In multicellular organisms, circadian oscillators are organized into multitissue systems which function as biological clocks that regulate the activities of the organism in relation to environmental cycles and provide an internal temporal framework. To investigate the organization of a mammalian circadian system, we constructed a transgenic rat line in which luciferase is rhythmically expressed under the control of the mouse Per1 promoter. Light emission from cultured suprachiasmatic nuclei (SCN) of these rats was invariably and robustly rhythmic and persisted for up to 32 days in vitro. Liver, lung, and skeletal muscle also expressed circadian rhythms, which damped after two to seven cycles in vitro. In response to advances and delays of the environmental light cycle, the circadian rhythm of light emission from the SCN shifted more rapidly than did the rhythm of locomotor behavior or the rhythms in peripheral tissues. We hypothesize that a self-sustained circadian pacemaker in the SCN entrains circadian oscillators in the periphery to maintain adaptive phase control, which is temporarily lost following large, abrupt shifts in the environmental light cycle.

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus has been referred to as the master circadian pacemaker that drives daily rhythms in behavior and physiology. There is, however, evidence for extra-SCN circadian oscillators. Neural tissues cultured from rats carrying the Per-luciferase transgene were used to monitor the intrinsic Per1 expression patterns in different brain areas and their response to changes in the light cycle. Although many Per-expressing brain areas were arrhythmic in culture, 14 of the 27 areas examined were rhythmic. The pineal and pituitary glands both expressed rhythms that persisted for >3 d in vitro, with peak expression during the subjective night. Nuclei in the olfactory bulb and the ventral hypothalamus expressed rhythmicity with peak expression at night, whereas other brain areas were either weakly rhythmic and peaked at night, or arrhythmic. After a 6 hr advance or delay in the light cycle, the pineal, paraventricular nucleus of the hypothalamus, and arcuate nucleus each adjusted the phase of their rhythmicity with different kinetics. Together, these results indicate that the brain contains multiple, damped circadian oscillators outside the SCN. The phasing of these oscillators to one another may play a critical role in coordinating brain activity and its adjustment to changes in the light cycle.

A conserved transcription-translation negative feedback loop forms the molecular basis of the circadian oscillator in animals. Molecular interactions within this loop have been relatively well characterized in vitro and in cell culture; however, in vivo approaches are required to assess the functional significance of these interactions. Here, regulation of circadian gene expression was studied in vivo by using transgenic reporter mouse lines in which 6.75 kb of the mouse Period1 (mPer1) promoter drives luciferase (luc) expression. Six mPer1-luc transgenic lines were created, and all lines express a daily rhythm of luc mRNA in the suprachiasmatic nuclei (SCN). Each mPer1-luc line also sustains a long-term circadian rhythm of luminescence in SCN slice culture. A 6-h light pulse administered during the early subjective night rapidly induces luc mRNA expression in the SCN; however, high luc mRNA levels are sustained, whereas endogenous mPer1 mRNA levels return to baseline, suggesting that posttranscriptional events mediate the down-regulation of mPer1 after exposure to light. This approach demonstrates that the 6.75-kb mPer1 promoter fragment is sufficient to confer both circadian and photic regulation in vivo and reveals a potential posttranscriptional regulatory mechanism within the mammalian circadian oscillator. N early all organisms express circadian (Ϸ24-h) rhythms in behavior, physiology, and cellular activity. In mice, Drosophila, Neurospora, and cyanobacteria, extensive studies indicate that the basic molecular circadian mechanism consists of a transcription-translation feedback loop (1, 2). Recent reviews describe the mammalian model in detail (3, 4); briefly, the transcription factors CLOCK and BMAL1 (also known as MOP3) activate transcription of the mouse Period (mPer1 and mPer2) and Cryptochrome (mCry1 and mCry2) genes. The PER and CRY proteins accumulate and translocate into the nucleus where they inhibit the activity of CLOCK and BMAL1. The turnover of the inhibitory PER and CRY proteins then leads to a new cycle of activation by CLOCK and BMAL1.Several genes in this transcriptional pathway exhibit circadian rhythms of expression, but they differ in characteristics of rhythmic expression such as circadian phase and response to light (3). For example, peak mRNA expression of mPer1, mPer2, and mCry1 occurs at different times in the suprachiasmatic nucleus (SCN), the site of the circadian pacemaker in mammals (5-7). However, the protein products of these three genes accumulate in SCN neurons around the same phase (8-10). In addition, a light pulse administered during the early subjective night leads to the rapid induction of mPer1, slower induction of mPer2, and no induction of mCry1 (11)(12)(13)(14). Clearly, regulated circadian gene expression remains an important component of the circadian mechanism.Transcriptional regulation of circadian promoter activity has been addressed initially in cell culture. In cell transfection͞ luciferase reporter assays, the CLOCK and BMAL1 proteins dimerize and bind thre...

Lithium treatment lengthens the period of circadian rhythms in most organisms. In the present study, we tested whether lithium acts directly on the mammalian suprachiasmatic nucleus (SCN) to lengthen rhythms of individual neurons. Lithium increased the circadian period of firing rate rhythms of cultured SCN neurons in a concentration-dependent manner. Lithium had no effect on the amplitude of these rhythms, but did affect the period of some cells more than others. The results indicate that lithium acts directly on the SCN to lengthen the free-running period of individual neurons.

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Glucocorticoids are involved in psychostimulant-induced hyperactivity, but the exact mechanism is not known. This study used the selective glucocorticoid receptor antagonist, RU-43044, to determine whether prefrontal neurotransmission is involved in glucocorticoid-mediated modulation of methamphetamine (METH)-induced hyperactivity in mice. Pretreatment with RU-43044 (10-30 mg/kg) attenuated the increased spontaneous locomotor activity induced by METH (1-2 mg/kg). The psychostimulant effect of METH was also attenuated by adrenalectomy. RU-43044 inhibited METH-induced increases in extracellular dopamine (DA), but not serotonin (5-HT), levels in the prefrontal cortex, but did not affect METH-induced increases in extracellular DA levels in the nucleus accumbens shell, although it inhibited increases in extracellular 5-HT levels. Adrenalectomy also attenuated the METH-induced increases in extracellular DA levels in the prefrontal cortex. RU-43044 did not affect METH-induced increases in plasma corticosterone levels. These findings suggest that glucocorticoid receptors are involved in METH-induced hyperactivity, and that prefrontal dopaminergic neurotransmission plays a role in glucocorticoid-mediated modulation of METH-induced behavioral changes.

Behavioral effects of 5-[3-[((2S)-1,4-benzodioxan-2-ylmethyl)amino]propoxy]-1,3-be nzodioxole HCl (MKC-242), a novel 5-HT1A-receptor agonist, were evaluated using animal models of anxiety and obsessive compulsive disorder and compared against reference compounds. MKC-242 suppressed foot shock-induced fighting behavior without loss of motor coordination in mice as the reference compounds did. The ED50 values of MKC-242, buspirone, tandospirone and diazepam were 1.7, 42, 80 and 2.0 mg/kg, p.o., respectively. The duration of the suppression of fighting by MKC-242 was longer than those of buspirone and tandospirone and comparable to that of diazepam. Similar results were also obtained with the water-lick conflict test in rats. The plasma concentration of MKC-242 in rats was much higher than the reported value of buspirone during 0.25-6 hr after oral administration. In addition, MKC-242 reduced marble burying behavior without reduction of motor activity. Fluoxetine, tandospirone and diazepam also reduced the behavior at non-sedative doses. These findings indicate that MKC-242 possesses a longer-lasting anxiolytic effect than azapirones. This might be due to the high concentration of the compound in plasma. In addition, it is also suggested that MKC-242 possesses an antiobsessional effect.