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The circadian clock controls daily oscillations of gene expression at the cellular level. We report the development of a high-throughput circadian functional assay system that consists of luminescent reporter cells, screening automation, and a data analysis pipeline. We applied this system to further dissect the molecular mechanisms underlying the mammalian circadian clock using a chemical biology approach. We analyzed the effect of 1,280 pharmacologically active compounds with diverse structures on the circadian period length that is indicative of the core clock mechanism. Our screening paradigm identified many compounds previously known to change the circadian period or phase, demonstrating the validity of the assay system. Furthermore, we found that small molecule inhibitors of glycogen synthase kinase 3 (GSK-3) consistently caused a strong short period phenotype in contrast to the well-known period lengthening by lithium, another presumed GSK-3 inhibitor, siRNA-mediated knockdown of GSK-3[beta] also caused a short period, confirming the phenotype obtained with the small molecule inhibitors. These results clarify the role of GSK-3[beta] in the period regulation of the mammalian clockworks and highlight the effectiveness of chemical biology in exploring unidentified mechanisms of the circadian clock. screening | small molecule library | kinase

Although it is accepted that extracellular fields generated by neuronal activity can influence the excitability of neighboring cells, whether this form of neurotransmission has a functional role remains open. In vivo field effects occur in the teleost Mauthner (M)-cell system, where a combination of structural features support the concept of inhibitory electrical synapses. A single spike in one M-cell evoked within as little as 2.2 ms of the onset of an abrupt sound, simulating a predatory strike, initiates a startle-escape behavior [Zottoli SJ (1977)J Exp Biol 66:243-254]. We show that such sounds produce synchronized action potentials in as many as 20 or more interneurons that mediate feed-forward electrical inhibition of the M-cell. The resulting action currents produce an electrical inhibition that coincides with the electrotonic excitatory drive to the M-cell; the amplitude of the peak of the inhibition is [approximately equal to] 40% of that of the excitation. When electrical inhibition is neutralized with an extracellular cathodal current pulse, subthreshold auditory stimuli are converted into ones that produce an M-spike. Because the timing of electrical inhibition is often the same as the latency of M-cell firing in freely swimming fish, we conclude that electrical inhibition participates in regulating the threshold of the acoustic startle-escape behavior. Therefore, a field effect is likely to be essential to the normal functioning of the neural network. acoustic startle | C-start | ephapse | field effect

Hemin, an oxidized form of heme, is an essential regulator of gene expression and cell cycle progression. Our laboratory previously reported (34) that chronic heroin treatment of spontaneously hypertensive rats reversed the eutrophic inward remodeling of small peripheral arteries. Whether long-term treatment of cultured vascular smooth muscle cells (VSMCs) with hemin alters the proliferation status of these cells has been unknown. In the present study, hemin treatment at 5 [micro]M for 4, 7, 14, and 21 days significantly inhibited the proliferation of cultured rat aortic VSMCs (A-10 cells) by arresting cells at [G.sub.0]/[G.sub.1] phases so as to decelerate cell cycle progression. Heme oxygenase (HO) activity and inducible HO-1 protein expression were significantly increased by hemin treatment. HO inhibitor tin protoporphyrin IX (SnPP) abolished the effects of hemin on cell proliferation and HO activity. Interestingly, heroin-induced HO-1 expression was further increased in the presence of SnPP. Heroin treatment had no significant effect on the expression of constitutive HO-2. Expression of p21 protein and the level of reactive oxygen species (ROS) were decreased by hemin treatment, which was reversed by application of SnPP. After removal of heroin from culture medium, inhibited cell proliferation and increased HO-1 expression in VSMCs were returned to control level within 1 wk. Transfection with HO-1 small interfering RNA significantly knocked down HO-1 expression and decreased HO activity, but had no effect on HO-2 expression, in cells treated with or without heroin for 7 days. The inhibitory effect of heroin on cell proliferation was abolished in HO-1 silenced cells. It is concluded that induction of HO-1 and, consequently, increased HO activity are responsible for the chronic inhibitory effect of hemin on VSMC proliferation. Changes in the levels of p21 and ROS might also participate in the cellular effects of hemin. vascular smooth muscle cells; heme oxygenase

Observers (N=3, all males, M age=30.7 yr.) were presented pairs of visual targets and instructed to fixate one as quickly as possible. The resulting saccades were of two types; bistable saccades accuratdy acquired one of the targets whereas averaging saccades landed at intermediate locations. The hypothesis was that averaging saccades result from a pooling of afferent activity prior to selection of saccadic direction and amplitude (i.e., coactive parallel processing) and that bistable saccades would show evidence of response competition between incompatible motor programs within a parallel architecture. Analysis of the distributions of saccadic reaction times showed that in all cases saccadic responses to double targets were too slow to be consistent with either the channel summation or any form of race model. Results indicate that inhibitory interactions operate during the selection of a target for a saccadic eye movement.

Ishii H, Niioka T, Watanabe H, Izumi H. Inhibitory effects of excess sympathetic activity on parasympathetic vasodilation in the rat masseter muscle. Am J Physiol Regul Integr Comp Physiol 293: R729-R736, 2007. First published May 30, 2007; doi: 10.1152/ajpregu.00866.2006.--The present study was designed to examine the effect of sympathetic tonic activity on parasympathetic vasodilation evoked by the trigeminal-mediated reflex in the masseter muscle in urethane-anesthetized rats. Sectioning of the superior cervical sympathetic trunk (CST) ipsilaterally increased the basal level of blood flow in the masseter muscle (MBF). Electrical stimulation of the peripheral cut end of the CST for 2 min using 2-ms pulses ipsilaterally decreased in a dependent manner the intensity (0.5-10 V) and frequency (0.1-5 Hz) of the MBF. The CST stimulation for 2 min at superior cervical sympathetic trunk; vasoconstrictor fiber; adrenoceptors; trigeminal-mediated reflex; jaw muscle

Physiological rhythmic activity in cortical circuits relies on GABAergic inhibition to balance excitation and control spike timing. With a focus on recent experimental progress in the hippocampus, here we review the mechanisms by which synaptic inhibition can control the precise timing of spike generation, by way of effects of GABAergic events on membrane conductance ('shunting' inhibition) and membrane potential ('hyperpolarizing' inhibition). Synaptic inhibition itself can be synchronized by way of interactions within networks of GABAergic neurons, and by excitatory neurons. The importance of GABAergic mechanisms for generation of cortical rhythms is now well established. What remains to be resolved is how such inhibitory control of spike timing can be harnessed for long-range fast synchronization, and the relevance of these mechanisms to network function. This review is part of the INMED/TINS special issue Physiogenic and pathogenic oscillations: the beauty and the beast, based on presentations at the annual INMED/TINS symposium (http://inmednet.com).