The calmodulin-binding transcription activator CAMTA1 is required for long-term memory formation in mice

Abstract

The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by calcium/calmodulin signaling in the nucleus. Recent studies have drawn attention to a new family of transcriptional regulators, the so-called calmodulin-binding transcription activator (CAMTA) proteins. CAMTAs are expressed at particularly high levels in the mouse and human brain, and we reasoned that, as calmodulin-binding transcription factors, CAMTAs may regulate the formation of long-term memory by coupling synaptic activity and calcium/calmodulin signaling to memory-related transcriptional responses. This hypothesis is supported by genetic studies that reported a correlation between Camta gene polymorphisms or mutations and cognitive capability in humans. Here, we show that acute knockdown of CAMTA1, but not CAMTA2, in the hippocampus of adult mice results in impaired performance in two memory tests, contextual fear conditioning and object-place recognition test. Short-term memory and neuronal morphology were not affected by CAMTA knockdown. Gene expression profiling in the hippocampus of control and CAMTA knockdown mice revealed a number of putative CAMTA1 target genes related to synaptic transmission and neuronal excitability. Patch clamp recordings in organotypic hippocampal slice cultures provided further evidence for CAMTA1-dependent changes in electrophysiological properties. In summary, our study provides experimental evidence that confirms previous human genetic studies and establishes CAMTA1 as a regulator of long-term memory formation.

Figure 1.

Analysis of CAMTA protein expression in mouse hippocampus. (A) Representative immunoblot showing expression of CAMTA1, CAMTA2, and β-Actin in mouse hippocampus at postnatal day (P) 1, 7, and 15, and at 3 mo. Position of molecular weight markers (kDa) is indicated. (B) Quantification of CAMTA1 and CAMTA2 protein expression (normalized to β-actin). Mean ± SEM and individual values are shown. N = 5 animals per group. (C,D) Specificity of CAMTA1 (C) and CAMTA2 (D) antibodies was confirmed by immunoblot analysis of primary mouse hippocampal neurons infected with control or Camta1 shRNA (C), or control or Camta2 shRNA (D).

Figure 2.

Acute Camta knockdown impairs long-term memory. (A) Verification of efficiency and specificity of different shRNA constructs by immunoblot analysis of primary mouse hippocampal neurons infected with the indicated rAAVs. Position of molecular weight markers (kDa) is indicated. (B–G) Gene expression analysis and behavioral phenotyping of mice after hippocampal stereotaxic delivery of rAAV-shCtrl2x or rAAV-shCamta1+2 (B–D) and rAAV-shCtrl or rAAV-shCamta1A or rAAV-shCamta1B (E–G). Contextual fear conditioning testing sessions performed at 1 h (STM) or 24 h (LTM) after conditioning are shown. In the object–place recognition test the training and 24-h testing sessions are shown. Equivalent exploration of the objects is indicated with a dashed line at 50%. The number of animals per group is indicated on the graphs. (STM) short-term memory, (LTM) long-term memory. Data are presented as mean + SEM. P values are indicated above the bars and were determined by two-tailed Student's t-test or Tukey's multiple comparison test when more than two groups were compared.

Figure 3.

Acute Camta1 knockdown does not affect neuronal morphology. The effect of Camta1+2 double knockdown (A–F), and Camta1 single knockdown (G–J) on CA1 pyramidal neuron morphology was assessed. (A) Representative photomicrographs of Golgi-impregnated CA1 pyramidal neurons. Focused projections of 3-D image stacks are shown. Scale bar, 20 µm. (B) Quantification of traced dendritic length of basal dendrites. Mean ± SEM and individual values are shown for n = 11 neurons from three animals (shCtrl2x) and n = 23 neurons from five animals (shCamta1+2). (C) Sholl analysis of traced basal dendrites. Mean ± SEM for n = 11 neurons from three animals (shCtrl2x) and n = 23 neurons from five animals (shCamta1+2) are shown. (D) Representative photomicrographs of Golgi-impregnated segments of CA1 pyramidal neuron basal dendrites. Focused projections of 3-D image stacks are shown. Scale bar, 5 µm. (E,F) Quantification of spine density (E) and spine length (F). Mean ± SEM and individual values are shown for n = 15 dendritic segments from three animals (shCtrl2x) and n = 20 dendritic segments from four animals (shCamta1+2). (G) Quantification of traced dendritic length of CA1 pyramidal neuron basal dendrites. Mean ± SEM and individual values are shown for n = 16 neurons from five animals per group. (H) Sholl analysis of traced basal dendrites. Mean ± SEM for n = 16 neurons from five animals per group are shown. (I,J) Quantification of spine density (I) and spine length (J). Mean ± SEM and individual values are shown for n = 16 dendritic segments from five animals (shCtrl) and n = 17 dendritic segments from five animals (shCamta1A). P-values were determined by two-tailed t-test (traced dendritic length, spine density, and spine length) or repeated-measures two-way ANOVA with Bonferroni post hoc test (Sholl analysis of dendritic complexity). Ctrl, shCtrl2x; KD, shCamta1+2.

Figure 4.

QRT-PCR validation of putative CAMTA1 target genes. (A) Relative mRNA expression of known activity-regulated genes in naïve animals (basal) and in animals that underwent object–place recognition training (trained). Hippocampal tissue was obtained from mice infected with shCtrl, shCamta1A, or shCamta1B. Number of animals per group is indicated in the legend. (B) Relative mRNA expression of indicated genes in the same animals as in A. Mean + 95%CI, as well as individual values are indicated.

Figure 5.

Validation of CAMTA1 knockdown efficiency in rat neurons. Primary rat hippocampal neurons and rat organotypic hippocampal slice cultures were infected with shCtrl or shCamta1. CAMTA1 protein expression was analyzed by Immunoblot. (A,B) Example photomicrographs of an organotypic slice culture infected with rAAV-shCtrl. Scale bar = 200 µm. (C,D), Representative immunoblots showing expression of CAMTA1 and Tubulin in rat hippocampal neurons (C) and rat organotypic cultures (D). Position of molecular weight markers (kDa) is indicated.