Tissue microdissection, microarray and qPCR

Microdissection of VPM from one litter corresponds to one biological replicate (n = 3 for each condition). For ThGrin1KO microarrays, collection of samples was done at P1 and WT littermates were used as controls. For IONS microarrays, collection of the samples was done at P3 and the VPM ipsilateral to the IONS was used as control. Fresh coronal brain sections (140 μm) were cut on a vibrating microtome (Leica, VT1000S) and thalamic nuclei were visually identified and microdissected using a Leica Dissecting Microscope (Leica, M165FC) in ice-cold oxygenated artificial cerebrospinal fluid under RNAse free conditions. Samples were stored in RNAlater (Sigma) at −80 °C.

For microarrays, RNA was extracted using an RNeasy kit (Qiagen) and two-cycle amplification and labeling were performed according to Affymetrix protocols using Superscript complementary DNA synthesis kit (Invitrogen), MEGAscript T7 kit and MessageAmp II aRNA amplification kit (Ambion). Experiments were performed blindly. Labeled cRNA was fragmented and hybridized to Affymetrix Mouse Genome 430 2.0 Array. GeneChips were incubated at 45 °C for 16 h with biotin-labeled cRNA probes, and then washed and stained using a streptavidin–phycoerythrin conjugate with antibody amplification as described in Affymetrix protocol, using Affymetrix GeneChip Fluidics Station 450. GeneChips were scanned on a GCS3000 scanner (Affymetrix). Microarray CEL files were read and normalized using ‘affy’ and ‘gcrma’ R packages, and transformed in log2. Data were analyzed as previously described¹⁸. Some of these arrays were used in ref. ¹⁷.

For quantitative PCR, cDNA was synthesized from 1 μg of cRNA (from the second amplification) using a mix of random hexamers—oligo d(T) primers and PrimerScript reverse transcriptase enzyme (Takara Bio Inc. Kit) following suppliers instructions. SYBR Green assays were designed using the program Primer Express v 2.0 (Applied Biosystems) with default parameters. Amplicon sequences were aligned against the mouse genome by BLAST, to ensure that they were specific for the gene being tested. Oligonucleotides were obtained from Invitrogen. The efficiency of each design was tested with serial dilutions of cDNA. PCR reactions (10 μl volume) contained diluted cDNA, 2 x Power SYBR Green Master Mix (Applied Biosystems), and 300 nM of forward and reverse primers. PCR were performed on a SDS 7900 HT instrument (Applied Biosystems) with the following parameters: 50 °C for 2 min, 95 °C for 10 min, and 45 cycles of 95 °C for 15 s/60 °C for 1 min. Each reaction was performed in three replicates on 384-well plates. Raw Ct values were obtained with SDS 2.2 (Applied Biosystems) and imported in Excel. Normalisation factor and fold changes were calculated using the GeNorm method³⁸.