• Effects of aging and fetal tissue transplantation on the patterns of expression of neuroendocrine and neurochemical outputs of the biological clock

      Cai, Aihua; Wise, Phyllis M. (1995)
      Circadian rhythmicity is a common physiological phenomenon. Many rhythms change with age. Since the suprachiasmatic nucleus (SCN) is the major neural circadian pacemaker, we hypothesized that changes in the circadian rhythms maybe due to changes in the SCN itself. In this dissertation, I asked two questions: (1) Does age affect the immediate early gene expression in the SCN? If so, can transplantation of fetal tissue containing the SCN restore normal function? (2) Does age change SCN-driven endocrine outputs, such as the hypothalamic-pituitary-adrenal cortex (HPA) axis; If so, can fetal SCN transplants restore normal function? The expression of immediate early genes (IEG), fos and jun, have been used as markers of neural activity in many studies. We used expression of IEG in the SCN as an index of neural activity of the SCN. Using immunocytochemistry, we detected a significant decrease in light-induced Fos and Jun-B expression in the SCN in middle-aged rats, and transplantation of fetal SCN tissue into middle-aged rats partially restored the IEG expression in hosts to that of young rats. We chose to study corticotropin releasing hormone (CRH) and proopiomelanocortin (POMC) gene expression in HPA axis. Using in situ hybridization histochemistry and solution hybridization-RNase protection assays, we detected that both diurnal rhythms of CRH mRNA in the paraventricular nucleus (PVN) and POMC mRNA in the anterior pituitary were abolished by the time animals were middle-aged. Transplantation of fetal SCN tissue into middle-aged rats restored a diurnal rhythm of CRH mRNA in the PVN, but the pattern was slightly altered. SCN transplants can restore a diurnal rhythm of POMC mRNA in the anterior pituitary, but the amplitude of the rhythm remained as low as that of middle-aged rats. In conclusion, aging affects rhythms of the SCN and SCN-driven endocrine outputs. These changes may not be permanent, since fetal SCN transplants can partially correct those changes.
    • Growth factor and cytokine regulation of the beta gene in rat astrocytes

      Hinkle, David Alan; Wise, Phyllis M. (1995)
      Basic fibroblast growth factor (FGF-2) and interleukin-l{dollar}\beta{dollar} (IL-1{dollar}\beta{dollar}) stimulate reactive gliosis and regulate neurotrophic factor expression in astrocytes. S100{dollar}\beta{dollar} is a putative neurotrophic factor which is over-expressed in reactive astrocytes. Therefore, we tested the hypothesis that FGF-2 and/or IL-1{dollar}\beta{dollar} would stimulate S100{dollar}\beta{dollar} gene expression. FGF-2 decreased S100{dollar}\beta{dollar} mRNA after 12 and 24 hours of treatment in cultured rat cortical astrocytes, but increased its levels after 7 days. IL-1{dollar}\beta{dollar} suppressed S100{dollar}\beta{dollar} mRNA levels after 24 and 48 hours, and continued to inhibit after 7 days of exposure. In combination, the effect of these two factors appeared synergistic. In C6 glioma cells, only FGF-2 suppressed gene expression. To assess indirectly whether alterations in transcriptional rate could explain the changes in mRNA, we measured levels of S100{dollar}\beta{dollar} primary transcript. FGF-2 decreased S100{dollar}\beta{dollar} nuclear primary transcript levels after 6 and 12 hours, but increased its levels after 48 hours. IL-1{dollar}\beta{dollar} decreased nuclear primary transcript after 48 hours. We further measured intracellular S100{dollar}\beta{dollar} protein levels to determine whether the alterations in mRNA were translated into parallel changes in the level of protein. FGF-2 did not suppress S100{dollar}\beta{dollar} protein levels after 1, 2, or 3 days of treatment, but increased it after 5 and 8 days. IL-1{dollar}\beta{dollar} and combination treatment did not significantly alter protein levels. Our results clearly demonstrate that FGF-2 and IL-1{dollar}\beta{dollar} influence the expression of the S100{dollar}\beta{dollar} gene, that this regulation appears to occur at the level of transcription, and that changes in mRNA are sometimes, but not always, reflected in changes at the level of protein. In a second study we determined whether the changes observed in vitro would also be seen in a lesion model for reactive gliosis in which both FGF-2 and IL-1{dollar}\beta{dollar} are elevated: the cortical contusion. Mild contusion bilaterally elevated S100{dollar}\beta{dollar} mRNA over sham levels in both the cortex and hippocampus of young adult, male rats. However, mRNA levels in sham animals decreased with time, making it unclear whether the contusions stimulated S100{dollar}\beta{dollar} or mitigated the inhibitory effect of sham. The contusion stimulated a robust elevation in GFAP mRNA, a "marker" of reactive gliosis, in both brain regions. Our data clearly demonstrate that contusion produces a vigorous glial response, and suggest that the mechanisms involved in the regulation of S100{dollar}\beta{dollar} and GFAP are different. This hypothesis is further supported by our in vitro finding that GFAP and S100{dollar}\beta{dollar} are differentially regulated in astrocytes by FGF-2 and IL-1{dollar}\beta.{dollar}* ftn*Originally published in DAI vol. 56, no. 9. Reprinted here with corrected author name.
    • Prolactin receptor gene expression in the hypothalamus

      Chiu, Sufen; Wise, Phyllis M. (1993)
      Changes in behavior and neuroendocrine secretions in response to prolactin treatment may be mediated by prolactin receptors in the brain. Autoradiographic binding studies using iodinated prolactin suggest that these receptors may be located in the rat choroid plexus and hypothalamus. It remains unclear if the prolactin binding sites represent classical prolactin receptors or other proteins from the prolactin/growth hormone receptor family that have retained an affinity for prolactin. Two prolactin receptors, a short and long form resulting from alternative splicing of one gene, were cloned recently. The objectives of this dissertation were to (1) examine if the prolactin receptor gene is expressed in brain areas that are believed to bind and/or respond to prolactin by reverse-transcription polymerase chain reaction (RT-PCR), (2) determine the cellular distribution of prolactin receptor mRNA by in situ hybridization, and (3) assess the influence of physiological conditions on prolactin receptor gene expression at the individual cell level by in situ hybridization. We demonstrated that both forms of the prolactin receptor mRNA are expressed in the pituitary gland and hypothalamus but not in cortical brain tissue or skeletal muscle by RT-PCR. Using in situ hybridization, prolactin receptor gene expression was detectable in medial preoptic nucleus, supraoptic nucleus, arcuate nucleus, lateral ventromedial nucleus and choroid plexus. Prolactin receptor gene expression in the periventricular area of the preoptic nucleus, arcuate nucleus, and choroid plexus is increased during aging {dollar}(p<0.05){dollar}. Therefore, we conclude that the classical prolactin receptors appear to mediate the binding and action of prolactin in the brain and are influenced by age-related changes.