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...

1 The novel 5-HT 7 receptor antagonist, SB-269970-A, potently displaced [ 3 H]-5-CT from human 5-HT 7(a) (pK i 8.9+0.1) and 5-HT 7 receptors in guinea-pig cortex (pK i 8.3+0.2). 2 5-CT stimulated adenylyl cyclase activity in 5-HT 7(a) /HEK293 membranes (pEC 50 7.5+0.1) and SB-269970-A (0.03 ± 1 mM) inhibited the 5-CT concentration-response with no signi®cant alteration in the maximal response. The pA 2 (8.5+0.2) for SB-269970-A agreed well with the pK i determined from [ 3 H]-5-CT binding studies. 3 5-CT-stimulated adenylyl cyclase activity in guinea-pig hippocampal membranes (pEC 50 of 8.4+0.2) was inhibited by SB-269970-A (0.3 mM) with a pK B (8.3+0.1) in good agreement with its antagonist potency at the human cloned 5-HT 7(a) receptor and its binding anity at guinea-pig cortical membranes. 4 5-HT 7 receptor mRNA was highly expressed in human hypothalamus, amygdala, thalamus, hippocampus and testis. 5 SB-269970-A was CNS penetrant (steady-state brain : blood ratio of ca. 0.83 : 1 in rats) but was rapidly cleared from the blood (CLb=ca. 140 ml min 71 kg 71). Following a single dose (3 mg kg 71 ) SB-269970 was detectable in rat brain at 30 (87 nM) and 60 min (58 nM). In guinea-pigs, brain levels averaged 31 and 51 nM respectively at 30 and 60 min after dosing, although the compound was undetectable in one of the three animals tested.

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.

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From histological investigations into the microstructure of conodont elements, a number of tissue types characteristic of the phosphatic skeleton of vertebrates have been identified. These include cellular bone, two forms of hypermineralized enamel homologs, and globular calcified cartilage. The presence of cellular bone in conodont elements provides unequivocal evidence for their vertebrate affinities. Furthermore, the identification of vertebrate hard tissues in the oral elements of conodonts extends the earliest occurrence of vertebrate hard tissues back by around 40 million years, from the Middle Ordovician (475 million years ago) to the Late Cambrian (515 million years ago).

Little is known regarding the internal anatomy of thelodonts, with most of the available information coming from a single specimen, the holotype of Turinia pagei (Powrie). Previous descriptions have led to many partial, or conflicting interpretations. Herein, we describe fully the anatomy of T. pagei based on the holotype and additional material. T. pagei possessed a branchial system composed of eight pairs of gills, a buccal/nasohypophyseal region lined with minute denticles, comparable to buccopharyngeal denticles of sharks, and possessed a stomach which is preserved by sediment infill in the holotype specimen. Contrast between the petrological character of the gut infill and the sediment in which the animal is preserved suggests that the gut was infilled in vivo and that T. pagei was probably a deposit feeder. Phylogenetic analysis resolves T. pagei and the Galeaspida as sister-taxa, comprising a sister-group to the Osteostraci plus jawed vertebrates. In contrast to the view that has prevailed hitherto, thelodonts with a dorsoventrally compressed cross-sectional profile comprise a monophyletic group, of which T. pagei is the least derived member. The furcacaudiforms are resolved as an unnatural group, one taxon being the sister taxon to the ‘conventional’ thelodont clade, and the other, the sister taxon to this clade plus galeaspids, osteostracans and jawed vertebrates. The analysis agrees with the earlier view that thelodonts lack distinct synapomorphies, but we argue that distinct synapomorphies are not a requisite of monophyly.

Three new specimens which preserve the soft parts of conodonts are described from the Lower Carboniferous of Granton, Edinburgh. The animal was apparently laterally flattened in life and the somites were V‐shaped. The nature of the preserved axial lines is equivocal; some may represent the walls of the gut. The elements of one of the new specimens show that it does not belong to Clydagnathus, to which the other soft‐bodied specimen from Granton was tentatively assigned. The possibility of a relationship between the euconodonts and the Chaetognatha is discounted. Nor do the conodonts constitute a phylum, but are a separate group of primitive jawless craniates.

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