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

Abstract

Early growth response 3 (<i>Egr3</i>) is an immediate early gene (IEG) that is regulated downstream of a cascade of genes associated with risk for psychiatric disorders, and dysfunction of <i>Egr3</i> 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 (<i>Bdnf</i>) in the hippocampus of WT mice. However, we found that this induction is absent in <i>Egr3</i>-/- 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 <i>Bdnf</i> exons demonstrated that ECS-induced expression of both exons IV and VI requires <i>Egr3</i>. <i>In situ</i> hybridization demonstrated high-level cellular expression of <i>Bdnf</i> in the hippocampal dentate gyrus following ECS in WT, but not <i>Egr3</i>-/-, mice. <i>Bdnf</i> promoter analysis revealed eight putative EGR3 binding sites in the <i>Bdnf</i> promoter, suggesting a mechanism through which EGR3 may directly regulate <i>Bdnf</i> gene expression. These findings do not appear to result from a defect in the development of hippocampal neurons in <i>Egr3</i>-/- mice, as cell counts in tissue sections stained with anti-NeuN antibodies, a neuron-specific marker, did not differ between <i>Egr3</i>-/- 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 <i>Egr3</i>-/-, compared to WT, mice. These findings indicate that <i>Egr3</i> is required for ECS-induced expression of <i>Bdnf</i> in the hippocampus and suggest that <i>Bdnf</i> may be a downstream gene in our previously identified biologically pathway for psychiatric illness susceptibility.