Stability and Function of Hippocampal Mossy Fiber Synapses Depend on Bcl11b/Ctip2

Abstract

Structural and functional plasticity of synapses are critical neuronal mechanisms underlying learning and memory. While activity-dependent regulation of synaptic strength has been extensively studied, much less is known about the transcriptional control of synapse maintenance and plasticity. Hippocampal mossy fiber (MF) synapses connect dentate granule cells to CA3 pyramidal neurons and are important for spatial memory formation and consolidation. The transcription factor Bcl11b/Ctip2 is expressed in dentate granule cells and required for postnatal hippocampal development. Ablation of Bcl11b/Ctip2 in the adult hippocampus results in impaired adult neurogenesis and spatial memory. The molecular mechanisms underlying the behavioral impairment remained unclear. Here we show that selective deletion of Bcl11b/Ctip2 in the adult mouse hippocampus leads to a rapid loss of excitatory synapses in CA3 as well as reduced ultrastructural complexity of remaining mossy fiber boutons (MFBs). Moreover, a dramatic decline of long-term potentiation (LTP) of the dentate gyrus-CA3 (DG-CA3) projection is caused by adult loss of Bcl11b/Ctip2. Differential transcriptomics revealed the deregulation of genes associated with synaptic transmission in mutants. Together, our data suggest Bcl11b/Ctip2 to regulate maintenance and function of MF synapses in the adult hippocampus.

Keywords: Bcl11b; hippocampus; mossy fiber boutons; synapses; transcription factor.

Figure 1

Verification of the tamoxifen-inducible CreER^T2 system. (A–F) Fluorescence staining using a Bcl11b-specific antibody (red) and Dapi (blue) on hippocampal sections of controls (A,C,E) and tamoxifen-induced Bcl11b mutants (B,D,F) at 2 (A,B) and 4 (C–F) weeks after induction. (G) Quantitative analysis of Bcl11b mRNA expression in the dentate gyrus of controls and tamoxifen-induced Bcl11b mutants at 4 weeks after induction (n = 3). Scale bar, 100 μm; error bars, SD; t-test, *p < 0.05.

Figure 2

Loss of Bcl11b/Ctip2 expression impairs mossy fiber (MF) terminals. (A) Quantification of overlapping puncta for vesicular glutamate transporter 1 (Vglut1; presynaptic marker of glutamatergic synapses) and Homer1 (postsynaptic marker of glutamatergic synapses) per optic field in the stratum lucidum (n = 3; 10 sections per animal). (B) Count of Zinc Transporter-3 (ZnT3)-labeled MF terminals per optic field in the stratum lucidum (n = 3; 10 sections per animal). (C) Representative pictures of control and mutant MFBs; scale bar, 500 nm. (D) Perimeter/area ratio of control and mutant MFBs (n = 6). (E) Average synapse score for mutants and controls (n = 4). (F) Number of postsynaptic densities per MFB (n = 6). Error bars, SEM; Mann-Whitney U test, *p < 0.05, **p < 0.01; MFB, mossy fiber bouton; PSD, postsynaptic density; a.i., after induction (of Bcl11b/Ctip2 mutation).

Figure 3

Analysis of short-term potentiation at 2 months after induction. (A) Representative traces of fEPSP recorded in the stratum lucidum in response to a stimulation of 20 volt. (B) Input/output curve showing the fEPSP amplitude depending on stimulation (n = 9). (C) Representative traces of fEPSP in the stratum lucidum induced by electrical stimulation of MFs at 5 Hz (left) and 40 Hz (right). (D) Paired-pulse analysis demonstrating the facilitation or the inhibition of the second fEPSP compared to the first one (n = 9). (E) Representative traces of fEPSP in response to five consecutive stimulations at 25 Hz. (F) Facilitation of the fEPSP in response to five consecutive stimulations at 25 Hz. n = 5, control; n = 7, mutant; error bars, SEM; t-test, *p < 0.05; fEPSP, field excitatory postsynaptic potential.

Figure 4

Analysis of LTP at 2 months after induction. (A) Representative average traces of baselines before LTP induction by HFS (pre-baseline, in black), 20–30 min after LTP induction (20–30 min post, in light blue), 30–40 min after LTP induction (30–40 min post, in green) and after DCG-IV application (1 μM DCG-IV, in red). (B) Time course of the fEPSP amplitude during LTP experiment. (C) Analysis of the facilitation of fEPSP at 0–10, 10–20, 20–30 and 30–40 min after HFS. n = 5, control; n = 7, mutant; error bars, SEM; t-test, *p < 0.05; fEPSP, field excitatory postsynaptic potential; HFS, high frequency stimulation; LTP, long-term potentiation; R.M.ANOVA, repeated measure ANOVA.

Figure 5

Transcriptome analysis of DG granule cells of adult-induced Bcl11b/Ctip2 mutants. (A) Gene ontology analysis reveals overrepresentation of “Cyclic nucleotide metabolite process GO:0009187; Regulation of catalytic activity GO:0050790; Synaptic transmission GO:0007268; Intracellular signal transduction GO:0035566” but not “Cell death GO:008219” as determined by PANTHER version 10.0. (B) Verification of potential Bcl11b/Ctip2 target genes by qRT-polymerase chain reaction (PCR) at 4 weeks after induction of the mutation (n = 4). (C) Representative data of 3 independent chromatin immunoprecipitation (ChIP) assays determining the direct interaction of Bcl11b/Ctip2 with C1ql2 and Sema5B promoter regions, respectively. ChIP assays were performed on hippocampal tissue of 3 months old wild-type animals employing a Bcl11b/Ctip2-specific antibody, IgG as a negative control and an RNA polymerase II (RNA pol)-specific antibody as positive control. Direct interaction was determined by qPCR using specific primers for the C1ql2, Sema5b and Gapdh promoters. Error bars, SD; *p < 0.05; **p < 0.01; ***p < 0.001.