middle-aged cohorts.

Methods

This study included three validation cohorts, i.e., the Rush Memory and Aging Study (MAP) (n = 903, age ≥53 years), the Kungsholmen Project (KP) (n = 905, age ≥75 years), and the Cardiovascular Health Cognition Study (CVHS) (n = 2496, age ≥65 years) that were each followed for dementia. Baseline data were collected on exposure to the 15 risk factors included in the ANU-ADRI of which MAP had 10, KP had 8 and CVHS had 9. Risk scores and C-statistics were computed for individual participants for the ANU-ADRI and the CAIDE index.

Results

For the ANU-ADRI using available data, the MAP study c-statistic was 0·637 (95% CI 0·596–0·678), for the KP study it was 0·740 (0·712–0·768) and for the CVHS it was 0·733 (0·691–0·776) for predicting AD. When a common set of risk and protective factors were used c-statistics were 0.689 (95% CI 0.650–0.727), 0.666 (0.628–0.704) and 0.734 (0.707–0.761) for MAP, KP and CVHS respectively. Results for CAIDE ranged from c-statistics of 0.488 (0.427–0.554) to 0.595 (0.565–0.625).

Conclusion

A composite risk score derived from the ANU-ADRI weights including 8–10 risk or protective factors is a valid, self-report tool to identify those at risk of AD and dementia. The accuracy can be further improved in studies including more risk factors and younger cohorts with long-term follow-up.     

Negative Regulation of Leptin-induced Reactive Oxygen Species (ROS) Formation by Cannabinoid CB1 Receptor Activation in Hypothalamic Neurons

The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB₁ receptor activity. Here we investigated the possibility of a negative regulation by CB₁ receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2′-chloroethylamide (ACEA) in a CB₁-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL^−/− mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB₁ activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels.     

Time-Dependent Effects of Training on Cardiovascular Control in Spontaneously Hypertensive Rats: Role for Brain Oxidative Stress and Inflammation and Baroreflex Sensitivity

Baroreflex dysfunction, oxidative stress and inflammation, important hallmarks of hypertension, are attenuated by exercise training. In this study, we investigated the relationships and time-course changes of cardiovascular parameters, pro-inflammatory cytokines and pro-oxidant profiles within the hypothalamic paraventricular nucleus of the spontaneously hypertensive rats (SHR). Basal values and variability of arterial pressure and heart rate and baroreflex sensitivity were measured in trained (T, low-intensity treadmill training) and sedentary (S) SHR at weeks 0, 1, 2, 4 and 8. Paraventricular nucleus was used to determine reactive oxygen species (dihydroethidium oxidation products, HPLC), NADPH oxidase subunits and pro-inflammatory cytokines expression (Real time PCR), p38 MAPK and ERK1/2 expression (Western blotting), NF-κB content (electrophoretic mobility shift assay) and cytokines immunofluorescence. SHR-S vs. WKY-S (Wistar Kyoto rats as time control) showed increased mean arterial pressure (172±3 mmHg), pressure variability and heart rate (358±7 b/min), decreased baroreflex sensitivity and heart rate variability, increased p47^phox and reactive oxygen species production, elevated NF-κB activity and increased TNF-α and IL-6 expression within the paraventricular nucleus of hypothalamus. Two weeks of training reversed all hypothalamic changes, reduced ERK1/2 phosphorylation and normalized baroreflex sensitivity (4.04±0.31 vs. 2.31±0.19 b/min/mmHg in SHR-S). These responses were followed by increased vagal component of heart rate variability (1.9-fold) and resting bradycardia (−13%) at the 4th week, and, by reduced vasomotor component of pressure variability (−28%) and decreased mean arterial pressure (−7%) only at the 8th week of training. Our findings indicate that independent of the high pressure levels in SHR, training promptly restores baroreflex function by disrupting the positive feedback between high oxidative stress and increased pro-inflammatory cytokines secretion within the hypothalamic paraventricular nucleus. These early adaptive responses precede the occurrence of training-induced resting bradycardia and blood pressure fall.     

Effects of Ambient Temperature on Sleep and Cardiovascular Regulation in Mice: The Role of Hypocretin/Orexin Neurons

The central neural pathways underlying the physiological coordination between thermoregulation and the controls of the wake-sleep behavior and cardiovascular function remain insufficiently understood. Growing evidence supports the involvement of hypocretin (orexin) peptides in behavioral, cardiovascular, and thermoregulatory functions. We investigated whether the effects of ambient temperature on wake-sleep behavior and cardiovascular control depend on the hypothalamic neurons that release hypocretin peptides. Orexin-ataxin3 transgenic mice with genetic ablation of hypocretin neurons (n = 11) and wild-type controls (n = 12) were instrumented with electrodes for sleep scoring and a telemetric blood pressure transducer. Simultaneous sleep and blood pressure recordings were performed on freely-behaving mice at ambient temperatures ranging between mild cold (20°C) and the thermoneutral zone (30°C). In both mouse groups, the time spent awake and blood pressure were higher at 20°C than at 30°C. The cold-related increase in blood pressure was significantly smaller in rapid-eye-movement sleep (REMS) than either in non-rapid-eye-movement sleep (NREMS) or wakefulness. Blood pressure was higher in wakefulness than either in NREMS or REMS at both ambient temperatures. This effect was significantly blunted in orexin-ataxin3 mice irrespective of ambient temperature and particularly during REMS. These data demonstrate that hypocretin neurons are not a necessary part of the central pathways that coordinate thermoregulation with wake-sleep behavior and cardiovascular control. Data also support the hypothesis that hypocretin neurons modulate changes in blood pressure between wakefulness and the sleep states. These concepts may have clinical implications in patients with narcolepsy with cataplexy, who lack hypocretin neurons.