DNA microarray analyses of circadian timing: the genomic basis of biological time.

Many aspects of physiology and behaviour are organized around a daily rhythm, driven by an endogenous circadian clock. Studies across numerous taxa have identified interlocked autoregulatory molecular feedback loops which underlie circadian organization in single cells. Until recently, little was known of (i) how the core clock mechanism regulates circadian output and (ii) what proportion of the cellular transcriptome is clock regulated. Studies using DNA microarray technology have addressed these questions in a global fashion and identified rhythmically expressed genes in numerous tissues in the rodent (suprachiasmatic nucleus, pineal gland, liver, heart, kidney) and immortalized fibroblasts, in the head and body of Drosophila, in the fungus Neurospora and the higher plant Arabidopsis. These clock controlled genes represent 0.5-9% of probed genes, with functional groups covering a broad spectrum of cellular pathways. There is considerable tissue specificity, with only approximately 10% rhythmic genes common to at least one other tissue, principally consisting of known clock genes. The remaining common genes may constitute genes operating close to the clock mechanism or novel core clock components. Microarray technology has also been applied to understand input pathways to the clock, identifying potential signalling components for clock resetting in fibroblasts, and elucidating the temperature entrainment mechanism in Neurospora. This review explores some of the common themes found between tissues and organisms, and focuses on some of the striking connections between the molecular core oscillator and aspects of circadian physiology and behaviour. It also addresses the limitations of the microarray technology and analyses, and suggests directions for future studies.

Genetic evidence for a family structure in stable social aggregations of the Australian lizard Egernia stokesii.

In this study we used data from six unlinked microsatellite loci to examine stable aggregations of Egernia stokesii, from a population in the southern Flinders Ranges of South Australia. We show that these aggregations are comprised of breeding partners, their offspring from two or more cohorts, and related adults, providing the first genetic evidence of a family structure in any lizard species. Despite this high level of relatedness within aggregations, most breeding pairs were unrelated and partners were less closely related to each other than they were to other potential within-group partners. Where individuals dispersed, both sexes usually moved to social groups close to their natal group. Although both sexes showed natal philopatry, there was some evidence that females in groups were more related than males in groups. These data suggest that an active choice of unrelated partners and male-biased dispersal may be the mechanisms used by E. stokesii to avoid inbreeding within groups.

Fitness to plead. A prospective study of the inter-relationships between expert opinion, legal criteria and specific symptomatology.

BACKGROUND: Psychiatrists are asked to give opinions as to fitness to plead, a legal concept. There is a dearth of research into fitness to plead in the UK, with no prospective studies and no studies involving the comparison of fit and unfit subjects. In particular, there have been no investigations into the meaning of 'unfit to plead' in terms of psychiatric symptomatology, or as to the relative importance of each legal fitness criterion in psychiatrists' conclusions as to fitness. METHOD: The study comprised a prospective evaluation of 479 consecutive referrals to psychiatrists at court. Individual legal fitness criteria were examined as predictors of unfitness. Associations of unfitness, and of individual legal fitness criteria, were examined with Brief Psychiatric Rating Scale (BPRS) symptom scores. RESULTS: The two most important of the legal criteria in clinical decisions as to unfitness were whether the person could follow the proceedings of the trial or give adequate instructions to their solicitor. The legal criteria concerning trial were more predictive of unfitness than those concerning plea. Unfitness was significantly associated with the presence of positive psychotic symptomatology, in particular conceptual disorganization and delusional thinking, but not with symptoms of anxiety, depression or withdrawal. CONCLUSION: Unfitness is most significantly associated with symptoms affecting comprehension and communication. The fitness criteria could be simplified without loss of power. These results, predominantly concerned with mental illness, may not generalize to the mentally impaired.

Anatomical and functional characterisation of a dopaminergic system in the suprachiasmatic nucleus of the neonatal Siberian hamster.

In altricial rodents, maternal influences entrain the developing circadian system in the perinatal period before the capacity to respond directly to photic cues develops. The aim of these studies was to investigate the potential role of dopamine in this process in the Siberian hamster. An initial study investigated the ontogeny of retinal innervation of the suprachiasmatic nuclei (SCN) by using cholera toxin B subunit as a tracer. This revealed that retinal fibres first innervate the SCN on postnatal day 3 (PD3), and ingrowth of fibres is extensive by PD6. In situ hybridisation studies revealed the presence of D1-dopamine receptor (D1-R) mRNA in the SCN on PD2, and levels of expression were similar in PD6 pups and adult hamsters. Immunocytochemical staining for tyrosine hydroxylase revealed abundant catecholaminergic fibres within the ventromedial zone of the SCN from the day of birth through PD20; however, in contrast, few fibres were present in adult SCN. Dopamine-beta-hydroxylase-immunoreactive fibres were absent from the neonatal and adult SCN, suggesting that the fibres in the SCN are dopaminergic. The function of this dopaminergic system was investigated by determining the effects of D1-R agonists on the expression of the immediate-early gene c-fos in the SCN. This was assessed in pups ages PD1- PD5 by in situ hybridisation and immunocytochemical localisation of its protein product. No induction was seen in the SCN, in marked contrast to studies in the developing rat. A final series of studies investigated dopaminergic function by determining whether a D1-agonist could induce phosphorylation of Ca2+/cyclic AMP response element-binding protein (CREB) on Ser133. Hypothalamic slices containing SCN taken from PD1 and PD2 hamsters were treated with D1-R agonists, and levels of phosphorylated CREB were assayed by Western blots. Phosphorylation of CREB was stimulated by D1-R agonists in both Syrian and Siberian hamster hypothalamus, but the response was far greater in Syrian hamster tissue (+138%+/-28%) than in Siberian hamster tissue (+43%+/-11%). Although the anatomical studies demonstrate the existence of a dopaminergic system in the SCN of the early postnatal Siberian hamster, the unresponsiveness of c-fos expression and the relative lack of phosphorylation of CREB after D1-R activation suggests a diminished role for dopamine in the regulation of circadian events during the postnatal period in this species.

CREB in the mouse SCN: a molecular interface coding the phase-adjusting stimuli light, glutamate, PACAP, and melatonin for clockwork access.

The suprachiasmatic nucleus (SCN) is a central pacemaker in mammals, driving many endogenous circadian rhythms. An important pacemaker target is the regulation of a hormonal message for darkness, the circadian rhythm in melatonin synthesis. The endogenous clock within the SCN is synchronized to environmental light/dark cycles by photic information conveyed via the retinohypothalamic tract (RHT) and by the nocturnal melatonin signal that acts within a feedback loop. We investigated how melatonin intersects with the temporally gated resetting actions of two RHT transmitters, pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate. We analyzed immunocytochemically the inducible phosphorylation of the transcription factor Ca2+/cAMP response element-binding protein (CREB) in the SCN of a melatonin-proficient (C3H) and a melatonin-deficient (C57BL) mouse strain. In vivo, light-induced phase shifts in locomotor activity were consistently accompanied by CREB phosphorylation in the SCN of both strains. However, in the middle of subjective nighttime, light induced larger phase delays in C57BL than in C3H mice. In vitro, PACAP and glutamate induced CREB phosphorylation in the SCN of both mouse strains, with PACAP being more effective during late subjective daytime and glutamate being more effective during subjective nighttime. Melatonin suppressed PACAP- but not glutamate-induced phosphorylation of CREB. The distinct temporal domains during which glutamate and PACAP induce CREB phosphorylation imply that during the light/dark transition the SCN switches sensitivity between these two RHT transmitters. Because these temporal domains are not different between C3H and C57BL mice, the sensitivity windows are set independently of the rhythmic melatonin signal.

Investigation into the regulation of the circadian system by dopamine and melatonin in the adult Siberian hamster (Phodopus sungorus).

Dopamine and melatonin have both been implicated in mediating maternal influences on the developing circadian system of altricial rodents. The aim of these studies was to investigate their role in the entrainment of the circadian system of the adult Siberian hamster (Phodopus sungorus). In-situ hybridization revealed that D1-dopamine receptor (D1-R) mRNA was expressed in the adult suprachiasmatic nucleus (SCN) at levels comparable to neonates. As dopamine has been postulated to mimic photic stimulation during early development, experiment 1 compared the effects of a D1-R agonist and a light pulse on free-running wheel running rhythms in hamsters maintained in constant dim red light. A phase response curve to light was generated, revealing clear phase delays early in the subjective night, and large phase advances in the late subjective night. However, the D1-R agonist (SKF 81297, 2 mg/kg, s.c.) did not produce consistent phase shifts at any circadian phase. Experiment 2 tested the ability of this dopaminergic agonist to modulate photic responses of the circadian system. Free-running animals were pre-treated with SKF 81297 (2 mg/kg, s.c.) 30 min before a 15 min light pulse given early or late in the subjective night. This agonist had no effect on the magnitude of phase shifts at either circadian time. In experiment 3, light pulses at CT13-15 induced expression of the immediate early gene c-fos in the SCN, as assessed by immunocytochemistry for the protein product. In contrast, SKF 81297 (2 mg/kg, s.c.) at the same phase did not induce c-fos in the SCN, despite marked c-fos induction in the caudate-putamen, nor did it affect photic induction of c-fos in the SCN. To investigate whether dopamine might be involved in nonphotic regulation of the circadian system in adult hamsters, experiment 4 compared the response of free-running hamsters to a series of injections of SKF 81297 (2 mg/kg, s.c.) or melatonin (1 mg/kg, s.c.), since melatonin receptor expression in the SCN also persists into adulthood. Animals were treated every 23.5 h for 6 days. The serial injections of melatonin produced cumulative phase advances of up to 3 h when delivered in late subjective day, but not when presented in late subjective night. Hamsters did not respond to SKF 81297 or vehicle treatment at either circadian phase. Moreover, pre-treatment with the dopaminergic agonist did not affect the phase-advancing effects of melatonin when both were given in the serial injection protocol. These results demonstrate clear phase-dependent effects of light pulses and melatonin on circadian rhythms in Siberian hamsters, but suggest that D1-Rs in the SCN no longer modulate photic or melatonin-dependent entrainment pathways in the adult.

Entrainment of the circadian system of mammals by nonphotic cues.

Although light is the principal zeitgeber to the mammalian circadian system, other cues can be shown to have a potent resetting effect on the clock of both adult and perinatal mammals. Nonphotic entrainment may have both biological and therapeutic significance. This review focuses on the effect of behavioral arousal as a nonphotic cue and the neurochemical circuitry that mediates arousal-induced entrainment in the adult rodent. In addition, it considers the role of nonphotic entrainment of the developing circadian system in perinatal life prior to the establishment of retinal input to the clock.

Maternal entrainment of the developing circadian system in the Siberian hamster (Phodopus sungorus).

The aim of these studies was to investigate maternal entrainment of developing circadian locomotor activity rhythms in the Siberian hamster. In Experiment 1, mothers were transferred from a 16:8 LD cycle into constant dim red light (DD) from the day of parturition, and wheel-running activity of the mother and pups was individually monitored from the time of weaning. The phases of the individual pups' rhythms were found to be synchronized both to the phase of the mother and to the phase of lights off (ZT 12) of the photo cycle that the mother was exposed to until the day of parturition. To investigate whether this synchrony might reflect direct effects of light acting upon the fetal circadian system in late gestation, the experiment was repeated but with mothers placed into DD early in pregnancy (< or = day 7 of gestation). The results were similar to the first study, suggesting that the mother rather than the photo cycle during the latter part of gestation entrains the developing circadian system. The third experiment investigated whether this entrainment occurred during the postnatal period. Breeding pairs were maintained on alternative light-dark cycles, LD and DL, that were 12 h out of phase. Litters born to mothers on one light-dark cycle were exchanged on the day of birth with foster mothers from the reversed light-dark cycle, then raised in DD. Control litters exchanged between mothers from the same light-dark cycle had similar litter synchrony as shown by nonfostered litters of Experiment 1. However, pups cross-fostered with mothers on reversed LD cycles showed a very different distribution of pup phases. Pups were not synchronized to their natural mother but to their foster mother. Moreover, pups were more scattered over the 24-h period and were found to be significantly synchronized to the phase of the reversed LD cycle. These results demonstrate the occurrence of postnatal entrainment in the Siberian hamster. The increased scatter produced by the cross-fostering paradigm results from some litters being completely entrained to the phase of the foster mother, some with an intermediate distribution between the phase of the natural and foster mothers, and a minority being associated with the phase of the natural mother. These results suggest that Siberian hamster pups are initially synchronized either prenatally or at birth but that the mother continues to provide entrainment signals during the postnatal period.

Non-photic signalling in the suprachiasmatic nucleus.

Scheduled arousal by handling and sub-cutaneous saline injection entrains the free-running clock of the adult Syrian hamster and outbred (ID(ICR)) but not inbred (C57B16) mice. Syrian hamsters bearing lesions of the intergeniculate leaflet of the thalamus remain able to entrain and phase-shift to light, but the lesions block completely entrainment by serial arousal, even though lesioned animals continue to respond acutely to the arousing cue. This suggests that the innervation from the IGL to the SCN is a necessary component of the pathways which signal an aroused state to the clock. Siberian hamsters do not entrain to serial arousal but they do entrain to serial injections of melatonin, whereas in adult Syrian hamster, systemic treatment with melatonin has no effect above that of arousal. In contrast to the adult, the foetal and neonatal Syrian hamster can be entrained by melatonin. These variations in sensitivity correlate with inter-specific and developmental differences in the pattern and level of expression of melatonin receptors in the SCN. The perinatal hamster can also be entrained by dopaminergic agonists. SCN tissue from neonatal Syrian hamsters was used to characterise the biochemical actions of dopamine and melatonin. In primary culture and tissue explants, forskolin, dopamine and glutamatergic agonists all stimulated the phosphorylation of the transcription factor CREB. This probably occurred via convergent actions through Ca2+ (glutamate) and cyclic AMP-dependent (forskolin, dopamine) signalling pathways. Dopamine induced phospho-CREB-ir exclusively in GABA-ir neurons and melatonin reversed this effect of dopamine, indicative of an inhibitory Gi protein linking via the Mel1a receptor to adenylyl cyclase. The regulation of phospho-CREB by multiple entraining cues in the SCN highlights its position as a point of convergence for regulators of the clock, and indicates a possible role in entrainment.

Ontogeny of a photic response in the suprachiasmatic nucleus in the Siberian hamster (Phodopus sungorus).

The ontogeny of photic responsiveness in the suprachiasmatic nucleus of the Siberian hamster (Phodopus sungorus) was studied using the enhanced expression of the immediate early gene c-fos as a marker of neuronal activation. c-fos expression was assessed by immunocytochemical localization of its protein product. Hamsters were kept on a 16 h light:8 h dark photocycle. The adult Siberian hamster showed a marked increase in the number of c-fos-immunoreactive (c-fos-ir) cells within the suprachiasmatic nuclei (SCN) in response to a 1 h light pulse delivered 1-3 h after lights off, in comparison to controls kept in the dark. This is consistent with previous studies in the Syrian hamster and rat. The development of the photic response was examined. The first study investigated the effects of a light pulse on c-fos induction in pups at 5, 9, 12 and 24 postnatal days of age (PD). The suprachiasmatic region was identified by immunocytochemical localization of peptide-histidine-isoleucine in adjacent sections, a peptide expressed early in the development of the rodent SCN. The distribution of c-fos-ir cells was also compared with the location of retinal efferents, as determined by intraocular injection of the tract tracer cholera toxin B subunit 24 h previously. At PD 9, 12 and 24, significant increases in the number of c-fos-ir cells occurred in the light pulsed animals in comparison to age-matched control animals which were moved within the non-illuminated room to provide a 'dark' pulse.(ABSTRACT TRUNCATED AT 250 WORDS)

A low-affinity nucleobase transporter in the protozoan parasite Giardia intestinalis.

A membrane transporter with general affinity for purine and pyrimidine bases has been identified in Giardia intestinalis trophozoites by measuring cellular influx of [3H]adenine, [3H]guanine and [3H]thymine at 0 degrees C. The base transporter is distinct from the thymine/uracil-specific (type 1) and broad-specificity (type 2) nucleoside transporters of G. intestinalis. Influx of each labelled base was retarded by unlabelled bases, with inhibition in the order: hypoxanthine greater than adenine greater than thymine greater than uracil. The IC50 values for these bases (measured for [3H]adenine influx) were 0.46 mM, 1.15 mM, 1.52 mM and 2.28 mM, respectively. Nucleosides did not inhibit base influx (less than or equal to 15% inhibition at 2 mM, a 400-fold molar excess, at which concentration [3H]nucleoside influx was inhibited by greater than 95%). The Michaelis-Menten constant (Km), calculated for adenine and thymine influx at 0 degrees C, was 1.44 +/- 0.08 mM and 1.61 +/- 0.37 mM, respectively, with corresponding Vmax of 383 +/- 16 and 498 +/- 112 pmol min-1 (10(6) cells)-1. The data demonstrate the existence of a low-affinity, facilitative base transporter with no detectable affinity for nucleosides. The inability of uridine or thymidine to significantly reduce the rate of thymine influx indicates that the previously described thymine/uracil-specific (type 1) thymidine transporter cannot transport thymine, despite its affinity for the base.

Premorbid emotional and behavioural adjustment in children with mild head injuries.

This study investigated the hypothesis that children sustaining a mild closed head injury have a higher prevalence of premorbid behavioural problems than children in the general community. The prevalence of emotional and behavioural problems among children with a mild closed head injury was compared with the prevalence of problems in children with a severe closed head injury, children in the general community, and children referred to a psychiatric outpatient clinic. The results showed that the children with a mild head injury did not have significantly more premorbid emotional and behavioural problems than other children in the community. The children with a mild head injury also had significantly fewer emotional and behavioural problems than children referred to a psychiatric outpatient clinic. The results suggest that the common assumption that children with a mild closed head injury have a higher prevalence of premorbid emotional and behavioural problems than other children in the general community may not be correct.