Hr Light-dark Cycle Research Articles

Expression of circadian regulatory genes is dysregulated by increased cytokine production in mice subjected to concomitant intestinal injury and parenteral nutrition.

We have developed a mouse model of Parenteral Nutrition Associated Cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of bile acid and sterol signaling and transport. In the liver, the master circadian gene regulators Bmal/Arntl and Clock drive circadian modulation of hepatic functions, including bile acid synthesis. Once activated, Bmal and Clock are downregulated by several transcription factors including Reverbα (Nr1d1), Dbp (Dbp), Dec1/2 (Bhlhe40/41), Cry1/2 (Cry1/2) and Per1/2 (Per1/2). The aim of this study was to examine the effects of PN on expression of hepatic circadian rhythm (CR) regulatory genes in mice. WT, IL1KO or TNFRKO mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN) and the expression of key CR regulatory transcription factors evaluated. Animals were NPO on a 14 hr light-dark cycle and were administered PN continuously over 24 hrs. Mice were sacrificed, and hepatic tissue obtained at 9-10AM (Zeitgeber Z+3/Z+4 hrs). PNAC was defined by increased serum aspartate aminotransferase, alanine aminotransferase, total bile acids, and total bilirubin and the effect of i.p. injection of recombinant IL-1β (200ng/mouse) or TNFα (200ng/mouse) on CR expression was examined after 4 hrs. In the PNAC model, DSS-PN increased serum biomarkers of hepatic injury (ALT, AST, serum bile acids) which was suppressed in both DSS-PN IL1KO and DSS-PN TNFRKO mice. In WT DSS-PN, mRNA expression of Arntl and Dec1 was suppressed corresponding to increased Nr1d1, Per2, Dbp and Dec2. These effects were ameliorated in both DSS-PN IL1KO and DSS-PN TNFRKO groups. Western analysis of the circadian transcription factor network revealed in WT mice DSS-PN significantly suppressed Reverbα, Bmal, Dbp, Per2 and Mtnr1b. With the exception of Dbp, DSS-PN mediated suppression was ameliorated by both IL1KO and TNFRKO. Intraperitoneal injection of IL-1β or TNFα into WT mice increased serum AST and ALT and suppressed mRNA expression of Nr1d1, Arntl and Clock and increased Dbp and Per2. Altered expression of CR-dependent regulatory genes during PNAC accompanies cholestasis and is, in part, due to increased cytokine (IL-1β and TNFα) production. Evaluation of the effects of modulating CR in PNAC thus deserves further investigation.

Corrigendum

European Journal of NeuroscienceVolume 56, Issue 5 p. 4699-4699 CORRIGENDUMFree Access Corrigendum First published: 02 September 2022 https://doi.org/10.1111/ejn.15795AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In Fifield and Vanderluit (2020), in the Material and Methods, section 2.1 Animals, page 3197 it reads: “Fifty-nine male C57BL/6 mice between 3 and 6 months of age were housed and maintained on a 12:12 hr light–dark cycle with food and water provided ad libitum.” The correct information should read as follows: “Fifty-one mice between 3 and 9 months of age were included in this study. Forty-six mice were male C57BL/6, and five mice were FVBN, which included three females and two males”. The five FVBN (test) mice were inadvertently included in the 4-h and 3-day time points. The complete data set of all C57BL/6 and FVBN mice included in the study are in the supporting information. The authors sincerely apologise for these inaccuracies and any inconvenience this may have caused. Supporting Information Filename Description ejn15795-sup-0001-supporting information.xlsxExcel 2007 spreadsheet , 25.4 KB Data S1. Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCE Fifield, K. E., & Vanderluit, J. L. (2020). Rapid degeneration of neurons in the penumbra region following a small, focal ischemic stroke. European Journal of Neuroscience, 52(4), 3196– 3214. https://doi.org/10.1111/ejn.14678Wiley Online LibraryPubMedWeb of Science®Google Scholar Volume56, Issue5September 2022Pages 4699-4699 ReferencesRelatedInformation

Heterochronic opsin expression due to early light deprivation results in drastically shifted visual sensitivity in a cichlid fish: Possible role of thyroid hormone signaling.

During early ontogeny, visual opsin gene expression in cichlids is influenced by prevailing light regimen. Red light, for example, leads to an early switch from the expression of short-wavelength sensitive to long-wavelength sensitive opsins. Here, we address the influence of light deprivation on opsin expression. Individuals reared in constant darkness during the first 14 days post-hatching (dph) showed a general developmental delay compared with fish reared under a 12:12hr light-dark cycle (control group). Several characters including pigmentation patterns and eye development, appeared later in dark-reared individuals. Quantitative real-time PCR and fluorescent in situ hybridization at six time points during the 14 days period revealed that fish from the control group expressed opsin genes from 5dph on and maintained a short-wavelength sensitive phenotype (sws1, rh2b, and rh2a). Onset of opsin expression in dark-reared Midas cichlids was delayed by 4 days and visual sensitivity rapidly progressed toward a long-wavelength sensitive phenotype (sws2b, rh2a, and lws). Shifts in visual sensitivities toward longer wavelengths are mediated by thyroid hormone (TH) in many vertebrates. Compared to control fish, dark-reared individuals showed elevated dio3 expression levels - a validated proxy for TH concentration - suggesting higher circulating TH levels. Despite decelerated overall development, ontogeny of opsin gene expression was accelerated, resulting in retinae with long-wavelength shifted predicted sensitivities compared to light-reared individuals. Indirect evidence suggests that this was due to altered TH metabolism.

Effect of Blue and Green Light Emitting Diode (LED) on Rat's Retina.

Our eyes are increasingly exposed to light from the light emitting diode (LED) of video display terminals (VDT) which contain much blue light. VDTs are equipped with televisions, personal computers, and smartphones.; hence it is so important to know the implications of LED radiations on the human visual system. The present study is designed to evaluate the influence of blue and green LEDs on rat's retina. Forty-six female Wistar albino rats weighing 250-300 g classified into 3 groups. Group (1) served as control. Group (II) exposed to (461nm) blue LED (12 hr light-dark cycle) for 3, 7, 14 and 21days. Group (III) exposed to (525.9 nm) green LED (12 hr light-dark cycle) for 3, 7, 14 and 21days. At the end of each period, electroretinogram (ERG) and microscopic examination are carried out. The results indicated that there was no significant effect for the green LED on the ERG, and microscopic examination illustrates mild changes. After exposure of rat's retina to blue LED, there is a reduction in a- and b- wave amplitudes, and the histopathological examination illustrates severe changes. The data suggest that the blue LED may cause retinal toxicity. Therefore, it is very important to recognize the hazard of LED radiations that affects vision and take appropriate precautions for the eye safetyKeywords: Light emitting diodes, Electroretinogram, histopathological examination.

Aberrant Light-Induced Depression is Associated with Impaired Sensorimotor Gating in Mice

Mice subjected to an irregular light-dark cycle are known to lose their capacity to synchronize their behavioral rhythm to environmental light, and to show endophenotypes related to depressive disorders. Here we observed that a susceptible strain of mice (C3H/HeJ) subjected to an irregular 3.5 hr:3.5 hr light-dark cycle showed an enhanced acoustic startle reflex and deficits in prepulse inhibition. As impaired sensorimotor gating is associated with the onset of a variety of mental disorders such as schizophrenia and major depressive disorder, irregular environmental light without circadian photo-entrainment may cause stress that has the potential to be involved in humans’ susceptibility to neuropsychiatric abnormalities.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

Ticking in Place for the Microbiome to Message Out

In a recent issue of Cell, Thaiss etal. (2016) report that gut microbiota exhibit diurnal rhythmicity in biogeographical localization and metabolomics during the 24hr light-dark cycle. This oscillation, together with the host circadian clock, co-regulates the colonic and hepatic epigenome and transcriptome.

Altered Body Weight Regulation in CK1ε Null and tau Mutant Mice on Regular Chow and High Fat Diets.

Disruption of circadian rhythms results in metabolic dysfunction. Casein kinase 1 epsilon (CK1ε) is a canonical circadian clock gene. Null and tau mutations in CK1ε show distinct effects on circadian period. To investigate the role of CK1ε in body weight regulation under both regular chow (RC) and high fat (HF) diet conditions, we examined body weight on both RC and HF diets in CK1ε −/− and CK1ε tau/tau mice on a standard 24 hr light-dark (LD) cycle. Given the abnormal entrainment of CK1ε tau/tau mice on a 24 hr LD cycle, a separate set of CK1ε tau/tau mice were tested under both diet conditions on a 20 hr LD cycle, which more closely matches their endogenous period length. On the RC diet, both CK1ε −/− and CK1ε tau/tau mutants on a 24 hr LD cycle and CK1ε tau/tau mice on a 20 hr LD cycle exhibited significantly lower body weights, despite similar overall food intake and activity levels. On the HF diet, CK1ε tau/tau mice on a 20 hr LD cycle were protected against the development of HF diet-induced excess weight gain. These results provide additional evidence supporting a link between circadian rhythms and energy regulation at the genetic level, particularly highlighting CK1ε involved in the integration of circadian biology and metabolic physiology.

Modulation of MAP2 and Tau Following Alpha Lipoic Acid Supplementation Ameliorates Arsenic Induced Adverse Effects in Developing Rat Cerebellum

The present study was designed to evaluate the effects of ALA on arsenic (iAs) induced alterations in developing rat cerebellum. Pregnant Wistar rats, procured from Experimental Animal Facility after obtaining ethical clearance from Institutional Ethical Committee, were maintained on a 12 hr light‐dark cycle with ad libitum access to food and water. The day of birth of pups was designated as postnatal day (PND) 0. Mother reared pups were divided into control and experimental groups, the later receiving either NaAsO2 alone or along with ALA from PND 1 to 21. Behavioral parameters (cliff avoidance, tail hung, negative geotaxis, Rota‐rod) were carried out at different time points. Animals were sacrificed on PND 22 and the fresh cerebella were processed for estimation of levels of iAs, malondialdehyde (MDA) and glutathione (GSH). Cerebella from perfusion fixed animals were processed for cryo‐sectioning and immunohistochemical localization of Microtubule associated protein (MAP2) and Tau. The observations of present study revealed behavioral deficits in pups exposed to NaAsO2 alone. Co‐administration of ALA with iAs resulted in improved behavioral parameters, decrease in iAs and MDA levels, increase in GSH levels along with upregulation of MAP2 and Tau expression suggesting thereby protective role of ALA in NaAsO2 induced neurotoxicity.DST INSPIRE Fellowship, Govt. of India (110053) to Parul Kaushal is acknowledged.

Impact of photoperiod on circadian trehalose and trehalase rhythms in the digestive system of silkworm, Bombyx mori

Circadian trehalose and trehalase rhythms were studied in the digestive system of Bombyx mori under 12 hr light-dark cycle (LD), continuous light (LL) and continuous dark (DD). The rhythmic changes were interpreted as synthetic cycles in gut wall and release cycles in gut lumen. The trehalose rhythm of gut wall comprised 8 trehalose synthetic cycles (TS cycles) under LD and LL and 7 under DD. The 24 hr trehalose rhythm of LD and LL was clock shifted to 27.2 hr under DD. The trehalose rhythm included 4 TR cycles under LD, 5 under LL and DD in the gut lumen and the 24 hr rhythm of LD was clock shifted to 19.2 hr under LL and DD. In the gut wall trehalase rhythm maintained 8 trehalase enzyme synthetic cycles (TES cycles) under LD, 10 LL and 7 under DD and the 24 hr rhythm of LD was clock shifted to 19.2 hr under LL and 27.2 hr under DD. In the gut lumen it included 4 TER cycles under LD and DD, 5 under LL and its 24-hr rhythm was advanced to 19.2 hr. Further analysis of data showed that LD favours trehalose synthesis, while LL and DD favour trehalase synthesis.

Impact of photoperiod on circadian sucrose and sucrase rhythms in the digestive system of silkworm, Bombyx mori.

The impact of photoperiod on circadian sucrose and sucrase rhythms were analyzed in the digestive system of Bombyx mori under 12 hr light-dark cycle (LD), continuous light (LL) and continuous dark (DD). The rhythmic changes were interpreted as synthetic cycles in gut wall and release or uptake cycles in gut lumen. The gut wall comprised 6 sucrose synthetic cycles (SS cycles) under LD, LL and 5 under DD. The 24 hr rhythm of LD and LL was clock shifted to 28.8 hr under DD. In gut content, the sucrose rhythm showed 7 sucrose uptake cycles (SUcycles) under LD, 6 under LL and 5 under DD and the 24 hr rhythm of LD was clock shifted to 28.0 hr under LL and 34 hr under DD. In the gut wall sucrase rhythm maintained 7 SES cycles under LD and DD and 9 cycles under LL and its 24-hr rhythm is advanced to 18.2 hr. In the gut lumen 5 SER cycles under LD, 8 under LL and 6 under DD and its rhythm is advanced to 15 hr under LL and 20 hr under DD. Further analysis of data showed that LD favoured both synthesis and uptake of sucrose while LL, favoured the sucrase synthesis and its release.

Impact of Photoperiod on the Circadian Cellulose and Cellulase Rhythms in the Digestive System of Silkworm, Bombyx Mori

Circadian cellulose and cellulase rhythms were studied in the digestive system of Bombyx mori under 12 hr light-dark cycle (LD), continuous light (LL) and continuous dark (DD) conditions. Since, the symbiotic microbes are associated with cellulose digestion in the silkworm; both the rhythms are presumed to have microbial origin. The rhythms were analyzed in terms of phase response curves and interpreted as cellulose synthetic cycles (CS cycles) and cellulase enzyme synthetic cycles (CES cycles) in gut wall and cellulose uptake cycles (CU cycles) and cellulase enzyme release cycles (CER cycles) in gut lumen. The cellulose rhythm in the gut wall maintained 7 CS cycles each with a duration of 3.4 hr and the 24 hr free running time remained unchanged under altered photoperiodic conditions. In gut lumen, the rhythm showed 7 CU cycles, each with a duration of 3.4 hr under LD and 6 CU cycles of 4.0 hr each under LL and DD, with the result, the 24 hr free running period of the rhythm was clock shifted to 28 hr both under LL and DD. The cellulase rhythm in the gut wall maintained 7 CES cycles with a duration of 3.4 hr each under LD and DD and 9 cycles each with a duration of 2.7 hr under LL. The 24 hr free running time of the cellulase rhythm of LD and DD, was thus advanced to 19 hr under LL. The cellulase in gut lumen maintained 6 CER cycles with a duration of 4.0 hr each, under LD, LL and DD and hence the 24 hr free running time of the rhythm was not affected. Further analysis of data showed that cellulose synthesis was stimulated by light cues, while its uptake from gut lumen through enhanced microbial cellulase activity was favoured by both diet and light cues.

Simulation analysis for the effect of light-dark cycle on the entrainment in circadian rhythm.

Circadian rhythms of the living organisms are 24hr oscillations found in behavior, biochemistry and physiology. Under constant conditions, the rhythms continue with their intrinsic period length, which are rarely exact 24hr. In this paper, we examine the effects of light on the phase of the gene expression rhythms derived from the interacting feedback network of a few clock genes, taking advantage of a computer simulation with Cell Illustrator. The simulation results suggested that the interacting circadian feedback network at the molecular level is essential for phase dependence of the light effects, observed in mammalian behavior. Furthermore, the simulation reproduced the biological observations that the range of entrainment to shorter or longer than 24hr light-dark cycles is limited, centering around 24hr. Application of our model to inter-time zone flight successfully demonstrated that 6 to 7 days are required to recover from jet lag when traveling from Tokyo to New York.

The Drosophila Circadian Network Is a Seasonal Timer

Previous work in Drosophila has defined two populations of circadian brain neurons, morning cells (M-cells) and evening cells (E-cells), both of which keep circadian time and regulate morning and evening activity, respectively. It has long been speculated that a multiple oscillator circadian network in animals underlies the behavioral and physiological pattern variability caused by seasonal fluctuations of photoperiod. We have manipulated separately the circadian photoentrainment pathway within E- and M-cells and show that E-cells process light information and function as master clocks in the presence of light. M-cells in contrast need darkness to cycle autonomously and dominate the network. The results indicate that the network switches control between these two centers as a function of photoperiod. Together with the different entraining properties of the two clock centers, the results suggest that the functional organization of the network underlies the behavioral adjustment to variations in daylength and season.

Tyrosine Phosphorylation of cGMP-Gated Ion Channels Is under Circadian Control in Chick Retina Photoreceptors

To investigate the role of tyrosine phosphorylation in circadian regulation of cGMP-gated cation channels (CNGCs) of chicken cone photoreceptors. Chick retinas were studied on the second day of constant darkness (DD) after several days of entrainment to 12:12 hr light-dark (LD) cycles in vitro. Inside-out patch recordings were made during the subjective day and subjective night to quantify circadian changes in the sensitivity of CNGCs to activation by cGMP after treatment with various tyrosine kinase and tyrosine phosphatase inhibitors. Immunoprecipitation and immunoblot analysis were also used to examine tyrosine phosphorylation of CNGCs and closely associated proteins after separation by conventional and two-dimensional SDS-PAGE. Treatment with tyrosine kinase inhibitors caused a significant decrease in K(1/2) for cGMP activation of CNGCs in patches excised from cones during the subjective day, but had no effect on K(1/2) during the subjective night. Conversely, treatment with a tyrosine phosphatase inhibitor caused a significant increase in the K(1/2) of CNGCs in patches excised during the subjective night but had no effect on channel K(1/2) during the subjective day. Broad spectrum serine-threonine phosphatase inhibitors had no effect. An 85-kDa tyrosine polypeptide that coimmunoprecipitated with CNGC alpha-subunits was detectable at higher levels during the subjective day than during the subjective night. CNGC alpha-subunits were not tyrosine phosphorylated as a function of the time of day. Circadian control of cone CNGCs appears to entail elevated daytime tyrosine phosphorylation of an approximately 85-kDa auxiliary protein or another subunit of the CNGCs.

Selection for early and late adult emergence alters the rate of pre-adult development in Drosophila melanogaster

BackgroundCircadian clocks have been implicated in the regulation of pre-adult development of fruit flies Drosophila melanogaster. It is believed that faster clocks speed up development and slower clocks slow it down. We established three sets of D. melanogaster populations (early, control and late). The early and late populations were raised by selecting for flies that emerged either in the morning or in the evening under 12:12 hr light/dark (LD) cycles. After 75 generations of selection, the time course and waveform of the adult emergence and activity rhythms of the early and the late populations diverged from each other as well as from the controls. In this paper, we report the consequence of this selection on the rate of pre-adult development.ResultsWe assayed the pre-adult development time of the selected and control populations under 12:12 hr LD cycles and constant darkness (DD). Under LD cycles, the early populations develop faster than the controls, while the late populations develop slower than the controls. Although flies take longer to develop under DD than in LD, the relative differences between the mean development times of the selected and control populations remain unaltered in DD. In a separate experiment designed to investigate the effect of time of egg collection and experimental conditions on the duration of pre-adult stage, we assayed the development time of the selected and control populations by collecting eggs at different times of the day (morning and evening) and by assaying their pre-adult development time under constant light (LL), LD, and DD conditions. Irrespective of the time of egg collection and assay light regime, the late flies continue to develop slower than the early flies.ConclusionThe results of our study clearly indicate that selection on the timing of adult emergence alters the rate of pre-adult development in D. melanogaster. The timing of egg collection as well as assay light regime does not have any measurable effect on the relative differences between the developmental rates of the early and the late flies. Taken together these results appear to suggest that pleiotropic effects of clock genes mediate correlated changes in the timing of adult emergence and the rate of pre-adult development in D. melanogaster.

A Pharmacodynamic Turnover Model Capturing Asymmetric Circadian Baselines of Body Temperature, Heart Rate and Blood Pressure in Rats: Challenges in Terms of Tolerance and Animal-handling Effects

This study presents development and behaviour of a feedback turnover model that mimics asymmetric circadian oscillations of body temperature, blood pressure and heart rate in rats. The study also includes an application to drug-induced hypothermia, tolerance and handling effects. Data were collected inn normotensive Sprague-Dawley rats, housed at 25 degrees C with a 12:12 hr light dark cycle (light on at 06:00 am) and with free access of food and water. The model consisted of two intertwined parallel compartments which captured a free-running rhythm with a period close to but not exactly 24 hrs. The free-running rhythm was synchronised to exactly 24 hrs by the environmental timekeeper (12:12 hr light on/off cycle) in experimental settings. The baseline model was fitted to a standardised 24-hr period derived from mean data of six animals over a period of nine consecutive days. The first-order rate constants related to the turnover of the baseline temperature, alpha and beta, were 0.026 min(-1) (+/-5%) and 0.0037 min(-1) (+/-3%). The alpha and beta parameters are approximately 2/transition time between day and night and 2/night time, respectively. The day:night timekeeper g(t), reference point T(ref) and amplitude were 0.053(+/-2%), 37.3(+/-0.02%) and 3.3% (+/-2%), respectively. Simulations with the baseline model revealed stable oscillations (free-running rhythm) in the absence of the timekeeper. This temperature-time profile was then symmetric and had a smaller amplitude, with a slightly shorter period and less pronounced temperature shift as compared to the profile in the presence of an external Timekeeper. Fitting the model to 96 hr mean profiles of blood pressure and heart rate from 10 control animals demonstrated the usefulness of the model. Simulations of the integrated temperature model succeeded in mimicking other modes of administration such as oral dosing.

Recoverin Improves Rod-Mediated Vision by Enhancing Signal Transmission in the Mouse Retina

Vision in dim light requires that photons absorbed by rod photoreceptors evoke signals that reliably propagate through the retina. We investigated how a perturbation in rod physiology affects propagation of those signals in the retina and ultimately visual sensitivity. Recoverin is a protein in rods that prolongs phototransduction and enhances visual sensitivity. It is not present in neurons postsynaptic to rods, yet we found that light-evoked responses of rod bipolar and ganglion cells were shortened when measured in recoverin-deficient retinas. Unexpectedly, the effect of recoverin on postsynaptic signals could not be explained by its effect on phototransduction. Instead, it is an effect of recoverin downstream of phototransduction in rods that prolongs signal transmission and enhances visual sensitivity. An important implication of our findings is that the recovery phase of the rod photoresponse does not contribute significantly to visual sensitivity near absolute threshold.

Forced Desynchronization of Dual Circadian Oscillators within the Rat Suprachiasmatic Nucleus

The circadian clock in the suprachiasmatic nucleus of the hypothalamus (SCN) [1] contains multiple autonomous single-cell circadian oscillators [2] and their basic intracellular oscillatory mechanism is beginning to be identified [3]. Less well understood is how individual SCN cells create an integrated tissue pacemaker that produces a coherent read-out to the rest of the organism. Intercellular coupling mechanisms must coordinate individual cellular periods to generate the averaged, genotype-specific circadian period of whole animals [4, 5]. To noninvasively dissociate this circadian oscillatory network in vivo, we (T.C. and A.D.-N.) have developed an experimental paradigm that exposes animals to exotic light-dark (LD) cycles with periods close to the limits of circadian entrainment [6]. If individual oscillators with different periods are loosely coupled within the network, perhaps some of them would be synchronized to the external cycle while others remain unentrained. In fact, rats exposed to an artificially short 22 hr LD cycle express two stable circadian motor activity rhythms with different period lengths in individual animals. Our analysis of SCN gene expression under such conditions suggests that these two motor activity rhythms reflect the separate activities of two oscillators in the anatomically defined ventrolateral and dorsomedial SCN subdivisions. Our “forced desychronization” protocol has allowed the first stable separation of these two regional oscillators in vivo, correlating their activities to distinct behavioral outputs, and providing a powerful approach for understanding SCN tissue organization and signaling mechanisms in behaving animals.

Entrainment of the Circatidal Swimming Activity Rhythm in the Cumacean Dimorphostylis asiatica (Crustacea) to 12.5-hour Hydrostatic Pressure Cycles

The cumacean Dimorphostylis asiatica (Crustacea) exhibits a circatidal swimming activity rhythm. The animals were exposed to a 12.5 hr sinusoidal change of hydrostatic pressure of 0.3 atm amplitude in the laboratory. Under constant dark conditions, most of the specimens were entrained to a daily bimodal swimming activity rhythm by the hydrostatic pressure cycle. A small number of individuals exhibited a unimodal daily rhythm, with no apparent entraining from the administered cycles. A marked feature was a flexible phase relationship between the entrained daily bimodal rhythm and the hydrostatic pressure cycles: the swimming activity of most of the specimens occurred around the pressure-decreasing phase, but for a small number of individuals it coincided with the pressure-increasing phase. Such flexibility suggests a weak entraining effect of hydrostatic pressure on the circatidal rhythm of this species. When exposed to 24 hr light-dark cycles and a hydrostatic pressure cycle simultaneously, the specimens exhibited a rhythmic activity entrained by the hydrostatic pressure cycle during the dark period, which closely resembles the temporal activity pattern of this species in the field. The light cycles entrained the swimming activity via direct inhibition and induction of activity (i.e., masking). Under light-dark conditions, the specimens exhibited activity on the pressure-increasing phase more frequently compared with specimens kept in constant darkness.