The lateral cortex containing TOM+ pyramidal neurons and GAD65-GF

The lateral cortex containing TOM+ pyramidal neurons and GAD65-GFP+ interneurons were trypsinised in Hanks’ medium for 10 min at 37 °C. After centrifugation the pellet was filtered using 40-μm-pore filters (Falcon). GFP+ and TOM+ cells were sorted using fluorescence-activated cell sorting (FACS). Total RNA from the sorted cells was extracted, amplified (MessageAMP™ II

aRNA Amplification kit; Ambion, Zug, Switzerland) in order to obtain at least 50 ng of RNA, and converted into cDNA. PCR was done using a REDtaq Ready-Mix (Sigma, Buchs, Switzerland) and PCR products were electrophoresed in a 2% agarose gel. For quantitative PCR, PCR reactions were performed in triplicate on cDNA from TOM+ cells and GAD65-GFP+ cells using SYBR green PCR Master Mix (Applied Biosystems, Rotkreuz, Switzerland) in an ABI Prism 7900 Sequence Detection system (Applied Biosystems).

Four genes were used as internal controls: beta-actin (actb), gamma-actin (actg1), eukaryotic elongation factor-1 (eef1a1) and beta-glucuronidase (Gusb). Primers for the different adrenergic receptors were designed using BMS-354825 ic50 the Ensembl database and the Primer3 software. Primer sequences were as follows: adra1a forward, 5′-CTGCCATTCTTCCTCGTGAT-3′ and reverse 5′-GCTTGGAAGACTGCCTTCTG-3′, adra1b, forward, 5′-AACCTTGGGCATTGTAGTCG-3′ and reverse 5′-CTGGAGCACGGGTAGATGAT-3′ adra1d forward, 5′-TCCGTAAGGCTGCTCAAGTT-3′ and reverse, 5′-CTGGAGCAGGGGTAGATGAG-3′, adra2a forward, 5′ TGCTGGTTGTTGTGGTTGTT-3′ and reverse, 5′-GGGGGTGTGGAGGAGATAAT-3′, adra2b, forward 5′-GCCACTTGTGGTGGTTTTCT-3′, reverse, 5′- TTCCCCAGCATCAGGTAAAC-3′, adra2c forward, 5′-TCATCGTTTTCACCGTGGTA-3′ and reverse, 5′-GCTCATTGGCCAGAGAAAAG-3′, adrb1 forward, 5′-TCGCTACCAGAGTTTGCTGA-3′ and reverse, 5′-GGCACGTAGAAGGAGACGAC-3′, adrb2, forward. 3-oxoacyl-(acyl-carrier-protein) reductase 5′-GACTACACAGGGGAGCCAAA-3′, and reverse, 5′-TGTCACAGCAGAAAGGTCCA-3′, adrb3 forward, 5′-TGAAACAGCAGACAGGGACA-3′,

reverse 5′-TCAGCTTCCCTCCATCTCAC-3′. Cortical slices were imaged in a thermoregulated chamber maintained at 37 °C and CO2 at 5% as previously described (Riccio et al., 2009). Time-lapse movies were acquired in parallel using two fluorescent microscopes (Eclipse TE2000; Nikon, Egg, Switzerland) equipped with a Nikon Plan 10×/0.30 objective connected to a digital camera (Retiga EX). Time-lapse imaging was performed 3–4 h after slice preparation over a period of 24 h. Images were acquired using the Open-lab software (version 5.0; Schwerzenbach, Switzerland) every 5 min for 200 min in short time-lapse sequences and for 600 min in washout experiments. A control time-lapse sequence of 95 min was acquired in each condition before the treatment condition. Time-lapse stacks were generated and analysed using Metamorph software (version 7.4; Visitron, Puchheim, Germany).

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