Evolving the EN 1500 test method for alcohol-based hand rub closer to clinical reality by reducing the organic load on hands and enabling product to be applied to dry hands.

BACKGROUND: The methods currently used in Europe and North America to evaluate the bactericidal efficacy of hand hygiene products have some limitations (e.g. selection of test organism, method of contamination), and none of the methods allow prediction of actual clinical efficacy. Therefore, the World Health Organization has proposed the development of methods that better reflect typical clinical reality. METHODS: In Experiment 1, two contamination methods (immersion method according to EN 1500 and low-volume method according to ASTM E2755) were tested with the EN 1500 test organism Escherichia coli using 60% v/v iso-propanol. Experiment 2 compared the two contamination methods with Enterococcus faecalis. Experiment 3 compared the two test organisms using the low-volume contamination method. Data within each experiment were compared using the Wilcoxon test for paired samples, and data from all experiments were combined and fit to linear mixed-effects models. RESULTS: Mixed-effects analysis confirmed that both the test organism and the contamination method impacted the pre-values, and all three factors influenced log10 reductions. Higher pre-values resulted in significantly higher log10 reductions, immersion contributed to significantly higher log10 reductions, and E. coli showed significantly lower log10 reductions. CONCLUSION: An efficacy evaluation against E. faecalis with a low-volume contamination method could be considered as an alternative to the EN 1500 standard. This could help to improve the clinical relevance of the test method by including a Gram-positive organism and reducing the soil load, allowing product application closer to reality.

Efficacy of alcohol-based hand sanitizers against human norovirus using RNase-RT-qPCR with validation by human intestinal enteroid replication.

Successful human norovirus (HuNoV) cultivation in stem cell-derived human intestinal enteroids (HIE) was recently reported. The purpose of this study was to evaluate the anti-HuNoV efficacy of two alcohol-based commercial hand sanitizers and 60% ethanol by suspension assay using RNase-RT-qPCR, with subsequent validation of efficacy by HuNoV cultivation using the HIE model. In suspension, when evaluated by RNase-RT-qPCR, 60% ethanol resulted in less than one log10 reduction in HuNoV genome equivalent copies (GEC) regardless of contact time (30 or 60s) or soil load. The two commercial products outperformed 60% ethanol regardless of contact time or soil load, providing 2·2-3·2 log10 HuNoV GEC reductions by suspension assay. Product B could not be validated in the HIE model due to cytotoxicity. Following a 60s exposure, viral replication in the HIE model increased 1·9 ± 0·2 log10 HuNoV GEC for the neutralization (positive) control and increased 0·9 ± 0·2 log10 HuNoV GEC in challenged HIE after treatment with 60% ethanol. No HuNoV replication in HIE was observed after a 60 s exposure to Product A.

Lateralized microstructural changes in early-stage Parkinson's disease in anterior olfactory structures, but not in substantia nigra.

Parkinson's disease (PD) is a progressive neurological disorder characterized by motor symptoms as well as severe deficits in olfactory function and microstructural changes in olfactory brain regions. Because of the evidence of asymmetric neuropathological features in early-stage PD, we examined whether lateralized microstructural changes occur in olfactory brain regions and the substantia nigra in a group of early-stage PD patients. Using diffusion tensor imaging (DTI) and the University of Pennsylvania Smell Identification Test (UPSIT), we assessed 24 early-stage PD patients (Hoehn and Yahr stage 1 or 2) and 26 healthy controls (HC). We used DTI and a region of interest (ROI) approach to study the microstructure of the left and right anterior olfactory structures (AOS; comprising the olfactory bulbs and anterior end of the olfactory tracts) and the substantia nigra (SN). PD patients had reduced UPSIT scores relative to HC and showed increased mean diffusivity (MD) in the SN, with no lateralized differences. Significant group differences in fractional anisotropy (FA) and MD were seen in the AOS, but these differences were restricted to the right side and were not associated with the primary side of motor symptoms amongst PD patients. No associations were observed between lateralized motor impairment and lateralized microstructural changes in AOS. Impaired olfaction and microstructural changes in AOS are useful for early identification of PD but asymmetries in AOS microstructure seem unrelated to the laterality of PD motor symptoms.

Management of arterial lines and blood sampling in intensive care: a threat to patient safety.

In 2008, the UK National Patient Safety Agency (NPSA) made recommendations for safe arterial line management. Following a patient safety incident in our intensive care unit (ICU), we surveyed current practice in arterial line management and determined whether these recommendations had been adopted. We contacted all 241 adult ICUs in the UK; 228 (94.6%) completed the survey. Some NPSA recommendations have been widely implemented - use of sodium chloride 0.9% as flush fluid, two-person checking of fluids before use - and their practice was consistent. Others have been incompletely implemented and many areas of practice (prescription of fluids, two-person checking at shift changes, use of opaque pressure bags, arterial sampling technique) were highly variable. More importantly, the use of the wrong fluid as an arterial flush was reported by 30% of respondents for ICU practice, and a further 30% for practice elsewhere in the hospital. Our survey provides evidence of continuing risk to patients.

Mapping the effects of the selective dopamine D2/D3 receptor agonist quinelorane using pharmacological magnetic resonance imaging.

Dopamine agonists with a high affinity for D2 and D3 receptors have a biphasic effect on rodent locomotion, inducing hypolocomotion at low doses and hyperlocomotion at higher doses. Controversy surrounds the role of the D3 receptor in mediating the hypolocomotor response to low agonist doses. This study examines patterns of neuronal activation induced by varying doses of the D2/D3 receptor agonist quinelorane using blood oxygen level dependent (BOLD) pharmacological magnetic resonance imaging (phMRI), and compares them with corresponding behavioural responses. Quinelorane (3 microg/kg) induced hypolocomotion in rats naive to the testing environment, and in phMRI experiments increased neuronal activity within the anterior olfactory nuclei, nucleus accumbens and islets of Calleja, regions containing a high density of D3 receptors. A 30 microg/kg dose of quinelorane resulted in biphasic locomotor effects, with initial hypolocomotion followed by sustained hyperlocomotion. phMRI indicated that this higher dose increased cerebral activity within limbic and olfactory regions, as did the lower drug dose, but induced additional activation in the caudate-putamen and globus pallidus, areas dense in D2 receptors but containing few D3 receptors. The more restricted pattern of activation at low agonist doses and close temporal relationship between behavioural and BOLD signal responses to quinelorane suggest that those nuclei most dense in D3 receptors play a key role in mediating the hypolocomotor effects of quinelorane. However, the presence of D3 receptors in activated brain regions may be coincidental, and further studies are required to show definitively which class of receptors mediates agonist-induced hypolocomotion. In contrast, the activation of D2 receptors within the striatum appears necessary for quinelorane-induced hyperlocomotion.

Diffusion-weighted MRI used to detect in vivo modulation of cortical spreading depression: comparison of sumatriptan and tonabersat.

Spreading cortical depolarization and depression of electroencephalographic activity (SD) may underlie the aura and spreading neurovascular events of migraine. Cortical depolarization may also precipitate the progressive development of cerebral pathology following ischemia. However, data on SD in the human brain are sparse, most likely reflecting the technical difficulties involved in performing such clinical studies. We have previously shown that the transient cerebral water disturbances during SD can be quantitatively investigated in the gyrencephalic brain using repetitive diffusion-weighted magnetic resonance imaging (DWI). To investigate whether DWI could detect modulation of the spatiotemporal properties of SD in vivo, the effects of the antimigraine drug sumatriptan (0.3 mg/kg iv) and the novel anticonvulsant tonabersat (10 mg/kg ip) were evaluated in the cat brain. Supporting previous findings, sumatriptan did not affect the numbers of events (range, 4-8), the duration of SD activity (39.8 +/- 4.4 min, mean +/- SEM), and event velocity (2.2 +/- 0.4 mm min(-1)); tonabersat significantly reduced SD event initiation (range, 0-3) and duration (13.2 +/- 5.0 min) and increased primary event velocity (5.4 +/- 0.7 mm min(-1)). However, both drugs significantly decreased, by >50%, the spatial extent of the first KCl-evoked SD event, and sumatriptan significantly increased event propagation across the suprasylvian sulcus (5.5 +/- 0.6 vs 2.4 +/- 0.4 events in controls). These results demonstrate (1) the feasibility of using DWI to evaluate therapeutic effects on SD, and (2) that sumatriptan may directly modulate the spatial distribution of SD activity in the gyrencephalic brain.

Appositions between cocaine and amphetamine-related transcript- and gonadotropin releasing hormone-immunoreactive neurons in the hypothalamus of the Siberian hamster.

Recent in vitro studies have provided evidence that cocaine and amphetamine-related transcript (CART) pathways in the hypothalamus mediate the effects of leptin upon gonadotropin releasing hormone (GnRH) secretion. The aim of the current study was to use dual label immunofluorescence to investigate the anatomical basis of such a pathway. CART-ir processes were found extensively in regions where GnRH cell bodies where located. Analysis using confocal microscopy showed that the majority of GnRH neurons (62%) had close appositions from CART-ir processes. The proportion of GnRH-ir perikarya with CART-ir appositions was significantly higher (P<0.05) in neurons located in the diagonal band of Broca (70%) compared to those more caudally located in the preoptic area (53%). This anatomical evidence for close appositions between CART-ir processes and GnRH cell bodies supports the hypothesis that one mechanism by which leptin causes its effect on the GnRH pulse generator is indirectly via CART neurons, thus allowing information about nutritional status and body fat stores to be conveyed to the reproductive system.

Detection of pharmacologically mediated changes in cerebral activity by functional magnetic resonance imaging: the effects of sulpiride in the brain of the anaesthetised rat.

Blood oxygenation level dependent (BOLD) contrast functional magnetic resonance imaging (fMRI) was used to study the effects of the D(2)-like receptor selective antagonist, sulpiride, at 2 Tesla in the brain of the alpha-chloralose anaesthetised rat. Region of interest (ROI) analysis indicated significant (P<0.05) bilateral increases in BOLD signal intensity in the frontal cortex following a single administration of sulpiride (10 mg/kg i.v.). BOLD signal changes were slow in onset and increased gradually during the experiment, reaching 8.0+/-0.5% (mean+/-S.E.M.) above pre-injection control values 165 min after drug administration. Signal increases remained high at the experiment end (3 h post sulpiride administration). Sulpiride (30 mg/kg i.v.) had a similar effect in the frontal cortex, increasing signal 5.2+/-1.8% above control values by 174 min; its effects were, however, more variable between rats, and were not statistically significant. Sulpiride (3 mg/kg i.v.) had no significant effect upon BOLD signal intensity in any brain region. No dose of sulpiride resulted in any significant BOLD signal changes in the striatum or cerebellum. These data are supportive of the notion that sulpiride causes an increase in frontal dopaminergic function by antagonism of presynaptically located dopamine D(2) receptors in this brain region, consistent with its therapeutic action. Furthermore, the utility of BOLD contrast fMRI as a means of detecting changes in neuronal activity contingent upon the administration of a psychoactive pharmacological agent has been demonstrated.

Comparative immunohistochemical localisation of GABA(B1a), GABA(B1b) and GABA(B2) subunits in rat brain, spinal cord and dorsal root ganglion.

GABA(B) receptors are G-protein-coupled receptors mediating the slow onset and prolonged synaptic actions of GABA in the CNS. The recent cloning of two genes, GABA(B1) and GABA(B2), has revealed a novel requirement for GABA(B) receptor signalling. Studies have demonstrated that the two receptor subunits associate as a GABA(B1)/GABA(B2) heterodimer to form a functional GABA(B) receptor. In this study we have developed polyclonal antisera specific to two splice variants of the GABA(B1) subunit, GABA(B1a) and GABA(B1b), as well as an antiserum to the GABA(B2) subunit. Using affinity-purified antibodies derived from these antisera we have mapped out the distribution profile of each subunit in rat brain, spinal cord and dorsal root ganglion. In brain the highest areas of GABA(B1a), GABA(B1b) and GABA(B2) subunit expression were found in neocortex, hippocampus, thalamus, cerebellum and habenula. In spinal cord, GABA(B1) and GABA(B2) subunits were expressed in the superficial layers of the dorsal horn, as well as in motor neurones in the deeper layers of the ventral horn. GABA(B) receptor subunit immunoreactivity in dorsal root ganglion suggested that expression of GABA(B1b) was restricted to the large diameter neurones, in contrast to GABA(B1a) and GABA(B2) subunits which were expressed in both large and small diameter neurones. Although expression levels of GABA(B1) and GABA(B2) subunits varied we found no areas in which GABA(B1) was expressed in the absence of GABA(B2). This suggests that most, if not all, GABA(B1) immunoreactivity may represent functional GABA(B) receptors. Although our data are in general agreement with functional studies, some discrepancies in GABA(B1) subunit expression occurred with respect to other immunohistochemical studies. Overall our data suggest that GABA(B) receptors are widely expressed throughout the brain and spinal cord, and that GABA(B1a) and GABA(B1b) subunits can associate with GABA(B2) to form both pre- and post-synaptic receptors.

Investigation of feline brain anatomy for the detection of cortical spreading depression with magnetic resonance imaging.

Cortical spreading depression (CSD) and peri-infarct depolarisation (PID) are related phenomena that have been associated with the human clinical syndromes of migraine (CSD), head injury and stroke (PID). Nevertheless the existence of CSD in man remains controversial, despite the detection of this phenomenon in the brains of most, if not all, other animal species investigated. This failure to unambiguously detect CSD clinically may be at least partly due to the anatomically complex, gyrencephalic structure of the human brain. This study was designed to establish conditions for the study of CSD in the brain of a gyrencephalic species using the noninvasive technique of magnetic resonance imaging (MRI). The 3-dimensional (3D) gyrencephalic anatomy of the cat brain was examined to determine the imaging conditions necessary to detect CSD events. Orthogonal transverse, sagittal and horizontal T1-weighted image slices showed that the marginal and suprasylvian gyri were the most appropriate cortical structures to study CSD. This was in view of (1) their simple geometry: (2) their lengthy extent of grey matter orientated rostrocaudally in the cortex: (3) their separation by a sulcus across which CSD spread could be studied and (4) the discontinuity in the grey matter in these regions between the right and left hemispheres dorsal to the corpus callosum. The structure suggested by the T1-weighted images was corroborated by systematic diffusion tensor imaging to map the fractional anisotropy and diffusion trace. Thus a single horizontal image plane could visualise the neighbouring suprasylvian and marginal gyri of both cerebral hemispheres, whereas its complex shape and position ruled out the ectosylvian gyrus for CSD studies. With the horizontal imaging plane, CSD events were reproducibly detected by animating successive diffusion-weighted MR images following local KCl stimulation of the cortical surface. In single image frames, CSD detection and characterisation required image subtraction or statistical mapping methods that, nevertheless, yielded concordant results. In repeat experiments, CSD events were qualitatively similar in appearance whether elicited by sustained or transient KCl applications. Our experimental approach thus successfully describes cat brain anatomy in vivo, and elucidates the necessary conditions for the application of MRI methods to detect CSD propagation.

Cortical spreading depression and migraine: new insights from imaging?

The possibility that spreading depression (SD) of cortical activity, a phenomenon observed in all vertebrates, causes the aura of migraine remains an open question in spite of nearly half a century of investigation. SD is also thought to be associated with the progressive neuronal injury observed during cerebral ischaemia. Thus, the ability to detect and investigate SD in humans might prove clinically significant. Animal studies of cortical spreading depression (CSD) have benefited greatly from the advent of relatively non-invasive imaging techniques. The use of these new imaging techniques for clinical studies will accelerate progress in this area of neurobiology.

Gene expression and protein distribution of the orexin-1 receptor in the rat brain and spinal cord.

Orexins-A and -B are neuropeptides derived from a single precursor prepro-orexin. The mature peptides are mainly expressed in the lateral hypothalamic and perifornical areas. The orexins have been implicated in the control of arousal and appear to be important messengers in the regulation of food intake. Two receptors for orexins have been characterised so far: orexin-1 and -2 receptors. To gain a further understanding of the biology of orexins, we studied the distribution of the orexin-1 receptor messenger RNA and protein in the rat nervous system. We first assessed the expression profile of the orexin-1 receptor gene (ox-r1) in different regions by using quantitative reverse transcription followed by polymerase chain reaction. Using immunohistochemical techniques, we investigated the distribution of orexin-1 receptor protein in the rat brain using a rabbit affinity-purified polyclonal antiserum raised against an N-terminal peptide. The orexin-1 receptor was widely and strongly expressed in the brain. Thus, immunosignals were observed in the cerebral cortex, basal ganglia, hippocampal formation, and various other subcortical nuclei in the hypothalamus, thalamus, midbrain and reticular formation. In particular, robust immunosignals were present in many hypothalamic and thalamic nuclei, as well as in the locus coeruleus. The distribution of the receptor protein was generally in agreement with the distribution of the receptor messenger RNA in the brain as reported previously by others and confirmed in the present study. In addition, we present in situ hybridisation and immunohistochemical data showing the presence of orexin-1 receptor messenger RNA and protein in the spinal cord and the dorsal root ganglia. Finally, due to the shared anatomical and functional similarities between orexins and melanin-concentrating hormone, we present a comparison between the neuroanatomical distribution of the orexin-1 receptor and melanin-concentrating hormone receptor protein-like immunoreactivities in the rat central nervous system, and discuss some functional implications. In conclusion, our neuroanatomical data are consistent with the biological effects of orexins on food intake and regulation of arousal. In addition, the data suggest other physiological roles for orexins mediated through the orexin-1 receptor.

Orexin-A immunoreactive neurons in the rat hypothalamus do not contain neuronal nitric oxide synthase (nNOS).

The lateral hypothalamic area (LHA), a key site involved in the central control of feeding and energy homeostasis, contains populations of neurons that produce the orexin peptides or nitric oxide, two chemical factors that increase food intake. In this study, we used immunohistochemistry to investigate the possibility that rat LHA neurons co-express orexin-A and neuronal nitric oxide synthase (nNOS). The orexin-A and nNOS cell populations in the LHA showed extensive overlap without co-localization, and no evidence of direct anatomic contact was found. The finding that LHA neurons do not co-localize orexin-A and nNOS may suggest that the actions of the orexins and nitric oxide on food intake are mediated via independent mechanisms, however, nitric oxide is a diffusible molecule and could potentially affect the activity of orexin neurons via a non-synaptic mechanism.

Hypoglycemia activates orexin neurons and selectively increases hypothalamic orexin-B levels: responses inhibited by feeding and possibly mediated by the nucleus of the solitary tract.

Orexins are novel appetite-stimulating peptides expressed in the lateral hypothalamic area (LHA), and their expression is stimulated by hypoglycemia in fasted rats. We investigated activation of orexin and other neurons during insulin-induced hypoglycemia using the immediate early gene product Fos. Insulin (50 U/kg) lowered plasma glucose by >50% after 5 h and stimulated feeding sixfold compared with saline-injected controls. Hypoglycemic rats allowed to feed and normoglycemic controls both showed sparse Fos-positive (Fos+) neurons in the LHA and the paraventricular nucleus (PVN) and arcuate nucleus (ARC) and showed none in the nucleus of the solitary tract (NTS), which relays visceral feeding signals to the LHA. In the LHA, total numbers of Fos+ neurons were comparable in fed hypoglycemic and control groups (60 +/- 6 vs. 52 +/- 4 cells/mm2, P > 0.05), as were Fos+ neurons immunoreactive for orexin (1.4 +/- 0.4 vs. 0.6 +/- 0.4 cells/mm2, P > 0.05). By contrast, hypoglycemic rats that were fasted showed significantly more Fos+ nuclei in the LHA (96 +/- 10 cells/mm2, P < 0.05, vs. both other groups) and Fos+ orexin neurons (8.4 +/- 3.3 cells/mm2, P < 0.001, vs. both other groups). They also showed two- to threefold more Fos+ nuclei (P < 0.001) in the PVN and ARC than both fed hypoglycemic rats and controls and showed strikingly abundant Fos+ neurons in the NTS and dorsal motor nucleus of the vagus. In parallel studies, whole hypothalamic orexin-A levels were not changed in hypoglycemic rats, whether fasted or freely fed, whereas orexin-B levels were 10-fold higher in hypoglycemic fasted rats than in control and hypoglycemic fed groups. These data support our hypothesis that orexin neurons are stimulated by falling glucose levels but are readily inhibited by signals related to nutrient ingestion and suggest that they may functionally link with neuronal activity in the NTS. Orexin-A and -B may play specific roles in behavioral or neuroendocrine responses to hypoglycemia.

Gepirone. Organon.

Gepirone, a pyridinyl piperazine 5-HT1A receptor agonist, has been developed by Fabre-Kramer as an antidepressant. Bristol-Myers Squibb (BMS) outlicensed the compound to Fabre-Kramer in 1993 and is no longer involved in its development [337393]. In May 1998, NV Organon (a subsidiary of Akzo Nobel) licensed the rights to the drug product for further development and marketing from Fabre-Kramer and, by October 1999, had submitted the drug for approval in the US [347133]. In December 2000, the company expected US and European launches in 2002 and 2003, respectively [402686]. Mechanism of action studies have demonstrated that gepirone, compared to buspirone, possesses a much greater selectivity for 5-HT1A receptors over dopamine D2 receptors. Long-term studies have shown that gepirone has a differential action at presynaptic (agonist) and post-synaptic (partial agonist) 5-HT1A receptors. However, further studies are still required to determine the relative contribution of pre- and post-synaptic 5-HT1A receptors to the therapeutic action of gepirone and related compounds. In March 2001, according to Schroder Salomon Smith Barney, Akzo Nobel targeted peak sales of Euro 300 million for gepirone [409013]. This amount was reiterated in an April 2001 report by HSBC Securities, which stated that gepirone was expected to achieve this figure in 2009 or 2010 [409014].

Pharmacological magnetic resonance imaging: a new application for functional MRI.

Various methods, including functional magnetic resonance imaging (fMRI), have recently been developed to allow investigators to study functional activity in the living brain. Such techniques are now being used to investigate regionally specific brain activity associated with the administration of CNS-active drugs. fMRI in particular is increasingly recognized as being a relatively non-invasive way to perform pharmacological investigations in experimental animals, healthy human volunteers, and individuals with CNS disease. This use of fMRI, dubbed 'pharmacological MRI' or 'phMRI', holds the promise of providing relatively straightforward pharmacodynamic assays and can be used to establish brain-penetrability parameters, or dose-ranging information for novel therapeutic compounds.

The distribution of the mRNA and protein products of the melanin-concentrating hormone (MCH) receptor gene, slc-1, in the central nervous system of the rat.

Melanin-concentrating hormone (MCH), a 19 amino acid cyclic peptide, is largely expressed in the hypothalamus. It is implicated in the control of general arousal and goal-orientated behaviours in mammals, and appears to be a key messenger in the regulation of food intake. An understanding of the biological actions of MCH has been so far hampered by the lack of information about its receptor(s) and their location in the brain. We recently identified the orphan G-protein-coupled receptor SLC-1 as a receptor for the neuropeptide MCH. We used in situ hybridization histochemistry and immunohistochemistry to determine the distribution of SLC-1 mRNA and its protein product in the rat brain and spinal cord. SLC-1 mRNA and protein were found to be widely and strongly expressed throughout the brain. Immunoreactivity was observed in areas that largely overlapped with regions mapping positive for mRNA. SLC-1 signals were observed in the cerebral cortex, caudate-putamen, hippocampal formation, amygdala, hypothalamus and thalamus, as well as in various nuclei of the mesencephalon and rhombencephalon. The distribution of the receptor mRNA and immunolabelling was in good general agreement with the previously reported distribution of MCH itself. Our data are consistent with the known biological effects of MCH in the brain, e.g. modulation of the stress response, sexual behaviour, anxiety, learning, seizure production, grooming and sensory gating, and with a role for SLC-1 in mediating these physiological actions.

Orexin A activates locus coeruleus cell firing and increases arousal in the rat.

The localization of orexin neuropeptides in the lateral hypothalamus has focused interest on their role in ingestion. The orexigenic neurones in the lateral hypothalamus, however, project widely in the brain, and thus the physiological role of orexins is likely to be complex. Here we describe an investigation of the action of orexin A in modulating the arousal state of rats by using a combination of tissue localization and electrophysiological and behavioral techniques. We show that the brain region receiving the densest innervation from orexinergic nerves is the locus coeruleus, a key modulator of attentional state, where application of orexin A increases cell firing of intrinsic noradrenergic neurones. Orexin A increases arousal and locomotor activity and modulates neuroendocrine function. The data suggest that orexin A plays an important role in orchestrating the sleep-wake cycle.

Differential distribution of orexin-A and orexin-B immunoreactivity in the rat brain and spinal cord.

The orexins are recently identified appetite-stimulating hypothalamic peptides. We used immunohistochemistry to map orexin-A and orexin-B immunoreactivity in rat brain, spinal cord, and some peripheral tissues. Orexin-A- and orexin-B-immunoreactive cell bodies were confined to the lateral hypothalamic area and perifornical nuclei. Orexin-A-immunoreactive fibers were densely distributed in the hypothalamus, septum, thalamus, locus coeruleus, spinal cord, and near the ventricles, but absent from peripheral sites investigated. In contrast, orexin-B-immunoreactive fibers were distributed sparsely in the hypothalamus. Orexin cells are strategically sited to contribute to feeding regulation, but their widespread projections suggest that orexins have other physiological roles.