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. 2012 Nov 28;32(48):17431-41.
doi: 10.1523/JNEUROSCI.4339-12.2012.

Enhanced cAMP Response Element-Binding Protein Activity Increases Neuronal Excitability, Hippocampal Long-Term Potentiation, and Classical Eyeblink Conditioning in Alert Behaving Mice

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Enhanced cAMP Response Element-Binding Protein Activity Increases Neuronal Excitability, Hippocampal Long-Term Potentiation, and Classical Eyeblink Conditioning in Alert Behaving Mice

Agnès Gruart et al. J Neurosci. .
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Abstract

The activity-regulated transcription factor cAMP response element-binding protein (CREB) is an essential component of the molecular switch that controls the conversion of short-term into long-term forms of plasticity, including those underlying long-term memory. Previous research in acute brain slices of transgenic animals expressing constitutively active CREB variants has revealed that enhancing CREB activity increases the intrinsic excitability of neurons and facilitates the late phase of long-term potentiation (LTP) in the Schaffer collateral pathway. Here, we report similar changes in plasticity at the Schaffer collateral pathway in alert behaving mice. Forebrain expression of a strong constitutively active CREB variant, VP16-CREB, enhanced in vivo LTP evoked in the Schaffer collateral pathway and caused significant changes in the input/output curve and paired-pulse facilitation in CA3-CA1 synapses, which could be explained by the increased excitability of hippocampal pyramidal neurons. In addition, classical eyeblink conditioning in transgenic mice and control littermates showed larger conditioned responses in mutant mice that were associated to a transient increase in the acquisition rate and in the concomitant learning-dependent change in synaptic strength. The sustained chronic activation of CREB activity, however, impaired the performance in this task. Our experiments demonstrate that the sustained enhancement of CREB function alters the physiology and plasticity of hippocampal circuits in behaving animals and that these changes have important consequences in associative learning.

Figures

Figure 1.
Figure 1.
I/O curves in VP16–CREB and control mice. A, Representative examples of fEPSPs (3 overlapping records) evoked in the stratum cellulare after paired pulses (40 ms of interpulse interval) presented to Schaffer collaterals (left) and in the stratum radiatum (right). B, C, I/O curves evoked at the CA1–CA3 synapse of control mice (n = 10) before (B) and after (C) dox removal. Note that no significant (p > 0.05) differences were observed. Stimulus consisted of paired pulses (40 ms of interstimulus interval) presented at increasing intensities in 20 μA steps. White circles indicate fEPSP amplitudes evoked by the first pulse, and black circles indicate fEPSPs evoked by the second pulse. Each circle represents the mean value computed from five stimulus presentations (10 animals per group). D, A scatter plot illustrating fEPSP values evoked by the paired pulses in control mice fed with dox (x-axis, on dox) and after dox removal (y-axis, off dox). The best linear fit is illustrated. E, F, I/O curves in the same hippocampal synapse of bitransgenic mice (n = 10) before (E) and after (F) dox removal. Note the increased excitability evoked in mutant mice after dox removal. G, A scatter plot illustrating fEPSP values evoked by the paired pulses in VP16–CREB mice fed with dox [x-axis; on dox (transgene Off)] and after dox removal [y-axis, off dox (transgene On)]. Note that values were shifted upward and that the slope of the linear fit was <1 (b = 0.72). In C and F, dox was removed from mouse diet 5 d before recording the I/O curve. The best sigmoid fit to data illustrated in B, C, E, and F is represented for each group of animals (r ≥ 0.98; p < 0.001).
Figure 2.
Figure 2.
fEPSP facilitation evoked in the pyramidal CA1 area by paired-pulse stimulation of Schaffer collaterals. A, Significant (p ≤ 0.05) EPSP facilitation was evoked at short (10–40 ms) interstimulus intervals in control mice (n = 10). The data shown are mean ± SEM slopes of the second fEPSP expressed as a percentage of the first for the six interpulse intervals. No differences (p > 0.05) were observed in control animals after dox removal. B, Averaged (five times) fEPSP paired traces collected from representative control mice at the indicated interstimulus intervals. C, D, Same data as in A and B, collected from mutant (VP16–CREB) mice (n = 10). Note in C the significant (p < 0.001) increase in PPF at 40 ms of interstimulus interval after dox removal.
Figure 3.
Figure 3.
Long-term potentiation evoked at the CA3–CA1 synapse with HFS of low (35%) intensity. A, Representative paired (40 ms of interval) fEPSPs recorded in the hippocampal CA1 area from control (white circles and triangles) and mutant (VP16–CREB; black circles and triangles) mice before (1, baseline) and 5–30 min (2) and 48 h (3) after HFS of Schaffer collaterals. HFS was set at 35% of the intensity (milliamperes) necessary to evoke a maximum fEPSP response. For comparative purposes, fEPSP amplitudes have been adapted to baseline value in control mice (100%). B, Graphs illustrating the time course of changes in fEPSPs (mean ± SEM) after HFS stimulation of the Schaffer collaterals in the two groups (n = 13 animals per group) of animals. The HFS train was presented after 15 min of baseline recordings, at the time indicated by the dashed line. fEPSP slopes are expressed as a percentage of the baseline (100%) slope. Both groups of animals presented a significant (p < 0.01) and long-lasting (∼24 h) increase in fEPSP slopes after HFS. In addition, significant differences between control and mutant animals were observed in the slopes of fEPSP evoked by the first (+p < 0.01) and the second (*p ≤ 0.05) pulses.
Figure 4.
Figure 4.
Long-term potentiation evoked at the CA3–CA1 synapses with HFS of high (50%) intensity. A, Schematic representation of the experimental design. The timeline shows the period of transgene induction and the two HFS sessions. B, Left shows the changes in fEPSPs (mean ± SEM) after HFS stimulation of the Schaffer collaterals in the four groups (n = 10 animals per group) of animals: group 1, control on dox (black circles); group 2, control off dox (black squares); group 3, VP16–CREB on dox (black triangles); and group 4, VP16–CREB off dox (black diamonds). The HFS train was presented after 15 min of baseline recordings, at the time indicated by the dashed line, and was set at 50% of the intensity (milliamperes) necessary to evoke a maximum fEPSP response. fEPSP slopes are expressed as a percentage of the corresponding baseline (100%) slope. Note that LTP evoked in group 4 (VP16–CREB off dox) was significantly (*p ≤ 0.01) larger and longer-lasting that those evoked in the other three groups of mice. As indicated at the top, the diet administered to each of the four groups was switched, and the same HFS session was repeated 20 d later. As shown in the right set of graphs, group 3 (VP16–CREB now with off dox) was the one presenting significantly larger and longer-lasting LTP (*p ≤ 0.01).
Figure 5.
Figure 5.
Electrode location and transgene expression. A, Photomicrographs illustrating the location (white arrows) of the stimulating (1) and recording (2) sites. Scale bars, 100 μm. DG, Dentate gyrus; D, dorsal; L, lateral; M, medial; V, ventral. B–D, Representative images of the double immunostaining of brain slices from animals used in the experiments described in Figures 1–3. We used antibodies against the VP16 domain in VP16–CREB (green) and c-fos (red), a well-characterized CREB target gene. Slices were counterstained using DAPI (blue). Strong expression of VP16–CREB and activation of downstream targets were observed in the CA1 and DG areas of the hippocampus (B), in striatum (C), and in specific layers of the cortex, including the premotor and motor area (D). Merge images (red and green channels) in the right panels of B–D correspond to the inset region labeled with a white square in the left panels. Arrowheads indicate examples of costained cells. Scale bar, 150 μm.
Figure 6.
Figure 6.
Evolution of CA3–CA1 synaptic field potentials and eyeblink conditioning learning in VP16–CREB mice. A, Schematic representation of this classical eyeblink conditioning experiment. Top lines indicate the time of gene induction and conditioning. B, Animals were implanted with hippocampal stimulating (St.) electrodes in the right Schaffer collaterals (Sch.) and recording (Rec.) electrodes in the ipsilateral pyramidal CA1 area. Additional EMG recording electrodes were also implanted in the left orbicularis oculi (O.O.) muscle and stimulating electrodes on the ipsilateral supraorbital nerve. A tone was used as CS, and an electric shock at the trigeminal nerve served as US. C, From top to bottom are illustrated the conditioning paradigm, and representative orbicularis oculi, EMG, and hippocampal recordings during paired CS–US presentations for control and mutant (VP16–CREB) mice. The time of stimulus presentation to Schaffer collaterals (St. Hipp.) is indicated, as are the times of presentation of CS and US (dotted lines). Data shown were collected during the ninth conditioning session. D, E, Quantification of the integrated EMG areas from the two groups (n = 10 per group) of animals for spontaneous activity (control, white squares; mutant, black squares) and for evoked conditioned responses (control, white circles; mutants, black circles). Spontaneous EMG activities were collected from the 200 ms preceding CS presentation. Although no significant differences (p = 0.23) were observed in the rectified EMG activities of the orbicularis oculi muscle collected before CS presentations, note the larger EMG areas presented by conditioned responses collected from mutant mice. *p < 0.05. F, Percentage of conditioned eyelid responses reached by the two experimental groups. The values presented by control mice (white circles) were significantly (p < 0.01) larger (from the 3rd to the 10th conditioning sessions and for 2 of the extinction sessions) than those reached by mutant mice (VP16–CREB; black circles), indicating that this associative learning was impaired by the chronic activation of CREB. G, Evolution of fEPSPs evoked at the CA3–CA1 synapse across conditioning for control (white circles) and mutant (VP16–CREB; black circles) mice. fEPSP slopes are expressed as a percentage of fEPSP slope recorded during habituation sessions for each of the two groups. Differences in fEPSP slopes between control and mutant groups were statistically significant from the fourth to the 10th conditioning sessions and for two extinction sessions (*p < 0.05), indicating that activity-dependent synaptic plasticity was severely impaired in mutant mice. The top insets show representative fEPSPs (averaged 5 times) collected from control (white circle) and mutant (VP16–CREB; black circle) mice during the second habituation (1), ninth conditioning (2), and fifth extinction (3) sessions.
Figure 7.
Figure 7.
Transient improvement of classical conditioning of eyelid responses in VP16–CREB mice. A, Schematic representation of the experimental design. B, C, Evolution of the percentage of conditioned eyelid responses (B) and of fEPSPs evoked at the CA3–CA1 synapse (C) after transgene activation the day proceeding the first habituation session (arrow). Note the earlier and significant (*p ≤ 0.05) increase in the percentage of conditioned responses (A) and in fEPSP slopes (B) observed in mutant mice, as well as the late significant depression for both parameters (n = 10 mice per group; *p ≤ 0.05).

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