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.

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.

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.

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.

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.

Localized changes in blood-brain barrier permeability following the administration of antineoplastic drugs.

The effects of four antineoplastic drugs on the permeability of the blood-brain barrier to Evans blue-albumin and to horseradish peroxidase were studied in cats. Extravasation of tracer in brain tissue was observed only rarely following the injection of methotrexate, cyclophosphamide, or vincristine. However, 5-fluorouracil (15 mg/kg) caused localized Evans blue-albumin exudation in various gray and white matter areas in 8 of 13 cats to which the Evans blue was administered 7 hr after the drug injection. Electron microscopy revealed that 5-fluorouracil stimulated pinocytotic vesicular transport of peroxidase across brain capillary endothelial cells and possibly that it widened endothelial tight junctions. Barrier leakage was not observed when time periods longer than 7 hr elapsed between 5-fluorouracil injection and tracer administration, and extravasation occurred only once after a shorter time interval. These results suggest that changes in blood-brain barrier permeability observed 7 hr after 5-fluorouracil administration are reversible and of fairly short duration. Such changes may be relevant to the development of secondary intracranial tumors following antineoplastic chemotherapy.

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.

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.

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.

Observations on the afferent and efferent organization of the vagus nerve and the innervation of the stomach in the squirrel monkey.

Horseradish peroxidase was injected into the cervical vagus nerve or stomach wall of adult squirrel monkeys. Following cervical vagus nerve injections, labelled afferent fibres were present in the tractus solitarius and labelled fibres and terminals were present in medial and lateral parts of the nucleus of the tractus solitarius (NTS) ipsilaterally. Afferent labelling was also seen in the ipsilateral commissural nucleus and in the area postrema. Labelling was present contralaterally in caudal levels of the medial parts of the NTS, in the commissural nucleus, and in the area postrema. Afferent projections to the ipsilateral pars interpolaris of the spinal trigeminal nucleus and to the substantia gelatinosa of the C1 segment of the spinal cord were also labelled. Following injections of HRP into the anterior and posterior stomach walls, the tractus solitarius was labelled bilaterally. Afferent labelling was concentrated bilaterally in the dorsal parts of the medial division of the NTS, i.e., in the subnucleus gelatinosus, and in the commissural nucleus. The regions of NTS immediately adjacent to the tractus solitarius were largely unlabelled. Injections of HRP into the cervical vagus nerve resulted in heavy retrograde labelling of neurons in the ipsilateral dorsal nucleus of the vagus (DMX) and in the nucleus ambiguus (NA). In addition a few neurones were labelled in the intermediate zone between these two nuclei. Retrogradely labelled neurons were also present in the nucleus dorsomedialis in the rostral cervical spinal cord and in the spinal nucleus of the accessory nerve. Injections of HRP into the left cricothyroid muscle in two cases resulted in heavy retrograde labelling of large neurons in the left NA. Following stomach wall injections of HRP retrograde labelling of neurons was seen throughout the rostrocaudal and mediolateral extent of the DMX; there was no apparent topographical organization of the projection. In these cases, a group of labelled smaller neurons was found lying ventrolateral to the main part of the NA through its rostral levels. This study in a primate indicates that a large vagal afferent projection originates in the stomach wall and terminates primarily in the subnucleus gelatinosus of the NTS and in the commissural nucleus with a distribution similar to that described previously in studies in several subprimate mammalian species. The present results and those of other studies suggest some degree of segregation of visceral input within different subnuclei of the NTS.(ABSTRACT TRUNCATED AT 400 WORDS)

The ultrastructure of the subnucleus gelatinosus of the nucleus of the tractus solitarius in the cat.

The dorsomedial region of the nucleus of the tractus solitarius termed the subnucleus gelatinosus (SNG) was studied at the light and electron microscopic level in the cat. In cresyl violet and luxol fast blue stained sections the SNG contained small neuronal somata that were scattered throughout a pale-staining neuropil containing few myelinated fibers. These neurons were difficult to impregnate with Golgi staining techniques, but in successful impregnations the somata were observed to be 10--19 micrometers in diameter and bore few sparsely branching primary dendrites. Spines were present on the dendrites of some neurons and were more numerous on distal portions of the dendritic tree. Ultrastructural examination of the SNG revealed that the neuronal complement consisted of round, oval, or spindle shaped neurons with little or no organized Nissl substance. Rare myelin-like ensheathments of neuronal perikarya were also observed. Bundles of fine unmyelinated axons that coursed mainly longitudinally were a prominent feature of the area. The most common type of axon terminal observed contained mainly round clear vesicles, approximately 31 nm in diameter, and made asymmetrical synaptic contact with a dendritic profile. Pleomorphic vesicle-containing terminals involved in symmetrical synaptic contact were also commonly seen. Axodendritic and axosomatic synapses were associated with terminals containing either round clear vesicles or pleomorphic vesicles. Less commonly, dendrodendritic and dendrosomatic synapses were seen, the presynaptic elements of which contained pleomorphic vesicles. Following removal of a nodose ganglion, degenerating terminals of vagal afferent fibers were observed throughout the neuropil. Such terminals contained round, clear vesicles with an occasional large, dense-cored vesicle, and made axodendritic and axosomatic synaptic contacts.

Paradoxical effects of lithium on serotonergic receptor function: an immunocytochemical, behavioural and autoradiographic study.

Lithium is the preferred treatment for bipolar affective disorder, yet its mechanism of action is poorly understood. Our study was designed to investigate the effect of lithium on the 5-HT2A or 5-HT2C (5-HT2A/2C) receptor subtypes, by comparing the consequences of chronic pre-treatment of rats with lithium on 5-HT2A/2C receptor-mediated behavioural responses, Fos expression, and the density of these receptors in the brain. In addition, the time-course and persistence of the effect of chronic lithium on 5-HT2A/2C receptor-mediated Fos expression was examined. Furthermore, the acute action of lithium on Fos expression was also examined. In an investigation of the dose response of Fos to the 5-HT2A/2C agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), rats received saline or 1, 2, 4, 8, 12, 16, 24 or 32 mg/kg DOI, then were sacrificed 3 h later for immunocytochemical localisation of Fos. In a chronic lithium study, rats received either control or lithium-containing (0.1% LiCO3) chow for 3 weeks prior to challenge with 8 mg/kg DOI. DOI-induced locomotor activity was measured for 30 min immediately following the drug challenge, then 150 min later, the animals were sacrificed for Fos immunocytochemistry. The brains of another group of rats, also receiving either control or lithium-containing diet for 3 weeks, were analysed for the distribution and density of 5-HT2A receptor binding sites by quantitative [3H]ketanserin autoradiography. One group of chronic lithium treated rats received ritanserin (0.4 mg/kg), a 5-HT2A/2C receptor antagonist, 40 min before DOI challenge and were sacrificed 3 h later for Fos localisation. In the time-course experiment, rats received lithium-containing diet for 3 weeks followed by normal, control diet for 48 h, 1, 2 or 4 weeks prior to DOI or saline challenge. A further group of animals received an injection of LiCl (3 mM/kg) before being challenged with DOI or saline 12, 24, 36 or 48 h later. The dose-response experiment revealed that little Fos-like immunoreactivity was evident above basal levels following administration of 1 mg/kg DOI. However, at all other doses examined, Fos-like immunoreactivity was elevated in a number of brain areas, particularly in cerebral cortex, olfactory tubercle and amygdala. Following 24 mg/kg DOI, the number of Fos-positive nuclei appeared to have reached a plateau level. Treatment of rats with chronic lithium significantly enhanced DOI-induced locomotor activity and Fos-like immunoreactivity throughout the cerebral cortex. This elevation in Fos-like immunoreactivity was completely abolished by prior treatment with ritanserin. In contrast, chronic lithium treatment had no effect on the density of [3H]ketanserin binding to 5-HT2A receptors in any brain region examined. The results of the time-course experiment demonstrated that the enhancing effect of lithium on 5-HT2A/2C receptor-mediated Fos expression was short-lived such that Fos-like immunoreactivity returned to untreated levels within 48 h. In the acute lithium experiment, administration of lithium to rats 12 or 24 h before DOI resulted in a similar elevation of Fos-like immunoreactivity to that seen in chronically treated animals. Administration of acute lithium 36 or 48 h before DOI had no effect. The effects of lithium on 5-HT2A/2C receptor function thus appear to be complex. In particular, the results of this study indicate that the enhancing effects of lithium on DOI-induced locomotor activity and Fos-like immunoreactivity are not accompanied by any alteration in the density of 5-HT2A receptor binding sites. If changes in receptor numbers therefore do not account for the physiological effect of chronic lithium, other explanations must be sought. The study also suggests that the inositol depletion hypothesis of lithium's therapeutic action does not adequately explain the mechanism of action of lithium in man.

Chronic electroconvulsive shock enhances 5-HT2 receptor-mediated head shakes but not brain C-fos induction.

Chronic electroconvulsive shock (ECS), a widely used treatment for intractable depression, increases the density of 5-HT2A receptor binding sites and mRNA in rat frontal cortex. In contrast, this treatment appears to have no significant effect on 5-HT-stimulated phosphatidyl inositol turnover in rat brain. To investigate the effect of chronic ECS on the 5-HT2 receptor family further, we determined its effects on head shakes and c-fos expression in the rat in response to the 5-HT2A/2C receptor agonist DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane]. Chronic ECS (5 electroconvulsive shocks over 10 days, via earclips under halothane anaesthesia) caused a significant enhancement in the number of head shakes counted in a 30 min period after administration of 2 or 8 mg/kg DOI. In contrast, this treatment had no effect on Fos expression, induced by either dose of DOI, in any region of rat forebrain examined. Fos expression was low-to-undetectable in the brains of animals treated with chronic ECS followed by saline and sham ECS animals that had been treated identically, but with no administration of electrocurrent. Thus the lack of any change in PI turnover, following chronic ECS administration, appears to be mirrored by the failure of this treatment to alter 5-HT2 receptor-mediated Fos expression.

BRL 46470 potently antagonizes neural responses activated by 5-HT3 receptors.

The effect of a novel 5-HT3 receptor antagonist, BRL 46470, has been studied on two electrophysiological models for 5-HT3 receptors: grease-gap recordings from rat isolated vagus nerve and whole-cell patch-clamp recordings from mouse neuroblastoma-rat glioma NG108-15 cells. Its action on the rat vagus nerve was compared to that of four other 5-HT3 receptor antagonists. On the rat vagus, BRL 46470 reduced the maximum depolarizing response to 5-HT in a concentration-dependent manner with an IC50 of 0.3-1.0 nM, but the EC50 for 5-HT was not appreciably affected. This action was similar to that of granisetron and ICS 205-930, but differed from that of GR38032F and (+)-tubocurarine which produced clear rightward shifts of the concentration-response curve to 5-HT. The 5-HT-induced fast inward current of voltage-clamped NG108-15 cells was also antagonized by 1 nM BRL 46470 in an insurmountable manner. In contrast to (+)-tubocurarine, the action of BRL 46470 on the rat vagus nerve and NG108-15 cells did not readily reverse on washing with antagonist-free medium. It is concluded that BRL 46470 is a potent, insurmountable 5-HT3 receptor antagonist on the rat vagus and NG108-15 cells.

P-chloroamphetamine induces c-fos in rat brain: a study of serotonin2A/2C receptor function.

Recent studies suggest that a class of substituted amphetamines, which includes p-chloroamphetamine, causes an acute release of serotonin (5-hydroxytryptamine) and appears to act preferentially on axons arising from the dorsal raphe nucleus. The postsynaptic targets of these axons are not well characterized, but they have been localized in close proximity to the distribution of serotonin2A receptor binding sites. In the present study, c-fos immunocytochemistry has been used to investigate this anatomical relationship further. Administration of p-chloroamphetamine or the serotonin2A/2C receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane to rats resulted in similar patterns of Fos-like immunoreactivity in some, but not all, forebrain areas. Areas which expressed Fos following either treatment included cerebral cortex, claustrum, amygdala and nucleus accumbens. A particularly close match was seen in layer Va of the somatosensory cortex. No specificity of p-chloroamphetamine for dorsal raphe nucleus-innervated areas was noted. Prior treatment of animals with p-chloroamphetamine two weeks before a second challenge with the same drug, or with the serotonin2A/2C receptor antagonist ritanserin 30 min before p-chloroamphetamine challenge, resulted in an attenuation of p-chloroamphetamine-induced Fos-like immunoreactivity in the olfactory tubercle, the islands of Calleja and the caudate-putamen. The reduction was most noticeable in layer Va of the somatosensory cortex. The results of this study indicate that a close anatomical correlation may exist between the fine serotonin axon terminals that show vulnerability to the neurotoxic effects of p-chloroamphetamine and serotonin2A receptors in some brain regions. This association may prove to be important in explaining the actions of certain psychotropic drugs, for example in the control of affective states.

Nodose ganglionectomy selectively reduces muscarinic cholinergic and delta opioid binding sites in the dorsal vagal complex of the cat.

The dorsal vagal complex of the medulla oblongata, comprising the nucleus tractus solitarii, the area postrema and the dorsal motor nucleus of the vagus nerve, is an important brainstem regulatory center for the autonomic nervous system. The major afferent input from abdominal and thoracic viscera to this region is via vagal sensory neurons which have their cell bodies in the nodose ganglion. Autoradiography has been used to study the effects of unilateral nodose ganglionectomy on receptor binding sites in this region of the brain for the neurotransmitters acetylcholine, norepinephrine, and opioids. Nodose ganglionectomy had no discernible effect on alpha 2 noradrenergic ([3H]p-aminoclonidine) or mu opioid [( 3H]Tyr-D-Ala-Gly-(NMePhe)-Gly-ol) binding sites. However, ganglionectomy did produce a 25% decrease in [3H]quinuclidinyl benzilate (muscarinic cholinergic) binding in the subnucleus gelatinosus of the solitary nucleus, and a marked decrease in [3H][D-Pen5]enkephalin (delta opioid) binding in the dorsomedial subnucleus of the nucleus tractus solitarii, ipsilateral to the lesion. These data suggest that muscarinic cholinergic and delta opioid receptors may be present on terminals of vagal afferent neurons that project to these specific brainstem regions. Since these vagal afferent neurons are known to arise, at least in part, from the gastrointestinal tract, it is possible that cholinergic and/or opioid receptors modulate specific autonomic functions associated with gastric sensory information such as satiety or nausea and emesis.

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.

Induction of c-fos in rat forebrain by pharmacological manipulation of 5-hydroxytryptamine levels.

The immunocytochemical localization of the immediate-early gene c-fos has been used to map sites of neuronal activity in the rat brain associated with 5-hydroxytryptamine function. Behavioural studies have shown that brain 5-hydroxytryptamine function is increased by treatment of animals with a combination of the 5-hydroxytryptamine precursor L-tryptophan (100 mg/kg) and the monoamine inhibitor tranylcypromine (20 mg/kg). We now report that such treatment induces a specific anatomical pattern of expression of c-fos in rat forebrain in many limbic, striatal and cortical areas which corresponds well with the distribution of 5-hydroxytryptamine-immunoreactive terminals. To investigate further the involvement of 5-hydroxytryptamine in this response, we pretreated animals with the tryptophan hydroxylase inhibitor p-chlorophenylalanine and observed the effects on Fos-like immunoreactivity after L-tryptophan and tranylcypromine challenge. Two-day pretreatment with p-chlorophenylalanine (300 mg/kg) prior to tranylcypromine and L-tryptophan resulted in a significant attenuation of Fos-like immunoreactivity in specific brain areas, including the piriform and frontal cortices, nucleus accumbens, caudate-putamen, paraventricular hypothalamus and paraventricular thalamic nucleus. A marked reduction of the hyperactivity syndrome was also seen, as has been reported in earlier studies. The results of this study suggest that the elevation in Fos-like immunoreactivity following treatment with tryptophan and a monoamine oxidase inhibitor is mainly due to increased 5-hydroxytryptamine synthesis and release. It is well known that 5-hydroxytryptamine mediates mood and affect, and this study indicates potential brain loci of action of serotonergic drugs.

Serotonin2/1C receptor activation causes a localized expression of the immediate-early gene c-fos in rat brain: evidence for involvement of dorsal raphe nucleus projection fibres.

Immunocytochemistry has been used to monitor the expression of the immediate-early gene c-fos in rat brain following administration of the serotonin2/1C receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane. At parenteral doses of 2 or 8 mg/kg the drug caused a highly localized expression of the Fos protein in frontal, parietal, cingulate and piriform cortex as well as in claustrum, mamillary bodies, globus pallidus, amygdala, nucleus accumbens and dorsomedial striatum. In particular, the location of heavy Fos immunoreactivity in the primary somatosensory cortex corresponds precisely to that region (layer Va) shown in other reports to receive a dense input of fine, non-varicose fibres which may arise from the dorsal raphe nucleus. All of the Fos-positive brain regions in the present study have been previously demonstrated to contain serotonin2 receptor ligand binding sites. Interestingly, no Fos-positive cells were found in the hippocampus, another brain region known to contain serotonin2 receptors. Pretreatment of animals with the serotonin2/1C receptor antagonist ritanserin (0.4 mg/kg) markedly attenuated Fos expression in all reactive areas of the brain. Counts of reactive cells indicated that this antagonism of the Fos response was statistically significant in these brain regions. Spiperone (1 mg/kg), a mixed serotonin2 and dopamine D2 antagonist, also attenuated the Fos response in the same regions, but had the effect of inducing Fos expression on its own in other extrapyramidal brain regions. Double labelling of reactive cells with different antisera recognizing Fos and neuron-specific enolase, and lack of double labelling with a glial fibrillary acidic protein antiserum, indicated that the Fos expression was in neurons within the brain nuclei examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for presynaptic 5-hydroxytryptamine3 recognition sites on vagal afferent terminals in the brainstem of the ferret.

Antagonists acting at the 5-hydroxytryptamine3 receptor are potent anti-emetic agents in cases of cytotoxic- and radiation-induced vomiting, and binding sites for these compounds have been described in brainstem areas known to be involved in mediation of nausea and vomiting. We have used autoradiography to examine the distribution of one of these antagonists, [3H]granisetron in the caudal brainstem of the ferret, a commonly used animal model for physiological investigations of emesis. The highest density of binding sites was found to be in the dorsomedial region of the nucleus of the solitary tract, the principal terminus for gastric vagal afferent fibres. Lower levels of binding were observed in the area postrema and the dorsal motor nucleus of the vagus. Following unilateral nodose ganglion excision, displaceable binding of [3H]granisetron in the nucleus of the solitary tract was attenuated on the ipsilateral side by 65%. Bilateral subdiaphragmatic vagotomy abolished binding of [3H]granisetron in the entire dorsal vagal complex. These results provide strong circumstantial evidence that 5-hydroxytryptamine3 receptors are located on vagal afferent terminals in the ferret brainstem.