Involvement of TRPC4 and 5 Channels in Persistent Firing in Hippocampal CA1 Pyramidal Cells

Abstract

Persistent neural activity has been observed in vivo during working memory tasks, and supports short-term (up to tens of seconds) retention of information. While synaptic and intrinsic cellular mechanisms of persistent firing have been proposed, underlying cellular mechanisms are not yet fully understood. In vitro experiments have shown that individual neurons in the hippocampus and other working memory related areas support persistent firing through intrinsic cellular mechanisms that involve the transient receptor potential canonical (TRPC) channels. Recent behavioral studies demonstrating the involvement of TRPC channels on working memory make the hypothesis that TRPC driven persistent firing supports working memory a very attractive one. However, this view has been challenged by recent findings that persistent firing in vitro is unchanged in TRPC knock out (KO) mice. To assess the involvement of TRPC channels further, we tested novel and highly specific TRPC channel blockers in cholinergically induced persistent firing in mice CA1 pyramidal cells for the first time. The application of the TRPC4 blocker ML204, TRPC5 blocker clemizole hydrochloride, and TRPC4 and 5 blocker Pico145, all significantly inhibited persistent firing. In addition, intracellular application of TRPC4 and TRPC5 antibodies significantly reduced persistent firing. Taken together these results indicate that TRPC4 and 5 channels support persistent firing in CA1 pyramidal neurons. Finally, we discuss possible scenarios causing these controversial observations on the role of TRPC channels in persistent firing.

Keywords: TRPC antagonists; TRPC channels; cholinergic modulation; hippocampus; intrinsic persistent activity; patch clamp; working memory.

Figure 1

TRPC4 and TRPC5 expression in mouse dorsal hippocampus. (A1) TRPC4 expression in a sagittal slice of the hippocampus. (A2) Low magnification image indicating the location of the image in A1. (B1) TRPC5 expression in CA1 in a sagittal slice of the hippocampus. (B2) Low magnification image indicating the location of the image in B1.

Figure 2

Carbachol dependent persistent firing in mice CA1 pyramidal cells. (A) Example of membrane potential response to 2 s depolarization current injection in the control condition (nACSF). Action potentials were fired only during the application of the stimulus, and no persistent firing was observed. (B) Example of long-lasting persistent firing during the application of 10 µM carbacol (CCh). (C) Example of self-terminating persistent firing in the presence of CCh (10 µM). (D) Current injection used in (A–C). The step depolarization current was 100 pA lasting for 2 s. (E) Percentages of cells that showed persistent firing in control condition (nACSF) and during the application of carbachol. (F) Post-stimulus firing frequency (Wilcoxon test, *** p < 0.001, n = 35). (G) Post-stimulus depolarization (paired t-test, *** p < 0.001, n = 35). All the recorded cells, shown in (E), were included in (F,G).

Figure 3

The effect of ML204 on persistent firing. (A) Example of persistent firing in CCh (10 µM). (B) Suppressed persistent firing in ML204 (10 µM) in the same cell shown in (A). (C) Current injection used in (A,B) (100 pA, 2 s). (D) Percentages of cells that showed persistent firing in CCh and during the application of ML204. (E) Post-stimulus firing frequency (Wilcoxon test, * p ≤ 0.05, n = 6). (F) Post-stimulus depolarization (paired t-test, ** p ≤ 0.01, n = 6). ML204 significantly suppressed persistent firing.

Figure 4

The effect of clemizole hydrochloride on persistent firing. (A) Example of persistent firing in CCh (10 µM). (B) Suppressed persistent firing in clemizole (CLE, 3 µM) in the same cell shown in (A). (C) Current injection used in (A,B) (100 pA, 2 s). (D) Percentages of cells that showed persistent firing in CCh and during the application of CLE. (E) Post-stimulus firing frequency (Wilcoxon test, * p ≤ 0.05, n = 9). (F) Post-stimulus depolarization (Wilcoxon test, * p ≤ 0.05, n = 9). Clemizole significantly suppressed persistent firing.

Figure 5

The effect of pico145 on persistent firing. (A) Example of persistent firing in CCh (10 µM). (B) Suppressed persistent firing in Pico145 (Pico, 100 nM) in the same cell shown in (A). (C) Current injection used in (A,B) (100 pA, 2 s). (D) Percentages of cells that showed persistent firing in CCh and during the application of Pico. (E) Post-stimulus firing frequency (Wilcoxon test, * p ≤ 0.05, n = 6). (F) Post-stimulus depolarization (Wilcoxon test, * p ≤ 0.05, n = 6). Pico145 significantly suppressed persistent firing.

Figure 6

The effect of intracellular application of TRPC4 antibody (1:100) on persistent firing. (A) Example of persistent firing recorded 5 min after the rupturing of the membrane. (B) Suppressed persistent firing recorded 15 min after the rupturing of the membrane in the same cell shown in (A). (C) Current injection used in (A,B) (100 pA, 2 s). (D) Percentages of cells that showed persistent firing in control (5 min) and 15 min after the rupturing of the membrane. (E) Post-stimulus firing frequency (Wilcoxon test, * p ≤ 0.05, n = 7). (F) Post-stimulus depolarization (Wilcoxon test, * p ≤ 0.05, n = 7). TRPC4 antibody significantly suppressed persistent firing.

Figure 7

The effect of intracellular application of TRPC5 antibody (1:100–500) on persistent firing. (A) Example of persistent firing recorded 5 min after the rupturing of the membrane. (B) Suppressed persistent firing recorded 15 min after the rupturing of the membrane in the same cell shown in (A). (C) Current injection used in (A,B) (100 pA, 2 s). (D) Percentages of cells that showed persistent firing in control (5 min) and 15 min after the rupturing of the membrane. (E) Post-stimulus firing frequency (Wilcoxon test, ** p ≤ 0.01). (F) Post-stimulus depolarization (paired t-test, *** p ≤ 0.001). TRPC5 antibody significantly suppressed persistent firing.

Figure 8

The effect of intracellular application of inactivated antibodies. (A–C) Inactivated TRPC4 antibody (In. Anti-TRPC4, 1:100). (A) Percentages of cells that showed persistent firing in control (5 min) and 15 min after the rupturing of the membrane. (B) Post-stimulus firing frequency (Wilcoxon test, ns, p = 0.11, n = 8). (C) Post-stimulus depolarization (paired t-test, * p ≤ 0.05, n = 8). (D–F) Inactivated TRPC5 antibody (In. Anti-TRPC5, 1:100). (D) Percentages of cells that showed persistent firing in control (5 min) and 15 min after the rupturing of the membrane. (E) Post-stimulus firing frequency (paired t-test, p = 0.82, n = 4). (F) Post-stimulus depolarization (paired t-test, p = 0.64, n = 4).