Three hundred μm thick coronal slices (male Sprague Dawley rats, 14–28 days old) were prepared using a vibratome, and bathed in aCSF of the following composition (mM); NaCl 125, NaHCO3 25, glucose 10, KCl 2.5, NaH2PO4 1.25, CaCl2 2, MgCl2 1, bubbled with 95% O2 and 5% CO2 gas mixture, and viewed with a Zeiss Axioskop microscope (Carl Zeiss Ltd., Welwyn Garden City, U.K.) fitted with a x64 water-immersion objective as previously described (Lee et al., 1998). The electrode buffer contained (in mM): K gluconate 120, NaCl 10, MgCl2 2, EGTA 0.5, HEPES 10, Na2ATP 4, Na2GTP 0.3, pH adjusted to 7.2 with KOH. In addition 0.5 μg ml−1 glycogen (Boehringer) and RNase inhibitor (Pharmacia, 0.1 u μl−1 were included to facilitate the harvesting of RNA from the cells. All solutions were made up in diethyl pyrocarbonate treated water (to inactivate RNases). Borosilicate recording electrodes were baked (2 h, 250°C) before being pulled to a resistance of between 3 and 5 MΩ. Electrophysiological signals were detected using an Axopatch-1D patch-clamp amplifier in the current clamp configuration and were recorded onto digital audiotape for later production. Membrane signals were filtered at 1 kHz and were digitized at 5 kHz through a Digidata 1200A/D converter using pClamp 6.0 software (Axon Instruments Inc, CA, U.S.A.).
The cytoplasm from large cells (>30 μm in one dimension) was gently aspirated under visual control into a patch-clamp recording electrode until at least 40% of the somatic cytoplasm had been collected. The electrode was then withdrawn from the cell to form an outside-out patch, or a nucleated patch, to prevent contamination on subsequent withdrawal from the slice. The contents of the electrode were forced into a microtube and reverse transcribed, subjected to 3′ cDNA amplification (TPEA–PCR), and 6% of the product used in each gene specific PCR reaction as described in Dixon et al. (1998) with the following modifications. Four defined pentameric sequences were present at the 3′ ends of the second-strand cDNA primers, each primer contained the following heel sequence at the 5′ end: CTG CAT CTA TCT AAT GCT CC. The four arbitrary 3′ sequences were: CGAGA, CGACA, CGTAC and ATGCG. 2.5 pg of each second strand primer were annealed at 50°C to the first strand cDNA, and second strand cDNA synthesis was performed at 72°C for 8 min using Taq DNA polymerase (0.35 units, Perkin Elmer) at pH 8.3 using 4.5 mM MgCl2 and 0.5 mM dNTPs. Subsequently, 1 ng of the 5′ and 3′ heel primers were added in 5 μl of PCR buffer containing 67 mM Tris HCl (pH 8.3), 4.5 mM MgCl2 0.5 mM dNTPs and 0.16 units of Taq and the reaction subjected to 10 cycles of 92°C (2.5 min, denaturation), 60°C (annealing, 1.5 min) and 72°C (extension, 1 min), followed by a final 10 min extension. A further 10 ng of each heel primer were then added in 20 μl of PCR buffer and subjected to a further 40 rounds of PCR (conditions as before). The final reaction mixture was then diluted to 100 μl with 10 mM Tris/ 0.1 mM EDTA (pH 8.1), and 6 μl samples used for subsequent gene specific PCR. Gene specific PCR was performed as described (Dixon et al., 1998) using the following primers (accession number, base number): choline acetyltransferase: forward primer (sequence from Brice et al., 1989, 2134–2155 TACTAAGCTCTGTTCCCATCCC, reverse primer 2303–2285, ACCCAGGTTGCTTCCAAAC), adenosine A1 receptor (Y12519, forward primer 2507–2526; reverse primer 2637–2619), adenosine A2A receptor (L08102, forward primer 1817–1835; reverse primer 1980–1960), NK1 receptor (J05097, forward primer 2991–3010; reverse primer 3195–3176; preproenkephalin (S49491, forward primer 1033–1050; reverse primer 1180–1159); preprotachykinin (M34184, forward primer 717–735; reverse primer 877–85). The genomic primers recognized two polymorphic repeats: STS ET3 (forward primer: GCCTGCATTCATCTTCATCTGC, reverse primer: AAAGGTGGAACTCGCCCGTTT) and STS RR1023 (forward primer: AGCCTCATCGATGCTCCTGT, reverse primer: CCAAGAGCTACCTGCACTCC).
All of the gene specific primers used had similar sensitivities, detecting cDNA derived from as little as 10 pg total whole brain RNA, while the genomic primers detected as little as 10 pg of rat genomic DNA.