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.Since the discovery of the orexins (1) investigations of their functions have been guided by evidence for their hypothalamic distribution (1, 2), focusing on feeding, energy homeostasis (1, 3), and neurocrine functions (3). Our studies now show the presence of orexin A immunoreactive fibers and varicosities in extrahypothalamic areas, particularly the locus coeruleus, and demonstrate that the functions of orexin A extend beyond the hypothalamus.Orexin A and B are derived from a 130-aa precursor, prepro-orexin, which is encoded by a gene localized to human chromosome 17q21 (1). Prepro-orexin, or preprohypocretin (2), was identified in the rat hypothalamus by directional tag PCR subtractive hybridization (2) and has been shown by Northern blot analysis to be abundant in the brain and detectable at low levels in testes but not in a variety of other tissues (1, 2). Hypocretins had been identified as hypothalamic neuropeptides, but their biological role was not described (2). Nucleotide sequence alignment shows that hypocretins 1 and 2 have sequence in common with orexins A and B, respectively, but additional amino acids are present in both hypocretins. In situ hybridization maps confirm dense prepro-orexin mRNA expression in the hypothalamus (1, 2). Immunocytochemical mapping of orexin A has identified a population of mediumsized neurones within the hypothalamus, median eminence (3), and ventral thalamic nuclei of rat brain (1, 3). This distribution has been confirmed in human tissue (4).Orexin A binds with high affinity to the novel G proteincoupled receptors orexin 1 (OX 1 ) (IC 50 20 nM) and orexin 2 (OX 2 ) (IC 50 38 nM). Calcium mobilization assays in transfected HEK293 cells confirm that orexin A is a potent agonist at both OX 1 (EC 50 30 nM) and OX 2 (EC 50 34 nM) (1). Emerging evidence suggests the existence of an extensive extrahypothalamic projection of orexin-immunoreactive neurones. Peyron et al. (5), in addition to confirming the presence of immunoreactive cell somata within the hypothalamus, reported immunolabeled fibers throughout extrahypothalamic regions, including septal nuclei, substantia nigra, and raphe nucle...

The hypothalamic peptide orexin-A and the orexin-1 receptor are localized in areas of the brain and spinal cord associated with nociceptive processing. In the present study, localization was confirmed in the spinal cord and demonstrated in the dorsal root ganglion for both orexin-A and the orexin-1 receptor. The link with nociception was extended when orexin-A was shown to be analgesic when given i.v. but not s.c. in mouse and rat models of nociception and hyperalgesia. The efficacy of orexin-A was similar to that of morphine in the 50 degrees C hotplate test and the carrageenan-induced thermal hyperalgesia test. However, involvement of the opiate system in these effects was ruled out as they were blocked by the orexin-1 receptor antagonist SB-334867 but not naloxone. Orexin-1 receptor antagonists had no effect in acute nociceptive tests but under particular inflammatory conditions were pro-hyperalgesic, suggesting a tonic inhibitory orexin drive in these circumstances. These data demonstrate that the orexinergic system has a potential role in the modulation of nociceptive transmission.

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

Summary:Alzheimer's disease (AD) is a devastating neurological condition characterized by a progressive decline in cognitive performance accompanied by behavioral and psychological syndromes, such as depression and psychosis. The neurochemical correlates of these clinical manifestations now appear to involve dysfunctions of multiple neurotransmitter pathways. Because of the extensive serotonergic denervation that has been observed in the AD brain and the important role played by serotonin (5-HT) in both cognition and behavioral control, this neurotransmitter system has become a focus of concerted research efforts to identify new treatments for AD. 5-HT exerts its diverse physiological and pharmacological effects through actions on multiple receptor subtypes. One of the newest members of this family is the 5-HT 6 receptor, a subtype localized almost exclusively in the CNS, predominating in brain regions associated with cognition and behavior. With the subsequent development of selective 5-HT 6 receptor antagonists, preclinical studies in rodents and primates have elucidated the function of this receptor subtype in more detail. It is increasingly clear that blockade of 5-HT 6 receptors leads to an improvement of cognitive performance in a wide variety of learning and memory paradigms and also results in anxiolytic and antidepressant-like activity. These actions are largely underpinned by enhancements of cholinergic, glutamatergic, noradrenergic, and dopaminergic neurotransmission, together with learning-associated neuronal remodeling. A preliminary report that the cognitive enhancing properties of a 5-HT 6 receptor antagonist (namely, SB-742457) extends into AD sufferers further highlights the therapeutic promise of this mechanistic approach.

Otitis media (OM), inflammation of the middle ear, remains the most common cause of hearing impairment in children. It is also the most common cause of surgery in children in the developed world. There is evidence from studies of the human population and mouse models that there is a significant genetic component predisposing to OM, yet nothing is known about the underlying genetic pathways involved in humans. We identified an N-ethyl-N-nitrosourea-induced dominant mouse mutant Junbo with hearing loss due to chronic suppurative OM and otorrhea. This develops from acute OM that arises spontaneously in the postnatal period, with the age of onset and early severity dependent on the microbiological status of the mice and their air quality. We have identified the causal mutation, a missense change in the C-terminal zinc finger region of the transcription factor Evi1. This protein is expressed in middle ear basal epithelial cells, fibroblasts, and neutrophil leukocytes at postnatal day 13 and 21 when inflammatory changes are underway. The identification and characterization of the Junbo mutant elaborates a novel role for Evi1 in mammalian disease and implicates a new pathway in genetic predisposition to OM.

Otitis media (OM), inflammation of the middle ear, is the most common cause of hearing impairment and surgery in children. Recurrent and chronic forms of OM are known to have a strong genetic component, but nothing is known of the underlying genes involved in the human population. We have previously identified a novel semi-dominant mouse mutant, Jeff, in which the heterozygotes develop chronic suppurative OM (Hardisty, R.E., Erven, A., Logan, K., Morse, S., Guionaud, S., Sancho-Oliver, S., Hunter, A.J., Brown, S.D. and Steel, K.P. (2003) The deaf mouse mutant Jeff (Jf) is a single gene model of otitis media. J. Assoc. Res. Otolaryngol., 4, 130-138.) and represent a model for chronic forms of OM in humans. We demonstrate here that Jeff carries a mutation in an F-box gene, Fbxo11. Fbxo11 is expressed in epithelial cells of the middle ears from late embryonic stages through to day 13 of postnatal life. In contrast to Jeff heterozygotes, Jeff homozygotes show cleft palate, facial clefting and perinatal lethality. We have also isolated and characterized an additional hypomorphic mutant allele, Mutt. Mutt heterozygotes do not develop OM but Mutt homozygotes also show facial clefting and cleft palate abnormalities. FBXO11 is one of the first molecules to be identified, contributing to the genetic aetiology of OM. In addition, the recessive effects of mutant alleles of Fbxo11 identify the gene as an important candidate for cleft palate studies in the human population.

The Notch signalling pathway has recently been implicated in the development and patterning of the sensory epithelium in the cochlea, the organ of Corti. As part of an ongoing large-scale mutagenesis programme to identify new deaf or vestibular mouse mutants, we have identified a novel mouse mutant, slalom, which shows abnormalities in the patterning of hair cells in the organ of Corti and missing ampullae, structures that house the sensory epithelia of the semicircular canals. We show that the slalom mutant carries a mutation in the Jagged1 gene, implicating a new ligand in the signalling processes that pattern the inner ear neuro-epithelium.

The robotic mouse is an autosomal dominant mutant that arose from a large-scale chemical mutagenesis program. It has a jerky, ataxic gait and develops adult-onset Purkinje cell loss in the cerebellum in a striking region-specific pattern, as well as cataracts. Genetic and physical mapping of the disease locus led to the identification of a missense mutation in a highly conserved region of Af4, a putative transcription factor that has been previously implicated in leukemogenesis. We demonstrate that Af4 is specifically expressed in Purkinje cells, and we hypothesize that the expression of mutant Af4 leads to neurodegeneration. This function was not identified through knock-out studies, highlighting the power of phenotype-driven mutagenesis in the mouse to identify new pathways involved in neurological disease.