in-vivo-visualization-spontaneous-activity-neonatal-mouse-sensory

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following ...

Kagoshima university

 Gain-of-function mutations in the L-type Ca channel Cav1.2 cause Timothy syndrome (TS), a multisystem disorder associated with neurologic symptoms, including autism spectrum disorder (ASD), seizures, and intellectual disability. Cav1.2 plays key roles in neural development, and its mutation can affect brain development and connectivity through Ca-dependent and -independent mechanisms. Recently, a gain-of-function mutation, I1166T, in Cav1.2 was identified in patients with TS-like disorder. Its channel properties have been analyzed  but  effects of this mutation on brain development remain unexplored.   electroporation was performed on ICR mice at embryonic day 15 to express GFP, wild-type, and mutant Cav1.2 channels into cortical layer 2/3 excitatory neurons in the primary somatosensory area. The brain was fixed at postnatal days 14-16, sliced, and scanned using confocal microscopy. Neuronal migration of electroporated neurons was examined in the cortex of the electroporated hemisphere, and callosal projection was examined in the white matter and contralateral hemisphere.  Expression of the I1166T mutant in layer 2/3 neurons caused migration deficits in approximately 20% of electroporated neurons and almost completely diminished axonal arborization in the contralateral hemisphere. Axonal projection in the white matter was not affected. We introduced second mutations onto Cav1.2 I1166T; L745P mutation blocks Ca influx through Cav1.2 channels and inhibits the Ca-dependent pathway, and the W440A mutation blocks the interaction of the Cav1.2 α1 subunit to the β subunit. Both second mutations recovered migration and projection.  This study demonstrated that the Cav1.2 I1166T mutation could affect two critical steps during cerebrocortical development, migration and axonal projection, in the mouse brain. This is mediated through Ca-dependent pathway downstream of Cav1.2 and β subunit-interaction.

Involvement of Calcium-Dependent Pathway and β Subunit-Interaction in Neuronal Migration and Callosal Projection Deficits Caused by the Cav1.2 I1166T Mutation in Developing Mouse Neocortex.

In vivo calcium imaging is essential to elucidate unique synchronous activities observed in the developing brain. Here, we present a protocol to image and analyze activity patterns in neonatal mouse neocortex in a single-cell level. We describe steps for in utero electroporation, cranial window surgery, two-photon imaging, and activity correlation analysis. This protocol facilitates the understanding of neuronal activities and activity-dependent circuit formation during development. For complete details on the use and execution of this protocol, please refer to Mizuno et al. (2014), Mizuno et al. (2018a), and Mizuno et al. (2018b)..

Article	Year
Involvement of Calcium-Dependent Pathway and β Subunit-Interaction in Neuronal Migration and Callosal Projection Deficits Caused by the Cav1.2 I1166T Mutation in Developing Mouse Neocortex. Frontiers in neuroscience\| PubMed ID: 34955712	2021
In vivo two-photon calcium imaging of cortical neurons in neonatal mice. STAR protocols\| PubMed ID: 37119143	2023

Nao Nakagawa-Tamagawa

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