Constant Dark Conditions Research Articles

Daily oscillation of phospholipase C β4 in the mouse suprachiasmatic nucleus

An endogenous biological clock located in the hypothalamic suprachiasmatic nucleus (SCN) regulates the timing of an organism's physiology and behavior. A variety of receptors are found on SCN pacemaker cells which permit the clock mechanism to respond to extra- and intra-SCN chemical messengers. A subset of these receptors is coupled to G-proteins, which when bound, lead to the activation of a variety of intracellular signaling cascades. One common signaling pathway employs the phosphotidylinositol-specific phospholipase C enzyme to increase intracellular calcium levels. A specific isoform of this enzyme, phospholipase C β4, is of particular interest to circadian biologists because in its absence, mice display a circadian phenotype. Moreover, it has been shown to be associated with receptor types that are involved in clock resetting. Despite compelling data that this enzyme could be a critical component of an intracellular signaling pathway in the SCN, no study to date has investigated the possible oscillation of phospholipase C in any mammalian tissue. In the present study, we analyzed the temporal variation in the number of phospholipase C β4 immunoreactive cells in the SCN. Herein, we show that PLCβ4 levels oscillate in the SCN of mice housed in a light:dark photoperiod. Protein levels reached a significant peak during the early night and a trough during the day. The oscillation was considerably damped in the SCN of mice housed in constant dark conditions indicating the cycle is photoperiod-dependent. These data are critical to understanding the temporal regulation of a variety of inputs to the mammalian central circadian clock.

Diurnal regulation of scent emission in rose flowers

Previous studies have shown diurnal oscillation of scent emission in rose flowers with a peak during the day (Helsper in Planta 207:88-95, 1998; Picone in Planta 219:468-478, 2004). Here, we studied the regulation of scent production and emission in Rosa hybrida cv. Fragrant Cloud during the daily cycle and focused on two terpenoid compounds, germacrene D and geranyl acetate, whose biosynthetic genes have been characterized by us previously. The emission of geranyl acetate oscillated during the daily light/dark cycle with a peak early in the light period. A similar daily fluctuation was found in the endogenous level of this compound and in the expression of its biosynthetic gene, alcohol acetyl transferase (RhAAT). The rhythmic expression of RhAAT continued under conditions of constant light or darkness, indicating regulation by the endogenous circadian clock. However, the accumulation and emission of geranyl acetate ceased under continuous light. Our results suggest that geranyl acetate production is limited by the level of its substrate geraniol, which is suppressed under constant light conditions. The emission of germacrene D also oscillated during the daily cycle with a peak early in the light period. However, the endogenous level of this compound and the expression of its biosynthetic gene germacrene D synthase (RhGDS) were constant throughout the day. The diurnal oscillation of germacrene D emission ceased under continuous light, suggesting direct regulation by light. Our results demonstrate the complexity of the diurnal regulation of scent emission: although the daily emission of most scent compounds is synchronized, various independently evolved mechanisms control the production, accumulation and release of different volatiles.

Effects of Unilateral Compound-Eye Removal on the Photoperiodic Responses of Nymphal Development in the Cricket Modicogryllus siamensis

The cricket, Modicogryllus siamensis, shows clear photoperiodic responses at 25 degrees C in nymphal development. Under long-day conditions (LD16:8), nymphs became adults about 50 days after hatching, while under short-day conditions (LD8:16) the duration of nymphal stage extended to more than 130 days. Under constant dark conditions, two developmental patterns were observed: about 60% of crickets became adults slightly slower than under the long-day conditions, and the rest at later than 100 days after hatching, like those under the short-day conditions. When the compound eye was unilaterally removed on the 2nd day of hatching, an increase of molting and an extension of the nymphal period were observed under the long-day conditions, while under the short-day conditions, some crickets developed faster and others slower than intact crickets. These results suggest that this cricket receives photoperiodic information through the compound eye, that a pair of the compound eyes is required for a complete photoperiodic response, and that interaction between bilateral circadian clocks may be also involved in the response.

Daily rhythm of aggregation in the haematophagous bug Triatoma infestans (Heteroptera: Reduviidae)

Triatomine bugs show a temporal modulation of many activities. Here, we analyse the daily modulation of the aggregation behaviour of Triatoma infestans larvae and its chronobiological basis. In the laboratory, groups of six bugs were released over an experimental arena during six consecutive days, where their aggregation behaviour was quantified every hour. When submitted to a 12/12 h photoperiod (L/D), the larvae of T. infestans exhibited a cyclic pattern of aggregation with a 24 h period, evincing the existence of a daily rhythm of aggregation in this species. Bugs exhibited the maximum aggregation tendency at the end of the scotophase (7:00 h), moment in which they naturally search for refuges. The minimum aggregation (i.e. maximal dispersion) was observed during the last part of the photophase and beginning of the scotophase (15:00 to 1:00 h). This cyclic pattern disappeared when constant conditions of illumination (L/L) or darkness (D/D) were imposed to the bugs, suggesting the absence of an endogenous circadian control of this behaviour. Insects submitted to L/L and D/D photoperiods presented lower global levels of aggregation than those submitted to L/D conditions. The lack of an endogenous control and the relevance of light cycles as a synchronization signal are discussed as the temporal modulation of this behaviour might play an important role in the nocturnal habits of this species.

Constant light housing during nursing causes human DSPS (delayed sleep phase syndrome) behaviour in Clock‐mutant mice

Delayed sleep phase syndrome (DSPS) is very often seen among patients with sleep-wake rhythm disorders. Humans with the 3111C allele of the human Clock gene tend to demonstrate a higher evening preference on the morningness-eveningness (ME) preference test. DSPS is thought to be an extreme form of this evening preference. Clock-mutant mice have been proposed as an animal model of evening preference. In this study, we looked at whether constant light (LL) housing of Clock-mutant mice during lactation would result in evening preference and/or DSPS. Housed under light-dark (LD) or constant dark (DD) conditions during the lactation period, both wild-type and Clock-mutant mice did not show a phase-delay in the locomotor activity measured under light-dark conditions, whereas constant light housing during lactation significantly caused a delayed onset. The magnitude of the delay during the light-dark cycle was positively associated with free-running period measured during constant darkness. Among wild, heterozygote, and homozygote pups born from heterozygous dams, only homozygote pups showed a delayed onset. Constant light-housed Clock-mutant mice exhibited a lower number and delayed peak of phospho-MAPK-immunoreactive cells in core regions of the suprachiasmatic nucleus (SCN) compared to light-dark housed wild-type or Clock-mutant mice. Activity onset returned to normal with daily melatonin injection at the lights-off time for 5 days. The present results demonstrate that Clock-mutant mice exposed to constant light during lactation can function as an animal model of DSPS and can be used to gain an understanding of the ethological aspects of DSPS as well as to find medication for its treatment.

Elevated rhythmic Ras activity in the suprachiasmatic nucleus of synRas transgenic mice: implications for the regulation of the mammalian circadian clock

Light is the main phase-adjusting stimulus of the circadian clock located in the suprachiasmatic nucleus (SCN). A candidate pathway transmitting photic information at the postsynaptic site in the SCN is the extracellular signalregulated kinase (ERK 1/2) which has been previously shown to be an essential element in the photoentrainment of the circadian rhythm. An upstream activator of the ERK signalling route is the small intracellular GTPase Ras. Here we observed that endogenous Ras activity in the SCN was subjected to rhythmic changes, reaching maximum levels at the late subjective day and minimum levels at the late subjective night (CT22). In order to investigate if Ras would modulate the circadian cycle, we used transgenic mice expressing constitutively activated Val-12 HaRas selectively in neurons (synRas mice). In these mice Ras activity was also cycling during the circadian rhythm yet, Ras activities were up-regulated at each time point measured. We investigated if this change in Ras activity translates into a behavioral phenotype by monitoring free-running activity rhythms under conditions of constant darkness. SynRas mice exhibited circadian rhythms in locomotor activities similar to WT mice. However, when challenged by applying a 15 minutes light pulse at CT22 to promote phase advance shifts, synRas mice were completely non-responsive. As a first step towards the possible intracellular mechanism of this behavioral change we analyzed ERK1/2 activities in more details: We found a 1,7-fold increase of circadian peak levels of ERK 1/2 activities at CT10 and CT14 in synRas mice, while at minimum levels (CT18, CT22) no differences were found between ERK1/2 activities of WT and synRas mice. In WT animals the 15 minutes light pulse at CT22 resulted in rapid up regulations of Ras, ERK1/2 and CREB activities as described previously by others. However, in correlation with the lack of a behavioral response, ERK1/2 but not Ras and CREB activities remained unchanged in synRas mice, suggesting that Ras-dependent and Ras-independent pathways may co-exist to regulate ERK1/2 and behavioral phase shifts in response to the acute light treatment.

Altered Circadian and Homeostatic Sleep Regulation in Prokineticin 2-Deficient Mice

Sleep is regulated by circadian and homeostatic processes. Recent studies with mutant mice have indicated that circadian-related genes regulate sleep amount, as well as the timing of sleep. Thus a direct link between circadian and homeostatic regulation of sleep may exist, at least at the molecular level. Prokineticin 2 (PK2), which oscillates daily with high amplitude in the suprachiasmatic nuclei (SCN), has been postulated to be an SCN output molecule. In particular, mice lacking the PK2 gene (PK2-/-) have been shown to display significantly reduced rhythmicity for a variety of circadian physiological and behavioral parameters. We investigated the role of PK2 in sleep regulation. EEG/EMG sleep-wake patterns were recorded in PK2-/- mice and their wild-type littermate controls under baseline and challenged conditions. PK2-/- mice exhibited reduced total sleep time under entrained light-dark and constant darkness conditions. The reduced sleep time in PK2-/- mice occurred predominantly during the light period and was entirely due to a decrease in non-rapid eye movement (NREM) sleep time. However, PK2-/- mice showed increased rapid eye movement (REM) sleep time in both light and dark periods. After sleep deprivation, compensatory rebound in NREM sleep, REM sleep, and EEG delta power was attenuated in PK2-/- mice. In addition, PK2-/- mice had an impaired response to sleep disturbance caused by cage change in the light phase. These results indicate that PK2 plays roles in both circadian and homeostatic regulation of sleep. PK2 may also be involved in maintaining the awake state in the presence of behavioral challenges.

Melanopsin changes in neonatal albino rat independent of rods and cones

Intrinsically photosensitive retinal ganglion cells employ the photopigment melanopsin and provide light information to brain areas responsible for the regulation of circadian rhythms. The expression of melanopsin is regulated by environmental illumination, but it remains to be clarified whether the rods and cones are involved. Here, we examined the influence of 5 days of constant light and dark conditions on melanopsin mRNA and protein expression in newborn albino rats, in which functional rods and cones have not yet been developed. We found that the melanopsin mRNA level was unaffected, whereas the melanopsin protein level was more than two-fold higher in the darkness-adapted group than in pups raised in constant light. In pups raised during 12 : 12 h light/dark cycles, the melanopsin protein level was significantly higher during the day than at night. Our findings indicate that melanopsin protein changes are independent of input from the rods and cones.

An automated tracking system for Caenorhabditis elegans locomotor behavior and circadian studies application

Automation of simple behavioral patterns, such as locomotor activity, is fundamental for pharmacological and genetic screening studies. Recently, circadian behaviors in locomotor activity and stress responses were reported in the nematode Caenorhabditis elegans, a well-known model in genetics and developmental studies. Here we present a new method for long-term recordings of C. elegans (as well as other similar-sized animals) locomotor activity based on an infrared microbeam scattering. Individual nematodes were cultured in a 96-well microtiter plate; we tested L15, CeMM and E. coli liquid cultures in long-term activity tracking experiments, and found CeMM to be the optimal medium. Treatment with 0.2% azide caused an immediate decrease in locomotor activity as recorded with our system. In addition to the validation of the method (including hardware and software details), we report its application in chronobiological studies. Circadian rhythms in animals entrained to light–dark and constant dark conditions ( n = 48 and 96 worms, respectively) at 16 °C, were analyzed by LS periodograms. We obtained a 24.2 ± 0.44 h period (52% of significantly rhythmic animals) in LD, and a 23.1 ± 0.40 h period (37.5% of significantly rhythmic animals) under DD. The system is automateable using microcontrollers, of low-cost construction and highly reproducible.

THE ACTIVITY RHYTHM OF BERRIED AND UNBERRIED FEMALES OF NEPHROPS NORVEGICUS (DECAPODA, NEPHROPIDAE)

Females of the Norway lobster, Nephrops norvegicus are usually excluded from behavioural research based on the assumption that their reproductive cycle alters the results. A seasonal fluctuation in the sex ratio of commercial catches suggests that females do not emerge from their burrows when berried. Burrow emergence (i.e., catches) and feeding rhythms of berried and unberried females were compared with data on their endogenous locomotor activity rhythm. Catch data and the percentage of empty stomachs already published for a population dwelling on the continental slope (400-430 m) in the western Mediterranean were reprocessed focusing on data for berried and unberried females in October, a month that falls within the breeding season of N. norvegicus in the study area. Catches for both groups significantly increased at daytime, when both displayed the lowest percentages of empty stomachs (i.e., diurnal feeding pattern). To assess the occurrence of alterations in endogenous locomotor patterns depending on the presence of eggs, locomotor rhythms of berried and unberried females were studied under conditions of constant darkness in the laboratory. All individuals showed endogenous circadian and nocturnal locomotion. Taken together, these data suggest that feeding, burrow emergence, and endogenous locomotion rhythms are not inhibited in berried females. A reduction in the duration and range of emergence (i.e., doorkeeping) is hypothesized as the basis for the reported fluctuations in the sex ratio in commercial catches.

Endothelial Nitric Oxide Is Not Involved in Circadian Rhythm Generation of Blood Pressure: Experiments in Wild‐Type C57 and eNOS Knock‐Out Mice under Light‐Dark and Free‐Run Conditions

Endothelial nitric oxide synthase knock out mice (eNOS‐/‐) are mildly hypertensive in comparison to wild‐type (WT) mice. Hypertension in eNOS‐/‐ mice is partly the result of an increase in peripheral resistance due to the absence of the vasodilatory action of NO. No data are available for these animals regarding the 24 h blood pressure profile under the 12:12 h light‐dark cycle (LD) and constant dark (DD) conditions. Therefore, this study aimed to investigate by radiotelemetry the circadian rhythms in systolic blood pressure (SBP) and diastolic blood pressure (DBP) of six eNOS‐/‐ mice and five wild‐type mice under LD and DD. Data were collected beginning 3 wks after operation (implantation of sensor) for 2 wks under LD and for another 2 wks thereafter under DD. Our results show that eNOS‐/‐ mice were hypertensive under all experimental conditions. SBP and DBP were significantly higher by about 15% in eNOS‐/‐ mice. No differences were found in the pattern of the circadian rhythms, rhythmicity, or period lengths during LD or DD. The genetic deletion of eNOS seems to lead to higher SBP and DBP, but the circadian blood pressure pattern is still preserved with higher values during the night (active phase) and lower values during the daytime (rest phase). Thus, endothelial‐derived NO plays an important role in the regulation of vascular tone and haemodynamics, but it is not important for the circadian organization of SBP and DBP.

Circadian rhythmicity during prolonged chemostat cultivation of Neurospora crassa

Following exposure to light and attainment of steady-state in the chemostat, Neurospora was grown in constant conditions of darkness at 25 °C for 6 days. Biomass samples were taken every 4 h for the extraction of RNA and protein, and the state of the circadian clock was assessed by assaying the levels of three rhythmically expressed mRNAs; frequency ( frq), antisense frq ( qrf) and clock-controlled gene-14 ( ccg-14), and by monitoring the clock-controlled rhythm of sporulation. Our results indicate that the Neurospora clock continued to run in the chemostat. This is the longest reported time that Neurospora has been grown in a chemostat in filamentous form and opens up the possibility of studying the response of Neurospora to a range of stimuli in the absence of confounding effects due to; alterations in growth rate, aging, and changing conditions of the growth medium.

Circadian rhythm of locomotor activity in the Japanese honeybee, Apis cerana japonica

Abstract To reveal circadian characteristics and entrainment mechanisms in the Japanese honeybee Apis cerana japonica, the locomotor‐activity rhythm of foragers is investigated under programmed light and temperature conditions. After entrainment to an LD 12 : 12 h photoperiodic regime, free‐running rhythms are released in constant dark (DD) or light (LL) conditions with different free‐running periods. Under the LD 12 : 12 h regime, activity offset occurs approximately 0.4 h after lights‐off transition, assigned to circadian time (Ct) 12.4 h. The phase of activity onset, peak and offset, and activity duration depends on the photoperiodic regimes. The circadian rhythm can be entrained to a 24‐h period by exposure to submultiple cycles of LD 6 : 6 h, as if the locomotive rhythm is entrained to LD 18 : 6 h. Phase shifts of delay and advance are observed when perturbing single light pulses are presented during free‐running under DD conditions. Temperature compensation of the free‐running period is demonstrated under DD and LL conditions. Steady‐state entrainment of the locomotor rhythm is achieved with square‐wave temperature cycles of 10 °C amplitude, but a 5 °C amplitude fails to entrain.

Germination and seedling growth requirements for propagation of Dioscorea dregeana (Kunth) Dur. and Schinz — A tuberous medicinal plant

Intensive collection of underground tubers of the medicinal plant Dioscorea dregeana from indigenous forests is a threat to the wild population. Domestication of this plant is the only possible alternative to fulfill the demand of formal and informal medicinal markets in South Africa. This study reports fundamental requirements for seed germination and seedling growth of D. dregeana. Germination responses of seeds were tested at different temperature regimes, light conditions and smoke treatments. The highest percentage germination (≥ 95%) and mean germination time (MGT) of 10 days was at 30/15 °C, followed by 25 °C. No germination was observed at 10 °C. Different light conditions did not significantly affect percentage germination at 25 °C; however, under constant dark conditions the MGT was reduced. Smoke-water (1:500 v/v) and a butenolide (10 − 7 M), isolated from smoke, stimulated germination and improved seedling vigour. Seedling growth was best at 25 °C and 30/15 °C, with large underground tubers forming on all seedlings. This indicates that temperature plays a significant role in regulating growth of tubers. Seedlings watered once a week with half-strength Hoagland's nutrient solution showed best growth performance, whereas, a deficiency of either nitrogen, phosphorous or potassium negatively affected seedling growth. Thus, we recommend application of moderate fertilizers and watering once per week at 25 °C for raising healthy D. dregeana seedlings.

Germination and post-germination response of Acacia seeds to smoke-water and butenolide, a smoke-derived compound

Fire plays a critical role in breaking hard-seeded dormancy and establishing seedlings of several Acacia species in arid and semi-arid regions. Numerous studies have reported an increase in seedling densities of some Acacias after fire without identifying the exact cause. However, it is generally believed and speculated that the effect is largely physical and heat related. Recent studies have revealed that smoke generated from wildfires has the ability to stimulate seed germination and also to improve seedling vigour. This stimulatory characteristic of smoke has now been confirmed by the isolation of a butenolide (3-methyl-2 H-furo[2,3- c]pyran-2-one) from plant smoke that actively promotes germination of many plant species. The species of Acacia investigated were A. hebeclada (deciduous shrub), A. mearnsii (invasive tree, native to Australia) and A. robusta (deciduous tree). Seeds of A. hebeclada germinated under different light conditions with smoke-derived butenolide solution (10 −7 M), exhibited a significantly ( p<0.05) greater germination percentage than untreated seeds. Whereas A. mearnsii seeds exposed to constant dark conditions showed a significantly ( p<0.05) better germination percentage than the control. However, there was a non-significant improvement for A. robusta seeds. All three species responded positively to the butenolide treatment (10 −7 M) after incubating for 10 days under constant dark conditions at 25±0.5 °C, achieving a higher vigour index and seedling mass in comparison to the controls. Smoke-water (1:500) had an intermediate effect on these species. This study shows that the butenolide, isolated from smoke, may have a significant effect on the post-fire seedling ecology of Acacias.

Clock, love and memory: Circadian and non-circadian regulation of Drosophila mating behavior by clock genes

Mating behavior is arguably the most important and fundamental behavior in animals. In the fruit fly, Drosophila melanogaster, both experience-dependent and independent aspects of mating behavior are regulated by the genes controlling circadian rhythms. Wild-type D. melanogaster show robust circadian rhythms in their mating activity, with the lowest point at zeitgeber time 12. These rhythms are intact under constant dark conditions and are abolished in mutants for the clock genes, period (per) and timeless (tim), indicating that the mating activity of D. melanogaster is under the control of an endogenous clock. Females are solely responsible for these rhythms, because the rhythms are lost when clock mutant females are paired with wild-type males, but not vice versa. Although the circadian clock might not regulate innate male courtship behavior, its plasticity is controlled by a clock gene, per. A male fly previously paired with a mated female that releases courtship-inhibiting cues subsequently reduces his courtship activities toward a virgin female. This experience-dependent depression of male courtship is based on associative learning, which can induce long-term memory (LTM) lasting at least five days. The courtship LTM is not under the influence of a circadian clock or most of the clock genes. Interestingly, however, mutants for per, whose expression is controlled by the cAMP responsible transcriptional factor, CREB, are defective in LTM, suggesting that per plays a key role in LTM formation, independent of the core oscillator of the circadian clock. Thus, clock genes are involved in both innate mating behavior and its plasticity.

Genome-wide screenings for circadian clock genes in Drosophila

A high-density oligonucleotide probe array (GeneChip) has been used to learn how gene expression is globally regulated by the circadian clock mechanism. Here I review five articles, including our own, describing the genome-wide identification of rhythmically expressed genes by a similar strategy using GeneChip in Drosophila under light-dark (LD) and constant dark (DD) conditions. These studies drew common conclusions in some aspects. A number of genes were cyclically expressed in fly heads and they were involved in diverse functions, suggesting the entire complexity of circadian gene expression in Drosophila. In addition, a global effect of clock mutations, ClkJrk, per0 and tim0, on gene expressions was commonly observed, implying that these clock genes are associated with the transcriptional regulation of a large number of genes.

A Model for the Circadian Rhythm of Cyanobacteria that Maintains Oscillation without Gene Expression

An intriguing property of the cyanobacterial circadian clock is that endogenous rhythm persists when protein abundances are kept constant either in the presence of translation and transcription inhibitors or in the constant dark condition. Here we propose a regulatory mechanism of KaiC phosphorylation for the generation of circadian oscillations in cyanobacteria. In the model, clock proteins KaiA and KaiB are assumed to have multiple states, regulating the KaiC phosphorylation process. The model can explain 1), the sustained oscillation of gene expression and protein abundance when the expression of the kaiBC gene is regulated by KaiC protein, and 2), the sustained oscillation of phosphorylated KaiC when transcription and translation processes are inhibited and total protein abundance is fixed. Results of this work suggest that KaiA and KaiB strengthen the nonlinearity of KaiC phosphorylation, thereby promoting the circadian rhythm in cyanobacteria.

Gemvid, an open source, modular, automated activity recording system for rats using digital video

BackgroundMeasurement of locomotor activity is a valuable tool for analysing factors influencing behaviour and for investigating brain function. Several methods have been described in the literature for measuring the amount of animal movement but most are flawed or expensive. Here, we describe an open source, modular, low-cost, user-friendly, highly sensitive, non-invasive system that records all the movements of a rat in its cage.MethodsOur activity monitoring system quantifies overall free movements of rodents without any markers, using a commercially available CCTV and a newly designed motion detection software developed on a GNU/Linux-operating computer. The operating principle is that the amount of overall movement of an object can be expressed by the difference in total area occupied by the object in two consecutive picture frames. The application is based on software modules that allow the system to be used in a high-throughput workflow. Documentation, example files, source code and binary files can be freely downloaded from the project website at .ResultsIn a series of experiments with objects of pre-defined oscillation frequencies and movements, we documented the sensitivity, reproducibility and stability of our system. We also compared data obtained with our system and data obtained with an Actiwatch device. Finally, to validate the system, results obtained from the automated observation of 6 rats during 7 days in a regular light cycle are presented and are accompanied by a stability test. The validity of this system is further demonstrated through the observation of 2 rats in constant dark conditions that displayed the expected free running of their circadian rhythm.ConclusionThe present study describes a system that relies on video frame differences to automatically quantify overall free movements of a rodent without any markers. It allows the monitoring of rats in their own environment for an extended period of time. By using a low-cost, open source hardware/software solution, laboratories can greatly simplify their data acquisition and analysis pipelines and improve their workload.

Distinct localization of prokineticin 2 and prokineticin receptor 2 mRNAs in the rat suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) is the master circadian clock that regulates physiological and behavioral circadian rhythms in mammals. Prokineticin 2 (PK2) is highly expressed in the SCN, and its involvement in the generation of circadian locomotor activity has been reported previously. In the present study, using in situ hybridization methods, we investigated the localization of PK2 and prokineticin receptor 2 (PKR2), a specific receptor for PK2, in the rat SCN. In steady light : dark (L : D = 12 : 12 h) and constant dark conditions, rPK2 mRNA displayed a robust circadian oscillation with a peak occurring during the day. Moreover, during peak expression, the rPK2 mRNA-positive neurons were scattered in both the dorsomedial and ventrolateral SCN, which are two functionally and morphologically distinct subregions. Furthermore, double-labeling in situ hybridization experiments revealed that greater than 50% of the rPK2 mRNA-containing neurons co-expressed either vasoactive intestinal peptide (VIP), gastrin-releasing peptide (GRP) or arginine vasopressin (AVP) in the SCN. In contrast, the rPKR2 mRNA levels did not show significant diurnal alterations. rPKR2 mRNA-containing neurons were also clustered in the dorsolateral part of the SCN, which shows negligible labeling of either rAVP, rVIP, rGRP or rPK2 transcripts. In addition, this region exhibited a delayed cycling of the rPer1 gene. These results suggest an intrinsic PK2 neurotransmission and functionally distinct roles for PKR2-expressing neurons in the SCN.