Nuclear speckles store and modify splicing factors that process pre-mRNAs

ernatively, the localization was either in the lateral hypothalamus at large or outside it. In ABA-ISH images of sagittal sections, the parvafox nucleus was more difficult to identify with certainty, and the data were consequently less unequivocal. The findings of the detailed evaluation are summarized as a whole in the 8 Szabolcsi et al. Parvalbumin-Neurons of the Parvafox Receive Glycinergic Input , Glra1, Glra2, Glra3, Npb and Npsr1 . These results coincide with the microarray fold change values; however, in some cases the expression levels were considerably PBTZ 169 biological activity higher when measured by qRT-PCR. Analysis of the Putative Roles of the Genes That Were Enriched in the Parvafox Nucleus We conducted a MetaCore -enrichment analysis on a set of genes that had been revealed to be expressed in the parvafox nucleus by the combined results of the gene-microarray, the ABA-ISH database and the B -database evaluations, as well as previous observations . We also examined the expression of these 9 genes in the BrainStars database and the ABA-ISH database, additionally we performed immunohistochemistry to validate the expression of Glra2 and Foxb1. Glra2 satisfies all five criteria for expression in the parvafox nucleus and represents the most robust result of this study. Cerebellin and the dopamine 2 receptor meet four of the five criteria for occurrence in the parvafox nucleus. Foxb1 and Npb are positive in our own gene-array and have been revealed by others to occur in the lateral hypothalamus, respectively in the parvafox nucleus but could not be confirmed neither in the ABA-ISH nor in the B databases. p values calculated in Student’s t-test. Positive in Foxb1-Cre-Egfp knock-in mice. by our strict selection criteria, was also expressed at high levels in the parvafox nucleus, with a mean fold-change of +11.92 in mouse 511. Screening of the B -microarray database revealed the glycine-receptor subunit alpha-1 to be likewise expressed at higher levels in the parvafox region than in the hippocampus. The expression of these three alpha-subunit isoforms of glycine receptors in the parvafox nucleus was confirmed by visual screening of the ABAISH database and for Glyr2 additionally also by immunofluorescence staining with specific antibodies. Immunohistochemistry Revealed Parv-Positive Neurons of the Parvafox Nucleus to Express Glycine Receptor 2 For an additional independent verification of the most robust and interesting results, an antiserum against the Glyr2-receptor was used to detect immunoreactivity in the hypothalamus. Positive immunolabeling was selectively observed in a few hypothalamic regions, one being the parvafox nucleus. At higher magnification, the immunoreactivity was revealed to be associated with the plasma membrane of the Parv-positive neurons in this region. Immunoblot analysis of murine hypothalamus protein extract with the antiserum against Glyr2 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19815860 resulted in a sharp band below 50 KDa, the expected molecular weight for the Glyr2 receptor, confirming the presence of Glyr2 in the hypothalamus, as well as the specificity of the Glyr2-antibody used in our study. In addition to the hypothalamus, we detected Glyr2 in hippocampal and cortical lysates as well, regions that served as reference structures in the microarray analysis. Quantitative Real-Time PCR Nine genes of particular interest because of their role as markers or neuromodulators were selected to verify the results of the gene microarray study and to confirm their expression in the r