Quantitative real-time PCR expression of the seven TRPC channels in rat brain
Results of real-time PCR performed on RNA isolated from adult rat whole brain (excluding the cerebellum) show that TRPC4 and 5 are the two predominantly expressed TRPC channels in the brain, comprising approximately 41% and 24% of the TRPC channel population respectively (). TRPC3, TRPC1, and TRPC6 are moderately expressed at 18%, 12%, and 5% of the population respectively. TRPC2 and TRPC7 show very little expression in the brain at less than 1%. The real-time PCR primers were designed to target all known splice variants of the TRPC channels and produce a single product (). The binding efficiency for each primer set was calculated in order to account for any differences in primer binding and the results were normalized to the housekeeping gene, GAPDH, in order to compare the expression of the different TRPC channels.
Expression of the TRPC channels in the rat brain.
Real-time PCR primer sequences for the seven TRPC channels in rat
Distribution of TRPC4 and 5 in the rodent brain
The qualitative expression of TRPC4 and 5 mRNA throughout the rat and mouse brain is presented in . The results of in situ hybridization of TRPC4 were observed to be similar between the species and are therefore shown together. Coronal and horizontal brain slices show that TRPC4 mRNA is highly expressed in the LS and cell body layer of the CA1-CA2 sub-regions of the HIP, vSUB, and dorsal tenia tecta (). Moderate expression was observed in the frontal cortex including the PFC (infralimbic and prelimbic), anterior cingulate, MCx, SCx, entorhinal cortex, piriform cortex, orbitofrontal cortex, amygdala, ventral hypothalamus, Purkinje and granule cell layers of the cerebellum. No specific labeling was detected in slices that were co- incubated with unlabeled control in situ hybridization probes for TRPC4 and 5. ()
Qualitative analysis of TRPC4 and TRPC5 mRNA expression in the rodent brain
Expression of TRPC4 and 5 in rat and mouse brain.
To examine the cortical layer-specific expression of TRPC4 and 5 in the frontal cortex, we performed quantitative analysis of the in situ hybridization signal in the superficial (layers 2/3) and deep (layer 5/6) cortical layers. Results show that TRPC4 mRNA is expressed at similar levels throughout layers 2,3,5, and 6 in all regions of the frontal cortex, including the PFC, anterior cingulate, MCx, SCx and orbitofrontal cortex. In layers lacking pyramidal neurons, such as layer 1 of the frontal cortex, there is no TRPC4 mRNA expression ().
Quantification of the cRNA labelling densities of TRPC4 and 5 in the cortical layers of rat.
Qualitative comparisons of TRPC4 mRNA expression in the cell body layer of the dorsal HIP of adult rat and mouse were similar. In both species TRPC4 expression is very high in CA1 while moderate expression in CA2 and DG, and little expression in CA3 and hilus is observed. Analysis of ventral HIP showed that TRPC4 is highly expressed in the vSUB with less expression in the entorhinal cortex (). In subcortical structures, there is moderate expression of TRPC4 mRNA in the amygdala, and ventral hypothalamus. In contrast, very low expression of TRPC4 mRNA is seen in either the dorsal striatum or nucleus accumbens, which matches the real-time PCR data indicating low expression of TRPC4 and 5 mRNA in the striatum relative to whole brain TRPC4 and 5 levels ().
Expression of TRPC4 and TRPC5 in the hippocampal formation.
Region-specific expression of TRPC4 and TRPC5 mRNA.
TRPC5 mRNA in situ hybridization indicates high expression concentrated in the deep layers 5/6 and superficial layers 2/3 of the PFC, vSUB, piriform, orbitofrontal, and entorhinal cortices (). As with TRPC4, no significant TRPC5 expression was detected in layer 1 of the PFC where there are few neuronal cell bodies. TRPC5 had little mRNA expression in the MCx or SCx. The cell body layer of the HIP had robust expression of TRPC5 in CA1, CA2, CA3, and hilus, and moderate expression in the DG and the vSUB ().
In subcortical structures high TRPC5 mRNA expression was detected in the Islands of Caleja, paraventricular hypothalamus, and dorsal and ventral hypothalamus. Little expression of TRPC5 is seen in the MCx, SCx and no expression is seen in the dorsal striatum although, interestingly, moderate expression of TRPC5 was detected in the nucleus accumbens (, ).
Relative TRPC channel expression in the prefrontal cortex and lateral septum
In situ hybridization results indicate that TRPC4 or 5 mRNA is highly expressed in the LS and PFC. To validate this using real-time PCR, we examined the relative expression of all TRPC channels in the PFC and LS of age matched adult rat. PCR of the seven TRPC channels in microdissections of the PFC indicate that TRPC4 and 5 mRNAs are the most highly expressed TRPC channels in PFC, representing 38% (TRPC4) and 34% (TRPC5) of the TRPC channel population. TRPC6, TRPC3, and TRPC1 had low mRNA expression in the PFC with 12%, 9%, and 6% respectively. TRPC7 and TRPC2 comprise <1% of the TRPC channel population in the PFC ().
Real-time PCR of the seven TRPC channels in microdissections of the LS of adult rat confirms that TRPC4 is the predominantly expressed channel in the LS, comprising 66% of the TRPC channel population. TRPC3 is moderately expressed in the LS with 22%, and TRPC5, TRPC6, and TRPC1 have lower expression with 4%, 4%, and 3% respectively. TRPC2 and TRPC7 comprise less than 1% of the TRPC channel population in the LS ().
Region-specific protein expression of TRPC4 and TRPC5
To examine whether the mRNA expression of TRPC4 and TRPC5 corresponds with the protein expression of these channels, we performed immunoblots for TRPC4 and TRPC5 protein in microdissections from striatum, PFC, and LS. We did not examine the relative protein expression of the TRPC4 and 5 in the brain due to potential differences in antibody affinities between the two channels. Due to these inherent limitations in immunoblotting, we restricted our protein analysis to a region-specific analysis 
. The antibody used for TRPC4 has previously been validated and shown to be specific 
. To verify the specificity of the TRPC5 commercial antibody we cloned TRPC5 into the mammalian transfection vector, pcDNA3.1-FLAG that inserts a FLAG tag on the C-terminal region of the protein. Immunoblots of protein harvested from HEK293 cells 48 hours after transfection with TRPC5-FLAG were blotted with both α-FLAG and α–TRPC5 antibodies. The detected bands were identical in size (inset, ), confirming that the FLAG-tagged protein expressed in the HEK293 cells is TRPC5 protein. No bands were detected on immunoblots of protein harvested from cells transfected with empty vector or from non-transfected cells (data not shown). In addition, immunoblots of protein harvested from TRPC5-FLAG transfected HEK293 cells and rat whole brain show identical bands, thus confirming the specificity of the TRPC5 antibody in rat brain lysates (inset, ).
Region-specific expression of TRPC4 and TRPC5 protein.
Results of TRPC5 protein expression from immunoblots from rat striatum, PFC, and LS show that TRPC4 protein is most highly expressed in the LS and moderately expressed in the PFC with lower expression in the striatum. TRPC5 protein is most highly expressed in the PFC and moderately expressed in the LS with lower expression in the striatum (). These results show that in general the protein expression of these channels match the mRNA expression levels in these regions.
Developmental expression of TRPC4 and 5 proteins
To determine if TRPC4 or 5 protein expression is developmentally regulated, we examined expression at different stages of development in the HIP. Immunoblots performed for TRPC4 and 5 protein in microdissections of HIP from post-natal day 0 (P0) and post-natal day 48 (P48) mice show that TRPC4 and 5 protein levels are higher at P48 compared to P0 (). HIP TRPC5 protein is present at low levels from embryonic day 18 (E18) E18 to P20 and increase robustly from P20 to P48 (). Similar results were observed in rat PFC, vSUB, and ECx comparing post-natal day 21 (P21) and post-natal day 63 (P63) rats ().
Developmental and surface expression of TRPC5 protein in the mouse hippocampus.
Developmental expression of TRPC5 protein in the rat brain.
To examine the surface expression of TRPC5 protein in P0 and P48 day old mice, we used the irreversible, membrane impermeable crosslinker, BS3
to cross-link surface-expressed TRPC proteins. Cross-linked samples were blotted and the ~100 kD band representing the remaining intracellular pool of TRPC5 was quantified. These methods are similar to those described by Grosshans et al 
. Cross-linking resulted in immunoblots that showed a TRPC5 labeled high molecular weight smear (>206 kD) in cross-linked samples that was not present in non-cross-linked samples, indicating successful surface cross-linking (). This procedure produces a reduction in the TRPC5 ~100 kD band in cross-linked samples compared to non cross-linked sample which reflects TRPC5 surface expression. (). To ensure that cross-linking produces no non-specific changes in protein we examined levels of the intracellular protein, beta-actin, and found that the total amount of protein was not significantly different across cross-linked and non cross-linked samples for P0 or P48 (Figure S1
). This confirms previous work demonstrating the lack of membrane permeability of BS3 
Expression of TRPC4 and TRPC5 channels is associated with a burst-induced delayed after-depolarization
Given the robust expression of these channels in adults and their presence on the surface of the membrane we attempted to identify a function for these channels in the corticolimbic system using whole-cell patch-clamp recording in the deep layer pyramidal neurons. Using the Group 1 metabotropic glutamate receptor (mGluR) agonist DHPG, we observed an action potential burst-induced delayed after-depolarization (dADP) lasting several seconds after the burst. This dADP correlated with the presence of TRPC4 and 5 expression. It was robust in areas such as layer 5 pyramidal neurons in the medial PFC, LS, and SUB where mRNA expression is high, yet it was absent in the nucleus accumbens and striatum where mRNA expression is low (, ). Na+
replacement by 80% with choline-Cl substantially reduced the dADP, but did not eliminate it, indicating a substantial Na+
ion component to the dADP (). Bath application of the voltage gated Na+
channel blocker, tetrodotoxin (1 µM), had no effect on the dADP amplitude, indicating that the current underlying the dADP is not a voltage-gated TTX-sensitive Na+
current (). Intracellular application of the Ca2+
chelator BAPTA (10 mM) significantly reduced the dADP demonstrating the role of intracellular Ca2+
in activation (). Application of the Na+
exchanger blocker benzamil (100 µM) failed to reduce the dADP (), ruling out the involvement of a Na+
exchanger. The dADP was reduced by the protein kinase C activator, PdBU (1 µM), which has also been shown to deactivate TRPC5 channels 
(). Application of the non-selective cation channel blocker, flufenamic acid (100 µM), and IP3
receptor blocker, heparin (2 mg/ml), significantly reduced the dADP (). A small dADP was induced in the absence of DHPG by the trivalent cation La3+
(100 µM), which has been shown to potentiate TRPC4 and 5 and block TRPC3, 6, and 7 current in transfected HEK293 cells 
( inset). This induction of the dADP by La3+
was blocked by the non-specific TRP channel blocker, SKF96365 (100 µM ) ( inset).
Deep layer 5 pyramidal PFC neurons show a burst-induced nonselective cation current-mediated slow afterdepolarization following activation of group 1 mGluR receptors.
Induction of a nonselective cation current-mediated dADP in brain regions expressing high levels of TRPC mRNA and protein.