Constant Dark Conditions Research Articles

Use of an intelligent CCD camera for the study of endogenous vertical migration rhythms in first zoeae of the crab Carcinus maenas

This paper describes an actographic system, based on video camera and image analysis software, which can detect small planktonic organisms and automatically determine their position in a bi-dimensional plane. This actograph was used to study swimming behaviour, under constant dark conditions in the laboratory, of the first zoea of the crab Carcinus maenas (L.), hatched from ovigerous females collected in the Ria de Aveiro, Portugal. An endogenous rhythm of vertical migration with a circatidal period was detected, in accordance with previous laboratory and field observations made on the species' first zoea from Wales and the Ria de Aveiro, respectively. The system also enabled the computation of dispersion indexes that supported the conclusion that crab zoeae use passive sinking during the descent phase of vertical migration. Mean swimming speed in the absence of light, irrespective of direction, was 0.0666 cm s–1. Vertical upward and downward components of swimming were 0.0437 and 0.0599 cm s–1, respectively. Although lower than values reported in the literature, these velocities are high enough to allow swimming throughout the water column of most estuaries during vertical migration in natural conditions at night.

Chicken pineal Cry genes: light-dependent up-regulation of cCry1 and cCry2 transcripts

Vertebrate cryptochrome homologs (CRYs) are negative regulators for the transcription/translation-based autoregulatory feedback loop of the circadian clock. In this study we identified two Cry genes in the chicken, cCry1 and cCry2, which are expressed in the pineal gland. Messenger RNA levels of both cCry1 and cCry2 displayed circadian oscillation in cultured pineal cells under light/dark and constant darkness conditions. Noticeably, their mRNA levels during the light period were significantly higher than those in the dark, indicating light-dependent up-regulation of the two Cry genes mediated by photoreceptor(s) intrinsic to the chick pineal cells. These cCRYs inhibited E-box element-dependent cBMAL1/2-cCLOCK-induced transcription, suggesting that the chick pineal circadian oscillator is composed of molecules that are functionally similar to those of mammals but are subject to light-regulation distinct from the mammalian clockwork.

Altered hormone levels and circadian rhythm of activity in the WKY rat, a putative animal model of depression.

The Wistar Kyoto (WKY) rat is hyperreactive to stress and exhibits depressive-like behavior in several standard behavioral tests. Because patients with depressive disorders often exhibit disruptions in the circadian rhythm of activity, as well as altered secretory patterns of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid hormones, we tested the hypothesis that these phenomena occur in the WKY rat. Plasma ACTH and corticosterone levels remained significantly higher after the diurnal peak for several hours in WKY rats relative to Wistar rats. Also, plasma levels of thyroid-stimulating hormone were significantly higher in WKY relative to Wistar rats across the 24-h period, despite normal or slightly higher levels of 3,5,3'-triiodothyronine. In addition, under constant darkness conditions, WKY rats exhibited a shorter free running period and a decreased response to a phase-delaying light pulse compared with Wistar rats. In several ways these results are similar to those seen in other animal models of depression as well as in depressed humans, suggesting that the WKY rat could be used to investigate the genetic basis for these abnormalities.

Rhythmic variation in γ-aminobutyric acid A-receptor subunit composition in the circadian system and median eminence of Syrian hamsters

Temporal changes in the level of expression of γ-aminobutyric acid (GABA) A receptor subunits α2, α5, β1 and β3 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 β1 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. α2 and β3 subunit levels peaked during the day in the median eminence. Finally, retinal α5 levels were maximal during the night. β1 temporal changes in the SCN and median eminence, as well as α2 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.

A null mutation in guanylate cyclase-1 alters the temporal dynamics and light entrainment properties of the iodopsin rhythm in cone photoreceptor cells

Guanylate cyclase-1 (GC1) plays a critical role in visual phototransduction and its absence severely compromises the ability of the photoreceptor cells to transduce light for vision. In this study we sought to determine if the absence of GC1 has any effect on light entrainment of the circadian oscillators located in these cells. We compared the rhythmic changes in transcript levels of iodopsin, a photoreceptor-specific gene whose expression is regulated by circadian oscillators, in retinas of normal chickens and GUCY1 ∗B ( ∗B) chickens that carry a null mutation in GC1. Our results show that iodopsin rhythms are present in ∗B retinas and that they can be entrained to light; however, the rise and fall of iodopsin transcript levels in ∗B retina under cyclic light conditions is significantly more rapid than that observed in normal retina, and under constant dark conditions, the phase of the iodopsin rhythm in ∗B retina is advanced by 6 h relative to that observed in normal retina. In addition, the rate of entrainment of the iodopsin rhythm in ∗B retina to a reversal of the light cycle is significantly slower than normal. The results of our study show that a functioning visual phototransduction cascade is not essential for light entrainment of the oscillators that drive the iodopsin rhythm in photoreceptor cells. We propose that the abnormal synthesis of cGMP in ∗B photoreceptors underlies the irregular iodopsin rhythms observed in post-hatch ∗B retina.

Light-induced phase-delay of the chicken pineal circadian clock is associated with the induction of cE4bp4, a potential transcriptional repressor of cPer2 gene.

The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. We searched for chicken pineal genes that are induced by light in a time-of-day-dependent manner, and isolated chicken homolog of bZIP transcription factor E4bp4 (cE4bp4) showing high similarity to vrille, one of the Drosophila clock genes. cE4bp4 was expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase was nearly opposite to the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays showed that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5'-flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies showed that the prolongation of the light period to early subjective night maintained the high level expression of the pineal cE4bp4, and presumably as a consequence delayed the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE4bp4 and cPer2 were followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene.

The locomotor activity rhythm of Drosophila melanogaster is controlled by a dual oscillator system

Wild-type fruit flies show a pronounced bimodal locomotor activity pattern under light–dark conditions. This pattern seems to be due to two oscillatory components — the morning and evening oscillators. Whereas the evening oscillator is controlled by the well studied clock gene period, the morning oscillator appears independent of the period gene. The present study shows that per 0 mutants retain the morning oscillator. The latter is apparently not strong enough to drive rhythmic activity under conditions of constant darkness. However, under light–dark conditions the activity of per 0 mutants is entrained whereby the onset of activity depends on the Zeitgeber period. This behaviour is characteristic of circadian oscillators. The nature of the morning oscillator and its putative relationship to the photoperiodic timing is discussed.

Constitutive expression and delayed light response of casein kinase Iepsilon and Idelta mRNAs in the mouse suprachiasmatic nucleus.

Casein kinase Iepsilon (CKIepsilon) and casein kinase Idelta (CKIdelta) phosphorylate clock oscillating mPER proteins, and play a key role in the transcription (post)translation feedback loop that generates circadian rhythm. In the present study, the expression profiles of CKIepsilon and CKIdelta mRNAs were examined in the mice clock center, suprachiasmatic nucleus (SCN). Moderate levels of CKIepsilon and CKIdelta mRNAs were constantly expressed in the SCN in both light:dark and constant dark conditions. This finding supports the hypothesis that CKI may form a constant threshold to the nuclear entry of mPER proteins as in the Drosophila homologue, double-time. Further, we demonstrated that the light exposure at subjective night induced a delayed increase in CKIepsilon and CKIdelta mRNAs in the SCN. CKIepsilon and CKIdelta proteins may play a role on light-induced phase-shift.

Light regulation of circadian clock-controlled gene expression in rice.

Using transgenic rice seedlings expressing a firefly luciferase (luc) gene under the control of a rice CAB (chlorophyll a/b binding protein) promoter, we demonstrated how light affects circadian clocks in the transcription of the CAB gene. Rhythmic luc expression was observed for more than 5 d under constant light and dark (DD) conditions after light/dark entrainment. After a light pulse was applied at different time points in DD various temporal patterns of CAB gene expression were individually observed. We first examined two distinct properties related to the entrainment mechanism of the circadian clock: fluence-rate dependence of free-running periods (FRPs) and phase resetting by a light pulse. Although fluence-rate dependent shortening of FRP was demonstrated, the FRP in DD was almost equal to that in constant light of a middle fluence-rate, indicating that this fluence-rate dependence may not fully describe the entrainment of the circadian clock in rice. Typical phase responses of the circadian clock by a single light pulse were also observed at the transcriptional level in rice seedlings. Thus, the phase resettings upon the light/dark transitions of daily cycles may be sufficient to explain the entrainment mechanisms of rice. We have further demonstrated that, in addition to having a gating effect to acute response, a light pulse can activate the circadian clock-controlled CAB1R gene expression at the first circadian peak in a phase-dependent manner. This suggests that light activates circadian clock activity in the diurnal CAB gene expression under daily light/dark cycles.

Circadian Clock Regulation of pH in the Rabbit Retina

Although it is generally accepted that the acid-base ratio of tissue, as represented by the pH, is strictly regulated to maintain normal function, recent studies in the mammalian nervous system have shown that neuronal activity can result in significant shifts in pH. In the mammalian retina, many cellular phenomena, including neuronal activity, are regulated by a circadian clock. We thus investigated whether a clock regulates retinal pH, using pH-sensitive microelectrodes to measure the extracellular pH (pH(o)) of the in vitro rabbit retina in the subjective day and night, that is, under conditions of constant darkness. These measurements demonstrated that a circadian clock regulates the pH(o) of the rabbit retina so that the pH(o) is lower at night than in the day. This day/night difference in retinal pH(o) was observed when the rabbits were maintained on a normal light/dark cycle and after they were maintained on a light/dark cycle that was phase-delayed by 9 hr. Continuous recordings of retinal pH(o) around subjective dusk indicated that the change from daytime to nighttime pH(o) is relatively fast and suggested that the clock that regulates pH(o) is located in the retina. The lowest pH(o) recorded in the retina in both the day and night was in the vicinity of the inner segments of photoreceptor cells, supporting the idea that photoreceptors serve as the primary source of protons. The circadian-induced shift in pH(o) was several times greater than light-induced pH(o) changes. These findings suggest that a circadian clock in the mammalian retina regulates retinal pH.

Circadian rhythm in NO synthase I transcript expression and its photoperiodic regulation in the rat pineal gland.

The photoneural regulation of nitric oxide synthase type I (NOS I) expression in the rat pineal was investigated using semiquantitative RT-PCR. NOS I transcript expression exhibited a daily rhythm with peak values during the night hours. The daily rhythm in NOS I transcript expression persisted under constant dark conditions and was abolished under constant light conditions. The extent of nocturnal NOS I expression was found to be dependent on the photoperiod. It was attenuated under 20 h light and 4 h dark (L:D 20:4) compared with 12 h light and 12 h dark (L:D 12:12). The present findings indicate that, in the rat pineal, NOS I transcript expression exhibits a true circadian rhythm. Further, photoperiod-induced changes in circadian transcript expression appear to be responsible for reported changes in NOS I protein expression.

Crayfish Procambarus clarkii shows circadian variations in different parameters of the GSH cycle.

The present study investigated the rhythmic changes in glutathione status in midgut gland and hemolymph as well as in glutathione reductase (GR) activity in the crayfish Procambarus clarkii. In order to determine the circadian nature of these rhythms different groups of crayfish were submitted to constant-darkness conditions for 24 or 72 h after they had spent 15 days under light-dark 12:12 cycles. The animals of the different batches were killed at 6 h intervals during a 24 h cycle. Reduced glutathione (GSH) and oxidized glutathione (GSSG) in hemolymph and midgut as well as midgut GR activity were determined in midgut gland and hemolymph by fluorometric and spectrophotometric method. Data analysis by chronogram and single Cosinor revealed circadian rhythmicity for GSH and GSSG concentration in both tissues as well as midgut GR activity. The rhythm parameters revealed oxidative stress induced by light. The possible correlation between the glutathione rhythm and other metabolic and behavioral rhythms of crayfish as well as the importance of the glutathione circadian temporal order in the adaptation of crayfish are discussed.

A Method for Harvesting and Shipping Live Citrus Rust Mites (Acari: Eriophyidae)

Field populations of citrus rust mite, Phyllocoptruta oleivora Ashmead in Florida have shown resistance to dicofol (Omoto et al. 1994) and shifts in susceptibility to abamectin (Bergh et al. 1999). Resistance monitoring of eriophyid mites has not been widely practiced, limiting the availability of reference strains and technical expertise. This study was prompted when a company expressed interest in testing the susceptibility of citrus rust mite populations from Texas to their acaricide. Under normal conditions, citrus rust mites do not live long off of the host (J. C. B., unpublished data), and importing them into Florida on citrus fruit or foliage requires quarantine. However, importing mites off of host plant tissue reduces the risk of introducing a new pest or plant pathogen and does not require their quarantine. Our objective was to develop methods for removing large numbers of citrus rust mites from their host and shipping them live to other locations. Toward this end, we capitalized on two aspects of their biology. First, mites disperse by leaping from the plant and are borne away on air currents (Bergh and McCoy 1997). This behavior also occurs on excised pieces of citrus leaf as the tissue deteriorates (Omoto et al. 1994). Second, rust mites in Florida are regularly submerged in rain or dew and can withstand extended periods of immersion, remaining motionless until dried (J. C. B., personal observation). Mites were harvested from 'Sunburst' mandarin leaves from seedlings maintained in a greenhouse at the Citrus Research and Education Center (CREC), Lake Alfred, FL. To examine the effect of temperature on the harvest of mites, heavily infested leaves were cut into 2 x 2 cm pieces, which were randomly assigned to 4 groups. Each piece of leaf was impaled on an insect pin and the pin was inserted into the side of a rubber stopper. Five stoppers with leaf pieces were placed in each of four translucent plastic boxes lined with wet paper towel. Each piece of leaf was positioned about 1.5 cm above the center of a filter paper disk (Whatman No. 50, 38 mm diam.) placed on the wet paper towel. The boxes were covered and placed in lighted environmental cabinets at 20, 25, 30, and 35?C. The filter paper disks were replaced at 2-h intervals from 09:00 to 17:00 hours, and the mites on each were counted using a dissecting microscope at 20x. The mean number of mites harvested on each of three days was compared among temperatures using PROC GLM of SAS (SAS Institute 1985) and the Tukey multiple range test at the 5% probability level. To examine the effect of light on the harvest of mites, rubber stoppers with impaled leaf pieces were placed in uncovered boxes in chambers set at 30?C, with bright, overhead lights and continuous darkness. The filter paper disks were replaced at 2-h intervals from 08:00-16:00 hours and the mites were counted as described above. The t-test was used to compare the mean number of mites harvested in constant light and dark conditions on each of three days.

Opioid induced non-photic phase shifts of hamster circadian activity rhythms

The phase of the circadian pacemaker in hamsters can be shifted by the application of certain non-photic stimuli late in the subjective day. A projection from the intergeniculate leaflet of the thalamus to the circadian pacemaker in the suprachiasmatic nucleus is believed to mediate some types of non-photic phase-shifting stimuli. In hamsters, this projection is immunoreactive to both Neuropeptide Y and enkephalin. Previous work in other laboratories has shown that Neuropeptide Y administration is capable of phase shifting circadian rhythms without the application of light. The present study was undertaken to determine if enkephalinergic compounds likewise have the ability to non-photically phase shift hamster activity rhythms. Hamsters were maintained under conditions of constant darkness and circadian wheel running activity was recorded. Agonists and antagonists selective for κ, μ, and δ opioid receptors were systemically applied without light to hamsters at circadian times 8 and 10 to determine if they were able to elicit phase shifts in wheel running activity rhythms. Of the compounds tested, only the delta opioid agonist BW373U86 significantly affected circadian phase. BW373U86 phase advanced hamster wheel running activity rhythms by approximately 45 min, although total activity levels following drug application were not significantly affected. Changes in the amount of wheel running activity were detected after administration of some μ and κ opioids, although the circadian phase was not altered. These results indicate that enkephalin-mimetic δ opioid agonists are capable of producing non-photic phase shifts in hamster activity rhythms, and that opioids can independently affect circadian phase and activity levels in hamsters.

Type II cAMP-dependent Protein Kinase-deficientDrosophila Are Viable but Show Developmental, Circadian, and Drug Response Phenotypes

We identified a unique type II cAMP-dependent protein kinase regulatory subunit (PKA-RII) gene in Drosophila melanogaster and a severely hypomorphic if not null mutation, pka-RII(EP(2)2162). Extracts from pka- RII(EP(2)2162) flies selectively lack RII-specific autophosphorylation activity and show significantly reduced cAMP binding activity, attributable to the loss of functional PKA-RII. pka-RII(EP(2)2162) shows 2-fold increased basal PKA activity and approximately 40% of normal cAMP-inducible PKA activity. pka-RII(EP(2)2162) is fully viable but displays abnormalities of ovarian development and multiple behavioral phenotypes including arrhythmic circadian locomotor activity, decreased sensitivity to ethanol and cocaine, and a lack of sensitization to repeated cocaine exposures. These findings implicate type II PKA activity in these processes in Drosophila and imply a common role for PKA signaling in regulating responsiveness to cocaine and alcohol.

Changes in circadian period and morphology of the hypothalamic suprachiasmatic nucleus in fyn kinase-deficient mice

Protein tyrosine phosphorylation is involved in intracellular signal transduction and plays important roles in various physiological events. To understand the role of Fyn, a non-receptor type tyrosine kinase of Src family kinases, in the mechanism of circadian rhythms, we analyzed the circadian locomotor behavior and morphology of the hypothalamic suprachiasmatic nucleus (SCN), a master circadian oscillator in Fyn mutant mice, because Fyn-like immunoreactive substance was observed in the SCN. Under constant dark (DD) condition the Fyn (−/−) mutant mice showed a free-running circadian rhythm, and the period of the circadian rhythm of the locomotor activity was significantly longer than that of the control mice. Fyn (−/−) mutant mice had abnormal distribution of neurons containing vasoactive intestinal polypeptide (VIP)-like immunoreactive substance in the SCN. These findings suggest that Fyn is involved in the mechanism of circadian oscillation and morphological formation of the SCN. The mechanism of the implication of Fyn discussed with the Fyn's roles in neural network formation and cellular signal transduction pathway.

Light-induced tyrosine phosphorylation of BIT in the rat suprachiasmatic nucleus.

Circadian changes of protein tyrosine phosphorylation in the hypothalamic suprachiasmatic nucleus have been studied using rats maintained under 12-h light/ 12-h dark cycles as well as constant dark conditions. We found that tyrosine phosphorylation of BIT (brain immunoglobulin-like molecule with tyrosine-based activation motifs), a transmembrane glycoprotein of 90-95 kDa, was higher in the light period than in the dark period and was increased after light exposure in the dark period. Similar changes in tyrosine phosphorylation were observed under constant dark conditions, but its amplitude was weaker than that in 12-h light/12-h dark cycles. As the tyrosine-phosphorylated form of BIT is able to bind to the Src homology 2 domain of a protein tyrosine phosphatase, SHP-2, we examined association of these proteins in suprachiasmatic nucleus extracts and found that SHP-2 was coprecipitated with BIT in parallel with its tyrosine phosphorylation. These results suggest that tyrosine phosphorylation of BIT might be involved in light-induced entrainment of the circadian clock.

Circadian rhythm of mt1 melatonin receptor expression in the suprachiasmatic nucleus of the C3H/HeN mouse.

This report studied the diurnal and circadian rhythms of mt1 melatonin receptor expression in the SCN of C3H/HeN mice maintained in either a light:dark (LD) cycle or in constant dark for a minimum of 6 wk. Diurnal times (ZT) were assessed with reference to the onset of the light period (ZT0) and circadian times (CT) were established by determining the phase of wheel running activity of each mouse before sacrifice. 2-[125I]-Iodomelatonin binding in the SCN revealed low amplitude diurnal and circadian rhythms with highest levels of binding 2 hr after lights on (41.3+/-1.7 fmol/mg protein, n = 5, at ZT2) or at the beginning of the subjective day (48.6+/-2.1 fmol/mg protein, n = 6, CT2), respectively. The expression of mt1 mRNA, determined by in situ hybridization with a 35S-labeled mouse mt1 riboprobe, showed robust diurnal and circadian rhythms. In animals housed under a LD cycle, low levels of expression were observed during the day, with a rapid rise in mt1 melatonin receptor expression at the beginning of the dark period (ZT14), coincident with an abrupt increase in levels of circulating melatonin measured by radioimmunoassay. In animals housed under constant dark conditions, a robust peak of mt1 mRNA expression occurred in the middle of the subjective night (CT18), 8 hr before the peak of protein expression, while the lowest levels of mt1 mRNA expression were observed during the day (CTI10). Results suggest that mt1 melatonin receptor rhythm in the C3H/HeN mouse SCN is regulated both by light and by the biological clock as distinct rhythms of both mRNA and protein are differentially expressed under a LD cycle and constant dark conditions.

Differential control of morning and evening components in the activity rhythm of Drosophila melanogaster--sex-specific differences suggest a different quality of activity.

The rhythms of locomotor activity of male and virgin or mated female flies were compared in the Drosophila melanogaster wild-type strains CantonS, Berlin, and OregonR. Under light-dark conditions, most flies showed a bimodal activity pattern with a morning peak around lights-on and an evening peak before lights-off. For all strains, a distinct sexual dimorphism was observed in the phase of the morning peak. Males had a significantly earlier morning peak than females and consequently a larger phase angle between morning and evening peak (psi(m, e)). Under constant dark conditions, the morning component merged with the evening component to a unimodal activity band in about half of the flies. In those flies who maintained bimodality, the sex-specific difference in psi(m, e) disappeared. Other sex-specific differences were now apparent: Males showed a shorter free-running period than females, and in two of the three strains, females were more active than males. Morning and evening components seem to contribute to the free-running period. Spontaneous or externally provoked change in psi(m, e) were correlated with period changes. In some flies, the morning and the evening components showed splitting, indicating that they are the output of two different oscillators. The sexual dimorphism in the phase of the morning peak under LD-conditions suggests that the function of activity during morning and evening peak might be different, for example, during the morning peak, males are active to find females. Overall, the results underline the multioscillatory nature of Drosophila's circadian system.

Impact of photoperiodic exposures during late gestation and lactation periods on the pineal and reproductive physiology of the Indian palm squirrel, Funambulus pennanti.

Studies on the maternal transfer of photoperiodic information in mammals indicate that the daily photoperiod perceived by the mother during the gestation-lactation period is communicated to the fetus either through the placenta or via the milk. However, the impact of photoperiodic exposures during gestation and lactation on the maternal pineal and reproductive physiology has not been reported for any tropical rodent. The exposure of pregnant female Indian palm squirrels (Funambulus pennanti) to constant light (24 h light:0 h dark), constant dark (0 h light:24 h dark), long daylength (14 h light:10 h dark) or short daylength (10 h light:14 h dark) during early gestation (< 30 days) resulted in the resorption of pregnancy, while during late gestation (> 30 days), it did not interfere with the maintenance of pregnancy. Alterations in photoperiodic condition during late gestation and lactation altered the postpartum recovery process. Pineal gland activity, as assessed by pineal mass, protein content and plasma melatonin, was lowest during the breeding phase, but increased gradually after parturition until the next breeding phase. During gestation and lactation, constant light, long daylength and short daylength conditions were less effective, while constant dark condition had a profound effect in depressing pineal gland activity, which subsequently advanced postpartum recovery. Hence, lactating females under constant darkness prepare themselves for next mating much earlier than females under natural daylength (12 h light:12 h dark) conditions. Therefore, photoperiodic information, mediated via the pineal gland, may be important for maintaining gestation physiology as well as postpartum recovery in female rodents.