Inhibition of N-methyl-D-aspartate receptor activity resulted in aberrant neuronal migration caused by delayed morphological development in the mouse neocortex.

Embryonic and neonatal neocortical neurons already express functional N-methyl-D-aspartate (NMDA) receptors before they form synapses. To elucidate the role of NMDA receptors in neuronal migration in the developing neocortex, we visualized radially migrating neurons by transferring the enhanced green fluorescent protein (EGFP) gene into the ventricular zone (VZ) of the mouse neocortex using in utero electroporation at E15.5. Two days later, we prepared neocortical slices and examined the EGFP-positive cells using time-lapse imaging in the presence of the NMDA receptor antagonist Cerestat. The EGFP-positive cells generated in the VZ in the control slices exhibited a multipolar morphology, but within several hours they became bipolar (with a leading process and an axon-like process) and migrated toward the pial surface. By contrast, many of the multipolar cells in the Cerestat-treated slices failed to extend either process and become bipolar, and frequently changed direction, although they ultimately reached their destination even after Cerestat-treatment. To identify the molecules responding for mediating NMDA signaling during neuronal migration and the changes in morphology observed above, we here focused on Src family kinases (SFKs), which mediate a variety of neuronal functions including migration and neurite extension. We discovered that the activity of Src and Fyn was reduced by Cerestat. These findings suggest that NMDA receptors are involved in neuronal migration and morphological changes into a bipolar shape, and in the activation of Src and Fyn in the developing neocortex.

Visualization of microglia in living tissues using Iba1-EGFP transgenic mice.

Microglia are thought to play important roles not only in repairing injured tissue but in regulating neuronal activity, and visualizing the cells is very useful as a means of further investigating the function of microglia in vivo. We previously cloned the ionized calcium-binding adaptor molecule 1 (Iba1) gene, which is expressed selectively in microglia/microphages. To generate new transgenic mice to visualize microglia with enhanced green fluorescent protein (EGFP), we here constructed a plasmid carrying EGFP cDNA under control of the Iba1 promoter. This construct was injected into C57B/6 mouse zygotes, and the Iba1-EGFP transgenic line was developed. Fluorescent in-situ hybridization analysis revealed that the Iba1-EGFP transgene was located on chromosome 11D. No obvious defects were observed during development or in adulthood, and the EGFP fluorescence remained invariant over the course of at least four generations. Judging from the immunoreactivity with anti-Iba1 antibody, all EGFP-positive cells in the adult brain were ramified microglia. In the developing transgenic embryos, EGFP signals were detected as early as embryonic Day 10.5. The most prominent EGFP signals were found in forebrain, spinal cord, eye, foreleg, yolk sac, liver, and vessel walls. At postnatal Day 6, clear EGFP signals were observed in the supraventricular corpus callosum, known as "fountain of microglia", where ameboid microglia migrate into the brain parenchyma and mature into ramified microglia. Iba1-EGFP transgenic mice thus permit observation of living microglia under a fluorescence microscope and provide a useful tool for studying the function of microglia in vivo.