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.
Highlights
The hippocampus and cortex are two brain areas extensively studied due to their implications in several important neuronal processes including cognition, learning, and memory
We thought that using BrainPhys medium (Bardy et al, 2015) instead of Neurobasal-A in the CM− would improve neuronal maintenance as this medium contains less neuroactive components that are likely detrimental for long term postnatal or mature neuronal cultures (Hogins et al, 2011; Maggioni et al, 2015)
Between DIV 7 and DIV 14, a strong ramification of the dendritic tree is observed (Figure 3ADIV 14) as well as the expression of synaptic markers (Figure 3D) consistent with the creation of a functional network. These experiments show that mouse postnatal hippocampal cultures kept in BrainPhys- or in Neurobasal-A-containing CM− undergo developmental stages which are similar to those previously described for embryonic culture (Meberg and Miller, 2003; Kaech and Banker, 2006)
Summary
The hippocampus and cortex are two brain areas extensively studied due to their implications in several important neuronal processes including cognition, learning, and memory. A commonly used method is the ‘sandwich’ method which requires growing neurons on coverslips on top of a layer of glia cells [for a detailed protocol see Kaech and Banker (2006)] This model provides cultures of almost pure neurons and is of particular interest to study interaction between astrocytes and neurons dissociated from two different mouse lines. To follow the development of neurons during the 14 days of culturing (Figure 3A), cells were plated on Ibidi dishes (81166) and representative images were taken at each time point using an Axiovert 40 CFL microscope with a 10X/0.25 objective. Remove one hippocampus (following the dotted line on Figure 1A-g) Repeat this step for the other hemisphere. Let decant by gravity (1 min), collect supernatant in a new 15 mL Falcon tube and repeat dissociation two times on remaining debris (one with 15 up and down and one with 10) by adding 1 mL of CM+. (21) Make a lot of bubbles (3 mL on graduations) in the 4% BSA aliquot (see Box 1), on top of which add delicately the supernatant
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