Hippocampal cells from E18 rat embryos were plated at a density of 500,000 cells per 60 mm poly-L-lysine (1mg/ml; Sigma) coated dish. Cultures were maintained at 37 C in 5% CO2 humidified incubator and used for experiments at 21 day in vitro (DIV). Control and experimental group cultures were incubated with 10 micro M TTX for 15 min followed by addition of 100 micro M baclofen or vehicle.
Total RNA preparation: Each experimental group was compared as replicates of three. For each replicate cultured hippocampal neurones were harvested after 2 hours incubation +/- baclofen and total RNA was isolated using RNeasy Mini kit (Qiagen, UK) according to the manufacturer's protocol.
Microarray Target Labelling and Hybridisation: Targets for cDNA microarrays were generated using 5 micro g of total RNA in a standard reverse transcription (RT) reaction. RNA was annealed, in 16 micro l water, with 1 micro g of 24-mer poly(dT) primer (Invitrogen, UK), by heating at 65oC for 10 min and cooling on ice for 2 min. The RT reaction was performed by adding 8 micro l of 5X first strand RT buffer (Life technologies, MD), 4 micro l of 20mM dNTPs minus dCTP (Pharmacia, UK), 4 micro l of 0.1M DTT, 40U of RNAse OUT (Life Technologies, MD), 6 micro l of 3000 Ci/mmol 33P-dCTP (ICN Biomedicals, UK) to the RNA/primer mixture to a final volume of 40 micro l. Two micro l (400 U) of Superscript II reverse transcriptase (Life Technologies, MD) was then added and the sample incubated for 30 min at 42oC followed by additional 2 micro l of Superscript II reverse transcriptase and another 30 min incubation. The reaction was stopped by addition of 5 micro l 0.5 M EDTA. Samples were incubated at 65oC for 30 min after addition of 10 micro l of 0.1M NaOH to hydrolyse and remove RNA. Samples were pH neutralized with 45 micro l 0.5M Tris, pH 8.0 and purified using Bio-Rad 6 purification columns (BioRad, UK). The NIA NeuroArray consists of 1152 cDNAs printed on nylon membrane in duplicate. The arrays were hybridised with alpha-33P-dCTP labelled cDNA probes overnight at 50oC in 4ml of hybridisation solution. Hybridised arrays were rinsed in 50ml of 2XSSC and 1%SDS twice at 55oC followed by two washes in 2X SSC and 1% SDS at 55oC for 15 min each. The microarrays were exposed to phosphorimager screens for 1-3 days. The screens were then scanned with Molecular Dynamics STORM PhosphorImager (Sunnyvale, CA) at 25 micro m resolution.
