The neural mechanisms through which the state of anesthesia arises and dissipates remain unknown. One common belief is that emergence from anesthesia is the inverse process of induction, brought about by elimination of anesthetic drugs from their CNS site(s) of action. Anesthetic-induced unconsciousness may result from specific interactions of anesthetics with the neural circuits regulating sleep and wakefulness. Orexinergic agonists and antagonists have the potential to alter the stability of the anesthetized state. In this report, we refine the role of the endogenous orexin system in impacting emergence from, but not entry into the anesthetized state, and in doing so, we distinguish mechanisms of induction from those of emergence. We demonstrate that isoflurane and sevoflurane, two commonly used general anesthetics, inhibit c-Fos expression in orexinergic but not adjacent melaninconcentrating hormone (MCH) neurons; suggesting that wakeactive orexinergic neurons are inhibited by these anesthetics. Genetic ablation of orexinergic neurons, which causes acquired murine narcolepsy, delays emergence from anesthesia, without changing anesthetic induction. Pharmacologic studies with a selective orexin-1 receptor antagonist confirm a specific orexin effect on anesthetic emergence without an associated change in induction. We conclude that there are important differences in the neural substrates mediating induction and emergence. These findings support the concept that emergence depends, in part, on recruitment and stabilization of wake-active regions of brain.anesthetic hypnosis ͉ arousal ͉ narcolepsy ͉ NREM sleep circuits ͉ volatile anesthetics

SUMMARY Circadian behavior in mammals is orchestrated by neurons within the suprachiasmatic nucleus (SCN), yet the neuronal population necessary for the generation of timekeeping remains unknown. We show that a subset of SCN neurons expressing the neuropeptide neuromedin S (NMS) plays an essential role in generation of daily rhythms in behavior. We demonstrate that lengthening period within Nms neurons is sufficient to lengthen period of the SCN and behavioral circadian rhythms. Conversely, mice without a functional molecular clock within Nms neurons lack synchronous molecular oscillations and coherent behavioral daily rhythms. Interestingly, we found that mice lacking Nms and its closely-related paralog, Nmu, do not lose in vivo circadian rhythms. However, blocking vesicular transmission from Nms neurons with intact cell-autonomous clocks disrupts the timing mechanisms of the SCN, revealing that Nms neurons define a subpopulation of pacemakers that control SCN network synchrony and in vivo circadian rhythms through intercellular synaptic transmission.

Death-associated protein kinase (DAPK) is a multidomain Ser/Thr protein kinase with an important role in apoptosis regulation. In these studies we have identified a DAPK-interacting protein called DIP-1, which is a novel multi-RING finger protein. The RING finger motifs of DIP-1 have E3 ligase activity that can auto-ubiquitinate DIP-1 in vitro. In vivo, DIP-1 is detected as a polyubiquitinated protein, suggesting that the intracellular levels of DIP-1 are regulated by the ubiquitin-proteasome system. Transient expression of DIP-1 in HeLa cells antagonizes the anti-apoptotic function of DAPK to promote a caspase-dependent apoptosis. These studies also demonstrate that DAPK is an in vitro and in vivo target for ubiquitination by DIP-1, thereby providing a mechanism by which DAPK activities can be regulated through proteasomal degradation.Regulation of protein degradation by the ubiquitin proteasome pathway is now known to be a major pathway through which cells modulate the expression levels of critical signaling proteins (1-6). This tightly regulated, complex pathway is a key regulator of many important signaling pathways and has an important role in many cellular processes including apoptosis, and recent studies have identified many apoptosis regulatory proteins as targets for ubiquitination (7-11). In addition to being targets for degradation, some apoptosis regulatory proteins have a more active role and act as components of the ubiquitin cascade via the ubiquitin ligase activity ascribed to the RING finger domains that is part of their primary structure. Targeting proteins for degradation by the ubiquitin proteasome pathway involves the covalent linkage of ubiquitin either to the amino terminus or specific lysine residues in the target protein through the action of three enzymes. In this process ubiquitin is first activated by an E1 ubiquitin-activating enzyme, transferred to an E2 ubiquitin-conjugating enzyme, and then ligated to the target protein by an E3 ubiquitin ligase (4,12) Recently the Ser/Thr protein kinase, death-associated protein kinase (DAPK) 1 has been implicated in apoptosis regulation. DAPK has a complex, multi-domain structure that includes a calcium/calmodulin-regulated kinase domain, a series of ankyrin repeats, and a carboxyl-terminal death domain (13-17). Although some of the regulatory features that directly control the catalytic activities of DAPK have been described, including the activation by calcium/calmodulin (17, 18) and the presence of an inhibitory autophosphorylation site (19), an understanding of how the cellular activities of DAPK are regulated in vivo is poorly understood. The presence of proteinprotein interaction domains within the primary structure of DAPK, including its ankyrin repeat motifs and death domain, suggests that additional interactions between DAPK and other cellular proteins will also be important for regulation of DAPK activities. In this study, we describe a new DAPK-interacting protein called DIP-1 (DAPK-interacting protein-1), which has a direct role in r...