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.

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.

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.

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.

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.