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. 2007 Jun 27;2(6):e573.
doi: 10.1371/journal.pone.0000573.

Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain

Melissa A Fowler  1 , Kyriaki Sidiropoulou, Emin D Ozkan, Christopher W Phillips, Donald C Cooper
Affiliations

Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain

Melissa A Fowler et al. PLoS One. .

Abstract

The canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that are activated by increases in intracellular Ca(2+) and G(q)/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6-9 weeks). In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS), pyramidal cell layer of the hippocampus (HIP), dentate gyrus (DG), and ventral subiculum (vSUB). TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2-6 of the prefrontal cortex (PFC), motor cortex (MCx), and somatosensory cortex (SCx). TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca(2+)and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. TRPC channel structure and mechanisms of activation.
Schematic showing the six transmembrane structure of the nonselective cation channels, TRPC4 and 5, and the conserved N-terminal ankryin and C-terminal TRP, CIRB, and PDZ domains. The channels are proposed to be activated by releases of intracellular Ca2+ stores from the ER, conformational changes following binding of IP3 to IP3R, and vesicular translocation. The channels are inhibited by PKC and potentiated by La3+.
Figure 2
Figure 2. Expression of the TRPC channels in the rat brain.
(A) Agarose gel showing a single product from real-time PCR of the TRPC channels (B) Pie chart showing the relative expression of the seven TRPC channels in rat whole brain determined using real-time PCR. Results was normalized to GAPDH mRNA levels. (n = 4; TRPC1: Avg = 0.0012, SEM = ±0.00011; TRPC3: Avg = 0.0018, SEM = ±0.00015; TRPC4: Avg = 0.0046, SEM = ±0.00037; TRPC5: Avg = 0.0022, SEM = ±0.000356; TRPC6: Avg = 0.00055, SEM = ±0.000055; TRPC7: Avg = 0.000027, SEM = ±0.0000054)
Figure 3
Figure 3. Expression of TRPC4 and 5 in rat and mouse brain.
(A1, B1, C1) In situ hybridization of TRPC4 in rat coronal brain slices. (Bregma, mm): A1, 3.70; B1, 1.00; C1, −4.16 (A2, B2, C1) In situ hybridization of TRPC5 in rat coronal brain slices. Coordinates same as A1,B1,C1. (D1, H1) Unlabeled S35-labeled probe control in situ hybridization of TRPC4 and TRPC5 in rat coronal brain slices. (E1, F1) In situ hybridization of TRPC4 in mouse coronal brain slices. (G1) In situ hybridization of TRPC4 in horizontal mouse brain slices. (E2,F2) In situ hybridization of TRPC5 in mouse coronal brain slices. (G2) In situ hybridization of TRPC5 in mouse horizontal brain slices. (D2, H2) Unlabeled S35-labeled probe control in situ hybridization of TRPC4 and TRPC5 in mouse coronal brain slices.
Figure 4
Figure 4. Quantification of the cRNA labelling densities of TRPC4 and 5 in the cortical layers of rat.
(A) (Left) In situ hybridization of TRPC4 in a rat coronal brain slice. (Right) In situ hybridization of TRPC5 in a rat coronal brain slice. Scale bars in µm. (B) (Upper) Magnification of TRPC4 5 cRNA labeling in the PFC layers. (Lower) Magnification of TRPC4 5 cRNA labeling in the SCx layers. (C) Quantification of the cRNA labelling densities of TRPC4 and 5 in the PFC and SCx layers expressed as a % change from background (n = 3). Scale bars in µm.
Figure 5
Figure 5. Expression of TRPC4 and TRPC5 in the hippocampal formation.
(A1, A2) cRNA labelling of TRPC4 mRNA in rat and mouse CA1-3, hilus, and dentate gyrus (B1, B2) cRNA labelling of TRPC5 mRNA in rat and mouse CA1-3, hilus, and dentate gyrus (C) cRNA labelling of TRPC4 and 5 in the ventral mouse hippocampal formation including the entorhinal cortex and ventral subiculum.
Figure 6
Figure 6. Region-specific expression of TRPC4 and TRPC5 mRNA.
(A–B) Pie charts showing the relative expression of the seven TRPC channels in rat prefrontal cortex (A) and lateral septum (B). Results was normalized to GAPDH mRNA levels. (n = 4; PFC TRPC1: Avg = 0.00055, SEM = ±0.000035; TRPC3: Avg = 0.00073, SEM = ±0.000066; TRPC4: Avg = 0.0031, SEM = ±0.00035; TRPC5 Avg = 0.0021, SEM = ±0.0010; TRPC6: Avg = 0.00010, SEM = ±0.000038; TRPC7: 0.000049, SEM = ±0.0000021; LS TRPC1: Avg = 0.0011, SEM = ±0.000020; TRPC3: Avg = 0.000082, SEM = ±0.000042; TRPC4: Avg = 0.0051, SEM = ±0.000011; TRPC5: Avg = 0.0015, SEM = ±0.00015; TRPC6: Avg = 0.00016, SEM = ±0.0000063; TRPC7: Avg = 0.00015, SEM = ±0.0000014) (C) Quantification of TRPC4 and TRPC5 mRNA levels in the striatum, prefrontal cortex (PFC), and lateral septum of adult rat using real-time PCR. Results was normalized to GAPDH mRNA levels. (n = 4)
Figure 7
Figure 7. Region-specific expression of TRPC4 and TRPC5 protein.
Graph shows the quantification of TRPC4 and TRPC5 protein levels in the striatum, prefrontal cortex, and lateral septum in the adult rat. Results was normalized to β-actin protein levels (Top) Representative bands of the immunoblots (n = 4). (Left inset) Immunoblot of rat brain tissue lysates and TRPC5 expressing HEK293 cells blotted with α-TRPC5 and of TRPC5 expressing HEK293 cells blotted with α-Flag.
Figure 8
Figure 8. Developmental and surface expression of TRPC5 protein in the mouse hippocampus.
(A) Quantification of TRPC5 protein levels in the HIP of E18 through P48 day old mice. (n = 4) (Top inset) Representative bands from the immunoblot showing TRPC5 expression in the HIP of E18-P48 mice. (B) Representative bands from an immunoblots showing surface (cross-linked) and intracellular TRPC5 protein in P0 and P48 mice (n = 6). (C) Quantification of TRPC4 and 5 protein levels in the HIP of P0 and P48 mice (n = 6; for P0, p = 0.002; for P48, p = 3.16e-5) (top inset) Representative bands from an immunoblots showing TRPC4 and TRPC5 protein in the HIP of P0 and P48 mice (n = 6)
Figure 9
Figure 9. Developmental expression of TRPC5 protein in the rat brain.
Quantification of TRPC5 protein levels in the prefrontal cortex (PFC), subiculum (SUB), and entorhinal cortex (ECx) in 21 and 63 day old rats. Results were normalized to β-actin protein levels. (Top left Inset) Representative bands of the immunoblot (n = 4)
Figure 10
Figure 10. Deep layer 5 pyramidal PFC neurons show a burst-induced nonselective cation current-mediated slow afterdepolarization following activation of group 1 mGluR receptors.
(A) Camera lucida reconstruction of a deep layer 5 pyramidal neuron from the PFC (left). (Right) schematic showing the PFC (infralimbic and prelimbic), shaded area where recordings were taken. (B) The group 1 mGluR agonist DHPG (50uM) induced a 20Hz burst-triggered (Inset) dADP (Red) compared to baseline before DHPG application to the bath. (C) (top) Intracellular Ca2+ chelation with BAPTA (10 mM; Blue) for 10 min after establishing whole-cell configuration substantially reduced the dADP induced by a 20 Hz burst and DHPG (50 uM)+BAPTA (10mM) immediately after establishing whole-cell configuration. (bottom) Bath application of the voltage-gated Na+ channel blocker, TTX (1 µM, Gray) for 10 min and elimination of action potentials at intensities 10 times the rheobase had no effect on the dADP induced by a 20 Hz burst and DHPG (50 uM)+TTX (1 µM). (D) The effects of the Na+/Ca2+ exchanger blocker benzamil (100 µM); Na+ ion 80% replacement with choline chloride; voltage-gated Na+ channel blocker, TTX (1 µM); intracellular Ca2+ chelation with BAPTA (10 mM); PKC activation with PdBU (1 µM),; IP3 receptor blockage with heparin (2 mg/ml); and nonselective cation channel blockade with flufenamic acid (100 µM) on the DHPG-(50 µM) and burst-induced dADP. (n>5, **p<0.01) (Inset) The TRPC4/5 potentiator, La3+ (100 µM) induced a small burst triggered (5 action potentials @ 20 Hz) dADP in the absence of mGluR activation and was blocked by the broad spectrum nonselective cation channel blocker, SKF96365 (100 µM). (n>5, **p<0.01)
Figure 11
Figure 11. Induction of a nonselective cation current-mediated dADP in brain regions expressing high levels of TRPC mRNA and protein.
(A) The group 1 mGluR agonist DHPG (50 uM; Red trace) induces a burst-triggered dADP in the prefrontal cortex (n = 6; p
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