in-vivo-two-photon-imaging-of-cortical-neurons-in-neonatal-mice

<em>In Vivo</em> Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

National Institute of Genetics

SOKENDAI (The Graduate University for Advanced Studies)

Here we describe "Supernova" series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood. Sparseness levels are adjustable. Labeled cell-specific gene knockout was accomplished by introducing Cre/loxP-based Supernova vectors into floxed mice. Furthermore, by combining with RNAi, TALEN, and CRISPR/Cas9 technologies, IUE-based Supernova achieved labeled cell-specific gene knockdown and editing/knockout without requiring genetically altered mice. Thus, Supernova system is highly extensible and widely applicable for single-cell analyses in complex organs, such as the mammalian brain.

Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo.

Establishment of precise neuronal connectivity in the neocortex relies on activity-dependent circuit reorganization during postnatal development; however, the nature of cortical activity during this period remains largely unknown. Using two-photon calcium imaging of the barrel cortex in vivo during the first postnatal week, we reveal that layer 4 (L4) neurons within the same barrel fire synchronously in the absence of peripheral stimulation, creating a "patchwork" pattern of spontaneous activity corresponding to the barrel map. By generating transgenic mice expressing GCaMP6s in thalamocortical axons, we show that thalamocortical axons also demonstrate the spontaneous patchwork activity pattern. Patchwork activity is diminished by peripheral anesthesia but is mostly independent of self-generated whisker movements. The patchwork activity pattern largely disappeared during postnatal week 2, as even L4 neurons within the same barrel tended to fire asynchronously. This spontaneous L4 activity pattern has features suitable for thalamocortical (TC) circuit refinement in the neonatal barrel cortex.

Patchwork-Type Spontaneous Activity in Neonatal Barrel Cortex Layer 4 Transmitted via Thalamocortical Projections.

Proper neuronal circuit function relies on precise dendritic projection, which is established through activity-dependent refinement during early postnatal development. Here we revealed dynamics of dendritic refinement in the mammalian brain by conducting long-term imaging of the neonatal mouse barrel cortex. By "retrospective" analyses, we identified "prospective" barrel-edge spiny stellate (SS) neurons in early neonates, which had an apical dendrite and primitive basal dendrites (BDs). These neurons retracted the apical dendrite gradually and established strong BD orientation bias through continuous "dendritic tree" turnover. A greater chance of survival was given to BD trees emerged in the barrel-center side, where thalamocortical axons (TCAs) cluster. When the spatial bias of TCA inputs to SS neurons was lost, BD tree turnover was suppressed, and most BD trees became stable and elaborated mildly. Thus, barrel-edge SS neurons could establish the characteristic BD projection pattern through differential dynamics of dendritic trees induced by spatially biased inputs.

Shingo Nakazawa

Division of Neurogenetics

National Institute of Genetics

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Shingo Nakazawa

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National Institute of Genetics

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Division of Neurogenetics