Treatment with FLX alone was observed to have no effect on IEG expression in the LA

synapses by puncta analyzer Synapses were analyzed with Puncta analyser plug-in for ImageJ as described. Maximum intensity projections of confocal microscopy images from 5 areas per cerebellum, 45 animals per group were quantified for the number of synaptic puncta using markers VGAT, VGLUT1, VGLUT2, GABRA6, synapsin 1 and gephyrin. Colocalization of these puncta was also analyzed. The areas analyzed covered different lobules of the cerebellum and were the same in all analyses and animals. The data were expressed as mean puncta number compared to control 6 SE. The unpaired Student’s t-test was used for statistical analysis ) with p,0.05 considered as statistically significant. Brains from P7 and P30 Cstb2/2 and control mice were dissected and frozen in ethanol-isopentane-bath. Sagittal 14 mm thick cryostat sections were mounted on gelatinchrome alum coated slides. Autoradiographic procedures have been described previously in detail. For muscimol binding to GABA agonist sites, sections were preincubated for 15 min in an icewater bath in 0.17 M Tris-HCl. The final DMXB-A site incubation in the same buffer was performed with 15 nM muscimol at 4uC for 30 min. Nonspecific binding was determined with 100 mM GABA. After incubation, the sections were washed in ice-cold incubation buffer twice for 30 s, dipped in distilled water and dried in airflow at room temperature. For Ro15-4513 binding to benzodiazepine sites of the GABAA receptors, sections were preincubated for 15 min in an ice-water bath in 50 mM TrisHCl, 120 mM NaCl. The final incubation in the same buffer was performed with 15 nM Ro15-4513 at 4uC for 60 min. Nonspecific binding was determined with 10 mM Ro151788. To detect a-6 subunit containing GABAA receptors, other GABAA receptor subtypes were blocked with 10 mM diazepam. After incubation, the sections were washed with icecold incubation buffer for 2660 s, dipped in distilled water and dried in airflow at room temperature. The sections were exposed with -plastic standards to Kodak Biomax MR films for 2 months. For quantification of binding densities, the imaging plates were analyzed with ImageJ and the data were expressed as mean radioactivity levels 6 SE with reference to -standards. The unpaired Student’s t-test was used for statistical analysis ) with p,0.05 considered as statistically significant. Autoradiography of GABAA receptor ligand binding sites Altered expression of genes involved in cellular biogenesis in Cstb2/2 mouse cerebellar granule cells To get insight into the gene expression changes in neurons, GO categories of 140 differentially expressed probe sets which correspond to 114 known genes were investigated from Cstb2/2 cerebellar granule cells. Of these, 128 probe sets were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19639069 upregulated and 12 downregulated. Biological processes comprised a number of upregulated genes involved in cell cycle, division and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19640475 growth, e.g. cell division cycle protein 20 homolog and its post-translational modifier, mitotic checkpoint serine/threonine-protein kinase. Differentially expressed genes linked to cellular architecture, such as genes of kinesin-like protein family were also detected. Categories of molecular function further indicated an increased expression of growth factors of the CCN family proteins, calcium-dependent annexins and protease inhibitors, as well as upregulation of several collagen type genes. In concordance with the gene function, protein products of majority of the differentially expressed genes localized into nucleus, althoug