Dopamine signaling in wake-promoting clock neurons is not required for the normal regulation of sleep in drosophila

Dopamine is a wake-promoting neuromodulator in mammals and fruit flies. In Drosophila melanogaster, the network of clock neurons that drives sleep/activity cycles comprises both wake-promoting and sleep-promoting cell types. The large ventrolateral neurons (l-LNvs) and small ventrolateral neurons (s-LNvs) have been identified as wake-promoting neurons within the clock neuron network. The l-LNvs are innervated by dopaminergic neurons, and earlier work proposed that dopamine signaling raises cAMP levels in the l-LNvs and thus induces excitatory electrical activity (action potential firing), which results in wakefulness and inhibits sleep. Here, we test this hypothesis by combining cAMP imaging and patch-clamp recordings in isolated brains. We find that dopamine application indeed increases cAMP levels and depolarizes the l-LNvs, but, surprisingly, it does not result in increased firing rates. Downregulation of the excitatory D1-like dopamine receptor (Dop1R1) in the l-LNvs and s-LNvs, but not of Dop1R2, abolished the depolarization of l-LNvs in response to dopamine. This indicates that dopamine signals via Dop1R1 to the l-LNvs. Downregulation of Dop1R1 or Dop1R2 in the l-LNvs and s-LNvs does not affect sleep in males. Unexpectedly, we find a moderate decrease of daytime sleep with downregulation of Dop1R1 and of nighttime sleep with downregulation of Dop1R2. Since the l-LNvs do not use Dop1R2 receptors and the s-LNvs also respond to dopamine, we conclude that the s-LNvs are responsible for the observed decrease in nighttime sleep. In summary, dopamine signaling in the wake-promoting LNvs is not required for daytime arousal, but likely promotes nighttime sleep via the s-LNvs.Fil: Fernández, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Hermann Luibl, Christiane. University Of Würzburg; AlemaniaFil: Peteranderl, Alina. University Of Wuerzburg; AlemaniaFil: Reinhard, Nils. University Of Wuerzburg; AlemaniaFil: Senthilan, Pingkalai R.. University Of Wuerzburg; AlemaniaFil: Hieke, Marie. University Of Wuerzburg; AlemaniaFil: Selcho, Mareike. University Of Wuerzburg; AlemaniaFil: Yoshii, Taishi. Okayama University; JapónFil: Shafer, Orie T.. City University of New York; Estados UnidosFil: Muraro, Nara Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Helfrich Förster, Charlotte. University Of Wuerzburg; Alemani

Stability of antibiotics under growth conditions for thermophilic anaerobes.

It was shown that the inhibitory effect of kanamycin and streptomycin in a growing culture of Clostridium thermohydrosulfuricum JW 102 is of limited duration. To screen a large number of antibiotics, their stability during incubation under the growth conditions of thermophilic clostridia was determined at 72 and 50 degrees C by using a 0.2% yeast extract-amended prereduced mineral medium with a pH of 7.3 or 5.0. Half-lives were determined in a modified MIC test with Escherichia coli, Staphylococcus aureus, and Bacillus megaterium as indicator strains. All compounds tested were similar at the two temperatures or more stable at 50 than at 72 degrees C. The half-life (t1/2) at pH 7.3 and 72 degrees C ranged from 3.3 h (k = 7.26 day-1, where k [degradation constant] = 1/t1/2) for ampicillin to no detectable loss of activity for kanamycin, neomycin, and other antibiotics. Apparently some compounds (e.g., lasalocid and neomycin) became more potent during incubation (k greater than 0). A change to pH 5.0 caused some compounds to become more labile (e.g., kanamycin) and others (e.g., streptomycin) to become more stable than at pH 7.3

Solution structure of the antitermination protein NusB of Escherichia coli: a novel all-helical fold for an RNA-binding protein.

The NusB protein of Escherichia coli is involved in the regulation of rRNA biosynthesis by transcriptional antitermination. In cooperation with several other proteins, it binds to a dodecamer motif designated rrn boxA on the nascent rRNA. The antitermination proteins of E.coli are recruited in the replication cycle of bacteriophage lambda, where they play an important role in switching from the lysogenic to the lytic cycle. Multidimensional heteronuclear NMR experiments were performed with recombinant NusB protein labelled with 13C, 15N and 2H. The three-dimensional structure of the protein was solved from 1926 NMR-derived distances and 80 torsion angle restraints. The protein folds into an alpha/alpha-helical topology consisting of six helices; the arginine-rich N-terminus appears to be disordered. Complexation of the protein with an RNA dodecamer equivalent to the rrn boxA site results in chemical shift changes of numerous amide signals. The overall packing of the protein appears to be conserved, but the flexible N-terminus adopts a more rigid structure upon RNA binding, indicating that the N-terminus functions as an arginine-rich RNA-binding motif (ARM)

J. Psychiatr. Res.

profound alterations of the hypothalamic-pituitary-thyroid (HPT) and the hypothalamic-pituitary-adrenal (HPA) systems at the hypophyseal level have been described in affective disorder. To precisely characterize the basal alterations of both axes during sleep. we simultaneously investigated sleep EEG and the secretion of thyrotropin. ACTH and cortisol in nine drug-free male patients with depression in comparison to 10 healthy age and sex matched controls. In depressed patients the nearly diametrical nocturnal secretion of thyrotropin and ACTH was disturbed by significantly blunted thyrotropin values (TSH AUC 51.96+/-5.68 vs. 87.23+/-13.63. P<0.05) and elevated ACTH values (ACTH AUC 1804+/-161 vs. 1538+/-130. P<0.05) compared to controls. Moreover, cross correlation analysis revealed a highly negative association of 0 lag between thyrotropin and ACTH and between thyrotropin and cortisol in the control sample, indicating a physiological nocturnal negative correlation of HPT and HPA system. In the patients sample these associations were weak and reached not statistical significance. Therefore, as a descriptive tool, the ratio TSH/ACTH revealed a significant group difference between controls and patients in the first half of the night (TSH/ACTH AUC 6.50+/-0.42 vs. 3.35+/-0.31. P<0.05). Sleep-EEG analysis showed a shortened REM latency, a decrease of stage 2 and an increase of awake time in the patients. Our data support the hypothesis that both hypophyseal hormones reflect a common dysregulation of both systems in depression probably due to impaired action of TRH-related corticotropin-release- inhibiting-factor (CRIF). The ratio TSH/ACTH might be a tool to characterize alterations of both the HPT and HPA axis in depression during the first half of the night, (C), 2002 Elsevier Science Ltd. All rights reserv