A Cannabinoid Receptor 1 Agonist Reduces Light-induced Phase Delays in Male But Not Female Mice.

Animals adapt to a changing environment by synchronizing their circadian rhythms to different stimuli, the strongest and most reliable being the daily light-dark cycle. Photic information reaches the central circadian pacemaker, the suprachiasmatic nucleus (SCN), which drives rhythms in physiology and behavior throughout the brain and body. The endocannabinoid system (ECS) is a neuromodulatory system that is present within the SCN, including the primary receptor, cannabinoid receptor 1 (CB1). Exogenous cannabinoids that target CB1 inhibit the phase-shifting effects of light in hamsters, mice, and rats. Furthermore, there is evidence in cultured microglial cells that cannabidiol (CBD), a constituent of Cannabis sativa, alters core circadian clock genes, while the CB1 agonist delta-9-tetrahydrocannabinol (THC) does not. The CB1 agonist studies were conducted using male animals only, but cannabinoids exhibit sex-dependent effects in various aspects of physiology and behavior. In addition, the effects of CBD on circadian behavioral rhythms have yet to be investigated. Therefore, we decided to test the effects of acute injections of CBD or the CB1 agonist CP 55,940 on light-induced phase delays in male and female C57BL/6J mice. Animals received a single injection at circadian time (CT) 15.5, followed by a 10-min light or dark (sham) pulse at CT 16. Running-wheel activity was monitored to determine activity levels and the behavioral phase shifts from different treatments. We observed a sex difference in the magnitude of phase delay size in response to CP 55,940 administration. Males had attenuated phase delays with increasing doses of CP 55,940, while females did not differ from control. Various doses of CBD had no effect on the phase-delaying effects of light in either sex. Our results show a sex difference in the gating of photic phase shifts by CB1 activation.

Regulation of Locomotor activity in fed, fasted, and food-restricted mice lacking tissue-type plasminogen activator.

BACKGROUND: Circadian rhythms of physiology and behavior are driven by a circadian clock located in the suprachiasmatic nucleus of the hypothalamus. This clock is synchronized to environmental day/night cycles by photic input, which is dependent on the presence of mature brain-derived neurotrophic factor (BDNF) in the SCN. Mature BDNF is produced by the enzyme plasmin, which is converted from plasminogen by the enzyme tissue-type plasminogen activator (tPA). In this study, we evaluate circadian function in mice lacking functional tPA. RESULTS: tPA-/- mice have normal circadian periods, but show decreased nocturnal wheel-running activity. This difference is eliminated or reversed on the second day of a 48-h fast. Similarly, when placed on daily cycles of restricted food availability the genotypic difference in total wheel-running activity disappears, and tPA-/- mice show equivalent amounts of food anticipatory activity to wild type mice. CONCLUSIONS: These data suggest that tPA regulates nocturnal wheel-running activity, and that tPA differentially affects SCN-driven nocturnal activity rhythms and activity driven by fasting or temporal food restriction.

Neural correlates of food anticipatory activity in mice subjected to once- or twice-daily feeding periods.

In rodents, restricted food access to a limited period each day at a predictable time results in the appearance of food anticipatory activity (FAA). Two shorter periods of food access each day can result in two FAA bouts. In this study, we examine FAA under 12:12 and 18:6 photoperiods in mice (Mus musculus) with one or two food access periods per day and measure the activation of the suprachiasmatic, dorsomedial and arcuate nuclei by assaying Fos protein expression, while making use of tissue-type plasminogen activator knockout mice to assess the role of neural plasticity in adaptation to restricted feeding cycles. Long days were utilised to allow for temporal separation of two restricted feeding periods during the light phase. Mice fed twice per day generally divided FAA into two distinct bouts, with mice lacking tissue-type plasminogen activator showing reduced FAA. Increases in Fos expression in response to one restricted feeding period per day were seen in the dorsomedial and arcuate nuclei in both 12:12 and 18:6 conditions, with an increase seen in the SCN in only the 12:12 condition. These increases were eliminated or reduced in the two feeding time conditions (done in 18:6 only). Both activity patterns and Fos expression differed for single restricted feeding times between 18:6 and 12:12 photoperiods. Fos activation was lower during RF in 18:6 than 12:12 across all three brain regions, a pattern not reflective of changes in FAA. These data suggest that involvement of these regions in FAA may be influenced by photoperiodic context.

Glycogen synthase kinase 3 regulates photic signaling in the suprachiasmatic nucleus.

Glycogen synthase kinase 3 (GSK3) is a serine-threonine kinase that regulates mammalian circadian rhythms at the behavioral, molecular and neurophysiological levels. In the central circadian pacemaker, the suprachiasmatic nucleus (SCN), inhibitory phosphorylation of GSK3 exhibits a rhythm across the 24 h day. We have recently shown that GSK3 is capable of influencing both the molecular clock and SCN neuronal activity rhythms. However, it is not known whether GSK3 regulates the response to environmental cues such as light. The goal of this study was to test the hypothesis that GSK3 activation mediates light-induced SCN excitability and photic entrainment. Immunofluorescence staining in the SCN of mice showed that late-night light exposure significantly increased GSK3 activity (decreased pGSK3ß levels) 30-60 min after the light-pulse. In addition, pharmacological inhibition of GSK3 blocked the expected light-induced excitability in SCN neurons; however, this effect was not associated with changes in resting membrane potential or input resistance. Behaviorally, mice with constitutively active GSK3 (GSK3-KI) re-entrained to a 6-h phase advance in the light-dark cycle in significantly fewer days than WT control animals. Furthermore, the behavioral and SCN neuronal activity of GSK3-KI mice was phase-advanced compared to WT, in both normal and light-exposed conditions. Finally, GSK3-KI mice exhibited normal negative-masking behavior and electroretinographic responses to light, suggesting that the enhanced photic entrainment is not due to an overall increased sensitivity to light in these animals. Taken together, these results provide strong evidence that GSK3 activation contributes to light-induced phase-resetting at both the neurophysiological and behavioral levels.

Urokinase-type plasminogen activator modulates mammalian circadian clock phase regulation in tissue-type plasminogen activator knockout mice.

Glutamate phase shifts the circadian clock in the mammalian suprachiasmatic nucleus (SCN) by activating NMDA receptors. Tissue-type plasminogen activator (tPA) gates phase shifts by activating plasmin to generate m(ature) BDNF, which binds TrkB receptors allowing clock phase shifts. Here, we investigate phase shifting in tPA knockout (tPA-/- ; B6.129S2-Plattm1Mlg /J) mice, and identify urokinase-type plasminogen activator (uPA) as an additional circadian clock regulator. Behavioral activity rhythms in tPA-/- mice entrain to a light-dark (LD) cycle and phase shift in response to nocturnal light pulses with no apparent loss in sensitivity. When the LD cycle is inverted, tPA-/- mice take significantly longer to entrain than C57BL/6J wild-type (WT) mice. SCN brain slices from tPA-/- mice exhibit entrained neuronal activity rhythms and phase shift in response to nocturnal glutamate with no change in dose-dependency. Pre-treating slices with the tPA/uPA inhibitor, plasminogen activator inhibitor-1 (PAI-1), inhibits glutamate-induced phase delays in tPA-/- slices. Selective inhibition of uPA with UK122 prevents glutamate-induced phase resetting in tPA-/- but not WT SCN slices. tPA expression is higher at night than the day in WT SCN, while uPA expression remains constant in WT and tPA-/- slices. Casein-plasminogen zymography reveals that neither tPA nor uPA total proteolytic activity is under circadian control in WT or tPA-/- SCN. Finally, tPA-/- SCN tissue has lower mBDNF levels than WT tissue, while UK122 does not affect mBDNF levels in either strain. Together, these results suggest that either tPA or uPA can support photic/glutamatergic phase shifts of the SCN circadian clock, possibly acting through distinct mechanisms.

Circadian rhythms of clock gene expression in the cerebellum of serotonin-deficient Pet-1 knockout mice.

Serotonin plays an important role in the central regulation of circadian clock function. Serotonin levels are generally higher in the brain during periods of high activity, and these periods are in turn heavily regulated by the circadian clock located in the suprachiasmatic nucleus. However, the role of serotonin as a regulator of circadian rhythms elsewhere in the brain has not been extensively examined. In this study, we examined circadian rhythms of clock gene expression in the cerebellum in mice lacking the Pet-1 transcription factor, which results in a developed brain that is deficient in serotonin neurons. If serotonin helps to synchronize rhythms in brain regions other than the suprachiasmatic nucleus, we would expect to see differences in clock gene expression in these serotonin deficient mice. We found minor differences in the expression of Per1 and Per2 in the knockout mice as compared to wild type, but these differences were small and of questionable functional importance. We also measured the response of cerebellar clocks to injections of the serotonin agonist 8-OH-DPAT during the early part of the night. No effect on clock genes was observed, though the immediate-early gene Fos showed increased expression in wild type mice but not the knockouts. These results suggest that serotonin is not an important mediator of circadian rhythms in the cerebellum in a way that parallels its regulation of the circadian clock in the suprachiasmatic nucleus.

Site-specific effects of gastrin-releasing peptide in the suprachiasmatic nucleus.

The effects of gastrin-releasing peptide (GRP) on the circadian clock in the suprachiasmatic nucleus (SCN) are dependent on the activation of N-methyl-d-aspartate (NMDA) receptors in the SCN. In this study, the interaction between GRP, glutamate and serotonin in the regulation of circadian phase in Syrian hamsters was evaluated. Microinjection of GRP into the third ventricle induced c-fos and p-ERK expression throughout the SCN. Coadministration of an NMDA antagonist or 8-hydroxy-2-di-n-propylamino-tetralin [a serotonin (5-HT)1A,7 agonist, DPAT] with GRP limited c-fos expression in the SCN to a region dorsal to GRP cell bodies. Similar to the effects of NMDA antagonists, DPAT attenuated GRP-induced phase shifts in the early night, suggesting that the actions of serotonin on the photic phase shifting mechanism occur downstream from retinorecipient cells. c-fos and p-ERK immunoreactivity in the supraoptic (SON) and paraventricular hypothalamic nuclei also increased following ventricular microinjection of GRP. Because of this finding, a second set of experiments was designed to test a potential role for the SON in the regulation of clock function. Syrian hamsters were given microinjections of GRP into the peri-SON during the early night. GRP-induced c-fos activity in the SCN was similar to that following ventricular administration of GRP. GRP or bicuculline (a γ-aminobutyric acidA antagonist) administered near the SON during the early night elicited phase delays of circadian activity rhythms. These data suggest that GRP-induced phase-resetting is dependent on levels of glutamatergic and serotonergic neurotransmission in the SCN and implicate activity in the SON as a potential regulator of photic signaling in the SCN.

Sex differences in behavioral circadian rhythms in laboratory rodents.

There is a strong bias in basic research on circadian rhythms toward the use of only male animals in studies. Furthermore, of the studies that use female subjects, many use only females and do not compare results between males and females. This review focuses on behavioral aspects of circadian rhythms that differ between the sexes. Differences exist in the timing of daily onset of activity, responses to both photic and non-photic stimuli, and in changes across the lifespan. These differences may reflect biologically important traits that are ecologically relevant and impact on a variety of responses to behavioral and physiological challenges. Overall, more work needs to be done to investigate differences between males and females as well as differences that are the result of hormonal changes across the lifespan.

Photic and nonphotic responses of the circadian clock in serotonin-deficient Pet-1 knockout mice.

The neurotransmitter serotonin plays an important role in the regulation of the circadian clock. To gain further insight into the mechanisms by which serotonin regulates rhythmicity, the authors investigated photic and nonphotic effects on the circadian clock in Pet-1 knockout mice. In these mice, the serotonergic system suffers a developmental loss of 70% of serotonin neurons, with the remaining neurons being deficient in serotonergic function as well. Pet-1 knockout mice show significantly decreased phase delays of the circadian clock in response to light pulses in the early night; however, this difference was not reflected in a difference in the expression of Fos protein in the suprachiasmatic nucleus. There were no genotypic differences detected in the phase-shifting response to injection of the 5-HT1A/7 (serotonin 1A and 7) agonist 8-OH-DPAT ((±)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide); however, there were small but significant differences in the phase-shifting responses to cages between genotypes and sexes. Several different patterns of wheel-running activity were observed in knockout mice that differed from those in wild-type mice, suggesting that normal serotonergic function is necessary for the proper consolidation of nocturnal activity. Overall, these data are consistent with other pharmacological and genetic studies demonstrating a significant role for serotonin in circadian clock function.

Klf15 orchestrates circadian nitrogen homeostasis.

Diurnal variation in nitrogen homeostasis is observed across phylogeny. But whether these are endogenous rhythms, and if so, molecular mechanisms that link nitrogen homeostasis to the circadian clock remain unknown. Here, we provide evidence that a clock-dependent peripheral oscillator, Krüppel-like factor 15 transcriptionally coordinates rhythmic expression of multiple enzymes involved in mammalian nitrogen homeostasis. In particular, Krüppel-like factor 15-deficient mice exhibit no discernable amino acid rhythm, and the rhythmicity of ammonia to urea detoxification is impaired. Of the external cues, feeding plays a dominant role in modulating Krüppel-like factor 15 rhythm and nitrogen homeostasis. Further, when all behavioral, environmental and dietary cues were controlled in humans, nitrogen homeostasis exhibited an endogenous circadian rhythmicity. Thus, in mammals, nitrogen homeostasis exhibits circadian rhythmicity, and is orchestrated by Krüppel-like factor 15.

Developmental disruption of the serotonin system alters circadian rhythms.

Serotonin (5-HT) plays an important role in circadian rhythms, acting to modulate photic input to the mammalian clock, the suprachiasmatic nucleus (SCN), as well as playing a role in non-photic input. The transcription factor Pet-1 is an early developmental indicator of neurons that are destined for a 5-HTergic fate. Mice lacking the Pet-1 gene show a 70% loss of 5-HT immunopositive cell bodies in adult animals. 5-HT neurotoxic lesion studies using 5,7-dihydroxytryptamine (5,7-DHT) have highlighted species-specific differences in response to 5-HT depletion and studies using knockout mice lacking various 5-HT receptors have helped to elucidate the role of individual 5-HT receptors in mediating 5-HT's effects on circadian rhythms. Here we investigate the effects of a developmental disruption of the 5-HT system on the SCN and circadian wheel-running behavior. Immunohistochemical analysis confirmed depletion of 5-HT fiber innervation to the SCN as well as greatly reduced numbers of cell bodies in the raphe nuclei in Pet-1 knockout mice. These mice also display significantly longer free-running periods than wildtype or heterozygote counterparts. In light-dark cycles, knockouts showed a shift in peak wheel running behavior towards the late night as compared to wildtype and heterozygote animals. When kept in constant darkness for 70 days, wildtype animals showed decreases in free-running period over time while the period of knockout animals remained constant. Immunohistochemical analysis for neuropeptides within the SCN indicates that the behavioral changes observed in Pet-1 knockout mice were not due to gross changes in SCN structure. These results suggest that developmental loss of serotonergic input to the clock has long-term consequences for both circadian clock parameters and the temporal organization of activity.

An NMDA antagonist inhibits light but not GRP-induced phase shifts when administered after the phase-shifting stimulus.

Brief light pulses or microinjection of gastrin-releasing peptide (GRP) into the third ventricle or near the suprachiasmatic nucleus (SCN) induce phase shifts of circadian rhythms during the subjective night. It has previously been reported that these effects are strongly influenced by the activation of N-methyl-d-aspartate (NMDA) receptors and the availability of glutamate. We hypothesized that the photic signaling pathway in the SCN was dependent on glutamate neurotransmission even after the completion of a photic stimulus. Adult male Syrian hamsters equipped with a surgically implanted guide cannula aimed at the SCN region were housed in constant darkness until stable free-running rhythms of wheel-running activity were apparent. Light pulses administered in the early night induced phase delays of circadian rhythms which were attenuated by the co-administration of (+/-)-2-amino-5-phosphonopentanoic acid (AP5), an NMDA antagonist. Microinjection of AP5 also inhibited light-induced shifts, to a lesser extent, immediately after and 15 min after, but not 30 min after the light pulse. A second experiment was designed to test whether AP5 would be able to attenuate GRP-induced shifts 15 min following microinjection of GRP. Phase shifts of animals that received microinjection of AP5 15 min after the administration of GRP were not different from those that received microinjection of GRP and vehicle. These data suggest that glutamate signaling remains necessary for a full photic response in the SCN even after the termination of the photic signal, but that this dependency ends once GRP-dependent signaling is complete.

Analysis of gene expression in prostate cancer epithelial and interstitial stromal cells using laser capture microdissection.

BACKGROUND: The prostate gland represents a multifaceted system in which prostate epithelia and stroma have distinct physiological roles. To understand the interaction between stroma and glandular epithelia, it is essential to delineate the gene expression profiles of these two tissue types in prostate cancer. Most studies have compared tumor and normal samples by performing global expression analysis using a mixture of cell populations. This report presents the first study of prostate tumor tissue that examines patterns of differential expression between specific cell types using laser capture microdissection (LCM). METHODS: LCM was used to isolate distinct cell-type populations and identify their gene expression differences using oligonucleotide microarrays. Ten differentially expressed genes were then analyzed in paired tumor and non-neoplastic prostate tissues by quantitative real-time PCR. Expression patterns of the transcription factors, WT1 and EGR1, were further compared in established prostate cell lines. WT1 protein expression was also examined in prostate tissue microarrays using immunohistochemistry. RESULTS: The two-step method of laser capture and microarray analysis identified nearly 500 genes whose expression levels were significantly different in prostate epithelial versus stromal tissues. Several genes expressed in epithelial cells (WT1, GATA2, and FGFR-3) were more highly expressed in neoplastic than in non-neoplastic tissues; conversely several genes expressed in stromal cells (CCL5, CXCL13, IGF-1, FGF-2, and IGFBP3) were more highly expressed in non-neoplastic than in neoplastic tissues. Notably, EGR1 was also differentially expressed between epithelial and stromal tissues. Expression of WT1 and EGR1 in cell lines was consistent with these patterns of differential expression. Importantly, WT1 protein expression was demonstrated in tumor tissues and was absent in normal and benign tissues. CONCLUSIONS: The prostate represents a complex mix of cell types and there is a need to analyze distinct cell populations to better understand their potential interactions. In the present study, LCM and microarray analysis were used to identify novel gene expression patterns in prostate cell populations, including identification of WT1 expression in epithelial cells. The relevance of WT1 expression in prostate cancer was confirmed by analysis of tumor tissue and cell lines, suggesting a potential role for WT1 in prostate tumorigenesis.

Temporal patterns of light-induced immediate-early gene expression in the suprachiasmatic nucleus.

Exposing an animal to light during the normal dark period of its daily cycle induces shifts in the animal's circadian rhythm of activity. These shifts are preceded by an increase in the expression of an array of immediate early genes in the suprachiasmatic nucleus, the location of the primary circadian clock in the brain. For most of these genes, little is known about the physiological significance of their expression in the SCN. In order to characterize the expression of these genes, laser capture microscopy, and real-time PCR were used to measure the time course of expression of immediate-early genes in the SCN after a 30-min light pulse during the early portion of the night. Most of the measured genes show peak expression shortly after the end of the stimulus and then decline back to baseline after 2h. However, a few genes, including Rrad, Egr3, and Jun, show a more sustained elevation in expression. Analysis of the function of light-induced genes in other cellular systems suggests a possible role for these genes in reducing the SCN to subsequent photic stimuli and in protecting the SCN from excitotoxicity.

The neonatal injury-induced spinal learning deficit in adult rats: central mechanisms.

Previous research has shown that small injuries early in development can alter adult pain reactivity and processing of stimuli presented to the side of injury. However, the mechanisms involved and extent of altered adult spinal function following neonatal injury remain unclear. The present experiments were designed to 1) determine whether the effects of neonatal injury affect processing contralateral to the injury and 2) evaluate the role of cells expressing the NK1 receptor, shown to be involved in central sensitization in adults, in the negative effects of neonatal injury. The present findings indicate that the effects of neonatal injury are primarily isolated to the injured hind limb and do not result in a bilateral alteration in adult spinal function. In addition, the effects of neonatal injury appear to be partially dependent on cells expressing the NK1 receptor as ablating these cells at the time of injury or in adulthood results in attenuation of the neonatal injury-induced spinal learning deficit.

Identification of novel light-induced genes in the suprachiasmatic nucleus.

BACKGROUND: The transmission of information about the photic environment to the circadian clock involves a complex array of neurotransmitters, receptors, and second messenger systems. Exposure of an animal to light during the subjective night initiates rapid transcription of a number of immediate-early genes in the suprachiasmatic nucleus of the hypothalamus. Some of these genes have known roles in entraining the circadian clock, while others have unknown functions. Using laser capture microscopy, microarray analysis, and quantitative real-time PCR, we performed a comprehensive screen for changes in gene expression immediately following a 30 minute light pulse in suprachiasmatic nucleus of mice. RESULTS: The results of the microarray screen successfully identified previously known light-induced genes as well as several novel genes that may be important in the circadian clock. Newly identified light-induced genes include early growth response 2, proviral integration site 3, growth-arrest and DNA-damage-inducible 45 beta, and TCDD-inducible poly(ADP-ribose) polymerase. Comparative analysis of promoter sequences revealed the presence of evolutionarily conserved CRE and associated TATA box elements in most of the light-induced genes, while other core clock genes generally lack this combination of promoter elements. CONCLUSION: The photic signalling cascade in the suprachiasmatic nucleus activates an array of immediate-early genes, most of which have unknown functions in the circadian clock. Detected evolutionary conservation of CRE and TATA box elements in promoters of light-induced genes suggest that the functional role of these elements has likely remained the same over evolutionary time across mammalian orders.

Short photoperiod and testosterone-induced modification of GnRH release from the hypothalamus of Peromyscus maniculatus.

Seasonally breeding animals undergo numerous physiological changes in response to changes in the length of the photoperiod. In most warm-weather breeding rodents, these changes result in reproductive quiescence during short photoperiods. It has been hypothesized that this change is mediated by changes in the activity of gonadotropin-releasing (GnRH) hormone neurons of the hypothalamus. This study was designed to test whether there are changes in the releasable pool of GnRH in the hypothalamus in response to changes in photoperiod, the presence of gonadal steroids, or the responsiveness of the individual animal to photoperiodic changes. Male deer mice (Peromyscus maniculatus) were maintained on long or short day photoperiod and either left intact, castrated, or castrated with testosterone replacement. KCl-evoked GnRH release was measured from hypothalamic explants from each animal and compared between long and short days, between castrated, intact, and castrated with testosterone replacement animals, and between animals that did or did not show gonadal regression in response to short day treatment. There was a significant decline in evoked release of GnRH in short day housed animals when comparing photoperiod responsive mice to nonresponsive mice. In addition, both reproductively nonresponsive and long day-housed mice release less GnRH following castration than their intact counterparts. When castrated long day-housed mice were provided with long day levels of testosterone, evoked GnRH release was restored to intact levels. Taken together, the results of this study suggest that variation in testicular response to short days is most likely due to differences in the release of GnRH from the hypothalamus.

Gastrin releasing peptide and neuropeptide Y exert opposing actions on circadian phase.

Microinjection of gastrin releasing peptide (GRP) into the third ventricle or the suprachiasmatic nucleus (SCN) induces circadian phase shifts similar to those produced by light. Administration of GRP during the day does not alter circadian phase. In contrast, neuropeptide Y (NPY) induces phase shifts of circadian rhythms during the day but has little effect when administered at night, similar to the effects of most non-photic stimuli. NPY inhibits the phase shifting effects of light, and GRP is thought to be part of the photic signaling system within the SCN. This experiment was designed to test whether GRP and NPY inhibit each other's effects on circadian phase. Adult male Syrian hamsters equipped with guide cannulas aimed at the SCN were housed in constant darkness until stable free-running rhythms of wheel running activity were apparent. Microinjection of GRP during the early subjective night induced phase delays that were blocked by simultaneous administration of NPY. During the middle of the subjective day, microinjection of NPY caused phase advances that were blocked by simultaneous administration of GRP. These data suggest that GRP and NPY oppose each other's effects on the circadian clock, and that the actions of NPY on the photic phase shifting mechanism in the SCN occur at least in part downstream from retinorecipient cells.

Glutamatergic activity modulates the phase-shifting effects of gastrin-releasing peptide and light.

Previous studies have established that microinjection of gastrin-releasing peptide (GRP) into the suprachiasmatic nucleus (SCN) region or third ventricle causes circadian phase shifts similar to those produced by light pulses. Activation of N-methyl-d-aspartate (NMDA) receptors in the SCN region also produces light-like phase shifts. This study was designed to test the effects of (+/-)-2-amino-5-phosphonopentanoic acid (AP5), an NMDA antagonist, and l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), a glutamate reuptake inhibitor, on GRP-induced phase shifts. Adult male Syrian hamsters equipped with a surgically implanted guide cannula aimed at the third ventricle were housed in constant darkness until stable free-running rhythms of wheel-running activity were apparent. Microinjection of GRP into the third ventricle at circadian time (CT)13 induced large phase delays. These GRP-induced phase delays were completely blocked by co-administration of AP5, suggesting that GRP-induced phase delays require concurrent activation of NMDA receptors. Microinjection of AP5 alone did not induce significant phase shifts. A second set of experiments was designed to test whether GRP-induced phase shifts would be enhanced by PDC. Co-administration of PDC and GRP elicited significantly larger phase delays at CT13 than GRP alone. However, administration of PDC alone did not induce a significant phase shift. Finally, when administered just prior to a light pulse, PDC elicited significantly larger phase delays than light pulse plus vehicle controls. These data suggest that the effects of GRP on the circadian clock phase are highly dependent on the level of excitation provided by activated NMDA receptors.

Modulation of photic response by the metabotropic glutamate receptor agonist t-ACPD.

Glutamate is the primary excitatory transmitter in the hypothalamus. It conveys photic information to the suprachiasmatic nucleus of the hypothalamus, thereby entraining the circadian clock to environmental light cycles. While ionotropic glutamate receptors have been implicated in the transduction of photic information in suprachiasmatic nucleus cells, there is evidence that metabotropic glutamate receptors play a significant modulatory role. We investigated the effects of the metabotropic glutamate agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) on light-evoked phase responses in Syrian hamsters at three phase points: circadian time 6, a time when light has no effect on the circadian timing system; circadian time 13.5, when light evokes the maximum phase delay; circadian time 19, the maximum phase advance. We found that ACPD significantly increased the light-evoked phase shift at circadian time 13.5, and had no effect at other phase points tested. These data support a role for metabotropic glutamate receptors in the circadian photic signal transduction system.