Hypocretin-1 modulates rapid eye movement sleep through activation of locus coeruleus neurons.

The hypocretins (hcrts), also known as orexins, are two recently identified excitatory neuropeptides that in rat are produced by approximately 1200 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.

Characterization of the promoter region and flanking sequences of the neuron-specific gene RC3 (neurogranin).

RC3 encodes a thyroid hormone-dependent, calmodulin-binding, protein kinase C substrate (neurogranin, p17) present in the dendritic spines of discrete neuronal populations in the forebrain. Its physiological role could be related to synaptic plasticity, memory, and other processes. In the present work we have isolated and sequenced 2.4 kbp of genomic DNA upstream from the origin of transcription and determined its nucleotide sequence. The major features of the RC3 promoter are the absence of TATA and CAAT boxes and the presence of an Initiator sequence surrounding the cap site. By sequence analysis we identified several cis-acting regulatory elements, among them response elements for retinoic acid and steroid (glucocorticoids/progesterone) hormone receptors. An oligonucleotide containing the retinoic acid responsive element bound to retinoic acid receptors specifically in vitro and conferred retinoic acid regulation to a heterologous promoter after transfection in COS-7 cells. Retinoic acid and dexamethasone, respectively, increased activity of the RC3 promoter in neuroblastoma cells when a deletion construct containing the retinoic acid and the glucocorticoid responsive elements was cotransfected with retinoic acid receptor or glucocorticoid receptor expression vectors. When added together all-trans retinoic acid and dexamethasone had additive effects. Despite the fact that RC3 expression in vivo is thyroid hormone-dependent, no evidence for the presence of a thyroid hormone responsive element was found within the 2.4 kbp flanking region analyzed and thyroid hormone did not increase reporter activity after cotransfection of suitable constructs with thyroid hormone receptor expression vectors. Our results suggest that the expression of RC3 in vivo could be subject to complex physiological signals, including retinoids and steroid hormones in addition to thyroid hormones.