CaMKII inhibitor 1 (CaMK2N1) mRNA is upregulated following LTP induction in hippocampal slices

Abstract

CaMK2N1 and CaMK2N2 (also known as CaMKIINα and β) are endogenous inhibitors of calcium/calmodulin-dependent kinase II (CaMKII), an enzyme critical for memory and long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning. CaMK2N1/2 mRNAs are rapidly and differentially upregulated in the hippocampus and amygdala after acquisition or retrieval of fear memory. Moreover, CaMK2N2 protein levels increase after contextual fear conditioning. Therefore, it was proposed that CaMK2N1/2 genes (Camk2n1/2) could be immediate-early genes transcribed promptly (30-60 min) after training. As a first approach to explore a role in synaptic plasticity, we assessed a possible regulation of Camk2n1/2 during the expression phase of LTP in hippocampal CA3-CA1 connections in rat brain slices. Quantitative PCR revealed that Camk2n1, but not Camk2n2, is upregulated 60 min after LTP induction by Schaffer collaterals high-frequency stimulation. We observed a graded, significant positive correlation between the magnitude of LTP and Camk2n1 change in individual slices, suggesting a coordinated regulation of these properties. If mRNA increment actually resulted in the protein upregulation in plasticity-relevant subcellular locations, CaMK2N1 may be involved in CaMKII fine-tuning during LTP maintenance or in the regulation of subsequent plasticity events (metaplasticity).

Keywords: CaMK2N1; CaMK2N2; CaMKII; LTP; gene expression.

Figure 1.. Camk2n1 expression levels increase after induction of LTP in the CA1 region.

(A) LTP induction in CA3-CA1 synapses. Top left, sketch representing the experimental setup: S1, S2 and R, stimulation and recording electrodes, placed in stratum radiatum. S1, S2 were located at 100–200 μm from R. LTP was induced by HFS (four tetani at 100 Hz, 1 s each, separated by 20 s). Top right, sample average traces evoked by basal stimulation at 0.05 Hz, before (−15–0 min) and after (50–60 min) induction. Bottom, summary plot of fEPSP slope relative to baseline for all experiments (N=6). (B) qPCR revealed an increase in Camk2n1 expression 60 min following LTP induction. Left, Camk2n1 expression relative to Gapdh in individual slices for each condition (LTP and Control). Right, relative expression fold-change of Camk2n2 in LTP slices compared to their control slices. (C) Camk2n2 expression did not change after LTP induction. Left, Right: same as B for Camk2n2. Experiments were conducted at 31°C ± 1 °C. Wilcoxon signed-rank test, *P=0.031; n.s.= not significant.

Figure 2.. Camk2n1 expression increase correlates with LTP magnitude in each slice.

Camk2n1 percent expression change in each LTP slice was calculated relative to the corresponding control slice, for each animal (N=6). LTP magnitude (last 20 min) relative to baseline. Spearman’s rank correlation coefficient indicates a statistically significant positive correlation between Camk2n1 expression increase and LTP magnitude (r_s=0.943, P=0.018).