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D R Weaver

Publications and source records attributed to D R Weaver.

At least 19 recordsLinked to original sources
PubMed1999

Photic induction of Period gene expression is reduced in Clock mutant mice.

The Clock mutation leads to abnormal circadian behavior and defective transcriptional activity of CLOCK, a basic helix-loop-helix (bHLH)/PAS protein. In situ hybridization was used to assess whether the Clock mutation affects the photic induction of mPer1, mPer2, and c-fos in the mouse suprachiasmatic nucleus (SCN). Exposure of wild-type mice to a 15 min light pulse at night rapidly induced expression of c-fos mRNA, with mPer1 and mPer2 mRNAs peaking later. Light exposure also increased c-fos, mPer1 and mPer2 mRNA levels in the SCN of homozygous Clock mutant mice, but the amplitude of the response to light was significantly reduced. Clock appears to play a role in circadian photoreception that is distinct from its role in the circadian oscillatory mechanism.

Animals↗
PubMed1999

A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock.

We examined the transcriptional regulation of the clock-controlled arginine vasopressin gene in the suprachiasmatic nuclei (SCN). A core clock mechanism in mouse SCN appears to involve a transcriptional feedback loop in which CLOCK and BMAL1 are positive regulators and three mPeriod (mPer) genes are involved in negative feedback. We show that the RNA rhythm of each mPer gene is severely blunted in Clock/Clock mice. The vasopressin RNA rhythm is abolished in the SCN of Clock/Clock animals, leading to markedly decreased peptide levels. Luciferase reporter gene assays show that CLOCK-BMAL1 heterodimers act through an E box enhancer in the vasopressin gene to activate transcription; this activation can be inhibited by the mPER and mTIM proteins. These data indicate that the transcriptional machinery of the core clockwork directly regulates a clock-controlled output rhythm.

3T3 Cells↗
PubMed1999

Expression of basic helix-loop-helix/PAS genes in the mouse suprachiasmatic nucleus.

The suprachiasmatic nuclei contain a circadian clock that drives rhythmicity in physiology and behavior. In mice, mutation of the Clock gene produces abnormal circadian behavior [Vitaterna M. H. et al. (1994) Science 264, 715-725]. The Clock gene encodes a protein containing basic helix-loop-helix and PAS (PER-ARNT-SIM) domains [King D. P. et al. (1997) Cell 89, 641-653]. The PAS domain may be an important structural feature of a subset of genes involved in photoreception and circadian rhythmicity. The expression and regulation of messenger RNAs encoding eight members of the basic helix-loop-helix/PAS protein superfamily were examined by in situ hybridization. Six of the genes studied (aryl hydrocarbon receptor nuclear transporter, aryl hydrocarbon receptor nuclear transporter-2, Clock, endothelial PAS-containing protein, hypoxia-inducible factor-1alpha and steroid receptor coactivator-1) were expressed in the suprachiasmatic nucleus of adult and neonatal mice. No evidence for rhythmicity of expression was observed when comparing brains collected early in the subjective day (circadian time 3) with those collected early in subjective night (circadian time 15). Neuronal PAS-containing protein-1 messenger RNA was expressed in the suprachiasmatic nucleus of adult (but not neonatal) mice, and a low-amplitude rhythm of neuronal PAS-containing protein-1 gene expression was detected in the suprachiasmatic nucleus. Neuronal PAS-containing protein-2 messenger RNA was not detected in adult or neonatal suprachiasmatic nucleus. Exposure to light at night (30 or 180 min of light, beginning at circadian time 15) did not alter the expression of any of the genes studied. The expression of multiple members of the basic helix-loop-helix/PAS family in the suprachiasmatic nucleus suggests a rich array of potential interactions relevant to the regulation of the suprachiasmatic circadian clock.

Aging↗
PubMed1998

Three period homologs in mammals: differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain.

We have cloned and characterized the mouse cDNA of a third mammalian homolog of the Drosophila period gene and designated it mPer3. The mPER3 protein shows approximately 37% amino acid identity with mPER1 and mPER2 proteins. The three mammalian PER proteins share several regions of sequence homology, and each contains a protein dimerization PAS domain. mPer3 RNA levels oscillate in the suprachiasmatic nuclei (SCN) and eyes. In the SCN, mPer3 RNA levels are not acutely altered by light exposure at different times during subjective night. This contrasts with the acute induction by light of mPer1 and mPer2 RNA levels during early and late subjective night. mPer3 is widely expressed in tissues outside of brain. In liver, skeletal muscle, and testis, mPer RNAs exhibit prominent, synchronous circadian oscillations. The results highlight the differential light responses among the three mammalian Per genes in the SCN and raise the possibility of circadian oscillators in mammals outside of brain and retina.

Animals↗
PubMed1998

Molecular analysis of mammalian timeless.

We cloned the mouse cDNA of a mammalian homolog of the Drosophila timeless (tim) gene and designated it mTim. The mTim protein shows five homologous regions with Drosophila TIM. mTim is weakly expressed in the suprachiasmatic nuclei (SCN) but exhibits robust expression in the hypophyseal pars tuberalis (PT). mTim RNA levels do not oscillate in the SCN nor are they acutely altered by light exposure during subjective night. mTim RNA is expressed at low levels in several peripheral tissues, including eyes, and is heavily expressed in spleen and testis. Yeast two-hybrid assays revealed an array of interactions between the various mPER proteins but no mPER-mTIM interactions. The data suggest that PER-PER interactions have replaced the function of PER-TIM dimers in the molecular workings of the mammalian circadian clock.

Amino Acid Sequence↗
PubMed1998

In-phantom neutron fluence measurements in the orthogonal Birmingham boron neutron capture therapy beam.

This paper presents the results of an experimental investigation into the performance of the Birmingham accelerator-based epithermal BNCT beam. In-phantom gold foil activation and boron trifluoride tube measurements have been used. The results have been compared with calculated response rates using Monte Carlo modeling of the entire neutron system from source to phantom and detector. The excellent agreement obtained gives us confidence in the validity of the simulations and our ability to predict accurately the neutronic performance of our BNCT facility.

Biophysical Phenomena↗
PubMed1998

The suprachiasmatic nucleus: a 25-year retrospective.

The suprachiasmatic nuclei (SCN) of the anterior hypothalamus contain the master circadian pacemaker in mammals. On the occasion of the 25th anniversary of the discovery of the SCN as the circadian clock, Charles A. Czeisler and Steven M. Reppert organized a meeting to review milestones and recent developments in the study of the SCN. The discovery that the SCN contain tissue necessary for generation of circadian rhythmicity was established by lesion studies published in 1972. The second phase of study demonstrated unequivocally that the SCN contain an autonomous circadian pacemaker. The principal studies in this period showed the presence of metabolic and electrical activity rhythms in the SCN in vivo and progressed to studies showing that the SCN maintain rhythmicity in vitro, demonstrating that the transplanted SCN can restore circadian function following destruction of the host SCN and ultimately showing that single SCN "clock cells" exhibit independent rhythms in firing rate. The third phase of study, aimed at identifying the biochemical and molecular mechanisms responsible for rhythmicity within the SCN, has begun with the identification of circadian mutants (tau mutant hamsters and Clock mutant mice) and the isolation of the Clock gene. This report traces the important steps forward in our understanding of the suprachiasmatic circadian clock by recounting the information presented at the SCN Silver Anniversary Celebration.

Animals↗
PubMed1997

Cellular construction of a circadian clock: period determination in the suprachiasmatic nuclei.

The circadian clock in the suprachiasmatic nuclei is composed of multiple, single-cell circadian oscillators (clock cells). We now test the hypothesis that the circadian period in behavior is determined by the mean period that arises from the coupling of clock cells with diverse circadian periods. For these studies, we monitored firing rate rhythms of individual suprachiasmatic nuclei neurons on fixed multielectrode plates and exploited the altered circadian periods expressed by heterozygous and homozygous tau mutant hamsters. The results show that circadian period in the whole animal is determined by averaging widely dispersed periods of individual clock cells. The data also demonstrate that the tau mutation affects circadian function in a cell-autonomous manner.

Animals↗
PubMed1997

Widespread expression of functional D1-dopamine receptors in fetal rat brain.

Maternal treatment with cocaine or the D1-dopamine receptor agonist, SKF 38393, induces expression of the immediate-early gene, c-fos, in fetal rodent brain. Our previous studies have focused on the suprachiasmatic nucleus late in gestation. In the present report, we examined the anatomical distribution of functional D1-dopamine receptors throughout fetal rat brain. Functional D1 receptors were defined using three complementary methods: in situ hybridization to detect D1 receptor mRNA, autoradiographic detection of 125I-SCH 23982 binding, and in situ hybridization to detect c-fos gene expression induced by maternal treatment with SKF 38393. D1-dopamine receptor binding, receptor mRNA, and SKF 38393-induced c-fos gene expression are widespread in fetal brain by late gestation. These data indicate that the fetal brain is sensitive to dopamine receptor activation, and suggest that gestational exposure to drugs of abuse acting via dopaminergic mechanisms may influence fetal brain function.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1997

[125I]4-aminobenzyl-5'-N-methylcarboxamidoadenosine (125I)AB-MECA) labels multiple adenosine receptor subtypes in rat brain.

Adenosine modulates neuronal activity and neurotransmitter release through interaction with cell surface receptors. Four adenosine receptor subtypes, A1, A2A, A2B, and A3 receptors, have been cloned and characterized. The agonist ligand, [125I]AB-MECA ([125I]4-aminobenzyl-5'N-methylcarboxamidoadenosine) has high affinity for recombinant A1 and A3 receptors [Olah et al., Mol. Pharmacol, 45 (1994) 978-982]. Rodent A3 receptors are relatively insensitive to xanthines; inhibition of A1 receptors with xanthines allows selective detection of A3 receptors despite the lack of selectivity of the ligand. We studied whether [125I]AB-MECA is useful for localization and characterization of A3 receptors in rat brain. The autoradiographic distribution of total [125I]AB-MECA (400 pM) binding closely resembled the pattern of A1 receptor binding, with highest levels in cerebellum, hippocampus, and thalamus, and moderate levels in cortex and striatum. Drug competition studies confirmed that almost all [125I]AB-MECA binding could be attributed to labeling of A1 receptors. Xanthine amine congener (1 microM) reduced specific [125I]AB-MECA binding by > 95%, indicating that xanthine-resistant A3 receptors represent a quantitatively minor subtype. Despite the use of a radioligand with high affinity and high specific activity, the low density of A3 receptors in rat brain appears insufficient to allow localization, or even consistent detection, of this receptor subtype. In the presence of DPCPX (50 nM, to block A1 receptors), residual [125I]AB-MECA binding to A2A receptors was observed in the striatum. Thus [125I]AB-MECA labels primarily A1 and A2A adenosine receptors in rat brain.

Adenosine↗
PubMed1997

Haloperidol regulates neurotensin gene expression in striatum of c-fos-deficient mice.

The immediate-early gene c-fos has been proposed to play a role in induction of neurotensin/neuromedin N (NT/N) gene expression in the striatum following acute haloperidol (HAL) treatment. We utilized mice with targeted disruption of the c-fos gene to directly test this hypothesis. A robust increase in NT/N gene expression was observed in the dorsolateral striatum (DLSt) in both wild-type (WT) and c-fos-deficient mice 4-6 h after a single injection of HAL (1 or 4 mg/kg) indicating that products of the c-fos gene are not absolutely required for induction of NT/N mRNA. The basal expression of preprotachykinin, preproenkephalin and preprocholecystokinin mRNAs did not differ between WT and c-fos knockout mice. HAL treatment first increased striatal NT/N mRNA on postnatal day (PD) 10. HAL-induced NT/N mRNA levels were significantly lower in c-fos knockout mice than in WT mice on PD 10 and 15. These findings indicate that reliance on c-fos may be greater earlier in development and that redundant molecular pathways can lead to induction of NT/N mRNA in mouse striatum.

Animals↗
PubMed1997

Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock.

The pineal hormone melatonin elicits two effects on the suprachiasmatic nuclei (SCN): acute neuronal inhibition and phase-shifting. Melatonin evokes its biological effects through G protein-coupled receptors. Since the Mel1a melatonin receptor may transduce the major neurobiological actions of melatonin in mammals, we examined whether it mediates both melatonin effects on SCN function by using mice with targeted disruption of the Mel1a receptor. The Mel1a receptor accounts for all detectable, high affinity melatonin binding in mouse brain. Functionally, this receptor is necessary for the acute inhibitory action of melatonin on the SCN. Melatonin-induced phase shifts, however, are only modestly altered in the receptor-deficient mice; pertussis toxin still blocks melatonin-induced phase shifts in Mel1a receptor-deficient mice. The other melatonin receptor subtype, the Mel1b receptor, is expressed in mouse SCN, implicating it in the phase-shifting response. The results provide a molecular basis for two distinct, mechanistically separable effects of melatonin on SCN physiology.

Animals↗
PubMed1997

Two period homologs: circadian expression and photic regulation in the suprachiasmatic nuclei.

We have characterized a mammalian homolog of the Drosophila period gene and designated it Per2. The PER2 protein shows >40% amino acid identity to the protein of another mammalian per homolog (designated Per1) that was recently cloned and characterized. Both PER1 and PER2 proteins share several regions of homology with the Drosophila PER protein, including the protein dimerization PAS domain. Phylogenetic analysis supports the existence of a family of mammalian per genes. In the mouse, Per1 and Per2 RNA levels exhibit circadian rhythms in the SCN and eyes, sites of circadian clocks. Both Per1 and Per2 RNAs in the SCN are increased by light exposure during subjective night but not during subjective day. The results advance our knowledge of candidate clock elements in mammals.

Amino Acid Sequence↗
PubMed1997

Reproductive safety of melatonin: a "wonder drug" to wonder about.

By some accounts, melatonin is the wonder drug of the 1990s. This previously obscure hormone came to the public's full attention as the result of a series of popular books claiming therapeutic benefits of melatonin ingestion. Some of these claims deserve serious consideration and investigation, whereas others appear unfounded. Without waiting for the outcome of the ongoing scientific debate, however, melatonin set astounding sales records. The hormone is now ingested on a daily basis by many thousands of people. There is little information on the potential adverse effects of melatonin ingestion in humans. Melatonin, its analogs, and its metabolites are not mutagenic, and melatonin possesses remarkably low acute toxicity in animals and humans. It is more difficult to exclude toxic effects of long-term melatonin treatment. The fact that melatonin is normally secreted each night does not ensure that exogenous melatonin, taken at other times and/or in supraphysiological doses, will not have adverse effects. Despite the well-recognized role of melatonin in the regulation of reproduction in photoperiodic species, it seems unlikely that chronic ingestion of moderate melatonin doses will have a profound impact on reproductive function in humans. Evidence that melatonin modulates steroid hormone action in some steroid-responsive tissues suggests that these tissues should be carefully examined when attempting to assess whether melatonin has chronic toxicity in humans. In the absence of sufficient information regarding the longterm safety of exogenous melatonin, the conservative course of action is to restrict melatonin use to those therapeutic applications in which a significant benefit is expected. The decision to ingest melatonin should be preceded by careful consideration of the expected benefits as well as the potential costs of treatment, with recognition of the fact that there has been exaggeration of the benefits and little attention paid to the potential costs in most discussions of this issue to date.

Animals↗
PubMed1996

The Mel1a melatonin receptor gene is expressed in human suprachiasmatic nuclei.

The pineal hormone melatonin influences circadian rhythmicity in many vertebrate species. The circadian effects of melatonin in humans have led to its use to treat jet lag and circadian-based sleep disorders. Melatonin is thought to influence circadian rhythmicity by acting in the suprachiasmatic nuclei (SCN). The recent cloning of two melatonin receptor subtypes with high affinity for melatonin allows molecular analysis of melatonin receptors in human SCN. We report that Mel1a receptor mRNA is detectable in neonatal human SCN by in situ hybridization. Mel1b and melatonin-related receptor mRNAs were not detected. The presence of Mel1a receptor mRNA in human SCN supports the hypothesis that the Mel1a receptor is responsible for the circadian effects of melatonin in humans.

Adult↗
PubMed1996

A1-adenosine receptor gene expression in fetal rat brain.

Adenosine influences neurotransmitter release, neuronal excitability, and firing rate, through A1-adenosine receptors (A1-R). Caffeine and related methylxanthines are adenosine receptor antagonists. Exposure of developing rodents to caffeine is associated with subtle, long-term changes in neurochemistry and behavior. The developmental appearance of A1-R gene expression was examined in rats by in situ hybridization. On gestational day (GD) 10, A1-R mRNA was expressed at very high levels in placental mesometrium. Expression of A1-R mRNA in brain was first detected on GD 14. Hybridization was restricted to portions of neuroepithelium, caudate-putamen, piriform cortex, hypoglossal nucleus, and ventral horn of spinal cord. Neuroepithelial A1-R mRNA increased in intensity and distribution at subsequent ages, reaching a maximum on GD 20 (the latest age studied). Hybridization signal was detected, with regional variation in intensity, throughout much of the brain by GD 16, with additional increases in extent and intensity through GD 20. Generally, a caudal > rostral gradient of hybridization intensity was apparent. The distribution on GD 20 resembled the widespread yet heterogeneous pattern observed in the adult, with high levels of A1-R gene expression in cortex, hippocampus, thalamus, cerebellum, pontine nuclei, brainstem motor nuclei, and spinal cord. Northern blot analysis confirmed the age-related increase in abundance of A1-R transcripts (ca. 3.5 and 5.5 kb). The early and widespread expression of A1-R mRNA, coupled with previous reports of prenatal A1-R binding, suggests that adenosine and adenosine antagonists, including caffeine, may influence neuronal differentiation, migration or synaptogenesis, thus producing long-lasting effects on brain and behavior.

Animals↗
PubMed1996

Cloning of a melatonin-related receptor from human pituitary.

We have cloned an orphan G protein-coupled receptor from a human pituitary cDNA library using a probe generated by PCR. The cDNA, designated H9, encodes a protein of 613 amino acids that is 45% identical at the amino acid level to the recently cloned human Mel(1a) and Mel(1b) melatonin receptors. Structural analyses of the encoded protein and its gene, along with phylogenetic analysis, further show that H9 is closely related to the G protein-coupled melatonin receptor family. Unusual features of the protein encoded by H9 include a lack of N-linked glycosylation sites and a carboxyl tail >300 amino acids long. H9 transiently expressed in COS-1 cells did not bind [125I]melatonin or [3H]melatonin. H9 mRNA is expressed in hypothalamus and pituitary, suggesting that the encoded receptor and its natural ligand are involved in neuroendocrine function.

Amino Acid Sequence↗