Research Articles

Control of Memory Formation Through Regulated Expression of a CaMKII Transgene

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Science  06 Dec 1996:
Vol. 274, Issue 5293, pp. 1678-1683
DOI: 10.1126/science.274.5293.1678

Figures

  • Fig. 1.

    Regulation of the CaMKII-Asp286 transgene with the tTA system. (A) Strategy used to obtain forebrain-specific doxycycline-regulated transgene expression. Two independent lines of transgenic mice are obtained, and the two transgenes are introduced into a single mouse through mating. (B) Quantitation by RT-PCR Southern blot of CaMKII-Asp286 expression from the tet-O promoter. RT-PCR was performed on total forebrain RNA and probed for expression of the CaMKII-Asp286 mutant mRNA as described (7, 21). Tg1, mouse carrying only the CaMKII promoter-tTA transgene (line B). Tg2, mouse carrying only the tet-O-CaMKII-Asp286 transgene (line 21). Tg1/Tg2, double transgenic mouse carrying both the CaMKII promoter-tTA transgene (line B) and tet-O-CaMKII-Asp286 (line 21) transgenes. Tg1/Tg2 + Dox, double transgenic mouse treated with doxycycline (2 mg/ml) plus 5% sucrose in the drinking water for 4 weeks.

  • Fig. 2.

    Forebrain-specific activation of a tet-O-lacZ transgene. (A) Coronal section of double transgenic line B lacl stained with X-Gal as described (42). Ctx, cerebral cortex; Str, striatum; Hip, hippocampus; Amy, amygdala. (B) X-Gal-stained coronal section of the hippocampus from double transgenic lines B lac1 and B lac2. CA1, CA1 cell body layer; CA3, CA3 cell body layer; DG, dentate gyrus.

  • Fig. 3.

    Regional distribution of the CaMKII-Asp286 mRNA determined by in situ hybridization (7). Medial sagittal sections of double transgenic lines B13, B21, and B22 showing CaMKII-Asp286 transgene expression. B21/Amygdala shows a close-up view of a coronal section from the B21 double transgenic line of mouse.

  • Fig. 4.

    Reversal of 10-Hz LTP deficit in CA1 of hippocampal slices. Field EPSP slopes before and after 10-Hz tetanic stimulation were recorded and expressed as the percentage of pre-tetanus baseline (22). Stimulation at 10 Hz for 1.5 min induced a transient depression followed by potentiation in wild-type mice (123 ± 9% at 60 min after tetanus; n = 12 slices, 6 mice) (□). Tetanus (10 Hz) induced a slight depression in B13 double transgenic mice (89 ± 6% at 60 min after tetanus; n = 9 slices, 3 mice) (▪). Doxycycline treatment reversed the defect in B13 mice (132 ± 10%; n = 8 slices, 4 mice) (•). Doxycycline treatment had no effect on synaptic potentiation in wild-type mice (122 ± 6%; n = 16 slices, 6 mice) (▿).

  • Fig. 5.

    Reversible deficits in explicit learning and memory in mice expressing the CaMKIIα transgene. (A) The Barnes circular maze. (B) Percentage of B22 transgenic and wild-type mice that met the learning criterion on the Barnes circular maze (24). A chi-square analysis revealed that the percentage of B22 transgenics acquiring the Barnes maze (0%) was significantly different from B22 transgenics on doxycycline and both wild-type groups (X2 = 53.05, P < 0.0001). Four groups of mice were tested: B22 transgenics (n = 6), B22 transgenics on doxycycline (1 mg/ml) for 4 weeks (n = 6), wild types (n = 8), and wild types on doxycycline (1 mg/ml) for 4 weeks (n = 7). (C) Mean number of errors across session blocks composed of five sessions. Values represent group means ± SEM. A three-way ANOVA revealed a main effect of genotype (F[1,23] = 4.28, P = 0.04). (D) The percentage of sessions in which the spatial search strategy was used across session blocks by B22 transgenic mice. Values represent group means ± SEM. A two-way ANOVA revealed a significant main effect of doxycycline (F[1,10] = 7.313, P = 0.02).

  • Fig. 6.

    Reversible deficits in implicit learning and memory in mice expressing the CaMKIIα transgene. Percentage of time spent freezing to context (A) and to cue (B) 24 hours after training in the B22 and B21 lines. Values represent group means ± SEM. A three-way ANOVA revealed a significant three-way interaction for context (genotype by line by doxycycline) (F[1,55] = 9.177, P = 0.0037) and a significant two-way interaction for cue (line by genotype) (F[1,55] = 5.087, P = 0.0281). Six groups of mice were tested: B22 transgenics (n = 6), B22 transgenics on doxycycline for 4 weeks (n = 11), B21 transgenics (n = 8), B21 transgenics on doxycycline for 4 weeks (n = 19), wild types (from both B22 and B21 lines) (n = 11), and wild types (from both B22 and B21 lines) on doxycycline for 4 weeks (n = 8). (C) Time line illustrating administration of doxycycline and behavioral training and testing. (D) Retention of context and cued conditioning. Percentage of time spent freezing to context and cue 6 weeks after training. Values represent group means ± SEM. Post hoc analysis by the Scheffe test revealed that B21 transgenic mice that were switched to water froze significantly less to context than B21 transgenic mice on doxycycline (P = 0.01) and wild types (P = 0.008) and significantly less to cue than B21 transgenic mice on doxycycline (P = 0.02) and wild types (P = 0.0088). Three groups of mice were tested: B21 transgenics on doxycycline for 4 weeks before training and 6 weeks after training (n = 8), B21 transgenics on doxycycline for 4 weeks before training that were switched to water for the 6 weeks after training (n = 8), and wild-type mice (from both B22 and B21 lines, n = 19). (E) The percentage of time spent freezing to an intruder during the first 120 s after the mouse was exposed to a rat. Values represent group means ± SEM.

Tables

  • Table 1.

    Effect of CaMKII-Asp286 mRNA expression on enzyme activity. Brains were removed and the striatum was dissected and immediately homogenized in 20 mM tris-HCl (pH 7.5), 0.5 mM EGTA, 0.5 mM EDTA, 2 mM leupeptin, 0.4 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.4 mM molybdate, and 10 mM sodium pyrophosphate. CaMKII enzyme activity was determined as described (7). B21 + Dox animals received doxycycline (1 mg/ml) plus 5% sucrose in the drinking water for 3 to 5 weeks. B21 + Dox withdrawal animals received doxycycline (1 mg/ml) for 3 to 5 weeks and were then switched to normal water for 6 weeks. The number of mice is given in parentheses.

    Mouse lineCaMKII activity
    Without Ca2+ (pmol min−1 μg−1)With Ca2+ (pmol min−1 μg−1)Ca2+-independent (%)
    Wild type0.13 ± 0.01 (5)10.4 ± 1.21.33 ± 0.21
    B210.90 ± 0.14 (5)20.9 ± 2.94.62 ± 1.02
    B21 + Dox0.16 ± 0.04 (5)12.9 ± 1.51.22 ± 0.30
    B21 + Dox withdrawal0.80 ± 0.03 (3)14.2 ± 0.75.70 ± 0.43