The coverslip containing the plated neurons was placed in a glass-bottomed chamber with 2 ml sterilized HBS. Isolated MNCs expressing AVP-eGFP were identified by their green fluorescence. Positive pressure was applied to the pipette (inner tip diameter, 10–20 μm), and the pipette was then targeted to AVP-eGFP neurons. After the pipette was attached to a cell, the positive pressure was removed and negative pressure applied to harvest cytoplasmic contents. Cytoplasmic contents of five AVP-eGFP neurons were harvested under visual control and pooled in a PCR tube with carrier RNA (poly-A RNA, Qiagen, Valencia, CA, USA). Total RNA was purified using an RNeasy Micro Kit (Qiagen), and heated with 5 μm Oligo dT primer (Takara Bio Inc., Shiga, Japan) and 1 mm dNTP mixture (Takara) at 65°C for 5 min then cooled on ice. The reverse transcription mixture (40 μl) contained these pooled cytoplasmic samples, 1× PrimeScript Buffer (Takara), 40 U RNase inhibitor (Takara), and 200 U PrimeScript RTase (Takara). Reverse transcription was carried out at 42°C for 60 min. After stopping the reaction at 70°C for 15 min, products were stored at −20°C until use. We performed PCR amplification of mRNA transcripts of AVP and ASIC subunits in a 50 μl reaction containing 3.3 μl of the reverse transcription mixture as template, 1× Ex Taq Buffer, 0.2 mm dNTP mixture, 0.12 μm forward and reverse primers, and 0.05 U Ex Taq (Takara) with the following amplification conditions: 94°C for 30 s; 40 cycles of 94°C for 30 s, 55°C for 1 min, 72°C for 1 min; and 72°C for 5 min. The amplified products were separated by electrophoresis on 2% agarose gels, stained with ethidium bromide, and visualized under UV irradiation. Gel images were captured using AE-6905H Image Saver HR (Atto, Tokyo, Japan). For ASICs, the following sets of primers were used: 5′-CACAGATGGCTGATGAAAAGCAG-3′ (forward)/5′-CATGGTAACAGCATTGCAGGTGC-3′ (reverse) for ASIC1a (Nagae et al. 2007) (GenBank accession no. NM_024154, product size 506 bp); 5′-ATGCCGTGCGGTTGTCCC-3′ (forward)/5′-CATGGTAACAGCATTGCAGGTGC-3′ (reverse) for ASIC1b (Nagae et al. 2007) (GenBank accession no. AJ309926, product size 563 bp); 5′-ATCTCTGCCTTGAATGCCAAGGTT-3′ (forward)/5′-AACTCCCCAGCGTGGTACAAGT-3′ (reverse) for ASIC2a (GenBank accession no. NM_001034014, product size 443 bp); 5′-CTGCCTTCATGGACCGTTTG-3′ (forward)/5′-CGAGTCCCATCTCTGAGGACCGG-3′ (reverse) for ASIC2b (Kawamata et al. 2006) (GenBank accession no. NM_012892, product size 429 bp); 5′-GATCCAGAGCCCTCTGATCCCTTGGG-3′ (forward)/5′-ATTCAACTCTTCCTGGAGCAGA-3′ (reverse) for ASIC3 (GenBank accession no. NM_173135, product size 603 bp); 5′-TCTGCCCGCCAAATATCTAC-3′ (forward)/5′-ATGCTAGCCCCAATGAACAG-3′ (reverse) for ASIC4 (Brockway et al. 2005) (GenBank accession no. NM_022234, product size 334 bp). As a positive control, we amplified glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) using the following primers: 5′-CATGCCGCCTGGAGAAACCTGCCA-3′ (forward)/5′-GGGCTCCCCAGGCCCCTCCTGT-3′ (reverse) (GenBank accession no. NM_017008.3, product size 429 bp). These primer sets were selected from previous reports (Brockway et al. 2005; Kawamata et al. 2006; Nagae et al. 2007) or were newly designed using Primer3 software (http://frodo.wi.mit.edu/) with confirmation of their specificity to the complementary sequences in the rat genome by NCBI BLAST (http://www.ncbi.nlm.nih.gov/blast/). We also performed the whole procedure without addition of reverse transcriptase (RT–) and obtained negative results. To confirm the specific expression of mRNA in AVP-eGFP neurons, we performed RT-PCR for GFP transcripts on five AVP-eGFP positive cells and five AVP-eGFP negative cells using the following primers: 5′-CACCATCTTCTTCAAGGACGAC-3′ (forward)/5′-ATGATATAGACGTTGTGGCTGTTGT-3′ (reverse) (GenBank accession no. YP002302326.1, product size 186 bp) (Ueta et al. 2005). We observed a band corresponding to GFP in all samples (n= 6) from five AVP-eGFP-positive cells, whereas no bands corresponding to GFP were detected in any of the samples (n= 3) from five AVP-eGFP-negative cells (data not shown).