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

Published: October 18th, 2018



1International Research Center for Medical Sciences (IRCMS), Kumamoto University, 2Division of Neurogenetics, National Institute of Genetics, 3Department of Genetics, SOKENDAI (The Graduate University for Advanced Studies)

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

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 methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

The precise wiring of neuronal circuits in the cerebral cortex is essential for higher brain functions including perception, cognition, and learning and memory. Cortical circuits are dynamically refined during postnatal development. Studies have investigated the process of cortical circuit formation using histological and in vitro culture analyses. However, the dynamics of circuit formation in living mammals has remained mostly unexplored.

Two-photon microscopy has been widely used for the in vivo analyses of neuronal circuits in the adult mouse brain1,2. However, ....

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Experiments should be performed in accordance with the animal welfare guidelines prescribed by the experimenter's institution.

1. Preparation of Pups for Imaging

NOTE: Pups with sparsely labeled cortical neurons can be obtained by in utero electroporation (IUE) of Supernova vectors5,6. The Supernova system consists of the following two vectors: TRE-Cre and CAG-loxP-STOP-loxP-Gene X-ires-tTA-WPRE. .......

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Figures 2D - 2F show representative results of two-photon time-lapse imaging of layer 4 cortical neurons using the present protocol. For the purpose of analysis, select neurons with clear dendritic morphology throughout the imaging periods. We analyzed the dendritic morphology of imaged neurons using morphological analysis software. Representative dendritic morphology reconstruction is shown in Figure 2F. Neurons showing disc.......

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Critical Steps in the Protocol and Troubleshooting:

The most critical step of the protocol is the removal of the skull (Protocol step 3.2). Upon insertion, the razor blade often adheres to the dura, causing dural bleeding and damage to the brain. This can be avoided by adding a drop of cortex buffer on the skull and removing the skull in cortex buffer.

Bleeding from the dura and the skin after cranial window preparation leads to occlusion of the window. To avoid this, .......

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The authors thank T. Sato, M. Kanbayashi, and S. Kouyama for their technical assistance. This work was supported by JSPS KAKENHI Grant Numbers JP15K14322 and JP16H06143, the Takeda Science Foundation, the Uehara Memorial Foundation, and the Collaborative Research Project of Niigata University Brain Research Institute 2017-2923 (H.M.) and by KAKENHI JP16K14559, JP15H01454, and JP15H04263 and Grant-in Scientific Research on Innovation Areas "Dynamic regulation of Brain Function by Scrap & Build System" (JP16H06459) from MEXT (T.I.).


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Name Company Catalog Number Comments
pK031. TRE-Cre Authors - Available from RIKEN BRC and Addgene
pK029. CAG-loxP-STOP-loxP-RFP-ires-tTA-WPRE Authors - Available from RIKEN BRC and Addgene
pK273. CAG-loxP-STOP-loxP-CyRFP-ires-tTA-WPRE Authors - Available from authors
Isoflurane Wako 099-06571
410 Anaesthesia Unit (isoflurane gas machine) Univentor 8323101
Vetbond (tissue adhesive) 3M 084-1469SB
MµltiFlex Round (loading tip) Sorenson 13810
Gelfoam (gelatin sponge) Pfizer 09-0353-01
Agarose Sigma A9793 Low melting point
Round-shaped coverslip Matsunami - Custom made
Unifast 2 (dental cement) GC -
Titanium bar Authors - Custom made (see Figure 1G)
Rimadyl (carprofen) Zoetis - Injectable
2-photon microscope Zeiss LSM7MP
Titanium-sapphire laser Spertra-Physics Mai-Tai eHPDS
Titanium plate Authors - Custom made (see Figure 2A)
IMARIS, FilamentTracer, MeasurementPro BITPLANE
Goniometer stage Thorlabs GN2/M

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