Role of the TRPC1 Channel in Hippocampal Long-Term Depression and in Spatial Memory Extinction

Abstract

Group I metabotropic glutamate receptors (mGluR) are involved in various forms of synaptic plasticity that are believed to underlie declarative memory. We previously showed that mGluR5 specifically activates channels containing TRPC1, an isoform of the canonical family of Transient Receptor Potential channels highly expressed in the CA1-3 regions of the hippocampus. Using a tamoxifen-inducible conditional knockout model, we show here that the acute deletion of the Trpc1 gene alters the extinction of spatial reference memory. mGluR-induced long-term depression, which is partially responsible for memory extinction, was impaired in these mice. Similar results were obtained in vitro and in vivo by inhibiting the channel by its most specific inhibitor, Pico145. Among the numerous known postsynaptic pathways activated by type I mGluR, we observed that the deletion of Trpc1 impaired the activation of ERK1/2 and the subsequent expression of Arc, an immediate early gene that plays a key role in AMPA receptors endocytosis and subsequent long-term depression.

Keywords: TRPC; long-term depression; mGluR; memory extinction.

Figure 1

Generation of the Trpc1 conditional knockout mice. A. Mice having the second exon of Trpc1 gene flanked with loxP site (panel A, middle line) were bred either with the PGK-Cre recombinase mice line to obtain a constitutive Trpc1 knockout mouse line (panel 1, upper line) or with mice expressing the CreERT2 fusion protein under the control of the regulatory elements of the CaMKIIα gene (CaMKII-CreERT2 transgene, panel 1, bottom line) to obtain Heterozygous transgenic mice (Trpc1^lox/− CaMKII^Cre+/− mice) (panel A, bottom line). B. Trpc1 expression measured by RT-qPCR analysis in hippocampal samples from Trpc1^lox/− CaMKII^Cre+/− and Trpc1^lox/− CaMKII^Cre−/− mice 10 days after the last IP injection of tamoxifen. *: p < 0.05, Student’s t-test, n = 9 samples of hippocampus from three different mice for each genotype (i.e., 3 independent experiments).

Figure 2

Trpc1 gene deletion impairs reference memory extinction. Morris Water Maze (MWM) test. A. The average escape latencies, i.e., the time required for CTRL (blue line, n = 15) and cKO mice (red line, n = 9) to reach the platform. All mice were treated with tamoxifen (5 days injection + 10 days of delay). B. Memory extinction evaluated by repeated probe tests (after the platform has been removed) at days 8 to 12. *: p < 0.05, two-way repeated measures ANOVA. C. The same experiment but the mice were injected with tamoxifen after the learning period, i.e., at days 8 to 12 and memory extinction evaluated at 10 days after, i.e., at days 22 to 26 (n = 7 vs 7). *: p < 0.05, two-way repeated measures ANOVA.

Figure 3

Pharmacological inhibition of TRPC1/4/5 impairs reference memory extinction. CTRL mice were submitted to a MWM training and thereafter were injected intraperitoneally with 0.1 mg kg⁻¹ weight pico145 (green line, n = 6) or with vehicle (blue line, n = 5). A. The average escape latencies (before injection). B. Memory extinction evaluated by repeated probe tests at days 8 to 12, one hour after injection of pico145 or vehicle. *: p < 0.05, two-way ANOVA.

Figure 4

Dihydroxyphenylglycine- (DHPG) induced long-term depression (LTD). A. The input–output relationship between field excitatory postsynaptic potentials (fEPSP) measured in CA1 stratum radiatum and the intensity of stimulation in brain slices from CTRL mice (blue lines) and cKO mice (red lines), both injected with tamoxifen. B. Time-course of fEPSP slopes (mean ± SEM) measured before and after stimulation with 50μM DHPG. CTRL brain slices (blue dots), cKO slices (red dots), and CTRL slices treated with 1 nM pico145 (green dots). Upper insets show representative traces of fEPSP before and 40 min after DHPG. ***: p < 0.001, one-way repeated measures ANOVA followed by Bonferroni test, n = 5 for each group).

Figure 5

DHPG-induced signaling pathways. Western Blot analysis of protein expression in hippocampal slices stimulated for 0, 5, 20, or 60 min with 50µM DHPG (two slices per sample, n= 3 to five independent experiments per group) A. The phosphorylation of ERK1/2 and the expression of Arc were significantly increased (by 3.6 times and 1.6 times, respectively) after a 5 and 20 min stimulation with DHPG in the Trpc1^+/+ brain slice. B. The expression of B56α and spectrin were significantly diminished (by 20 and 22% respectively) after a 60 min stimulation with DHPG in Trpc1^+/+ brain slices. These effects were not observed in Trpc1^−/− mice.