To study the function of the neuropeptide pigment-dispersing factor (PDF) in the circadian system of Drosophila, we misexpressed the pdf gene from the grasshopper Romalea in the CNS of Drosophila and investigated the effect of this on behavioral rhythmicity. pdf was either ectopically expressed in different numbers of neurons in the brain or the thoracical nervous system or overexpressed in the pacemaker neurons alone. We found severe alterations in the activity and eclosion rhythm of several but not all lines with ectopic pdf expression. Only ectopic pdf expression in neurons that projected into the dorsal central brain severely influenced activity rhythms. Therefore, we conclude that PDF acts as a neuromodulator in the dorsal central brain that is involved in the rhythmic control of behavior. Overexpression of pdf in the pacemaker neurons alone or in the other neurons that express the clock genes period (per) and timeless (tim) did not disturb the activity rhythm. Such flies still showed a rhythm in PDF accumulation in the central brain terminals. This rhythm was absent in the terminals of neurons that expressed PDF ectopically. Probably, PDF is rhythmically processed, transported, or secreted in neurons expressing per and tim, and additional PDF expression in these cells does not influence this rhythmic process. In neurons lacking per and tim, PDF appears to be continuously processed, leading to a constant PDF secretion at their nerve terminals. This may lead to conflicting signals in the rhythmic output pathway and result in a severely altered rhythmic behavior.

Despite its pharmacological relevance, the mechanism of the development of tolerance to the action of benzodiazepines is essentially unknown. The acute sedative action of diazepam is mediated via ␣ 1 -GABA A receptors. Therefore, we tested whether chronic activation of these receptors by diazepam is sufficient to induce tolerance to its sedative action. Knock-in mice, in which the ␣ 1 -, ␣ 2 -, ␣ 3 -, or ␣ 5 -GABA A receptors had been rendered insensitive to diazepam by histidine-arginine point mutation, were chronically treated with diazepam (8 d;) and tested for motor activity. Wild-type, ␣ 2 (H101R), and ␣ 3 (H126R) mice showed a robust diminution of the motor-depressant drug action. In contrast, ␣ 5 (H105R) mice failed to display any sedative tolerance. ␣ 1 (H101R) mice showed no alteration of motor activity with chronic diazepam treatment. Autoradiography with [ 3 H]flumazenil revealed no change in benzodiazepine binding sites. However, a decrease in ␣ 5 -subunit radioligand binding was detected selectively in the dentate gyrus with specific ligands. This alteration was observed only in diazepam-tolerant animals, indicating that the manifestation of tolerance to the sedative action of diazepam is associated with a downregulation of ␣ 5 -GABA A receptors in the dentate gyrus. Thus, the chronic activation of ␣ 5 -GABA A receptors is crucial for the normal development of sedative tolerance to diazepam, which manifests itself in conjunction with ␣ 1 -GABA A receptors.

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Classical benzodiazepines such as diazepam are widely used tranquillisers and hypnotics in various neuropsychiatric diseases including alcohol-related disorders. One of the major drawbacks of benzodiazepine therapy, however, is an exacerbation of the sedative and hypnotic effects associated with alcohol intake, even at low doses. Even though the gamma-aminobutyric acid (GABA)A receptor complex is a common target for the actions of both classes of drugs, the molecular mechanisms underlying the enhanced pharmacological properties of the combined use of benzodiazepines and alcohol remain to be identified. The present experiments aimed at clarifying which of the GABAA receptor subtypes mediate the augmented hypnotic-like and sedative effects of combined diazepam and alcohol using the righting reflex and motor activity assays, respectively, in histidine-to-arginine point mutated mice that possess diazepam-insensitive alpha1-, alpha2-, alpha3- or alpha5-GABAA receptors. The combination of diazepam (2 or 3 mg/kg) and ethanol (3 g/kg) induced loss of righting reflex with a significantly dose-dependent increase of the latency to its full recovery in wild-type, alpha1(H101R), alpha3(H126R) and alpha5(H105R) but not in alpha2(H101R) mice. A combined treatment with diazepam (1 mg/kg) and ethanol (2.5 g/kg) precipitated motor inhibition similarly in wild-type and alpha2(H101R) mice. Responsiveness of the alpha2(H101R) mice to ethanol alone was similar to that of wild-type mice. These results demonstrate that induction of loss of righting reflex by combined diazepam and alcohol is closely dependent on the activation of the alpha2-GABAA receptors by the benzodiazepine whereas precipitation of sedation involves GABAA receptors other than the alpha2-GABAA receptors.

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