Family-based association of FKBP5 in bipolar disorder.

The FKBP5 gene product forms part of a complex with the glucocorticoid receptor and can modulate cortisol-binding affinity. Variations in the gene have been associated with increased recurrence of depression and with rapid response to antidepressant treatment. We sought to determine whether common FKBP5 variants confer risk for bipolar disorder. We genotyped seven tag single-nucleotide polymorphisms (SNPs) in FKBP5, plus two SNPs previously associated with illness, in 317 families with 554 bipolar offspring, derived primarily from two studies. Single marker and haplotypic analyses were carried out with FBAT and EATDT employing the standard bipolar phenotype. Association analyses were also conducted using 11 disease-related variables as covariates. Under an additive genetic model, rs4713902 showed significant overtransmission of the major allele (P=0.0001), which was consistent across the two sample sets (P=0.004 and 0.006). rs7757037 showed evidence of association that was strongest under the dominant model (P=0.001). This result was consistent across the two datasets (P=0.017 and 0.019). The dominant model yielded modest evidence for association (P<0.05) for three additional markers. Covariate-based analyses suggested that genetic variation within FKBP5 may influence attempted suicide and number of depressive episodes in bipolar subjects. Our results are consistent with the well-established relationship between the hypothalamic-pituitary-adrenal (HPA) axis, which mediates the stress response through regulation of cortisol, and mood disorders. Ongoing whole-genome association studies in bipolar disorder and major depression should further clarify the role of FKBP5 and other HPA genes in these illnesses.

Electrophysiological actions of orexins on rat suprachiasmatic neurons in vitro.

The study of neural arousal mechanisms has been greatly aided by the discovery of the orexin peptides (orexin A and orexin B), the subsequent identification of the neurons that synthesize these peptides, their projections in the brain, and the distribution of orexin receptors in the central nervous system. Orexin neuron activation is partly controlled by circadian signals generated in the brain's main circadian pacemaker, the suprachiasmatic nuclei (SCN). The SCN clock is in turn reset by arousal-promoting stimuli and, intriguingly, orexin fibers and receptor expression are detected in the SCN region. It is unclear, however, if orexin can alter SCN neuronal activity. Here using a coronal brain slice preparation, we found that orexin A and orexin B (0.1-1 microM) elicited significant changes in the extracellularly recorded firing rate and firing pattern in approximately 80% of rat SCN cells tested; the most common response was suppression of firing rate. Co-application of orexin A with a cocktail of ionotropic GABA and glutamate receptor antagonists did not alter the actions of this peptide on firing rate, but did change some its effects on firing pattern. We conclude that orexins can alter SCN neurophysiology and may influence the transmission of information through the SCN to other CNS regions.

Comparative analyses of multi-species sequences from targeted genomic regions.

The systematic comparison of genomic sequences from different organisms represents a central focus of contemporary genome analysis. Comparative analyses of vertebrate sequences can identify coding and conserved non-coding regions, including regulatory elements, and provide insight into the forces that have rendered modern-day genomes. As a complement to whole-genome sequencing efforts, we are sequencing and comparing targeted genomic regions in multiple, evolutionarily diverse vertebrates. Here we report the generation and analysis of over 12 megabases (Mb) of sequence from 12 species, all derived from the genomic region orthologous to a segment of about 1.8 Mb on human chromosome 7 containing ten genes, including the gene mutated in cystic fibrosis. These sequences show conservation reflecting both functional constraints and the neutral mutational events that shaped this genomic region. In particular, we identify substantial numbers of conserved non-coding segments beyond those previously identified experimentally, most of which are not detectable by pair-wise sequence comparisons alone. Analysis of transposable element insertions highlights the variation in genome dynamics among these species and confirms the placement of rodents as a sister group to the primates.

Kinetics of metabolism and degradation of mometasone furoate in rat biological fluids and tissues.

Mometasone furoate (MF) is a potent glucocorticoid developed for the treatment of glucocorticoid-responsive inflammatory disorders. The in-vitro and ex-vivo kinetics of the degradation and metabolism of MF were studied in selected biological fluids of rat and subcellular fractions of different rat tissues. In-vitro, MF was found to degrade slowly into four products in serum and urine, and metabolized rapidly and extensively in rat liver, minimally in extrahepatic tissues, including intestine, stomach, lung and kidney. Further investigation found that the microsomal fraction was the major intracellular site of MF 6 beta-hydroxylation in rat liver. Using chemical inhibitors, CYP3A was found to be the major enzyme involved in the in-vitro MF 6 beta-hydroxylation in rat liver microsomes. Enzyme kinetic studies in rat liver microsomes showed that the overall metabolic process of MF followed biphasic Michaelis-Menten kinetics, while 6 beta-hydroxylation obeyed monophasic Michaelis-Menten kinetics. The kinetic parameters derived from the kinetic models along with the enzyme inhibition studies suggest that MF is mainly metabolized via 6 beta-hydroxylation mediated by CYP3A primarily, and also biotransformed via other pathway(s) catalysed by other enzymes in rat liver in-vitro.

The roles of vasoactive intestinal polypeptide in the mammalian circadian clock.

Biological oscillations with an endogenous period of near 24 h (circadian rhythms) are generated by the master circadian pacemaker or clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus. This clock is synchronised to recurring environmental signals conveyed by selective neural pathways. One of the main chemical constituents of SCN neurones is vasoactive intestinal polypeptide (VIP). Such neurones are retinorecipient and activated by light. Exogenous application of VIP resets the SCN circadian clock in a light-like manner, both in vivo and in vitro. These resetting actions appear to be mediated through the VPAC2 receptor (a type of receptor for VIP). Unexpectedly, genetically ablating expression of the VPAC2 receptor renders the circadian clock arrhythmic at the molecular, neurophysiological and behavioural levels. These findings indicate that this intrinsic neuropeptide acting through the VPAC2 receptor participates in both resetting to light and maintenance of ongoing rhythmicity of the SCN.

Effects of vasoactive intestinal polypeptide on neurones of the rat suprachiasmatic nuclei in vitro.

The suprachiasmatic nuclei (SCN) of the hypothalamus house the main circadian pacemaker in mammals. Vasoactive intestinal polypeptide (VIP) is the most abundant neuropeptide in the SCN and has been shown to phase-shift the electrical activity rhythm of SCN cells in vitro. However, the effects of VIP on the cellular activity of rat SCN neurones are unknown. In this study, we examined the acute effects of VIP on the extracellularly recorded spontaneous firing rate of SCN neurones in an in-vitro hypothalamic slice preparation. Furthermore, with the use of receptor-selective agonists and antagonists, we determined which receptors might mediate the effects of VIP in the SCN. Approximately 50% of cells responded to VIP; the main type of response was suppression in firing rate, although a few cells were activated. Suppression responses to VIP were mimicked by the VPAC(2) receptor agonist Ro 25-1553 and blocked by the selective VPAC(2) receptor antagonist PG 99-465. The PAC(1) receptor agonist maxadilan evoked responses from 40% of SCN cells, and activations to this agonist were not altered by PG 99-465. Responses to VIP were not blocked by antagonists to ionotropic glutamate receptors, but the duration of suppression was modulated by the GABA(A) receptor antagonist bicuculline. Our data indicate that VIP alters the electrical activity of rat SCN neurones in vitro, via both VPAC(2) and PAC(1) receptors.

High-throughput variation detection and genotyping using microarrays.

The genetic dissection of complex traits may ultimately require a large number of SNPs to be genotyped in multiple individuals who exhibit phenotypic variation in a trait of interest. Microarray technology can enable rapid genotyping of variation specific to study samples. To facilitate their use, we have developed an automated statistical method (ABACUS) to analyze microarray hybridization data and applied this method to Affymetrix Variation Detection Arrays (VDAs). ABACUS provides a quality score to individual genotypes, allowing investigators to focus their attention on sites that give accurate information. We have applied ABACUS to an experiment encompassing 32 autosomal and eight X-linked genomic regions, each consisting of approximately 50 kb of unique sequence spanning a 100-kb region, in 40 humans. At sufficiently high-quality scores, we are able to read approximately 80% of all sites. To assess the accuracy of SNP detection, 108 of 108 SNPs have been experimentally confirmed; an additional 371 SNPs have been confirmed electronically. To access the accuracy of diploid genotypes at segregating autosomal sites, we confirmed 1515 of 1515 homozygous calls, and 420 of 423 (99.29%) heterozygotes. In replicate experiments, consisting of independent amplification of identical samples followed by hybridization to distinct microarrays of the same design, genotyping is highly repeatable. In an autosomal replicate experiment, 813,295 of 813,295 genotypes are called identically (including 351 heterozygotes); at an X-linked locus in males (haploid), 841,236 of 841,236 sites are called identically.

Distribution of substance P and neurokinin-1 receptor immunoreactivity in the suprachiasmatic nuclei and intergeniculate leaflet of hamster, mouse, and rat.

The circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) receives photic information directly via the retinohypothalamic tract (RHT) and indirectly from retinally innervated cells in the thalamic intergeniculate leaflet (IGL) that project to the SCN. Using standard immunohistochemical methods, we examined the presence and distribution of substance P (SP) and the neurokinin-1 receptor (NK-1) in the SCN and IGL of rat and determined whether the patterns of immunostaining generalized to the SCN and IGL of Syrian hamster, Siberian hamster, and mouse. Terminals immunoreactive for SP were sparse within the SCN of Siberian and Syrian hamsters and mouse but were intense in the ventral, retinally innervated portion of the rat SCN. Immunostaining for the NK-1 receptor was mainly absent from the SCN of hamster and mouse. In contrast, a plexus of NK-1-ir cells and processes that was in close proximity to SP-ir terminals was found in the ventral SCN of the rat. Substance P-ir terminals were observed in the IGL of all four species, as were NK-1-ir cells and fibres. Double-labelled IGL sections of hamster or rat revealed SP-ir terminals in close apposition to NK-1-immunostained cells and/or fibres. These data indicate that SP could be a neurotransmitter of the RHT in rat, but not in hamster or in mouse, and they highlight potential species differences in the role of SP within the SCN circadian pacemaker. Such species differences do not appear to exist at the level of the IGL, where SP-ir and NK-1-ir were similar in all species studied.

High-performance liquid chromatographic analysis of mometasone furoate and its degradation products: application to in vitro degradation studies.

A method of analysis of mometasone furoate in pharmaceutical formulations and biological fluids is necessary to study the degradation kinetics and determine its stability. A simple high-performance liquid chromatographic method was developed for simultaneous determination of mometasone furoate and its degradation products in human plasma. Plasma (0.5 ml) was extracted with dichloromethane after addition of the internal standard, dexamethasone 21-acetate. Separation was achieved on a Beckman C(8) column with UV detection at 248 nm. The calibration curve was linear ranging from 0.2 to 100 microg/ml. The mean extraction efficiency was >86%. Precision of the assay was <10% (CV), and was within 10% at the limit of quantitation (0.2 microg/ml). Bias of the assay was lower than 7%. The limit of detection was 50 ng/ml for a 0.5-ml sample. The assay was applied successfully to the in vitro kinetic study of degradation of mometasone furoate in human plasma and simulated biological fluids.

Orexin-A immunoreactive neurons in the rat hypothalamus do not contain neuronal nitric oxide synthase (nNOS).

The lateral hypothalamic area (LHA), a key site involved in the central control of feeding and energy homeostasis, contains populations of neurons that produce the orexin peptides or nitric oxide, two chemical factors that increase food intake. In this study, we used immunohistochemistry to investigate the possibility that rat LHA neurons co-express orexin-A and neuronal nitric oxide synthase (nNOS). The orexin-A and nNOS cell populations in the LHA showed extensive overlap without co-localization, and no evidence of direct anatomic contact was found. The finding that LHA neurons do not co-localize orexin-A and nNOS may suggest that the actions of the orexins and nitric oxide on food intake are mediated via independent mechanisms, however, nitric oxide is a diffusible molecule and could potentially affect the activity of orexin neurons via a non-synaptic mechanism.

Vasoactive intestinal polypeptide (VIP) phase-shifts the rat suprachiasmatic nucleus clock in vitro.

In mammals, the principal circadian pacemaker is housed in the hypothalamic suprachiasmatic nuclei (SCN). The SCN exhibit high levels of vasoactive intestinal polypeptide (VIP) immunoreactivity and two of the three VIP receptors, VPAC(2) and PAC(1), are found in the rat SCN. However, the role of VIP in the SCN remains unclear. In this study, we examined the phase-resetting actions of VIP and selective VIP receptor agonists on the electrical activity rhythm of rat SCN neurons in vitro. Application of VIP during the subjective day did not shift the peak in the firing rate rhythm. However, VIP treatment during the early or late subjective night evoked a small phase delay or a large phase advance, respectively. The phase-advancing effect of VIP was reproduced by the novel VPAC(2) receptor agonist RO 25-1553, but not by pituitary adenylate cyclase-activating peptide (a potent PAC(1) receptor agonist), or by [K15,R16,L27]VIP(1-7)/GRF(8-27), a novel, selective VPAC(1) receptor agonist. These data show that VIP phase-dependently phase-resets the rodent SCN pacemaker in vitro, presumably via the VPAC(2) receptor. As the pattern of phase-shifting evoked by VIP and RO 25-1553 resembles the phase-resetting actions of light on rodent behavioural rhythms, these data support a role for VIP and the VPAC(2) receptor in photic entrainment of the rodent circadian pacemaker.

Estimating divergence times in the presence of an overdispersed molecular clock.

Molecular loci that fail relative-rate tests are said to be "overdispersed." Traditional molecular-clock approaches to estimating divergence times do not take this into account. In this study, a method was developed to estimate divergence times using loci that may be overdispersed. The approach was to replace the traditional Poisson process assumption with a more general stationary process assumption. A probability model was developed, and an accompanying computer program was written to find maximum-likelihood estimates of divergence times under both the Poisson process and the stationary process assumptions. In simulation, it was shown that confidence intervals under the traditional Poisson assumptions often vastly underestimate the true confidence limits for overdispersed loci. Both models were applied to two data sets: one from land plants, the other from the higher metazoans. In both cases, the traditional Poisson process model could be rejected with high confidence. Maximum-likelihood analysis of the metazoan data set under the more general stationary process suggested that their radiation occurred well over a billion years ago, but confidence intervals were extremely wide. It was also shown that a model consistent with a Cambrian (or nearly Cambrian) origination of the animal phyla, although significantly less likely than a much older divergence, fitted the data well. It is argued that without an a priori understanding of the variance in the time between substitutions, molecular data sets may be incapable of ever establishing the age of the metazoan radiation.

Metabolism kinetics of beclomethasone propionate esters in human lung homogenates.

PURPOSE: The purposes of this study were to characterize the kinetics of beclomethasone dipropionate (BDP) and its 17-monopropionate ester (17-BMP) in human lung 1000g supernatant (HLu) at 37 degrees C, and to analyze the interindividual variability in the metabolism of BDP in HLu. METHODS: The concentrations of BDP and its metabolites were determined by HPLC with UV detection at 242 nm. Kinetics of BDP and 17-BMP decomposition were characterized by least-squares fitting of rate equations. RESULTS: The active metabolite 17-BMP was rapidly formed following the incubation of BDP in HLu. Kinetics of BDP and 17-BMP in HLu were nonlinear owing to product inhibition and enzyme saturation. A model taking into account the product inhibition provides a kinetic basis for understanding the in vivo behavior of BDP and its metabolites in human lung. There was approximately a 3.5-fold difference in the initial half-life of BDP in HLu observed in seven subjects. CONCLUSIONS: An effective activation of BDP was demonstrated in HLu through the rapid formation of 17-BMP. Kinetics of BDP and 17-BMP in HLu were well characterized by the nonlinear kinetic model. Interindividual difference in the initial half-life of BDP was due mainly to esterase metabolizing activity rather than binding affinity.

The hypothalamus and the regulation of energy homeostasis: lifting the lid on a black box.

The hypothalamus is the focus of many peripheral signals and neural pathways that control energy homeostasis and body weight. Emphasis has moved away from anatomical concepts of 'feeding' and 'satiety' centres to the specific neurotransmitters that modulate feeding behaviour and energy expenditure. We have chosen three examples to illustrate the physiological roles of hypothalamic neurotransmitters and their potential as targets for the development of new drugs to treat obesity and other nutritional disorders. Neuropeptide Y (NPY) is expressed by neurones of the hypothalamic arcuate nucleus (ARC) that project to important appetite-regulating nuclei, including the paraventricular nucleus (PVN). NPY injected into the PVN is the most potent central appetite stimulant known, and also inhibits thermogenesis; repeated administration rapidly induces obesity. The ARC NPY neurones are stimulated by starvation, probably mediated by falls in circulating leptin and insulin (which both inhibit these neurones), and contribute to the increased hunger in this and other conditions of energy deficit. They therefore act homeostatically to correct negative energy balance. ARC NPY neurones also mediate hyperphagia and obesity in the ob/ob and db/db mice and fa/fa rat, in which leptin inhibition is lost through mutations affecting leptin or its receptor. Antagonists of the Y5 receptor (currently thought to be the NPY 'feeding' receptor) have anti-obesity effects. Melanocortin-4 receptors (MC4-R) are expressed in various hypothalamic regions, including the ventromedial nucleus and ARC. Activation of MC4-R by agonists such as alpha-melanocyte-stimulating hormone (a cleavage product of pro-opiomelanocortin which is expressed in ARC neurones) inhibits feeding and causes weight loss. Conversely, MC4-R antagonists such as 'agouti' protein and agouti gene-related peptide (AGRP) stimulate feeding and cause obesity. Ectopic expression of agouti in the hypothalamus leads to obesity in the AVY mouse, while AGRP is co-expressed by NPY neurones in the ARC. Synthetic MC4-R agonists may ultimately find use as anti-obesity drugs in human subjects Orexins-A and -B, derived from prepro-orexin, are expressed in specific neurones of the lateral hypothalamic area (LHA). Orexin-A injected centrally stimulates eating and prepro-orexin mRNA is up regulated by fasting and hypoglycaemia. The LHA is important in receiving sensory signals from the gut and liver, and in sensing glucose, and orexin neurones may be involved in stimulating feeding in response to falls in plasma glucose.

Concentration-effect relationship of hydroxychloroquine in patients with rheumatoid arthritis--a prospective, dose ranging study.

OBJECTIVE: A 6 month prospective randomized double blind study was conducted to investigate hydroxychloroquine dose concentration-effect relationships in people with rheumatoid arthritis. METHODS: Patients were randomized in 2 groups: one group received 200 mg hydroxychloroquine sulfate daily (A) and one group received 400 mg daily (B). Each month, 8 disease variables were assessed, adverse events recorded, and hydroxychloroquine blood concentrations determined. RESULTS: Twenty-three patients were included: 10 in group A and 13 in group B. After 6 months of therapy, a significant improvement in disease activity was noted for 6 criteria with no statistical differences between groups: pain (assessed by a visual analog scale), joint scores (swelling and tenderness), impairment in daily living activity (18 activities graded 0 to 8), patient assessment of disease state, and erythrocyte sedimentation rate. Hydroxychloroquine steady-state blood concentrations (Month 6) were significantly different between groups (mean +/- SD): 450.6 +/- 285.3 ng/ml (A) vs 870.3 +/- 329.3 ng/ml (B) (p = 0.0001). Steady-state concentrations were correlated with the daily dose (r = 0.63, p = 0.005), the improvement in activity of daily living (r = 0.49, p = 0.03), and the improvement in joint tenderness score (r = 0.47, p = 0.038). CONCLUSION: The data indicate that hydroxychloroquine is an effective therapy, but there were no further improvements observed in the group receiving 400 mg daily compared to those receiving 200 mg. There were some correlations between hydroxychloroquine steady-state blood concentrations and effects.

The index of dispersion of molecular evolution: slow fluctuations.

The most simple neutral model of molecular evolution predicts that the number of substitutions within a lineage in T generations ought to be Poisson distributed. Therefore, the variance in the number of substitutions ought to equal the mean number. The ratio of the variance to the mean number of substitutions is called the index of dispersion, R(T). Assuming infinite sites, no recombination model of the gene, and a haploid, Moran population structure, R(T) is derived for a general stationary model of molecular evolution. R(T) is shown to be affected by fluctuations in parameters only when they occur on a very slow time scale. In order for parameter fluctuations to cause R(T) to deviate significantly from one, the time between parameter changes must be roughly as large, or larger, than the time between substitutions.

Understanding the overdispersed molecular clock.

Rates of molecular evolution at some protein-encoding loci are more irregular than expected under a simple neutral model of molecular evolution. This pattern of excessive irregularity in protein substitutions is often called the "overdispersed molecular clock" and is characterized by an index of dispersion, R(T) > 1. Assuming infinite sites, no recombination model of the gene R(T) is given for a general stationary model of molecular evolution. R(T) is shown to be affected by only three things: fluctuations that occur on a very slow time scale, advantageous or deleterious mutations, and interactions between mutations. In the absence of interactions, advantageous mutations are shown to lower R(T); deleterious mutations are shown to raise it. Previously described models for the overdispersed molecular clock are analyzed in terms of this work as are a few very simple new models. A model of deleterious mutations is shown to be sufficient to explain the observed values of R(T). Our current best estimates of R(T) suggest that either most mutations are deleterious or some key population parameter changes on a very slow time scale. No other interpretations seem plausible. Finally, a comment is made on how R(T) might be used to distinguish selective sweeps from background selection.

The evolution of an alpha-esterase pseudogene inactivated in the Drosophila melanogaster lineage.

Previous analyses of the alpha-esterase cluster of Drosophila melanogaster revealed 10 active genes and the DmalphaE4a-Psi pseudogene. Here, we reconstruct the evolution of the pseudogene from the sequences of 12 alleles from widely scattered D. melanogaster populations and single alleles from Drosophila simulans and Drosophila yakuba. All of the DmalphaE4a-Psi alleles contain numerous inactivating mutations, suggesting that pseudogene alleles are fixed in natural populations. Several lines of evidence also suggest that DmalphaE4a is now evolving without selective constraint in the D. melanogaster lineage. There are three polymorphic indels which result in frameshifts; a key nucleotide of the intron splice acceptor is polymorphic; the neutral mutation parameter is the same for replacement and silent sites; one of the nonsilent polymorphisms results in a stop codon; only 1 of the 13 replacement polymorphisms is biochemically conservative; residues that are conserved among active esterases have different states in DmalphaE4a-Psi; and there are about half as many transitional polymorphisms as transversional ones. In contrast, the D. simulans and D. yakuba orthologs DsalphaE4a and DyalphaE4a do not have the inactivating mutations of DmalphaE4a-Psi and appear to be evolving under the purifying selection typical of protein- encoding genes. For instance, there have been more substitutions in the introns than in the exons, and more in silent sites than in replacement sites. Furthermore, most of the amino acid substitutions that have occurred between DyalphaE4a and DsalphaE4a are located in sites that typically vary among active alpha-esterases rather than those that are usually conserved. We argue that the original alphaE4a gene had a function which it has lost since the divergence of the D. melanogaster and D. simulans lineages.

Patterns of genetic variation in Mendelian and complex traits.

This review discusses the prospects for understanding the genetic basis of complex traits in humans. We take the view that work done on Drosophila melanogaster can serve as a model for understanding complex traits in humans, and the literature on this model system, as well as on humans, is reviewed. The prospects for success in understanding the genetic basis of complex traits depend, in part, on the nature of the forces acting on genetic variation. We suggest that different experimental approaches should be undertaken for traits caused by common genetic variants versus those arising from rare genetic variants.

Electrophysiological effects of opioid receptor activation on Syrian hamster suprachiasmatic nucleus neurones in vitro.

Entrainment of the dominant circadian pacemaker localised to the hypothalamic suprachiasmatic nuclei (SCN) is mediated partially via the indirect retino-geniculo-hypothalamic projection to the SCN, which is presumed to utilise enkephalin and other neurotransmitters, to modulate circadian rhythmicity. In the present study, we have investigated electrophysiologically the currently unknown functional effects of enkephalin, and another opioid receptor agonist morphine, on hamster SCN neuronal activity in vitro. Basal or N-methyl-D-aspartate-evoked firing rates of SCN neurones were generally unresponsive (86%) to the opioid receptor agonists leucine-enkephalin, methionine-enkephalin, or morphine. Washout of the enkephalins or morphine resulted in a rebound excitatory response ("withdrawal activation") in 39% of neurones tested. Withdrawal activation was also elicited by administration of the opioid receptor antagonist naloxone, following pre-exposure to morphine, in 59% of neurones tested. These withdrawal responses were blocked or attenuated by the alpha2-adrenoceptor agonist clonidine, results which suggest a functional interaction exists between opioid receptors and alpha2-adrenoceptors in the SCN. Our observations show that opioid receptor agonists are largely devoid of actions on normal hamster SCN circadian pacemaker activity, while the occurrence of withdrawal responses may have implications on circadian function during withdrawal from opiate abuse.