TRPC4 ion channel protein is selectively expressed in a subpopulation of dopamine neurons in the ventral tegmental area

Abstract

The nonselective cation channel TRPC4 has been shown to be present in high abundance in the corticolimbic regions of the brain and play a pivotal role in modulating cellular excitability due to their involvement in intracellular Ca 2+ regulation. Recently we reported their involvement in socialization and regulating anxietylike behaviors in rats. Given the important role for dopamine in modulating emotions involved in social anxiety we investigated whether TRPC4 protein and mRNA was found on dopaminergic neurons of the ventral tegmental area (VTA). Using emulsion autoradiography we found that TRPC4 mRNA is indeed present in the VTA and the substantia nigra. Additionally, immunohistochemistry verified it's presence on a subpopulation of dopamine neurons in the VTA. We confirmed these findings by testing Trpc4 knock-out rats in addition to wildtype animals. This novel finding suggests that TRPC4 plays a pivotal role in regulating dopamine release in a subpopulation of neurons that may modulate emotional and cognitive responses in social situations.The TRPC family of nonselective channels are made up of seven members (TRPC1-7) and the TRPC4 channel is one of the two most abundant in the mammalian brain 1 . Our recent findings indicate that the TRPC4 channel is important for sociability in a rodent model of social interaction 3 . For example, Trpc4 knock-out rats exhibit significantly decreased social interaction compared to their wild-type counterparts 3 . Previous experiments in our lab indicate that TRPC4 channels are highly expressed in the corticolimbic regions including the lateral septum, hippocampus, prefrontal cortex (PFC) and the amygdala 3 . These brain regions receive extensive input from dopamine (DA) neurons in the VTA. Given the importance of DA in modulating reward and stress we tested for the presence of TRPC4 protein in the cells of the VTA. We were particularly interested in whether TRPC4 channels were found in GABAergic interneurons and projection neurons or within tyrosine hydroxylase (TH) expressing DA neurons. RESULTS Trpc4 Knock-out RatsThe Sleeping Beauty (SB) gene-trap transposon method was used to create the Trpc4 knock-out animals 4 . The SB method uses cutand-paste transposable elements to generate heritable loss-offunction mutations. Fig. 1a shows the location of the trpc4 gene on the rat genome and where the transposon was inserted. By inserting the SB transposon into the first intron of the trpc4 gene, the full-length protein product is completely eliminated. Using primers for the Trpc4 knock-out and wildtype alleles, we were able to confirm the deletion using PCR and gel electrophoresis (Fig 1b). TRPC4 LocalizationAs shown Fig. 2, TRPC4 expression was found on a subpopulation of dopamine neurons in the VTA, in addition to positive expression in the substantia nigra. TRPC4 Fig. 1: a. Schema of the trpc4 gene in the rat genome and the Sleeping Beauty (SB) gene knock-out system. The trpc4 gene is located on chromosome 2 of the rat genome, between 143.35 Mb and 143.49 Mb. The Sleeping Be...