Single-cell RT-PCR gene expression profiling of acutely dissociated and immunocytochemically identified central neurons

Abstract

Identification of neurons for single-cell mRNA profiling is difficult when cells of interest are located in heterogeneous brain regions. We developed a protocol in which acutely dissociated neurons are immunocytochemically labeled prior to single-cell reverse transcription-polymerase chain reaction (RT-PCR). We tested the protocol on hypothalamic melanin-concentrating hormone (MCH) and prepro-orexin (PPO) neurons, which are similarly distributed but functionally different. Cells dissociated from the perifornical region of the posterior hypothalamus of juvenile or adult rats were incubated with anti-MCH or anti-PPO primary antibodies, followed by washout and incubation with fluorescein-tagged secondary antibodies. Individual labeled cells were subjected to RT-PCR with primers for PPO and MCH. MCH mRNA was detected in 26 out of the 38 successfully reverse-transcribed cells identified as MCH-containing, and 28 cells out of the 42 identified as PPO-containing expressed PPO mRNA. No cell expressed both mRNAs. Most MCH neurons tested (five out of six) expressed the adrenergic α 2A receptor mRNA, whereas it was absent from all seven PPO neurons tested. Neither PPO ( n=11) nor MCH ( n=6) cells expressed the type 2 orexin receptor mRNA. Thus, the method allows, with at least 66% confidence, immunocytochemical cell identification prior to mRNA studies of single neurons located in heterogeneous brain regions.