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. 2019 Apr 26;3(2):NS20180207.
doi: 10.1042/NS20180207. eCollection 2019 Jun.

Culturing primary neurons from rat hippocampus and cortex

Madhusmita Priyadarshini Sahu  1 , Outi Nikkilä  1 , Seija Lågas  1 , Sulo Kolehmainen  1 , Eero Castrén  1
Affiliations

Culturing primary neurons from rat hippocampus and cortex

Madhusmita Priyadarshini Sahu et al. Neuronal Signal. .

Abstract

Primary neurons from rodent brain hippocampus and cortex have served as important tools in biomedical research over the years. However, protocols for the preparation of primary neurons vary, which often lead to conflicting results. This report provides a robust and reliable protocol for the production of primary neuronal cultures from the cortex and hippocampus with minimal contribution of non-neuronal cells. The neurons were grown in serum-free media and maintained for several weeks without any additional feeder cells. The neuronal cultures maintained according to this protocol differentiate and by 3 weeks develop extensive axonal and dendritic branching. The cultures produced by this method show excellent reproducibility and can be used for histological, molecular and biochemical methods.

Keywords: cerebral cortex; hippocampus; primary neuron.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. The procedure for extracting neuronal cells from the intact animal tissue
(A) Flowchart summarizing the complete procedure. (B) This part of the procedure was performed in the animal facility (a) opening the visceral cavity of the rat, (b) extracting the pups, (c) collecting the pups into sterile PBS. (C) This procedure was performed in the sterile laminar hood. (a–c) Extraction of the brain from the pups, (d,e) dissecting the cortex and hippocampus from the brain. (D) Trituration of the tissue to produce homogeneous cells, (a–c) hippocampal neurons and (d–f) cortical neurons.
Figure 2
Figure 2. Cultured cortical and hippocampal primary neurons stained at three different time points for neuronal markers Map2, NeuN and GFAP
(A) (a–f) Cortical neurons at 7 DIV (a,d), 14 DIV (b,e) and 21 DIV (c,f). (B) Hippocampal neurons at 7 DIV (a,d,g), 14 DIV (b,e,h) and 21 DIV (c,f,i). (C) Cell counting in cortical neurons using markers for neurons (NeuN), astrocytes (GFAP) and a dendritic cell marker, Map2. (D) Cell counting of the representative markers in hippocampal neurons. The neurons are stained with NeuN (green), Map2 (red) and GFAP (gray). Scale bar = 10 µm. Data represented as mean ± SEM. Abbreviations: DIV, days in vitro.
Figure 3
Figure 3. A representative sample image used for Sholl analysis of 21 DIV hippocampal neuron. The graphs show retrieved metrics of the linear Sholl plot for the bitmap image using ImageJ
See this image and copyright information in PMC

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