The CREB/CREM transcription factors negatively regulate early synaptogenesis and spontaneous network activity

Abstract

The family of CREB (cAMP response element-binding protein) transcription factors are involved in a variety of biological processes including the development and plasticity of the nervous system. In the maturing and adult brain, CREB genes are required for activity-dependent processes, including synaptogenesis, refinement of connections and long-term potentiation. Here, we use CREB1(Nescre)CREM(-/-) (cAMP-responsive element modulator) mutants to investigate the role of these genes in stimulus-independent patterns of neural activity at early stages. We show that lack of CREB/CREM genes specifically in neural tissue leads to increased synaptogenesis and to a dramatic increase in the levels of spontaneous network activity at embryonic stages. Thus, the functions of CREB/CREM genes in neural activity differ in distinct periods of neural development.

Figure 1.

CREB/CREM mutant mice display normal fiber cytoarchitectonics but increased synaptogenesis. A–F, Coronal sections of E18 forebrains immunoreacted for L1 and TAG1 showing that the trajectories of the main fiber tracts of the forebrain are unaltered in CREB1^NescreCREM^−/− embryos. CC, Corpus callosum; CTT, corticothalamic tract; F, fimbria; H, hippocampus; S, striatum; T, thalamus. G–H, Hippocampal sections immunoreacted for L1 showing similar patterns of fiber distribution (arrows) in CREB1^+/+CREM^+/− and CREB1^NescreCREM^−/− embryos. DG, Dentate gyrus; CA1–CA3, hippocampal subfields. I, J, Electron micrographs showing axon terminals displaying synaptic contacts (arrows) in the stratum radiatum of CREB1^+/+CREM^+/− and CREB1^NescreCREM^−/− hippocampi. K, Density of synaptic terminals in the stratum radiatum and stratum lacunosum moleculare in the genotypes showing increased number of contacts in CREB1^+/+CREM^−/−, CREB1^NescreCREM^+/−, and CREB1^NescreCREM^−/− embryos, compared with CREB1^+/+CREM^+/− brains. *p < 0.05, **p < 0.01). Scale bars: A–H, 200 μm; I, J, 0.5 μm.

Figure 2.

Spontaneous correlated network activity is enhanced in the hippocampus of E18 CREB1^NescreCREM^+/− and CREB1^NescreCREM^−/− embryos. A–G, Examples of network activity in the hippocampal CA1 region of CREB1^+/+CREM^+/− and CREB1^NescreCREM^−/− embryos. A, B, Fura-2-loaded hippocampal slices showing active neurons (squares). C, Representative plots of ΔF/F over time illustrating spontaneous [Ca⁺²]_i changes in a hippocampal neuron. The initiation of [Ca⁺²]_i transients is indicted by bars on the x-axis. D, E, Raster plots of all active cells shown in A and B in a CREB1^+/+CREM^+/− (D) and CREB1^NescreCREM^−/− (E) slice. Horizontal lines represent the activity profile over 800 s of each active neuron, and vertical thick lines mark the onset of [Ca⁺²]_i oscillations. The long vertical lines crossing the horizontal lines in D and E mark the times where the onset of the oscillations is synchronous across multiple cells. F, G, Correlation maps illustrating a significant (p < 0.01) spatiotemporal coactivation among all cell pairs shown in A–D and B–E assayed with χ² contingency tables. Each pair of synchronous cells is connected by lines. The thickness of the lines is proportional to the degree of significance. The p value reflects the overall level of coactivation obtained by Monte Carlo simulation and represents the probability that the coactivations present in this field are caused by chance. The complexity of spatiotemporal correlations is enhanced in CREB1^NescreCREM^−/− embryos, as denoted by the increased number of cells involved in correlated networks. H–K, Histograms showing significant increases in the percentage of active neurons (H), the activation rates Ca²⁺ transients/cell/10⁴ s (I), and the number of active cells involved in correlated networks in CREB1^NescreCREM^−/− hippocampal slices (K), whereas durations of Ca²⁺ oscillation do not differ (J). *p < 0.01. Scale bar, 50 μm.

Figure 3.

A, Histograms showing the percentage of active neurons (with respect to controls) in the E18 CA1 hippocampal region, after incubations with EGTA, thapsigargin, BMI, and CNQX/APV. White bars represent control CREB1^+/+CREM^+/− slices and black bars represent CREB1^nescreCREM^−/− double-mutant embryos. B, Average of Monte Carlo p values representing the probability that the overall degree of synchronous correlation present in the network is caused by chance. CREB1^NescreCREM^−/− hippocampal slices exhibit significant values in basal conditions, whereas neurotransmitter receptor antagonists decorrelate spontaneous activity. C, Western blots showing protein expression levels of neurotransmitter receptors (GABA_Aα1 subunit, glutamate receptor NMDA receptor subunit NR1 and GluR2/3) in brain lysates of E18 embryos. Densitometric analyses revealed no significant differences among groups in neurotransmitter receptor protein expression levels (p > 0.05).