Single-cell RT-PCR Analysis and CRF Effects on TRPC Channels Expressed in Rat Brain

The canonical transient receptor potential (TRPC) channels comprise a family of nonselective cation channels composed of seven members (TRPC1-7). TRPC channels are widely distributed in the nervous system and contribute to neuronal excitation. In the present study, using the single-cell RT-PCR method, the distribution of TRPC channel mRNA was analyzed in cholinergic neurons in the nucleus basalis (NB), serotonergic neurons in the dorsal raphe nucleus (DRN), noradrenergic neurons in the locus coeruleus (LC), dopaminergic neurons in the substantia nigra (SN), and dopaminergic neurons in the ventral tegmental area (VTA). NB, LC, SN and VTA neurons were cultured from 3-5 day-old and DRN from 10-12 day-old Long Evans rats. Single-cell RT-PCR was performed on these cultured neurons using the previously described method (Kawano et al., 2004, Neuroscience letters, 358:63). Tyrosine hydroxylase primers were used to identify noradrenergic neurons in LC and dopaminergic neurons in SN and VTA. Choline acetyltransferase primers were used to identify cholinergic neurons in NB and tryptophan hydroxylase primers were used to identify serotonergic neurons in DRN. TRPC1 mRNA was most frequently detected in all neuron types. TRPC2, involved in pheromone sensing, was not present in any of the five brain areas examined. TRPC3, 4 and 5 existed most frequently in cholinergic neurons in NB (80-90%). TRPC6 was relatively more frequent in dopaminergic neurons in VTA (58%) and SN (46%). TRPC7 was most frequently found in noradrenergic neurons (80%) in LC and cholinergic neurons (80%) in the NB. Interestingly, cholinergic neurons in the NB showed the highest frequency of TRPC mRNA expression. Present results demonstrate that each type of neuron expresses specific combination of TRPCs, suggesting that the specific TRPCs are responsible for excitation of each type of neurons. Secondly, I have investigated the effects of corticotrophin-releasing factor (CRF) on nucleus basalis (NB) cholinergic neurons with emphasis on non-selective cation (TRPC) channels. I have identified PKC as a necessary signal transducer in the activation of TRPC channels Finally, I have studied the effects of CRF on TRPC5 using HEK293A cell lines that have been transfected with TRPC5 and CRHR1 cDNAs. I found PKC activation a requirement for the opening of TRPC5 channels.