Does cAMP response element-binding protein have a pivotal role in hippocampal synaptic plasticity and hippocampus-dependent memory?

Abstract

Previous studies addressing the role of the transcription factor cAMP response element-binding protein (CREB) in mammalian long-term synaptic plasticity and memory by gene targeting were compromised by incomplete deletion of the CREB isoforms. Therefore, we generated conditional knock-out strains with a marked reduction or complete deletion of all CREB isoforms in the hippocampus. In these strains, no deficits could be detected in lasting forms of hippocampal long-term potentiation (LTP) and long-term depression (LTD). When tested for hippocampus-dependent learning, mutants showed normal context-dependent fear conditioning. Water maze learning was impaired during the early stages, but many mutants showed satisfactory scores in probe trials thought to measure hippocampus-dependent spatial memory. However, conditioned taste aversion learning, a putatively hippocampus-independent memory test, was markedly impaired. Our data indicate that in the adult mouse brain, loss of CREB neither prevents learning nor substantially affects performance in some hippocampus-dependent tasks. Furthermore, it spares LTP and LTD in paradigms that are sensitive enough to detect deficits in other mutants. This implies either a species-specific or regionally restricted role of CREB in the brain and/or a compensatory upregulation of the cAMP response element modulator (CREM) and other as yet unidentified transcription factors.

Figure 5.

Strategy choice by Creb^CamKCre7 and Creb^NesCre mice in the water maze. The path recordings on the left exemplify the six exclusive categories of swim patterns that have been defined to classify the gradual improvement of spatial precision and efficiency during the learning process (from bottom to top). Note that these categories describe the successive stages of water maze learning occurring “on average,” across a group of mice. Thus, not every category will be seen during an individual learning trial. DF, Degrees of freedom.

Figure 1.

Schematic representation of the targeting strategy used for the generation of mouse strains with a progressive reduction of CREB in the brain. A, Genomic structure of the Creb gene. Coding exons are indicated by filled boxes; noncoding exons are indicated by open boxes. 5′- and 3′-flanking sequences and introns are given as lines. Q1, KID, Q2, bZIP, Domains of the CREB protein; Q, glutamine-rich transactivation domain; b, basic region; ZIP, leucine zipper dimerization domain; ATG, start condon. B, Creb^CamKCre7, LoxP-flanked Creb exon 10 was excised by a constitutively active Cre-recombinase fused with the C-terminal-truncated ligand binding domain of the progesterone receptor fusion (Kellendonk et al., 1996) expressed under the control of a CamKIIα promoter. Filled triangles indicate the loxP site remaining after excision of exon 10. pA, Polyadenylation signal. C, Creb^NesCre, Exon 10 was deleted under the control of the brain-specific promoter nestin (Nes). enhII, Enhancer II. D, CREB immunostaining in the different Creb mutant and wild-type lines at 8–12 weeks of age. WT, Wild type; DG, dentate gyrus.

Figure 2.

LTP in the CA1 region is unchanged in mutant strains with a progressive deletion of all CREB isoforms in the hippocampus. A, Scheme of electrode placement in the hippocampal CA1 region. SC, Schaffer collaterals; MF, mossy fibers; PP, perforant path; DG, dentate gyrus; stimul., stimulation; fimbr, fimbria; fiss. hipp, fissura hippocampi. B, LTP in Creb^CamKCre7 mice lacking CREB in ∼80% of CA1 neurons. Both groups expressed a stable LTP of ≥6 hr duration (mutant, 156.2 ± 19.8%, n = 4; wild type, 139 ± 10.4%, n = 4). C, Normal LTP was even obtained in Creb^NesCre mice, which do not have any residual CREB in the CA1 area (mutants, n = 6; wild type, n = 7). D, Creb^αΔ mice (n = 9) with a genetic background of an undefined mixture of C57BL/6 and 129SvEv displayed robust LTP (wild types, n = 5). Insets show representative recordings of a mutant (top row) and a wild-type mouse (bottom row) taken during baseline recording (1), 10 min after the third 100 Hz train (2), and at the end of the recording time (3). HFS, High-frequency stimulation.

Figure 3.

LTD in the CA1 region of the hippocampus is not altered by a progressive reduction of Creb gene dosage. A, Creb^comp mice with only one allele coding for the β-isoform of CREB display a robust depression. The depression obtained after the first LFS train was larger in Creb^comp mice, as in wild-type controls (Creb^comp, 45.4 ± 4.4%, n = 7; wild type, 55.4 ± 3.3%, n = 11; p < 0.05; Mann–Whitney U test). B, LTD in Creb^CamKCre7 mice. Note that recordings of wild-type mice (n = 9) displayed a stepwise decline with every additional LFS train, whereas the depression of mutants (n = 12) was already saturated after the second LFS train. C, Normal LTD in Creb^NesCre mice (n = 7). The depression was already saturated after the second LFS train and significantly larger, as in wild-type littermates (n = 7) (p < 0.05; Mann–Whitney U test). Insets show representative LTD recordings arranged as in Figure 2.

Figure 4.

CREB deficiency impairs water maze learning, which is predominantly attributable to a marked increase in wall hugging (thigmotaxis). A, Water maze learning in Creb^CamKCre7 was indistinguishable from wild-type littermates. B, Creb^NesCre mice displayed a significantly increased swim path length. C, Marked reduction of CREB in the forebrain did not impair spatial memory. D, Apparent deficits of spatial memory in Creb^NesCre mice during probe trial 1. E, Pooling of four Creb mutant strains (Creb^CamKCre7, Creb^NesCre, Creb^αΔ, and Creb^comp) and three wild-type strains, respectively, contrasts the performance deficits of CREB-deficient mutants with their littermate controls. F, In mutant strains, the percentage of thigmotaxis is markedly increased. Two-way ANOVA with repeated measures with days 1–14 and genotype (mutant and wild type) as factors was used for the analysis of the values depicted in Figure 4. The p values represent the genotype effect.

Figure 6.

Loss of CREB does not affect hippocampus-dependent learning but severely impairs associative learning that depends on the activation of extrahippocampal brain regions. A, Reduction of CREB does not result in significant changes in context-dependent fear conditioning, a task that is contingent on the functional integrity of the hippocampus. The two mutant strains show freezing scores similar to those obtained for their respective littermate groups, indicating unimpaired LTM. B, Creb mutants display a significant attenuation of conditioned taste aversion, a hippocampus-independent associative learning paradigm. Creb^NesCre mice (filled bars) avoided saccharin less than wild types (p < 0.0001) during three choice tests separated by 24 hr (ct-1–ct-3). Animals of both genotypes developed the same preference for the saccharin solution when its first consumption during conditioning was followed by vehicle injection not inducing malaise (saccharin preference is indicated by the gray bar). Mean ± SEM are shown.