Heteromeric canonical transient receptor potential 1 and 4 channels play a critical role in epileptiform burst firing and seizure-induced neurodegeneration

Abstract

Canonical transient receptor potential channels (TRPCs) are receptor-operated cation channels that are activated in response to phospholipase C signaling. Although TRPC1 is ubiquitously expressed in the brain, TRPC4 expression is the most restrictive, with the highest expression level limited to the lateral septum. The subunit composition of neuronal TRPC channels remains uncertain because of conflicting data from recombinant expression systems. Here we report that the large depolarizing plateau potential that underlies the epileptiform burst firing induced by metabotropic glutamate receptor agonists in lateral septal neurons was completely abolished in TRPC1/4 double-knockout mice, and was abolished in 74% of lateral septal neurons in TRPC1 knockout mice. Furthermore, neuronal cell death in the lateral septum and the cornu ammonis 1 region of hippocampus after pilocarpine-induced severe seizures was significantly ameliorated in TRPC1/4 double-knockout mice. Our data suggest that both TRPC1 and TRPC4 are essential for an intrinsic membrane conductance mediating the plateau potential in lateral septal neurons, possibly as heteromeric channels. Moreover, excitotoxic neuronal cell death, an underlying process for many neurological diseases, is not mediated merely by ionotropic glutamate receptors but also by heteromeric TRPC channels activated by metabotropic glutamate receptors. TRPC channels could be an unsuspected but critical molecular target for clinical intervention for excitotoxicity.

Fig. 1.

TRPC1 and TRPC4 are the predominant TRPCs expressed in septum and hippocampus. A, quantitative real-time PCR comparison of the relative expression of TRPCs in the septum and hippocampus of wild-type mice. The relative fold expression is plotted on a log scale as mRNA molecules per 1000 GAPDH mRNA molecules. B, Western blot analysis of TRPC4 expression in protein fractions obtained from brains of wild-type and TRPC4-knockout mice, respectively, with in-house generated anti-TRC4 antibody 781. In mouse tissues, two variants of the TRPC4 protein are expressed, the “full-length” TRPC4 (TRPC4α) protein and a slightly smaller variant, called TRPC4_Δ (or TRPC4β). C and D, immunofluorescence labeling of TRPC4 in the septum of wild-type (C) and TRPC4 knockout mice (D). Scale bar, 300 μm (C and D). LS, lateral septum; MS, medial septum. E, colocalization of TRPC4 (red) with the neuronal marker NeuN (green) in the lateral septum. Scale bar, 12 μm. F and G, pre-embedding immunogold labeling for TRPC4 in the mouse lateral septum. Note that TRPC4 is predominantly localized at the postsynaptic plasma membrane in close vicinity to the unlabeled presynaptic terminals. Scale bars: F, 0.5 μm; G, 0.2 μm. H, colocalization of TRPC4 (red) and mGluR1 (green) in the septum. I, colocalization of TRPC4 (red) and syntaxin (green) in the hippocampal CA3 region. Scale bars: H, 12 μm; I, 60 μm.

Fig. 2.

Group I mGluRs mediate the plateau potentials induced by 1S,3R-ACPD in the dorsolateral septum. A, in wild-type dorsolateral septal neurons, injection of a series of current step pulses (20 ms, 0.1–1.0 nA, −80 mV) was followed by a simple decay of the membrane potential. After superfusion of the group I mGluR agonist 1S,3R-ACPD (30 μM), injection of the same series of current step pulses consistently induced the appearance of subthreshold (open arrowhead) and prolonged plateau (arrow) depolarizing afterpotentials (n = 13). Cells were manually clamped at −80 mV to offset the agonist-induced membrane depolarization. B, action potentials were evoked by a brief depolarizing current step (0.8 nA, 20 ms) applied every 10 s. Superfusion of LY367385 (100 μM) resulted in a complete block of the 1S,3R-ACPD-plateau depolarizing responses (left, four of four dorsolateral septal neurons). Likewise, superfusion of 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt; 10 μM) also blocked the 1S,3R-ACPD-induced plateau potential (right; four of six dorsolateral septal neurons).

Fig. 3.

TRPC1 and TRPC4 mediate the plateau potentials underlying mGluR agonist-induced epileptiform burst firing in the dorsolateral septum. A, action potentials were evoked by a brief depolarizing current step (0.8 nA, 20 ms) applied every 10 s. Superfusion of SKF96365 (20 μM) resulted in a complete block of the 1S,3R-ACPD-induced subthreshold and plateau depolarizing responses (n = 3) (red trace). The SKF96365 block was fully reversible upon washout of the drug (blue trace). B, a brief depolarizing current step (0.8 nA, 20 ms) was applied every 10 s to evoke action potentials, and 20 consecutive traces during the wash-in of mGluR agonists were shown. Note the transition from normal firing to subthreshold responses and the full plateaus as the agonist concentration increases during the wash-in. Superfusion of 1S,3R-ACPD (30 μM) in TRPC1/4 double-knockout (DKO) mice (n = 10) failed to induce the subthreshold or plateau depolarizing responses seen in age-matched wild-type controls (i.e., 100% of the dorsolateral septal neurons in TRPC1/4 DKO mice were nonresponding cells). Consistent with the complete absence of mGluR agonist-induced plateau responses, the dorsolateral septal neurons in TRPC1/4 DKO mice failed to exhibit the spontaneous bursting seen in age-matched wild-type control cells after depolarizing the cells during 1S,3R-ACPD superfusion by injecting a constant holding current. The input resistances of the dorsolateral septal neurons in the TRPC1/4 DKO mice were not significantly different from that of wild-type neurons (78.5 ± 4.2 and 68.5 ± 5.2 MΩ, respectively) (p > 0.05, unpaired t test). Scale bars for insets, 20 mV, 500 ms. C, the percentage of cells exhibiting mGluR agonist-induced full plateau potential in wild-type, TRPC1 knockout, and TRPC1/4 DKO mice. D, neurons in the dorsolateral septum were recorded in whole-cell patch-clamp configuration (V_h, −65 mV). The voltage-gated sodium and calcium channels were blocked by tetrodotoxin (1 μM) and Cd²⁺ (30 μM). The I-V relationship was then determined by a slow voltage ramp (−100 to +40 mV; 2 mV/s). The current induced by 30 μM 1S,3R-ACPD (I-ACPD) or by 30 μM 1S,3R-ACPD and 20 μM SKF96365 (I-ACPD,SKF) was determined by subtracting the control current from the current in the presence of 1S,3R-ACPD or 30 μM 1S,3R-ACPD and 20 μM SKF96365. Note the significant reduction of responses to 1S,3R-ACPD by SKF96365 in wild-type mice and the minimal response to 1S,3R-ACPD in TRPC1/4 DKO mice.

Fig. 4.

Seizure-induced neuronal cell death in the dorsolateral septum is significantly reduced in TRPC1/4 double-knockout mice. A, pilocarpine induces comparable intensities of seizures in wild-type and TRPC1/4 double-knockout (DKO) mice (p > 0.05, two-way analysis of variance). Pooled data (mean ± S.E.M.) were plotted (n = 24, 16 for WT at 175 and 280 mg/kg pilocarpine; n = 12, 6 for TRPC1/4 DKO at 175 and 280 mg/kg pilocarpine). See Materials and Methods for description of seizure scale. B, the mortality after pilocarpine injections within the first 24 h is significantly reduced in TRPC1/4 DKO mice (n = 12, 6) compared with wild-type mice (n = 24, 10, 16). C, representative images of FJC stained neurons in the dorsolateral septal nucleus (DLSN) of wild-type and TRPC1/4 DKO mice (2-day survival: wild-type, 175 mg/kg; TRPC1/4 DKO, 280 mg/kg). Scale bar, 0.20 mm (0.05 mm for insets). D, serial coronal sections containing septum (50 μm) were stained with FJC. FJC-positive neurons in the DLSN (see C) were counted in three sections approximately 300 μm apart. Mice (five WT treated with 175 mg/kg pilocarpine; five TRPC1/4 DKO treated with 280 mg/kg pilocarpine) included in analysis showed comparable seizures. TRPC1/4 DKO mice exhibit a significant reduction in FJC-positive neurons in the DLSN (two-tailed unpaired t test; **, P < 0.01).

Fig. 5.

Sparing of hippocampal neurons from pilocarpine seizure induced neurodegeneration in TRPC1/4 double-knockout mice. A and B, representative FJC stained transverse sections through the hippocampus in wild-type (A) and TRPC1/4 double-knockout (B) mice (2-day survival: wild-type, 175 mg/kg; TRPC1/4 KO, 280 mg/kg). C and D, Nissl staining in representative transverse sections through the hippocampus in wild-type (C) and TRPC1/4 KO mice (D). There is a noticeable reduction of Nissl-stained cell bodies in CA1 (open arrowhead) and CA3 (closed arrowhead) regions in wild-type but not TRPC1/4 KO mice. E to H, high-power photomicrographs illustrating the reduced gliosis and sparing of CA1 and CA3 neurons in the TRPC1/4 KO hippocampus (G and H) versus the same regions in wild-type hippocampus (E and F). The photomicrographs were taken from regions demarcated by the arrowheads in C and comparable regions in D. Scale bars: A to D, 200 μm; E–H, 50 μm.

Fig. 6.

Neuronal cell death after pilocarpine-induced seizure is reduced in TRPC1/4 double-knockout mice. Serial coronal sections (50 μm) from mice with similar pilocarpine-induced seizures (see Fig. 4D) were stained with Nissl and surviving neurons were counted using Stereologer with a 100× oil-immersion objective. Note that number of the surviving neurons in the CA1 region is significantly higher in TRPC1/4 double-knockout (DKO) mice (p < 0.05, two-tail unpaired t test).