The Immediate Early Gene Egr3 Is Required for Hippocampal Induction of Bdnf by Electroconvulsive Stimulation

Abstract

Early growth response 3 (Egr3) is an immediate early gene (IEG) that is regulated downstream of a cascade of genes associated with risk for psychiatric disorders, and dysfunction of Egr3 itself has been implicated in schizophrenia, bipolar disorder, and depression. As an activity-dependent transcription factor, EGR3 is poised to regulate the neuronal expression of target genes in response to environmental events. In the current study, we sought to identify a downstream target of EGR3 with the goal of further elucidating genes in this biological pathway relevant for psychiatric illness risk. We used electroconvulsive stimulation (ECS) to induce high-level expression of IEGs in the brain, and conducted expression microarray to identify genes differentially regulated in the hippocampus of Egr3-deficient (-/-) mice compared to their wildtype (WT) littermates. Our results replicated previous work showing that ECS induces high-level expression of the brain-derived neurotrophic factor (Bdnf) in the hippocampus of WT mice. However, we found that this induction is absent in Egr3-/- mice. Quantitative real-time PCR (qRT-PCR) validated the microarray results (performed in males) and replicated the findings in two separate cohorts of female mice. Follow-up studies of activity-dependent Bdnf exons demonstrated that ECS-induced expression of both exons IV and VI requires Egr3. In situ hybridization demonstrated high-level cellular expression of Bdnf in the hippocampal dentate gyrus following ECS in WT, but not Egr3-/-, mice. Bdnf promoter analysis revealed eight putative EGR3 binding sites in the Bdnf promoter, suggesting a mechanism through which EGR3 may directly regulate Bdnf gene expression. These findings do not appear to result from a defect in the development of hippocampal neurons in Egr3-/- mice, as cell counts in tissue sections stained with anti-NeuN antibodies, a neuron-specific marker, did not differ between Egr3-/- and WT mice. In addition, Sholl analysis and counts of dendritic spines in golgi-stained hippocampal sections revealed no difference in dendritic morphology or synaptic spine density in Egr3-/-, compared to WT, mice. These findings indicate that Egr3 is required for ECS-induced expression of Bdnf in the hippocampus and suggest that Bdnf may be a downstream gene in our previously identified biologically pathway for psychiatric illness susceptibility.

Keywords: brain-derived neurotrophic factor; early growth response 3; electroconvulsive therapy; immediate early genes; psychosis treatment; schizophrenia.

FIGURE 1

Electroconvulsive seizure (ECS)-induced hippocampal Bdnf expression is Egr3-dependent. Expression microarray analysis (A), and validation using qRT-PCR (B–D), of hippocampal Bdnf expression from WT, and Egr3–/–, mice at baseline (no ECS) and 1 h after ECS. Two-way ANOVAs showed significant interaction of genotype and ECS treatment on Bdnf expression in (A) male microarray data (p = 0.004) and qRT-PCR data in (C) female cohort 1 (p = 0.0005) and (D) female cohort 2 (p = 0.0003), and (B) significant effect of treatment in original male cohort (p = 0.0098; n = 4–5 animals/group; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 controlled for multiple comparisons).

FIGURE 2

Electroconvulsive seizure (ECS)-mediated hippocampal induction of Bdnf exon IV and VI requires Egr3. Exon IV (A–C) and VI (D–F) hippocampal Bdnf expression determined using qRT PCR from WT, and Egr3–/–, mice at baseline (no ECS) and 1 h after ECS. Two-way ANOVAs showed significant interactions of genotype and ECS treatment on Bdnf exon IV expression in (A) original male cohort data (p = 0.0041), (B) female cohort 1 (p = 0.0019), and (C) female cohort 2 (p = 0.0001). Two-way ANOVAs showed significant effect of treatment on Bdnf exon VI expression in original (D) male cohort (p = 0.0099), (E) female cohort 1 (p = 0.0064), and (F) female cohort 2 (p = 0.0237; n = 4–5 animals/group; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 controlled for multiple comparisons).

FIGURE 3

Bdnf induction in the hippocampal dentate gyrus requires Egr3. Representative tissue sections demonstrating in situ hybridization labeling of Bdnf mRNA expressing cells in the hippocampus of WT and Egr3–/– mice at baseline (A,B) and 1 h following ECS (C,D). (A) Bdnf is expressed sparsely in CA3, CA2, and the DG of WT mice at baseline. (C) ECS induces high-level Bdnf expression in the DG, and increased expression in CA3, CA2, and CA1 of WT mice. (B) Bdnf expression in Egr3–/– mice is limited to rare cells in the CA3 region of the hippocampus, with no clear expression above background in the DG, CA2, or CA1, at baseline. (D) ECS produces little to no increase in Bdnf expression in the DG, CA3, CA2, and CA1 regions of Egr3–/– mice.

FIGURE 4

Map of predicted EGR3 binding sites in Bdnf upstream region. EGR3 binding sites identified in Table 1 are indicated in red. Positions are shown relative to exon 1 of the mouse Bdnf gene as identified in the NCBI Refseq database. Numbers under each binding site correspond to numbered rows in Table 1.

FIGURE 5

Egr3 is not required for development of normal numbers of hippocampal neurons. Sample montaged photomicrographs from the hippocampus of anti-NeuN stained (A) WT and (B) Egr3–/– mice show no obvious signs of changes in gross morphology (scale bar = 100 μm). Consistent with this observation, (C) quantification of NeuN+ cells (n = 6 animals/genotype) demonstrated no significant differences in neuron density in any hippocampal region between WT (black) and Egr3–/– (gray) mice.

FIGURE 6

Egr3 is not required for normal development of neuronal morphology. Samples of Golgi-Cox staining in (A) granule cells and (D) pyramidal cells shows impregnation of both gross dendritic structure (scale bar = 20 μm) as well as spines (inset, scale bar = 10 μm). Quantitative data show no statistically significant differences in either dendritic morphology or spine number between WT (black) and Egr3–/– (gray) mice in the (B) DG_sp, (C) DG_ip, (E) CA1, (F) CA3a/b, or (G) CA3c regions.