Nuclear calcium/calmodulin regulates memory consolidation

Abstract

The neuronal response to a Ca2+ stimulus is a complex process involving direct Ca2+/calmodulin (CaM) actions as well as secondary activation of multiple signaling pathways such as cAMP and ERK (extracellular signal-regulated kinase). These signals can act in both the cytoplasm and the nucleus to control gene expression. To dissect the role of nuclear from cytoplasmic Ca2+/CaM signaling in memory formation, we generated transgenic mice that express a dominant inhibitor of Ca2+/CaM selectively in the nuclei of forebrain neurons and only after the animals reach adulthood. These mice showed diminished neuronal activity-induced phosphorylation of cAMP response element-binding protein, reduced expression of activity-induced genes, altered maximum levels of hippocampal long-term potentiation, and severely impaired formation of long-term, but not short-term, memory. Our results demonstrate that nuclear Ca2+/CaM signaling plays a critical role in memory consolidation in the mouse.

Figure 5.

CaMBP transgene disrupts spatial learning and memory formation in the water-maze task. A, Four groups of mice were tested: CaMBP transgenics (n = 15), CaMBP transgenics on Dox (n = 21), wild types (WT) (n = 11), and wild types on Dox (n = 14). They showed normal visible platform learning. The average time to reach the platform was calculated for each day, and values represent group means ± SEM. A two-way ANOVA with repeated measures between CaMBP and wild-type mice off Dox showed no significant effect of genotype. B, CaMBP transgenics are impaired during hidden platform training. A probe trial was given on every training day before starting the training session (days 1-6) and 24 hr after completing training (day 7). Comparison of mean ± SEM percentage time spent in target (TAR) quadrant is shown. Three-way ANOVA with repeated measures revealed a significant two-way interaction (genotype by Dox) (F_(1,57) = 4.38; p < 0.05). Planned comparison confirmed that CaMBP mice off Dox spent significantly less time than controls in the target quadrant on the last 3 d (days 5-7) (p < 0.05). C, CaMBP transgenics show impaired spatial memory in the 24 hr probe trial. Bars represent the percentage of time spent swimming in the target, opposite, right, and left of target quadrants. A two-way ANOVA revealed a significant effect of genotype (F_(1,57) = 7.82; p < 0.01) and Dox (F_(1,57) = 9.13; p < 0.01). Planned comparisons between the four animal groups confirmed a significant difference between CaMBP transgenic mice off Dox and all control groups (^*p < 0.02). All three control groups spent significantly more time in the target quadrant than in each of the other quadrants (t test; p < 0.02 for all groups). D, Transgene expression was activated by withdrawal of Dox in the group of CaMBP transgenic mice that had acquired the water-maze task with the transgene suppressed. The mice were placed off Dox immediately after completing the training and the 24 hr probe trial and then retested 2 weeks later; transgene activation after spatial memory acquisition had no effect on recall performance. A two-way ANOVA revealed a significant two-way interaction (genotype by Dox) (F_(1,56) = 7.33; p < 0.01). Planned comparisons between the four animal groups confirmed a significant difference between CaMBP mice trained with the gene activated and the three control groups (^*p < 0.01).

Figure 1.

Generation and characterization of the CaMBP mice. A, Transgenic mice expressing the tTA with the CaMKIIα promoter were crossed with mice carrying the tetO promoter linked to the CaMBP gene. The CaMBP is expressed only in double transgenic mice in the absence of Dox. B, Regional distribution of CaMBP mRNA was analyzed by in situ hybridization. CaMBP expression was detected in the cortex (Cx), hippocampus (Hip), and striatum (St). Amy, Amygdala. Dox suppresses CaMBP expression. C, Western blot analysis of hippocampal nuclear and cytoplasmic fractions was performed to evaluate CaM distribution. No difference was detected between wild-type (WT) and CaMBP transgenic mice in either nuclear or cytoplasmic CaM levels. A nuclear antigen [neuronal-specific nuclear protein (NeuN)] and a cytoplasmic antigen [Syntaxin 13 (Syn13)] were used as controls. D, Immunostaining of brain sections confirmed that CaM distribution is not altered in CaMBP transgenic mice. The CA1 region of the hippocampus is shown for a wild-type and a transgenic mouse. DAPI staining was used to label nuclei. Scale bar, 5 μm.

Figure 2.

CaMBP immunolocalization. A, CaMBP immunostaining in cell culture. COS-7 cells were transiently transfected with CaMBP and GFP. Cells that are positive for GFP are also positive for CaMBP; nuclear counterstaining confirms CaMBP nuclear localization. Scale bar, 30 μm. B, The nuclear localization of CaMBP was confirmed by staining brain sections. DAPI staining labels nuclei. CaMBP-positive cells were detected only in brain areas in which CaMBP mRNA is expressed and was exclusively nuclear. Immunostaining is shown for the CA1 region of the hippocampus and thalamus of CaMBP transgenic mice and in the CA1 region of the hippocampus of wild-type (WT) animals. Scale bar, 25 μm.

Figure 3.

Altered S133-CREB phosphorylation and c-Fos levels in CaMBP mice after seizure induction. A, CREB phosphorylation was detected by immunofluorescence with a phospho-S133-specific antibody in wild-type (WT) and CaMBP transgenics at baseline or 10 min after seizure onset. Representative images from the CA1 hippocampal subregion are shown. Scale bar, 50 μm. B, Quantification of phospho-S133 CREB and total CREB in the CA1 region of hippocampus. Values represent group mean intensity of phospho-S133 CREB or CREB immunoreactivity and have been normalized to baseline of the wild-type group. Seizure induced CREB phosphorylation in wild-type mice (t test; ^*p < 0.05; n = 7) but not in CaMBP transgenics. There was no significant difference in baseline pCREB levels between wild types and CaMBP mice (CaMBP, 119 ± 14% of wild type). Total CREB levels were not affected by seizures and did not differ between wild types and transgenics (CaMBP, 105.1 ± 12% of wild-type; n = 4). C, Western blot quantification of phospho-MAPK in the hippocampus. Values represent the ratio of phosphorylated to total p42 and p44 MAPK levels in whole-cell hippocampal extracts from wild-type and mutant mice with and without seizure (n = 4). Seizure caused an increase in p42 and p44 phosphorylation, but no difference was detected between wild-type and CaMBP transgenic mice. D, Protein levels of c-fos in the striatum. Seizure caused an increase in c-fos in the wild-type but not in the transgenic mice. See Results for quantification.

Figure 4.

CaMBP mice exhibit LTP deficits. A, Analysis of input-output curves plotting the fEPSP slopes and their corresponding presynaptic fiber volley amplitudes of wild-type (WT) (n = 5) and CaMBP transgenic (n = 5) mice shows no significant differences. B, Paired-pulse facilitation in slices from wild-type (n = 8) and CaMBP transgenic mice (n = 9). Percentage of facilitation, measured by taking a ratio of the second fEPSP slope over the first fEPSP slope between 50 and 250 msec, was quantified. Paired-pulse facilitation in the CaMBP transgenic mice does not differ from wild type. C, LTP induced by one-train tetanic stimulation (100 Hz for 1 sec) in wild-type mice (n = 7) and CaMBP transgenic mice (n = 7). The level and time course of potentiation in the two groups was indistinguishable. D, Late-phase LTP induced by three-train tetanic stimulation was decreased in CaMBP transgenic mice. Potentiation at 3 hr after tetanus: wild type, 220 ± 22%, n = 8; CaMBP, 150 ± 29%, n = 11 (t test; p < 0.05).

Figure 6.

LTM is impaired in the CaMBP transgenic mice. A, Object recognition. Four groups of mice were tested: wild types (WT) off (n = 17) and on (n = 16) Dox and CaMBP transgenics off (n = 20) and on (n = 19) Dox. Values represent group means ± SEM. All mice spent more time exploring the novel object at the immediate and 1 hr delay tests. When tested 24 hr after training, the control mice spent significantly more time exploring the novel object compared with the mutants off Dox. A three-way ANOVA with repeated measures revealed a significant effect of genotype (F_(1,68) = 8.22; p < 0.01). Planned comparisons for the CaMBP mice off Dox showed a significant difference between the 24 hr test and both the immediate and 1 hr tests (p < 0.01) and in 24 hr memory from each of the control groups (p < 0.02 in all cases). There was no difference in total exploration time between the groups (WT off Dox, 16.6 ± 1.8 sec; CaMBP off Dox, 19.1 ± 2.7 sec; WT on Dox, 20.1 ± 3.3 sec; CaMBP on Dox, 23.8 ± 2.5 sec). B, Time spent freezing during 1 hr contextual, 24 hr contextual, and 24 hr cued fear conditioning. There was no difference in 1 hr context memory between CaMBP transgenic mice off Dox (n = 22) and wild-type mice off Dox (n = 17). Four groups (CaMBP off Dox, n = 29; WT off Dox, n = 26; CaMBP on Dox, n = 17; WT on Dox, n = 17) were trained and tested for 24 hr contextual and cued memory. A two-way ANOVA for context freezing revealed a significant effect of genotype (F_(1,85) = 5.89; p < 0.02) and Dox (F_(1,85) = 6.76; p < 0.02). Post hoc Scheffe showed that the CaMBP transgenic mice off Dox froze less than all of the control groups (^*p < 0.025 in all cases). There was no significant effect of genotype in 24 hr cued conditioning. C, CaMBP transgenic (n = 5) and wild-type (n = 5) mice were assayed for conditioned taste aversion using saccharin/water discrimination. Both groups preferred saccharin to water before conditioning. Mice showed a marked aversion to saccharin both 48 hr and 8 d after conditioning. A two-way ANOVA with repeated measures showed a significant effect of conditioning (F_(2,20) = 28.7; p < 0.0001) but no effect of genotype. There was no difference in the total water intake between the two groups (CaMBP, 1.98 ± 0.3; WT, 1.72 ± 0.1).