Project description:Pooled Immunoflourescent guided laser capture microdisection of calyceal vestibular primary afferent neurons (calretinin positive) and dimorphic-bouton vestibular primary afferent neurons (calretinin negative) were used for microarray expression profiling. Transcription analysis of each of these biologically diverse pools was completed.
Project description:The functional diversity of neuropeptides and/or their receptors may provide a complex means to modulate vestibular primary afferent neuronal function. The precise role of these neuropeptides in the physiology of the vestibular neuroepithelium is poorly understood. The vestibular caliceal afferent neurons when compared to the dimorphic and bouton afferent neurons have different functional properties. Immunofluorescent laser capture microdissection utilizing calretinin (calbindin 2, Calb2) antibodies allowed selective acquisition of these two primary afferent neuronal populations from the rat (Rattus norvegicus). After capturing the caliceal afferent neurons, as well as the dimorphic and bouton afferent neurons, microarray expression profiling was completed. Analysis of the resulting data revealed that there were 732 genes involved in synaptic/signaling, calcium binding/solute transport, and other. Of those genes, 52 were related to afferent modulation, including 21 genes representing neuropeptides or their receptors. The observed expression of 52 genes related to afferent modulation identified using microarray were confirmed using PCR from the Wackym-Soares normalized rat vestibular periphery cDNA library or by RT-PCR from fresh ganglia tissue. The majority of the neuropeptides and/or receptors were found to be expressed in both groups, although there were neuropeptides and/or receptors that were unique to the dimorphic and bouton afferent neuron pool. The expression of selected neuropeptides or their receptors was confirmed using immunohistochemistry in the crista ampullares. Our results suggest that the unexpected neuropeptide diversity and their differences in expression pattern could serve unique roles in the physiology of the vestibular neuroepithelium. Keywords: expression profiling, neuropeptides, vestibular neuroepithelium, calretinin
Project description:A series of two color gene expression profiles obtained using Agilent 44K expression microarrays was used to examine sex-dependent and growth hormone-dependent differences in gene expression in rat liver. This series is comprised of pools of RNA prepared from untreated male and female rat liver, hypophysectomized (‘Hypox’) male and female rat liver, and from livers of Hypox male rats treated with either a single injection of growth hormone and then killed 30, 60, or 90 min later, or from livers of Hypox male rats treated with two growth hormone injections spaced 3 or 4 hr apart and killed 30 min after the second injection. The pools were paired to generate the following 6 direct microarray comparisons: 1) untreated male liver vs. untreated female liver; 2) Hypox male liver vs. untreated male liver; 3) Hypox female liver vs. untreated female liver; 4) Hypox male liver vs. Hypox female liver; 5) Hypox male liver + 1 growth hormone injection vs. Hypox male liver; and 6) Hypox male liver + 2 growth hormone injections vs. Hypox male liver. A comparison of untreated male liver and untreated female liver liver gene expression profiles showed that of the genes that showed significant expression differences in at least one of the 6 data sets, 25% were sex-specific. Moreover, sex specificity was lost for 88% of the male-specific genes and 94% of the female-specific genes following hypophysectomy. 25-31% of the sex-specific genes whose expression is altered by hypophysectomy responded to short-term growth hormone treatment in hypox male liver. 18-19% of the sex-specific genes whose expression decreased following hypophysectomy were up-regulated after either one or two growth hormone injections. Finally, growth hormone suppressed 24-36% of the sex-specific genes whose expression was up-regulated following hypophysectomy, indicating that growth hormone acts via both positive and negative regulatory mechanisms to establish and maintain the sex specificity of liver gene expression. For full details, see V. Wauthier and D.J. Waxman, Molecular Endocrinology (2008)