Shuttle mission effects on glutamate receptor expression in the developing rodent spinal cord.

BACKGROUND: Within the mammalian central nervous system (CNS), glutamate receptors play a fundamental role in excitatory neurotransmission and synaptic plasticity. Studies of the neonatal cerebral cortex suggests that rearing environment can influence the dynamic patterns of glutamate receptor subunit expression during development. We examined this issue in the developing spinal cord, a well studied region of the CNS in which activity-dependent synaptic plasticity is known to occur. METHODS: We compared the abundance (by immunoblot analysis) and tissue distribution (by immunohistology) of glutamate receptor subunits in neonatal animals who participated in the Neurolab Space Shuttle mission. Flight animals were either postnatal day 8 or 13 at launch and spent the next 16 d in microgravity; tissues were recovered within 12 h of landing. Littermate control animals were reared on Earth at 1 G. RESULT: Using semi-quantitative immunoblot assays, no statistically significant differences were found in the overall abundance of any glutamate receptor subunit in the spinal cords of the two groups of animals. Similarly, immunohistological examination of spinal cords revealed no evidence for differences in the distribution of glutamate receptor subunits between the two groups of animals. CONCLUSIONS: These results indicate that the developmental regulation of glutamate receptor subunit expression in the spinal cord is not appreciably affected by the conditions associated with this space shuttle mission and prolonged rearing period in microgravity.

Developmental regulation of N-methyl-D-aspartate- and kainate-type glutamate receptor expression in the rat spinal cord.

Spinal motor neurons undergo experience-dependent development during a critical period in early postnatal life. It has been suggested that the repertoire of glutamate receptor subunits differs between young and mature motor neurons and contributes to this activity-dependent development. In the present study we examined the expression patterns of N-methyl-D-aspartate- and kainate-type glutamate receptor subunits during the postnatal maturation of the spinal cord. Young motor neurons express much higher levels of the N-methyl-D-aspartate receptor subunit NR1 than do adult motor neurons. Although there are eight potential splice variants of NR1, only a subgroup is expressed by motor neurons. With respect to NR2 receptor subunits, young motor neurons express NR2A and C, while adult motor neurons express only NR2A. Young motor neurons express kainate receptor subunits GluR5, 6 and KA2 but we are unable to detect these or any other kainate receptor subunits in the adult spinal cord. Other spinal cord regions display a distinct pattern of developmental regulation of N-methyl-D-aspartate and kainate receptor subunit expression in comparison to motor neurons. Our findings indicate a precise spatio-temporal regulation of individual subunit expression in the developing spinal cord. Specific combinations of subunits in developing neurons influence their excitable properties and could participate in the emergence of adult neuronal form and function.

Identification and characterization of basal and cyclic AMP response elements in the promoter of the rat GTP cyclohydrolase I gene.

5812 base pairs of rat GTP cyclohydrolase I (GTPCH) 5'-flanking region were cloned and sequenced, and the transcription start site was determined for the gene in rat liver. Progressive deletion analysis using transient transfection assays of luciferase reporter constructs defined the core promoter as a highly conserved 142-base pair GC-rich sequence upstream from the cap site. DNase I footprint analysis of this region revealed (5' --> 3') a Sp1/GC box, a noncanonical cAMP-response element (CRE), a CCAAT-box, and an E-box. Transcription from the core promoter in PC12 but not C6 or Rat2 cells was enhanced by incubation with 8-bromo-cyclic AMP. Mutagenesis showed that both the CRE and CCAAT-box independently contribute to basal and cAMP-dependent activity. The combined CRE and CCAAT-box cassette was also found to enhance basal transcription and confer cAMP sensitivity on a heterologous minimal promoter. The addition of the Sp1/GC box sequence to this minimal promoter construct inhibited basal transcription without affecting the cAMP response. EMSA showed that nuclear proteins from PC12 but not C6 or Rat2 cells bind the CRE as a complex containing activating transcription factor (ATF)-4 and CCAAT enhancer-binding protein beta, while both PC12 and C6 cell nuclear extracts were recruited by the CCAAT-box as a complex containing nuclear factor Y. Overexpression of ATF-4 in PC12 cells was found to transactivate the GTPCH promoter response to cAMP. These studies suggest that the elements required for cell type-specific cAMP-dependent enhancement of gene transcription are located along the GTPCH core promoter and include the CRE and adjacent CCAAT-box and the proteins ATF-4, CCAAT enhancer-binding protein beta, and nuclear factor Y.

Regulation of GTP cyclohydrolase I gene expression and tetrahydrobiopterin content in cultured sympathetic neurons by leukemia inhibitory factor and ciliary neurotrophic factor.

Cultures of neonatal rat superior cervical ganglia (SCG) were used to test the hypothesis that the cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) control GTP cyclohydrolase I (GTPCH) gene expression and 5,6,7,8-tetrahydrobiopterin (BH4) content as traits of the noradrenergic phenotype. Treatment for 7 days with 1 ng/ml of LIF was found to produce the characteristic switch in the SCG neurotransmitter phenotype reported by others, as evidenced by a 60% decline in tyrosine hydroxylase. (TH) activity and a 75% increase in choline acetyltransferase activity. This LIF treatment paradigm decreased BH4 levels in a concentration-dependent manner, with a maximal decline of 60% observed at 1 ng/ml. Analysis of the time course of this response indicated that LIF decreased BH4 levels by 60% following 3-7 days of treatment. Treatment of cultures with CNTF (2 ng/ml) resulted in a decline in BH4 levels that was of equal magnitude and followed the same time course as that produced by LIF. The LIF-dependent decline in BH4 levels resulted from a reduction in GTPCH enzyme activity, which decreased by 75% following 7 days of treatment. Nuclease protection assays of RNA extracted from cells treated for 7 days with 2 ng/ml of LIF or CNTF detected a 78-96% reduction in GTPCH mRNA content relative to beta-actin mRNA content. Concomitant decreases in TH and GTPCH gene expression in response to LIF or CNTF demonstrate a coordinated regulation of gene expression for this BH4-dependent enzyme and the rate-limiting enzyme in the synthesis of its essential cofactor, BH4. Moreover, these results indicate that GTPCH gene expression in SCG neurons should be regarded as a trait of the noradrenergic phenotype.

Role of insulin-like growth factor I in regulating growth hormone release and feedback in the male rat.

In vivo and in vitro (static incubation and perifusion) procedures were used to examine the role of insulin-like growth factors (IGFs) in growth hormone (GH) feedback. An alpha 2-adrenergic agonist, clonidine (CLON; 2 x 10(-8) M in vitro or 30 micrograms/ml/kg body weight i.v. in vivo), which mimics the hypothalamic mechanism triggering GH release, was injected to induce a GH surge. Feedback was initiated by human GH (hGH; 2 x 10(-6) M) in vitro or ovine GH (oGH) (20 micrograms/2 microliters intraventricularly) in vivo. GH-releasing factor (GRF; 1 x 10(-8) M) was added at the end of in vitro experiments to test pituitary responsiveness. The involvement of somatostatin (SRIF), GRF and IGFs in mediating GH feedback was evaluated in hypothalamic-pituitary coperifusion. CLON-induced GH release in this system was associated with increased GRF and decreased SRIF release, and the pattern was reversed by hGH. The influence of hGH was mimicked by IGF-I (1.5 x 10(-8) M), except that the GH release was depressed below baseline levels, suggesting a direct effect of IGF-I on the pituitary. Furthermore, the inhibitory effect of hGH on the CLON-induced GH surge and hypothalamic releasing factors (increased SRIF and decreased GRF) was reversed by antisera to IGF-I (1:100), IGF-II (1:100), or both. To determine whether IGF-I is released from hypothalamus or pituitary in response to GH, tissues were tested separately in static incubation. As compared with basal levels, incubation of hypothalami with hGH increased IGF-I and SRIF and decreased GRF release. Because GH and IGF-I release remained unchanged when pituitaries were incubated alone with hGH, the site of IGF-I release and GH feedback is most likely at the hypothalamic level. To evaluate the role of IGFs on GH feedback in vivo, male rats were prepared with permanently implanted 3rd-ventricular and jugular cannulae. CLON was administered intravenously, and oGH, IGF-I (0.5 microgram/2 microliters), and IGF-I and -II antisera (1:100) were injected intraventricularly. In this as in in vitro studies, IGF-I mimicked the inhibitory feedback effect of GH on the CLON-induced GH surge, and IGF antisera blocked GH feedback. We propose that these studies suggest that endogenous hypothalamic IGF-I mediates the influence of GH in the feedback mechanism by increasing SRIF and depressing GRF release.

Differential metabolism of tetrahydrobiopterin in monoamine neurons: a hypothesis based upon clinical and basic research.

This chapter has attempted to describe and integrate some of the clinical and basic research that support our hypothesis that the metabolism of BH4 is normally heterogeneous across different populations of monoamine-containing neurons. Based upon this hypothesis, there may now be reason to support the idea that certain neuropsychiatric illnesses, which are though to be the result (at least in part) of altered monoamine metabolism, might find their roots in an abnormal metabolism of BH4 within specific monoaminergic cell groups. Such a specific dysfunction might not be apparent in the rest of the brain or peripheral nervous system, thereby being difficult to detect. Perhaps the application of molecular biological techniques to studies of BH4 metabolism in man will shed new light on these problems.