Single-Cell Visualization Deep in Brain Structures by Gene Transfer.

A projection neuron targets multiple regions beyond the functional brain area. In order to map neuronal connectivity in a massive neural network, a means for visualizing the entire morphology of a single neuron is needed. Progress has facilitated single-neuron analysis in the cerebral cortex, but individual neurons in deep brain structures remain difficult to visualize. To this end, we developed an in vivo single-cell electroporation method for juvenile and adult brains that can be performed under a standard stereomicroscope. This technique involves rapid gene transfection and allows the visualization of dendritic and axonal morphologies of individual neurons located deep in brain structures. The transfection efficiency was enhanced by directly injecting the expression vector encoding green fluorescent protein instead of monitoring cell attachment to the electrode tip. We obtained similar transfection efficiencies in both young adult (≥P40) and juvenile mice (P21-30). By tracing the axons of thalamocortical neurons, we identified a specific subtype of neuron distinguished by its projection pattern. Additionally, transfected mOrange-tagged vesicle-associated membrane protein 2-a presynaptic protein-was strongly localized in terminal boutons of thalamocortical neurons. Thus, our in vivo single-cell gene transfer system offers rapid single-neuron analysis deep in brain. Our approach combines observation of neuronal morphology with functional analysis of genes of interest, which can be useful for monitoring changes in neuronal activity corresponding to specific behaviors in living animals.

Genome-Wide Target Analyses of Otx2 Homeoprotein in Postnatal Cortex.

Juvenile brain has a unique time window, or critical period, in which neuronal circuits are remodeled by experience. Mounting evidence indicates the importance of neuronal circuit rewiring in various neurodevelopmental disorders of human cognition. We previously showed that Otx2 homeoprotein, essential for brain formation, is recaptured during postnatal maturation of parvalbumin-positive interneurons (PV cells) to activate the critical period in mouse visual cortex. Cortical Otx2 is the only interneuron-enriched transcription factor known to regulate the critical period, but its downstream targets remain unknown. Here, we used ChIP-seq (chromatin immunoprecipitation sequencing) to identify genome-wide binding sites of Otx2 in juvenile mouse cortex, and interneuron-specific RNA-seq to explore the Otx2-dependent transcriptome. Otx2-bound genes were associated with human diseases such as schizophrenia as well as critical periods. Of these genes, expression of neuronal factors involved in transcription, signal transduction and mitochondrial function was moderately and broadly affected in Otx2-deficient interneurons. In contrast to reported binding sites in the embryo, genes encoding potassium ion transporters such as KV3.1 had juvenile cortex-specific binding sites, suggesting that Otx2 is involved in regulating fast-spiking properties during PV cell maturation. Moreover, transcripts of oxidative resistance-1 (Oxr1), whose promoter has Otx2 binding sites, were markedly downregulated in Otx2-deficient interneurons. Therefore, an important role of Otx2 may be to protect the cells from the increased oxidative stress in fast-spiking PV cells. Our results suggest that coordinated expression of Otx2 targets promotes PV cell maturation and maintains its function in neuronal plasticity and disease.