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Procedures for Culturing and Genetically Manipulating Murine Hippocampal Postnatal Neurons.

ABSTRACT: Neuronal hippocampal cultures are simple and valuable models for studying neuronal function. While embryonic cultures are widely used for different applications, mouse postnatal cultures are still challenging, lack reproducibility and/or exhibit inappropriate neuronal activity. Yet, postnatal cultures have major advantages such as allowing genotyping of pups before culture and reducing the number of experimental animals. Herein we describe a simple and fast protocol for culturing and genetically manipulating hippocampal neurons from P0 to P3 mice. This protocol provides reproducible cultures exhibiting a consistent neuronal development, normal excitatory over inhibitory neuronal ratio and a physiological neuronal activity. We also describe simple and efficient procedures for genetic manipulation of neurons using transfection reagent or lentiviral particles. Overall, this method provides a detailed and validated protocol allowing to explore cellular mechanisms and neuronal activity in postnatal hippocampal neurons in culture.

SUBMITTER: Moutin E

PROVIDER: S-EPMC7204911 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Procedures for Culturing and Genetically Manipulating Murine Hippocampal Postnatal Neurons.

Moutin Enora E Hemonnot Anne-Laure AL Seube Vincent V Linck Nathalie N Rassendren François F Perroy Julie J Compan Vincent V

Frontiers in Synaptic Neuroscience 20200430

Neuronal hippocampal cultures are simple and valuable models for studying neuronal function. While embryonic cultures are widely used for different applications, mouse postnatal cultures are still challenging, lack reproducibility and/or exhibit inappropriate neuronal activity. Yet, postnatal cultures have major advantages such as allowing genotyping of pups before culture and reducing the number of experimental animals. Herein we describe a simple and fast protocol for culturing and genetically ...[more]

PMID: 32425766

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Project description:Exposure to radiofrequency electromagnetic fields (RF-EMFs) has increased rapidly in children, but information on the effects of RF-EMF exposure to the central nervous system in children is limited. In this study, pups and dams were exposed to whole-body RF-EMF at 4.0 W/kg specific absorption rate (SAR) for 5 h per day for 4 weeks (from postnatal day (P) 1 to P28). The effects of RF-EMF exposure on neurons were evaluated by using both pups' hippocampus and primary cultured hippocampal neurons. The total number of dendritic spines showed statistically significant decreases in the dentate gyrus (DG) but was not altered in the cornu ammonis (CA1) in hippocampal neurons. In particular, the number of mushroom-type dendritic spines showed statistically significant decreases in the CA1 and DG. The expression of glutamate receptors was decreased in mushroom-type dendritic spines in the CA1 and DG of hippocampal neurons following RF-EMF exposure. The expression of brain-derived neurotrophic factor (BDNF) in the CA1 and DG was significantly lower statistically in RF-EMF-exposed mice. The number of post-synaptic density protein 95 (PSD95) puncta gradually increased over time but was significantly decreased statistically at days in vitro (DIV) 5, 7, and 9 following RF-EMF exposure. Decreased BDNF expression was restricted to the soma and was not observed in neurites of hippocampal neurons following RF-EMF exposure. The length of neurite outgrowth and number of branches showed statistically significant decreases, but no changes in the soma size of hippocampal neurons were observed. Further, the memory index showed statistically significant decreases in RF-EMF-exposed mice, suggesting that decreased synaptic density following RF-EMF exposure at early developmental stages may affect memory function. Collectively, these data suggest that hindered neuronal outgrowth following RF-EMF exposure may decrease overall synaptic density during early neurite development of hippocampal neurons.

Dataset Information

Procedures for Culturing and Genetically Manipulating Murine Hippocampal Postnatal Neurons.

Publications

Procedures for Culturing and Genetically Manipulating Murine Hippocampal Postnatal Neurons.

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