Abstract
Primary culture of mouse hippocampal neurons is a very useful in vitro model for studying neuronal development, axonal and dendritic morphology, synaptic functions, and many other neuronal features. Here we describe a step-by-step process of generating primary neurons from mouse embryonic hippocampi (E17.5/E18.5). Hippocampal neurons generated with this protocol can be plated in different tissue culture dishes according to different experimental aims and can produce a reliable source of pure and differentiated neurons in less than one week. This protocol covers all the steps necessary for the preparation, culture and characterization of the neuronal culture, including the illustration of dissection instruments, surgical procedure for embryos' isolation, culturing conditions and assessment of culture's purity and differentiation. Evaluation of neuronal activity was performed by analysis of calcium imaging dynamics at six days in culture.
Highlights
The murine hippocampus has a well-defined, C-shaped structure that is easy to locate and isolate. It is mainly composed of pyramidal cells with fewer interneurons and glial cells compared to other brain regions (Kaech and Banker, 2006)
The hippocampus is an ideal region for generating primary neuron cultures of high purity from wild-type or genetically engineered mouse models that can be used for disease modeling or to investigate multiple aspects of neuronal function such as synaptic transmission and electrophysiological properties, sensitivity to neurotoxicity, differentiation and aging (Busche, 2018; Koyama and Ikegaya, 2018; Molnar, 2011; Wu et al, 2019; Rush et al, 2020)
We report analysis of neuronal differentiation and function after 6 days in culture by testing for neuronal differentiation markers and neuronal activity by cellular calcium imaging dynamics
Summary
2. Evenly coat the culture dish surface with 0.1 mg/ml poly-D-lysine solution and incubate at room temperature for 20 min in a laminar flow hood. Transfer poly-D-lysine/laminin coated coverslips directly into wells of a 24-well plate by using sterile forceps in a sterile laminar flow hood before plating neurons. Note: Use forceps (Figure 1B) to pinch and pull up the skin layer while performing the incision with surgical scissors. This will help to avoid accidental damage to the embryos located immediately underneath. 4. Carefully expose the whole uterus (uterine horns) containing the embryos (Figure 2B) by using forceps and surgical scissors (Figures 1A-1B) and immediately transfer the uterus to a 100 mm dish containing ice-cold PBS (Figure 3A). In the figures shown in this protocol PBS was intentionally used instead of medium containing phenol red only for the purpose of obtaining more clear images (Figure 3A, Figure 4, Figure 5 and Figure 6)
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