Factors that influence magnetic orientation in Caenorhabditis elegans.

Magnetoreceptive animals orient to the earth's magnetic field at angles that change depending on temporal, spatial, and environmental factors such as season, climate, and position within the geomagnetic field. How magnetic migratory preference changes in response to internal or external stimuli is not understood. We previously found that Caenorhabditis elegans orients to magnetic fields favoring migrations in one of two opposite directions. Here we present new data from our labs together with replication by an independent lab to test how temporal, spatial, and environmental factors influence the unique spatiotemporal trajectory that worms make during magnetotaxis. We found that worms gradually change their average preferred angle of orientation by ~ 180° to the magnetic field during the course of a 90-min assay. Moreover, we found that the wild-type N2 strain prefers to orient towards the left side of a north-facing up, disc-shaped magnet. Lastly, similar to some other behaviors in C. elegans, we found that magnetic orientation may be more robust in dry conditions (< 50% RH). Our findings help explain why C. elegans accumulates with distinct patterns during different periods and in differently shaped magnetic fields. These results provide a tractable system to investigate the behavioral genetic basis of state-dependent magnetic orientation.

Deficits in temporal processing correlate with clinical progression in Huntington's disease.

OBJECTIVES: Precise temporal performance is crucial for several complex tasks. Time estimation in the second-to-minutes range-known as interval timing-involves the interaction of the basal ganglia and the prefrontal cortex via dopaminergic-glutamatergic pathways. Patients with Huntington's disease (HD) present deficits in cognitive and motor functions that require fine control of temporal processing. The objective of the present work was to assess temporal cognition through a peak-interval time (PI) production task in patients with HD and its potential correlation with the Unified Huntington's Disease Rating Scale (UHDRS). MATERIALS AND METHODS: Patients with molecular diagnosis of HD and controls matched by age, sex and educational level (n=18/group) were tested for interval timing in short- (3 seconds), medium- (6 seconds) and long (12 seconds)-duration stimuli. RESULTS: Significant differences were observed in the PI task, with worse performance in HD compared to controls. Patients underestimated real time (left-shifted Peak location) for 6- and 12-second intervals (P<.05) and presented decreased temporal precision for all the intervals evaluated (P<.01). Importantly, a significant correlation was found between time performance and the UHDRS (P<.01). Patients' responses also deviated from the scalar property. CONCLUSIONS: Our results contribute to support that timing functions are impaired in HD in correlation with clinical deterioration. Recordings of cognitive performance related to timing could be a potential useful tool to measure the neurodegenerative progression of movement disorder-related pathologies.

Circadian disruption promotes tumor-immune microenvironment remodeling favoring tumor cell proliferation.

Circadian disruption negatively affects physiology, posing a global health threat that manifests in proliferative, metabolic, and immune diseases, among others. Because outputs of the circadian clock regulate daily fluctuations in the immune response, we determined whether circadian disruption results in tumor-associated immune cell remodeling, facilitating tumor growth. Our findings show that tumor growth rate increased and latency decreased under circadian disruption conditions compared to normal light-dark (LD) schedules in a murine melanoma model. Circadian disruption induced the loss or inversion of daily patterns of M1 (proinflammatory) and M2 (anti-inflammatory) macrophages and cytokine levels in spleen and tumor tissues. Circadian disruption also induced (i) deregulation of rhythmic expression of clock genes and (ii) of cyclin genes in the liver, (iii) increased CcnA2 levels in the tumor, and (iv) dampened expression of the cell cycle inhibitor p21WAF/CIP1 , all of which contribute to a proliferative phenotype.

Circadian rhythms in the vegetative state.

OBJECTIVE: To evaluate whether vegetative state patients maintain circadian rhythms. RESEARCH DESIGN: An observational study of five single cases. METHODS AND PROCEDURES: Five chronic vegetative state patients underwent clinical and neurological evaluations and 2-week continuous temperature measurements. MAIN OUTCOMES AND RESULTS: The two patients with traumatic brain injury showed well-formed circadian temperature rhythms and had more reflexive behaviours and relatively low cortical and sub-cortical atrophy, whereas the three patients from anoxic-hypoxic origin demonstrated no cycles or rhythmic behaviour. CONCLUSIONS: The presence of periods of wakefulness does not imply preserved sleep-wake cycling capacity, nor preserved circadian rhythms and it should not be taken as a distinguishing feature for the definition of the vegetative state.

Inhibition of GABA transaminase enhances light-induced circadian phase delays but not advances.

The CNS neurotransmitter GABA is distributed extensively throughout the suprachiasmatic nucleus, the site of circadian pacemaker cells in mammals. Pharmacological agents that act at GABAA receptors alter specific circadian responses to light and may induce phase shifts of circadian rhythms. In the present study, the role of endogenously released GABA in rhythm regulation was investigated using vigabatrin (gamma-vinyl GABA), an agent that has been shown to increase chronically or acutely the CNS levels of this neurotransmitter by inhibiting GABA transaminase. In Experiment 1, hamsters in constant darkness (DD) received a saline or a vigabatrin injection 1 hr before a 15-min, 700-lux light pulse. Vigabatrin increased photic phase delays but did not affect advances. In Experiment 2, vigabatrin delivered chronically via osmotic minipump treatment did not affect locomotor activity period in DD. However, after 14 days of infusion, photic phase delays (but not advances) were greatly increased in the vigabatrin group. In Experiment 3, in constant light (LL), chronic vigabatrin-treated animals showed an increased period that returned to pretreatment values after the 14-day drug infusion. The results are consistent with the phase-dependent effects of other agents that alter GABA neurotransmission. The data support the general hypothesis that GABA modulates the circadian responses to light in a phase-dependent manner, and may participate in entrainment to light-dark cycles by influencing the relative responsiveness to light in the early and late subjective night.

Regulation of circadian photic responses by nitric oxide.

A role for nitric oxide in circadian responses to light has been indicated in previous studies. To determine the specific function of NO-, the authors manipulated NO- and nitric oxide synthase (NOS) activity prior to light pulses that would normally induce phase shifts. The NOS inhibitor, L-NAME, selectively attenuated phase advances of locomotor rhythms and had no effect on phase delays. The NO- donor, SNAP, potentiated both photic responses, and phase delays were larger than the maximum responses that could be obtained with light alone. The date suggest a model in which NO- participates in the adaptation of the system to environmental lighting conditions by regulating in a phase-dependent manner responsiveness to light.

Glial and light-dependent glutamate metabolism in the suprachiasmatic nuclei.

The suprachiasmatic nuclei, the main circadian clock in mammals, are entrained by light through glutamate released from retinal cells. Astrocytes are key players in glutamate metabolism but their role in the entrainment process is unknown. We studied the time dependence of glutamate uptake and glutamine synthetase (GS) activity finding diurnal oscillations in glutamate uptake (high levels during the light phase) and daily and circadian fluctuations in GS activity (higher during the light phase and the subjective day). These results show that glutamate-related astroglial processes exhibit diurnal and circadian variations, which could affect photic entrainment of the circadian system.

Pigment-dispersing factor signaling in the circadian system of Caenorhabditis elegans.

The neuropeptide pigment-dispersing factor (PDF) is important for the generation and entrainment of circadian rhythms in the fruitfly Drosophila melanogaster. Recently two pdf homologs, pdf-1 and pdf-2, and a PDF receptor, pdfr-1, have been found in Caenorhabditis elegans and have been implicated in locomotor activity. In this work, we have studied the role of the PDF neuropeptide in the circadian system of C. elegans and found that both pdf-1 and pdf-2 mutants affect the normal locomotor activity outputs. In particular, loss of pdf-1 induced circadian arrhythmicity under both light-dark (LD) and constant dark (DD) conditions. These defects can be rescued by a genomic copy of the pdf-1 locus. Our results indicate that PDF-1 is involved in rhythm generation and in the synchronization to LD cycles, as rhythmic patterns of activity rapidly disappear when pdf-1 mutants are recorded under both entrained and free-running conditions. The role of PDF-2 and the PDF receptors is probably more complex and involves the interaction between the two pdf paralogues found in the nematode.

Melatonin effects on behavior: possible mediation by the central GABAergic system.

The best described function of the pineal hormone melatonin is to regulate seasonal reproduction, with its daily production and secretion varying throughout the seasons or the photoperiod. Additionally, a number of behavioral effects of the hormone have been found. This review describes the effects of melatonin in rodent behavior. We focus on: (a) inhibitory effects (sedation, hypnotic activity, pain perception threshold elevation, anti-convulsive activity, anti-anxiety effects); and (b) direct effects on circadian rhythmicity (entrainment, resynchronization, alleviation of jet-lag symptoms, phase-shifting). Most of these effects are clearly time-dependent, with a peak of melatonin activity during the night. One of the possible mechanisms of action for melatonin in the brain is the interaction with the GABAergic system, as suggested by neurochemical and behavioral data. Finally, some pineal hormone effects might be candidates as putative therapies for several human disorders.

Neuropeptide Y and glutamate block each other's phase shifts in the suprachiasmatic nucleus in vitro.

The suprachiasmatic nuclei contain a circadian clock whose activity can be recorded in vitro for several days. Photic information is conveyed to the nuclei primarily via a direct projection from the retina, the retinohypothalamic tract, utilizing an excitatory amino acid neurotransmitter. Photic phase shifts may be mimicked by application of glutamate in vitro. A second, indirect pathway to the suprachiasmatic nuclei via the geniculohypothalamic tract utilizes neuropeptide Y as a transmitter. Phase shifts to neuropeptide Y in vitro are similar to those seen to non-photic stimuli in vivo. We have used the hypothalamic slice preparation to examine the interactions of photic and non-photic stimuli in the suprachiasmatic nuclei. Coronal hypothalamic slices containing the suprachiasmatic nuclei were prepared from Syrian hamsters and 3 min recordings of the firing rate of individual cells were performed throughout a 12 h period. Control slices receiving either no application or application of artificial cerebrospinal fluid to the suprachiasmatic nucleus showed a consistent daily peak in their rhythms. Glutamate produces phase shifts of the circadian clock in the hamster hypothalamic slice preparation during the subjective night but not during the subjective day. These phase shifts were similar in timing and direction to the photic phase response curve in vivo confirming previous work with the rat slice preparation. Neuropeptide Y produces phase shifts of the circadian clock during the subjective day but not during the subjective night. The phase shifts are similar in timing and direction to the non-photic phase response curve in vivo, confirming previous in vitro work. We then examined the interaction of these neurochemicals with each other at various times during the circadian cycle. We found that both advances and delays to glutamate in the slice are blocked by application of neuropeptide Y. We also found that phase shifts to neuropeptide Y in the slice are blocked by application of glutamate. These results indicate that photic and non-photic associated neurochemicals can block each others phase shifting effects within the suprachiasmatic nucleus in vitro. These experiments demonstrate the ability of photic and non-photic associated neurochemicals to interact at the level of the suprachiasmatic nucleus. It is clear that neuropeptide Y antagonizes the effect of glutamate during the subjective night, and that glutamate antagonizes the effect of neuropeptide Y during the subjective day. Great care must be taken when devising treatments where photic and non-photic signals may interact.

KN-62, an inhibitor of Ca2+/calmodulin kinase II, attenuates circadian responses to light.

Expression of immediate early genes and phosphorylation of the transcription factor CREB are induced in the suprachiasmatic nucleus after light pulses that cause phase shifts of circadian rhythms. To test for a direct role of this signalling pathway in mediating circadian responses to light in hamsters, we used KN-62 to inhibit the activity of CaM kinase II (known to phosphorylate CREB) prior to giving light pulses at times that would normally induce phase shifts. Central administration of KN-62 significantly inhibited phase delays and advances induced by bright pulses of light. The data support a model for photic responses of the circadian clock in the SCN that includes the phosphorylation of CREB by activation of CaM kinase II.

Neuropeptide Y phase shifts the circadian clock in vitro via a Y2 receptor.

The suprachiasmatic nuclei (SCN) contain a circadian clock whose activity can be recorded in vitro for several days. This clock can be reset by the application of neuropeptide Y. In this study, we focused on determination of the receptor responsible for neuropeptide Y phase shifts of the hamster circadian clock in vitro. Coronal hypothalamic slices containing the SCN were prepared from Syrian hamsters housed under a 14 h:10 h light:dark cycle. Tissue was bathed in artificial cerebrospinal fluid (ACSF), and the firing rates of individual cells were sampled throughout a 12 h period. Control slices received either no application or application of 200 nl ACSF to the SCN at zeitgeber time 6 (ZT6; ZT12 was defined as the time of lights off). Application of 200 ng/200 nl of neuropeptide Y at ZT6 resulted in a phase advance of 3.4 h. Application of the Y2 receptor agonist, neuropeptide Y (3-36), induced a similar phase advance in the rhythm, while the Y1 receptor agonist, [Leu31, Pro34]-neuropeptide Y had no effect. Pancreatic polypeptide (rat or avian) also had no measurable phase-shifting effect. Neuropeptide Y applied at ZT20 or 22 had no detectable phase-shifting effect. These results suggest that the phase-shifting effects of neuropeptide Y are mediated through a Y2 receptor, similar to results found in vivo.

Cyclic AMP and protein kinase A rhythmicity in the mammalian suprachiasmatic nuclei.

The levels of cyclic AMP and protein kinase A, as well as the activity of this enzyme, were measured in the hamster suprachiasmatic nuclei at different time points throughout the daily or circadian cycle. Significant diurnal variations for levels of AMPc and the catalytic subunit of protein kinase A and the activity of this enzyme were found. All of these parameters tended to increase throughout the nocturnal phase, reaching higher values at the end of the night and the beginning of the day and minimal values around the time of lights off. This rhythmicity appears to be under exogenous control, since constant darkness abolished fluctuations throughout the circadian cycle. In vitro incubation in the presence of melatonin during the day significantly decreased cyclic AMP levels and basal protein kinase A activity in the SCN, while neither neuropeptide Y nor light pulses affected these parameters. These results suggest a significant diurnal regulation of the cyclic AMP-dependent system in the hamster circadian clock.

Photic control of nitric oxide synthase activity in the hamster suprachiasmatic nuclei.

Circadian rhythms are controlled by an endogenous clock, which in mammals is located in the hypothalamic suprachiasmatic nuclei (SCN). A role for nitric oxide in circadian responses to light has been indicated. To test the role of nitric oxide synthase (NOS) in the SCN and in circadian responses to light, we examined NOS specific activity at different time points and photic conditions. NOS activity was determined by the conversion of 3H-arginine to 3H-citrulline. NOS enzymatic activity in the SCN was significantly higher during the dark phase than during the day, without any changes in the levels of the NOS protein. However, this difference disappeared when animals were placed under constant darkness, and NOS activity was similar at CT 8 and CT 18 (with CT 12 defined as the onset of the subjective night). When 5-min light pulses were administered at these time points (when light would induce no phase shift or a phase advance, respectively), NOS activity was significantly increased almost equally. A spectrophotometric assay was used to determine NO content in the SCN, showing relatively high constitutive levels enhanced by 100 microM glutamate. These results suggest that NOS activity is not controlled by the circadian clock, although it might mediate some of the effects of light on biological rhythms.

Melatonin as an anxiolytic in rats: time dependence and interaction with the central GABAergic system.

Anxiolytic and pro-exploratory melatonin properties were assessed in rats using a plus-maze procedure. Both melatonin (1 mg/kg) and diazepam (0.5 mg/kg) showed a significant diurnal variation to decrease anxiety and to promote exploratory behavior. Melatonin displayed anxiolytic activity at night, with absence of effects at noon and a weak activity at the beginning of the light phase. Melatonin pro-exploratory activity was found only at night. Diazepam exerted significant anxiolysis during the night, with less activity during the day. Diazepam pro-exploratory activity was found during the night only. A dose-response study carried out by injecting 1-20 mg/kg melatonin at 12:00 or 18:00 h indicated that melatonin activity was greatest at 18:00 h. Diazepam was anxiolytic at both times, and pro-exploratory at 18:00 h only. Melatonin activity was blunted by administration of the benzodiazepine antagonist, flumazenil.

Time-dependent anticonvulsant activity of melatonin in hamsters.

The objective of the present study was to assess whether the anticonvulsant activity of melatonin displays diurnal variability in hamsters. Convulsions were induced by administering 3-mercaptopropionic acid (3-MP). There was a significant diurnal variation in 3-MP-induced convulsions, hamsters being more prone to exhibit seizures during the night than during the day. Melatonin (50 mg/kg i.p.) had a maximal anticonvulsive effect in the early evening (20:00 h). The administration at 20:00 h of the central-type benzodiazepine antagonist, Ro 15-1788, although unable by itself to modify seizure threshold, blunted the anticonvulsant response to melatonin. The results indicate that the time-dependent anticonvulsant activity of melatonin is sensitive to central-type benzodiazepine antagonism.

Neonatal clomipramine treatment of Syrian hamsters: effect on the circadian system.

The circadian behavior of male Syrian hamsters injected with the serotonin/norepinephrine reuptake inhibitor clomipramine (15 mg/kg from postnatal days 8 to 21) was examined. Clomipramine treatment significantly augmented mean activity values of wheel running rhythm, as well as delayed its acrophase. After a 6-h phase advance of the light-dark cycle, reentrainment of clomipramine-treated hamsters took significantly longer than controls. Clomipramine-treated hamsters exhibited a shorter circadian period than controls in constant light conditions, but no differences were found in constant darkness. Light pulses applied at late subjective night to clomipramine-treated hamsters caused significantly reduced phase advances as compared to controls, while no differences were found in phase delay magnitudes when light pulses were applied during early subjective night. Administration of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) at circadian time 8 significantly advanced the onset of activity to a greater extent in clomipramine-treated hamsters than in controls. The results indicate that neonatal clomipramine treatment of hamsters causes long-lasting changes in the circadian system, by increasing activity levels and by partially inhibiting light-evoked responses. An enhancement of a non-photic, serotonergic-induced response was also unveiled.

Time-dependent melatonin analgesia in mice: inhibition by opiate or benzodiazepine antagonism.

The aim of this study was to determine whether melatonin-induced analgesia in mice exhibits the time dependency known to occur for several other effects of the hormone, and to analyze to what extent the activity of melatonin can be inhibited by the opiate antagonist naloxone or the central-type benzodiazepine (BZP) antagonist Ro 15-1788. Analgesia was assessed with the hot plate procedure. There was a significant diurnal variation in the pain threshold, with an increase in latency during the dark phase of the daily photo period. Melatonin (20-40 mg/kg i.p.) exhibited maximal analgesic effects at late evening (20:00 h). The administration of naloxone or Ro 15-1788 at 20:00 h, although unable by themselves to modify pain threshold, blunted the analgesic response to melatonin. Significant increases in the latency of the hot plate response were found after diazepam injection, an effect blocked by Ro 15-1788 or naloxone. These results indicate that time-dependent melatonin analgesia is sensitive to opioid or central-type BZP antagonism.

Circadian responses to light: the calmodulin connection.

KN-62, an inhibitor of CaM kinase II, attenuated phase shifts induced by low intensity light pulses and reduced light-induced phosphorylation of the transcription factor, CREB, in the suprachiasmatic nucleus. The calmodulin inhibitor, W-7, had similar effects: neither drug produced a complete block of photic responses. The results support the hypothesis that circadian responses to light are mediated in part by CaM kinase activity and CREB, and suggest that other signal transduction pathways also take part.

Rhythmic variation in gamma-aminobutyric acid(A)-receptor subunit composition in the circadian system and median eminence of Syrian hamsters.

Temporal changes in the level of expression of gamma-aminobutyric acid (GABA)(A) receptor subunits alpha2, alpha5, beta1 and beta3 were characterized by Western blot analysis in the hamster suprachiasmatic nuclei, retina and median eminence. A nocturnal maximum in the level of GABA(A) receptor beta1 subunit at midday and midnight (12:00 and 00.00 h) was found in the suprachiasmatic nucleus (SCN), the retina and the median eminence of Syrian hamsters. Alpha2 and beta3 subunit levels peaked during the day in the median eminence. Finally, retinal alpha5 levels were maximal during the night. beta1 temporal changes in the SCN and median eminence, as well as alpha2 variations in the median eminence were maintained under constant dark conditions, suggesting an endogenous control, while the other variations were only observed under light-dark cycle conditions.