Selective blockade of the discriminative stimulus effects of pentobarbital in pigeons

The ability of CNS stimulants to block the discriminative effects of pentobarbital was studied in pigeons trained to discriminate IM pentobarbital (5 mg/kg) from saline. Pentobarbital, when administered alone, consistently produced greater than 90% pentobarbital-appropriate responding. The concomitant administration of pentobarbital and increasing doses of bemegride or pentylenetetrazol resulted in a dose-related decrease in pentobarbital-appropriate responses. In contrast, picrotoxin, another CNS stimulant, had little or no effect on pentobarbital-appropriate responding produced by pentobarbital.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46426/1/213_2004_Article_BF00432447.pd

The clock genes Period 2 and Cryptochrome 2 differentially balance bone formation

Background: Clock genes and their protein products regulate circadian rhythms in mammals but have also been implicated in various physiological processes, including bone formation. Osteoblasts build new mineralized bone whereas osteoclasts degrade it thereby balancing bone formation. To evaluate the contribution of clock components in this process, we investigated mice mutant in clock genes for a bone volume phenotype. Methodology/Principal Findings: We found that Per2Brdm1 mutant mice as well as mice lacking Cry2-/- displayed significantly increased bone volume at 12 weeks of age, when bone turnover is high. Per2Brdm1 mutant mice showed alterations in parameters specific for osteoblasts whereas mice lacking Cry2-/- displayed changes in osteoclast specific parameters. Interestingly, inactivation of both Per2 and Cry2 genes leads to normal bone volume as observed in wild type animals. Importantly, osteoclast parameters affected due to the lack of Cry2, remained at the level seen in the Cry2-/- mutants despite the simultaneous inactivation of Per2. Conclusions/Significance: This indicates that Cry2 and Per2 affect distinct pathways in the regulation of bone volume with Cry2 influencing mostly the osteoclastic cellular component of bone and Per2 acting on osteoblast parameters

Disturbed Clockwork Resetting in Sharp-1 and Sharp-2 Single and Double Mutant Mice

BACKGROUND: The circadian system provides the basis to anticipate and cope with daily recurrent challenges to maintain the organisms' homeostasis. De-synchronization of circadian feedback oscillators in humans causes 'jet lag', likely contributes to sleep-, psychiatric-, metabolic disorders and even cancer. However, the molecular mechanisms leading to the disintegration of tissue-specific clocks are complex and not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Based on their circadian expression and cell culture experiments, the basic Helix-Loop-Helix (bHLH) transcription factors SHARP-1(Dec2) and SHARP-2(Stra13/Dec1) were proposed as novel negative regulators of the molecular clock. To address their function in vivo, we generated Sharp-1 and Sharp-2 single and double mutant mice. Our experiments reveal critical roles for both factors in regulating period length, tissue-specific control of clock gene expression and entrainment to external cues. Light-pulse experiments and rapid delays of the light-dark cycle (experimental jet lag) unravel complementary functions for SHARP-1 and SHARP-2 in controlling activity phase resetting kinetics. Moreover, we show that SHARP-1 and 2 can serve dual functions as repressors and co-activators of mammalian clock gene expression in a context-specific manner. This correlates with increased amplitudes of Per2 expression in the cortex and liver and a decrease in the suprachiasmatic nucleus (SCN) of double mutant mice. CONCLUSIONS/SIGNIFICANCE: The existence of separate mechanisms regulating phase of entrainment, rhythm amplitude and period length has been postulated before. The differential effects of Sharp-deficiency on rhythmicity and behavioral re-entrainment, coupled to tissue-dependent regulatory functions, provide a new mechanistic basis to further understand the complex process of clock synchronizations

Antagonism of the discriminative effects of ethylketazocine, cyclazocine, and nalorphine in macaques

dl -Ethylketazocine (EKC, 0.01 mg/kg) and saline were established as discriminative stimuli for food-maintained responding in macaque monkeys. Thirty consecutive presses on a right or left lever were reinforced with food, contingent on whether EKC or saline were administered before the session. For tests of antagonism, naltrexone, or UM 979 [( l )-5,9-alpha-dimethyl-2-(3-furylmethyl)-2′-hydroxy-6,7-benzomorphan] was administered concomitantly with EKC, dl -cyclazocine, or nalorphine. Both antagonists blocked completely the EKC discriminative stimulus. The antagonism of the stimulus and rate-altering effects of EKC was surmountable, with considerable intersubject variability in the magnitude of the EKC dose increase required to overcome the blockade. Cyclazocine and nalophine, mixed agonist-antagonist opioids that share stimulus properties with EKC, were also susceptible to antagonism. Naltrexone antagonized completely the EKC stimulus effects of nalorphine; naltrexone and UM 979 antagonized completely the EKC stimulus effects of cyclazocine. Naltrexone antagonism of the cyclazocine stimulus was not surmountable, due to a lack of antagonism of the rate-decreasing effects of high cyclazocine doses.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46433/1/213_2004_Article_BF00555213.pd

Genetic and Molecular Analysis of Wild-Derived Arrhythmic Mice

A new circadian variant was isolated by screening the intercross offspring of wild-caught mice (Mus musculus castaneus). This variant was characterized by an initial maintenance of damped oscillations and subsequent loss of rhythmicity after being transferred from light-dark (LD) cycles to constant darkness (DD). To map the genes responsible for the persistence of rhythmicity (circadian ratio) and the length of free-running period (τ), quantitative trait locus (QTL) analysis was performed using F2 mice obtained from an F1 cross between the circadian variant and C57BL/6J mice. As a result, a significant QTL with a main effect for circadian ratio (Arrhythmicity; Arrh-1) was mapped on Chromosome (Chr) 8. For τ, four significant QTLs, Short free-running period (Sfp-1) (Chr 1), Sfp-2 (Chr 6), Sfp-3 (Chr 8), Sfp-4 (Chr 11) were determined. An epistatic interaction was detected between Chr 3 (Arrh-2) and Chr 5 (Arrh-3). An in situ hybridization study of clock genes and mouse Period1::luciferase (mPer1::luc) real-time monitoring analysis in the suprachiasmatic nucleus (SCN) suggested that arrhythmicity in this variant might not be attributed to core circadian mechanisms in the SCN neurons. Our strategy using wild-derived variant mice may provide a novel opportunity to evaluate circadian and its related disorders in human that arise from the interaction between multiple variant genes

Circadian oscillator proteins across the kingdoms of life : Structural aspects 06 Biological Sciences 0601 Biochemistry and Cell Biology

Circadian oscillators are networks of biochemical feedback loops that generate 24-hour rhythms and control numerous biological processes in a range of organisms. These periodic rhythms are the result of a complex interplay of interactions among clock components. These components are specific to the organism but share molecular mechanisms that are similar across kingdoms. The elucidation of clock mechanisms in different kingdoms has recently started to attain the level of structural interpretation. A full understanding of these molecular processes requires detailed knowledge, not only of the biochemical and biophysical properties of clock proteins and their interactions, but also the three-dimensional structure of clockwork components. Posttranslational modifications (such as phosphorylation) and protein-protein interactions, have become a central focus of recent research, in particular the complex interactions mediated by the phosphorylation of clock proteins and the formation of multimeric protein complexes that regulate clock genes at transcriptional and translational levels. The three-dimensional structures for the cyanobacterial clock components are well understood, and progress is underway to comprehend the mechanistic details. However, structural recognition of the eukaryotic clock has just begun. This review serves as a primer as the clock communities move towards the exciting realm of structural biology

Discriminative stimulus effects of a centrally administered, delta-opioid peptide (d-Pen2-d-Pen5-enkephalin) in pigeons

 The present study assessed the discriminative stimulus effects of the delta-opioid agonist [ d -Pen 2 - d -Pen 5 ]enkephalin (DPDPE) in pigeons. Food-restricted pigeons were trained to discriminate between ICV injections of 100 μg [ d -Pen 2 - d -Pen 5 ]enkephalin (DPDPE) and saline in a two-key operant procedure; acquisition of discriminative control was rapid (14–28 daily sessions). [ d -Ser 2 , Leu 5 , Thr 6 ]enkephalin (DSLET) and [ d -Ala 2 ]deltorphin II, peptides selective for delta-opioid receptors, produced discriminative stimulus effects similar to DPDPE, and were approximately equipotent to DPDPE. The non-peptidic, delta-opioid agonist BW373U86 (0.032–100 mg/kg, IM) partially generalized to DPDPE. The kappa-opioid agonist U69,593 (0.01–1 mg/kg, IM), and the mu-opioid agonists, DAMGO (0.1–3.2 μg, ICV) and morphine (1–10 mg/kg, IM), did not produce discriminative stimulus effects similar to DPDPE, up to doses that markedly decreased response rates. Naltrindole (0.1 mg/kg, IM), an antagonist selective for delta-opioid receptors, produced approximately a 30-fold reduction in the potency of DPDPE. DPDPE’s discriminative stimulus effect in pigeons appears to be mediated through a delta-opioid receptor; this effect may provide a procedure for assessing delta-opioid receptor function in vivo.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41956/1/213-127-3-225_61270225.pd