The novel neuropeptides called hypocretins (orexins) have recently been identified as being localized exclusively in cell bodies in a subregion of the tuberal part of the hypothalamus. The structure of the hypocretins, their accumulation in vesicles of axon terminals, and their excitatory effect on cultured hypothalamic neurons suggest that the hypocretins function in intercellular communication. To characterize these peptides further and to help understand what physiological functions they may serve, we undertook an immunohistochemical study to examine the distribution of preprohypocretin-immunoreactive neurons and fibers in the rat brain. Preprohypocretin-positive neurons were found in the perifornical nucleus and in the dorsal and lateral hypothalamic areas. These cells were distinct from those that express melanin-concentrating hormone. Although they represent a restricted group of cells, their projections were widely distributed in the brain. We observed labeled fibers throughout the hypothalamus. The densest extrahypothalamic projection was found in the locus coeruleus. Fibers were also seen in the septal nuclei, the bed nucleus of the stria terminalis, the paraventricular and reuniens nuclei of the thalamus, the zona incerta, the subthalamic nucleus, the central gray, the substantia nigra, the raphe nuclei, the parabrachial area, the medullary reticular formation, and the nucleus of the solitary tract. Less prominent projections were found in cortical regions, central and anterior amygdaloid nuclei, and the olfactory bulb. These results suggest that hypocretins are likely to have a role in physiological functions in addition to food intake such as regulation of blood pressure, the neuroendocrine system, body temperature, and the sleep-waking cycle.

We describe a hypothalamus-specific mRNA that encodes preprohypocretin, the putative precursor of a pair of peptides that share substantial amino acid identities with the gut hormone secretin. The hypocretin (Hcrt) protein products are restricted to neuronal cell bodies of the dorsal and lateral hypothalamic areas. The fibers of these neurons are widespread throughout the posterior hypothalamus and project to multiple targets in other areas, including brainstem and thalamus. Hcrt immunoreactivity is associated with large granular vesicles at synapses. One of the Hcrt peptides was excitatory when applied to cultured, synaptically coupled hypothalamic neurons, but not hippocampal neurons. These observations suggest that the hypocretins function within the CNS as neurotransmitters.The hypothalamus acts as a major regulatory center for autonomic and endocrine homeostasis. Structurally, it is a confederation of nuclei that regulate a broad array of physiological and behavioral activities. For some of these activities, particular peptides have been identified as major products of individual nuclei (1). These peptides exert their actions by transport to the pituitary, by entering the general circulation, or by secretion within the CNS. However, the hypothalamus has been implicated in the regulation of activities beyond those for which factors have been identified.We recently used directional tag PCR subtraction to identify 38 rat mRNAs selectively expressed within the hypothalamus (2). Preliminary in situ hybridization studies revealed that one of these, called clone 35 in that work, was expressed exclusively by a bilaterally symmetric structure within the posterior hypothalamus. Here we show that the clone 35 mRNA encodes the precursor of two putative peptides, the hypocretins, that share substantial amino acid identities with each other and with the gut hormone secretin. The Hcrt mRNA, which accumulates primarily after postnatal week 3 and in mouse is a product of a gene on chromosome 11, is restricted to neuronal cell bodies of the dorsal and lateral hypothalamus. Its protein product, visualized immunocytochemically, is sorted into secretory vesicles in fibers that project within the hypothalamus and to other brain areas. At least one of the peptides has neuroexcitatory activity. Cumulatively, these observations suggest that the Hcrt mRNA encodes peptides that act endogenously within the central nervous system as homeostatic regulators. The circuitry revealed by the immunohistochemistry suggests a role in nutritional homeostasis. MATERIALS AND METHODSProduction of Antisera. Antiserum 2050 was generated by coupling the synthetic 17-mer CPTATATALAPRGGSRV to the carrier keyhole limpet hemocyanin with glutaraldehyde and immunizing rabbits as described (3). In Western transfer blots using as target electrophoretically separated proteins from bacteria transformed with the plasmid pRSET B engineered to express preprohcrt, we observed a single prominent immunoreactive band with a migration of Ϸ19 kDa with the hyperi...

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I have determined the nucleotide sequence of the ampicillin resistance gene of pBR322, an Escherichia coli plasmid that encodes a penicillin beta-lactamase. This gene codes for a protein of 286 amino acid residues. The first 23 amino acids presumably form a signal for secretion, because they do not appear in the mature enzyme, whose partial amino acid sequence has been determined independently.

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The hypocretins (hcrts), also known as orexins, are two recently identified excitatory neuropeptides that in rat are produced by ϳ1200 neurons whose cell bodies are located in the lateral hypothalamus. The hypocretins/orexins have been implicated in the regulation of rapid eye movement (REM) sleep and the pathophysiology of narcolepsy. In the present study, we investigated whether the locus coeruleus (LC), a structure receiving dense hcrtergic innervation, which is quiescent during REM sleep, might be a target for hcrt to regulate REM sleep. Local administration of hcrt1 but not hcrt2 in the LC suppressed REM sleep in a dose-dependent manner and increased wakefulness at the expense of deep, slow-wave sleep. These effects were blocked with an antibody that neutralizes hcrt binding to hcrt receptor 1. In situ hybridization and immunocytochemistry showed the presence of hcrt receptor 1 but not the presence of hcrt receptor 2 in the LC. Iontophoretic application of hcrt1 enhanced the firing rate of LC neurons in vivo, and local injection of hcrt1 into the LC induced the expression of c-fos in the LC area. We propose that hcrt receptor 1 in the LC is a key target for REM sleep regulation and might be involved in the pathophysiological mechanisms of narcolepsy. Key words: norepinephrine; orexin; orexin receptors; c-fos; arousal; microinjection; immunocytochemistryThe hypocretins (hcrt1 and hcrt2), also called orexins, are two neuropeptides derived from the same precursor, which are expressed in a small set of neurons in the perifornical area of the hypothalamus Sakurai et al., 1998). The hypocretins are neuroexcitatory and bind to two different G-protein-coupled receptors, hcrt receptors 1 and 2 (hcrtr1 and hcrtr2, also known as OX1 and OX2 receptors) with different affinities (Sakurai et al., 1998). Recently, evidence has emerged that confirms a role for the hypocretins in arousal states. Lin et al. (1999) mapped the canine narcolepsy mutation (canarc-1) to hcrtr2. Knock-out experiments in mice demonstrated that the absence of hypocretin causes alterations in sleep architecture, particularly on the amount of rapid eye movement (REM) sleep during the dark period (Chemelli et al., 1999). In addition, hcrt-deficient mice display electroencephalographic patterns and behaviors that resemble those of narcoleptic attacks. Intracerebroventricular infusion of nanomolar amounts of hypocretin has recently been shown to increase arousal, reduce REM sleep, and affect neuroendocrine balance (Hagan et al., 1999). Nishino and colleagues (2000) found that seven of nine patients with narcolepsy had undetectable hcrt1 in CSF. These independent studies indicate that the hypocretins have a major role in the regulation of sleep, but the role of different brain structures and the contribution of each of the hcrt receptors remain unknown.The projections of hcrt-containing neurons extend widely throughout the brain Date et al., 1999). Four main hcrtergic afferent regions can be recognized from anatomical studies : an intrahypothalamic field...

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