Biomedical subjects

D A Golombek

Publications and source records attributed to D A Golombek.

At least 19 recordsLinked to original sources

PubMed2004

Participation of transcription factors from the Rel/NF-kappa B family in the circadian system in hamsters.

We have studied the presence and activity of components of the nuclear factor-kappaB (NF-kappaB) transcription factor in the hamster circadian system analyzing wheel-running activity, protein expression and DNA binding activity by electrophoresis mobility shift assays (EMSA). Non-rhythmic specific immunoreactive bands corresponding to a NF-kappaB subunit (p65) were found in hamster suprachiasmatic nuclei (SCN) homogenates. The active form of NF-kappaB evidenced by EMSA was clear and specific in SCN nuclear extracts. The administration of the NF-kappaB inhibitor pyrrolidine-dithiocharbamate (PDTC) blocked the light-induced phase advance at circadian time 18 (vehicle+light pulse: 2.08+/-0.46 h, PDTC+light: 0.36+/-0.35 h). These results demonstrate the presence and activity of Rel/NF-kappaB family proteins in the hamster SCN and suggest that these proteins may be related to the entrainment and regulation of circadian rhythms.

Animals↗

PubMed2002

Circadian rhythms in airline pilots submitted to long-haul transmeridian flights.

BACKGROUND: Circadian rhythms shift out of phase after transmeridian flights. Desynchronization between body rhythms and the environment is linked to jet lag, which depends on age, flight direction, and number of time zones crossed. METHODS: To investigate this problem in airline pilots, we performed a multivariate analysis of their circadian systems during Madrid-Mexico-Madrid flights (-7 time zones, n = 12) and Madrid-Tokyo-Madrid flights (+8 time zones, n = 21). Telemetry was used to record pilots' activity, skin temperature, and heart rate, obtaining 6 d of continuous data, including 2 d before the flight, the flights themselves, 2 d at the stopover, and 1 d after the return flight. Time series were analyzed by cosinor, and the resulting parameters of the rhythms were compared by ANOVA and Tukey contrasts in every category formed by the age groups (under and over 50 yr old) and flight direction groups. Subjective time estimation of short, intermediate, and long intervals was recorded. Other psychological variables were measured, including anxiety, tiredness, and performance. RESULTS AND CONCLUSIONS: Activity/rest and heart rate rhythms appeared to be linked to a "weak oscillator." Temperature rhythms manifested a rigid response after the phase shifts of the light/dark cycle, closely related to the biological clock. Subjective time appreciation tended to be overestimated without exhibiting a clear circadian component, but attributable to fatigue and stress. Psychometric evaluation showed that desynchronization affected all the pilots. Some results showed an age-related variability with a more marked influence in younger pilots. No consistent effects regarding flight direction were found.

Activity Cycles↗

PubMed2001

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.

Animals↗

PubMed2001

Circadian urinary 6-sulphatoxymelatonin, cortisol excretion and locomotor activity in airline pilots during transmeridian flights.

Airline pilots divided into two groups of age (over and under 50 years) were studied before, during and after westbound (Madrid-Mexico City-Madrid, n = 12) and eastbound (Madrid-Tokyo-Madrid, n = 21) flights. A group of 10 age-matched people staying in Madrid were submitted to the same tests and served as a control group. Changes in urinary 6-sulphatoxymelatonin (6-aMTs) and free cortisol excretion (determined in 6-hr intervals) were measured by radioimmunoassay. Using wrist actigraphy, the circadian locomotor activity rhythm (LAR) was also monitored. Maximal baseline excretion of 6-aMTs occurred between 00:00 and 12:00 hr and maximal excretion of cortisol took place between 6:00 and 12:00 hr in the control group. Analysed globally, older pilots exhibited significantly lower values of 6-aMTs than younger ones. In both flight directions, pilots maintained the pattern of excretion of 6-aMTs, corresponding to baseline. The return flight to Madrid from Mexico and Tokyo coincided with a maximum in 6-aMTs excretion. Pilots kept the cortisol pattern found in the control group, with those over 50 years of age exhibiting significantly lower cortisol values than the younger ones. A 7-hr delay in acrophase of LAR after 2 days in Mexico City was found after cosinor analysis, and similar pre-flight values were found after returning to Madrid. An 8-9-hr acrophase advance of LAR was observed after arriving in Tokyo, with acrophase on the post-return flight day still being advanced 3 4 hr as compared to pre-flight values. Decreases in the amplitude of LAR in older pilots were found at Mexico City, as well as at Tokyo stopover and on post-flight day. Data confirm the occurrence of internal desynchronization in airline crewmembers after transmeridian flights.

Adult↗

PubMed2001

Rhythmicity of the cGMP-related signal transduction pathway in the mammalian circadian system.

Entrainment of mammalian circadian rhythms requires the activation of specific signal transduction pathways in the suprachiasmatic nuclei (SCN). Pharmacological inhibition of kinases such as cGMP-dependent kinase (PKG) or Ca2+/calmodulin-dependent kinase, but not cAMP-dependent kinase, blocks the circadian responses to light in vivo. Here we show a diurnal and circadian rhythm of cGMP levels and PKG activity in the hamster SCN, with maximal values during the day or subjective day. This rhythm depends on phosphodiesterase but not on guanylyl cyclase activity. Five-minute light pulses increased cGMP levels at the end of the subjective night [circadian time 18 (CT18)], but not at CT13.5. Western blot analysis indicated that the PKG II isoform is the one present in the SCN. Inhibition of PKG or guanylyl cyclase in vivo significantly attenuated light-induced phase shifts at CT18 (after 5-min light pulses) but did not affect c-Fos expression in the SCN. These results suggest that cGMP and PKG are related to SCN responses to light and undergo diurnal and circadian changes.

1-Methyl-3-isobutylxanthine↗

PubMed2000

Delay model of the circadian pacemaker.

We present a simple and realistic model of the circadian pacemaker that can be interpreted in molecular terms. The model, which consists of a single time-delay differential equation, simulates the expression of a generic clock protein that inhibits its own expression through a feedback mechanism. Despite its simplicity, this model fulfils most of the necessary characteristics of a realistic representation of natural circadian clocks: robust and stable oscillations with circadian free-running periods, typical phase response curves and entrainment to environmental zeitgebers. The present model reduces the molecular mechanism necessary to sustain stable oscillations to its bare bones, suggesting that the essential factor is the time-delayed negative feedback of the oscillating protein on its own expression.

Animals↗

PubMed2000

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.

Animals↗

PubMed1999

Neuroactive steroids alter the circadian system of the Syrian hamster in a phase-dependent manner.

Several steroid compounds affect neuronal function, primarily by modulating the GABAA receptor complex. A circadian variation in the brain concentration of neurosteroids has been reported in rats and humans. We have previously reported that natural occurring or synthetic neuroactive steroids such as androsterone and alphaxalone also have a rhythmic effect on behavior (anesthetic and anticonvulsant activity) and GABAergic activity. In the present work, we have tested the ability of neuroactive steroids to phase shift circadian rhythms in hamsters. The GABA(A) negative modulator dehydroepiandrosterone sulphate (DHEAS) elicited phase advances when administered at CT 6, while the positive modulator androsterone lacked any effect at this time. A complete phase response curve for DHEAS revealed a nonphotic-like effect. DHEAS also blocked the circadian effects of light, while androsterone induced photic-like responses. There is also evidence that neurosteroids may be present and even synthesized in the SCN. Collectively, the results so far indicate that some neuroactive steroids might modulate the activity of the circadian clock.

Androsterone↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

Conservation of locomotor behavior in the golden hamster: effects of light cycle and a circadian period mutation.

Locomotor activity in rodents is restricted temporally by the animal' s circadian system. The relative stability of both the species-specific pattern and the amount of locomotor activity per cycle suggested that this behavior may be regulated by conservative mechanisms. In these experiments, the wheel-running behavior of golden hamsters carrying the circadian period mutation, tau, was analyzed in animals housed in a 24-h light:dark cycle (LD) and in constant dark (DD) conditions to determine which aspects of this behavior were conserved. In DD, apart from the change in period which defines the mutation, no main effects of allele combination were found in either average amount of activity, activity profile, or length of the activity phase. In LD, wild-type behavior did not differ from that in DD; however, heterozygous mutants exhibited early onsets of activity, significant fragmentation of both activity and rest, an increase in the duration of the active phase, and an overall decrease in the amount of activity. Despite these differences, the total amount of time spent on the wheel in LD or DD was the same for all environment/genotype combinations. The data show that a conservative mechanism that may influence daily patterns of locomotor behavior is related more to a drive to perform the behavior than the quantity or timing of the behavior itself.

Animals↗

PubMed1998

The rhythmic GABAergic system.

GABA is the major inhibitory neurotransmitter in the mammalian brain, and has been implicated in the regulation of a variety of behavioral functions, including biological rhythms. The focus of this minireview is the rhythmic variation of the central GABAergic system, comprising fluctuations of GABA levels and turnover, GABA receptor affinity and postsynaptic activity on the chloride ionophore in rodent's brain. Neurochemical rhythms correlated with diurnal and circadian changes in several behaviors associated with the GABA(A) receptor, e.g., anxiolysis-related behavior. GABA is considered to be the principal neurotransmitter of the mammalian circadian system, being present in the suprachiasmatic nuclei and the intergeniculate leaflet. Pharmacological manipulations of GABA(A) receptors phase shift circadian rhythms and alter circadian responses to light. Administration of putative modulators of GABA function, like melatonin or neuroactive steroids, affects the timing of biological rhythms. Therefore, not only does the GABAergic system exhibit strong diurnal and circadian variations, but it also serves as one of the key modulators of the circadian apparatus.

Animals↗

PubMed1997

Circadian phase shifts to neuropeptide Y In vitro: cellular communication and signal transduction.

Mammalian circadian rhythms originate in the hypothalamic suprachiasmatic nuclei (SCN), from which rhythmic neural activity can be recorded in vitro. Application of neurochemicals can reset this rhythm. Here we determine cellular correlates of the phase-shifting properties of neuropeptide Y (NPY) on the hamster circadian clock in vitro. Drug or control treatments were applied to hypothalamic slices containing the SCN on the first day in vitro. The firing rates of individual cells were sampled on the second day in vitro. Control slices exhibited a peak in firing rate in the middle of the day. Microdrop application of NPY to the SCN phase advanced the time of peak firing rate. This phase-shifting effect of NPY was not altered by block of sodium channels with tetrodotoxin or block of calcium channels with cadmium and nickel, consistent with a direct postsynaptic site of action. Pretreatment with the glutamate receptor antagonists (DL-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione disodium) also did not alter phase shifts to NPY. Blocking GABAA receptors with bicuculline (Bic) had effects only at very high (millimolar) doses of Bic, whereas blocking GABAB receptors did not alter effects of NPY. Phase shifts to NPY were blocked by pretreatment with inhibitors of protein kinase C (PKC), suggesting that PKC activation may be necessary for these effects. Bathing the slice in low Ca2+/high Mg2+ can block phase shifts to NPY, possibly via a depolarizing action. A depolarizing high K+ bath can also block NPY phase shifts. The results are consistent with direct action of NPY on pacemaker neurons, mediated through a signal transduction pathway that depends on activation of PKC.

2-Amino-5-phosphonovalerate↗

PubMed1997

Aging attenuates diurnal variation in hamster locomotion, anxiolysis and GABA turnover.

A decrease in amplitude of wheel running circadian rhythms was found in old (18 month old) Syrian hamsters, as compared with young (3 month old.) animals. In a plus-maze paradigm, amplitude of variation of anxiety-related variables (2400 vs. 1600 h) was significantly impaired in aged hamsters. Cerebral cortex, hypothalamic and pineal gamma-aminobutyric acid (GABA) turnover was higher at night, amplitude of variation being significantly smaller in aged hamsters. The results further support the existence of impaired amplitude of circadian rhythms in aged Syrian hamsters.

Age Factors↗

PubMed1997

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.

Animals↗

PubMed1997

Melatonin site and mechanism of action: single or multiple?

By affecting the entrainment pathways of the biologic clock, melatonin has a major influence on the circadian and seasonal organization of vertebrates. In addition, a number of versatile functions that far transcend melatonin actions on photoperiodic time measurement and circadian entrainment have emerged. Melatonin is a free radical scavenger and antioxidant and it has a significant immunomodulatory activity, being presumably a major factor in an organism's defense toxic agents and invading organisms. Besides affecting specific receptors in cell membranes to exert its effects, the interaction of melatonin with nuclear receptor sites and with intracellular proteins, like calmodulin or tubulin-associated proteins, as well as the direct antioxidant effects of melatonin, may explain many general functions of the pineal hormone.

Animals↗

PubMed1997

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.

Animals↗

PubMed1996

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.

Animals↗