Primary neuronal culture, immunocytochemistry and Sholl analysis

Neurons can be taken out of the brain, separated from one another and kept alive on glass for weeks. In such a dissociated primary culture, each neuron grows an axon and several dendrites on a flat surface where every branch can be seen and measured. Banker and Cowan (1977) grew isolated hippocampal neurons from fetal rats on polylysine-coated coverslips, and Dotti, Sullivan and Banker (1988) followed single neurons day by day and described a stereotyped sequence: several short, similar processes appear first, one of them then grows rapidly and becomes the axon, and the others elongate a few days later and become dendrites. A serum-free medium, B27-supplemented Neurobasal, keeps embryonic hippocampal neurons alive with very little glial growth (Brewer et al., 1993); a widely used protocol is that of Kaech and Banker (2006).

Human neurons can be made in the same way from induced pluripotent stem cells (iPSCs), which are reprogrammed from adult cells such as skin fibroblasts (Takahashi et al., 2007). Forcing iPSCs to express a single transcription factor, NGN2, converts them into neurons within about two weeks (Zhang et al., 2013). Human neurons mature more slowly than rodent neurons, so cultures are kept for longer.

To see which process is which, cultures are fixed and stained by immunofluorescence: MAP2 marks dendrites (Cáceres et al., 1984), Tau marks axons (Binder et al., 1985), the antibody SMI-312 recognises phosphorylated neurofilaments in axons (Ulfig et al., 1998), GFAP marks astrocytes (Eng et al., 2000) and DAPI marks every nucleus. Expressing green fluorescent protein, GFP (Chalfie et al., 1994), from a plasmid in a small fraction of neurons fills single cells completely, so that their whole arbor can be traced even in a crowded dish.

The branching of a traced dendritic tree is often summarised by a Sholl analysis: circles of increasing radius are drawn around the cell body and the number of times the dendrites cross each circle is counted (Sholl, 1953). Software for this is freely available (Ferreira et al., 2014). Many neurons are traced from each dish, but neurons from the same culture share its history, so the independent culture is the unit of analysis; treating every neuron as independent is pseudoreplication (Lazic, 2010; Aarts et al., 2014).

Learning objectives

• Plan a culture experiment with the independent culture (or, for human cells, the independent differentiation) as the unit of analysis, with randomised and coded coverslips and a vehicle control
• Explain why coverslips are coated, why tissue is digested and triturated gently, and work at a biosafety cabinet with sterile technique
• Count live and dead cells with trypan blue on a haemocytometer, calculate the concentration and the volume needed for a chosen seeding density, and explain the trade-off between survival and traceability
• Choose a serum-free medium and a feeding schedule, recognise contamination and the developmental stages of neurons in culture, and choose when to fix
• Design an immunocytochemistry panel for MAP2, Tau or SMI-312, GFAP and DAPI with matching secondary antibodies and filter cubes, and include a no-primary control
• Trace dendrites without the axon or neighbouring cells, run a Sholl analysis, and compare conditions with a paired t-test on culture means

Ethics and safety

Rodent neurons come from embryos or newborn pups. The pregnant animal or the pups are killed humanely by an approved method carried out by a trained, competent person, and the work is approved by an Institutional Animal Ethics Committee (in India, the CCSEA under the Prevention of Cruelty to Animals Act 1960; elsewhere, frameworks such as EU Directive 2010/63/EU, the US Animal Welfare Act and PHS Policy, and the UK Animals (Scientific Procedures) Act 1986). The 3Rs apply: one litter supplies many coverslips, which reduces the number of animals, and a well-planned design avoids repeating cultures for lack of power. Human iPSC lines are made from cells donated with informed consent and are obtained from documented sources; their use follows the consent terms and institutional approval. Cell culture is done in a class II biosafety cabinet under the local biological safety rules; paraformaldehyde is toxic and is handled in a fume hood with gloves and eye protection. Experiments with animals are reported following the ARRIVE guidelines, developed by the NC3Rs in the United Kingdom and endorsed by journals internationally (Percie du Sert et al., 2020).

Key terms

• Primary culture: Cells taken directly from an animal or person and grown in a dish, as opposed to a cell line that divides indefinitely.
• Coverslip: A thin glass disc on which the cells grow; after staining it is mounted on a microscope slide.
• Poly-D-lysine: A positively charged synthetic polymer coated on glass so that cells attach; the D form resists digestion by the cells' own enzymes.
• Trituration: Gently pipetting digested tissue up and down to separate it into single cells.
• Trypan blue: A blue dye that enters only cells with a damaged membrane, so dead cells look blue and live cells look clear.
• Haemocytometer: A thick glass slide with a counting grid of known area and depth, used to count cells and calculate their concentration.
• Days in vitro (DIV): The number of days since the cells were plated.
• MAP2: Microtubule-associated protein 2, found in dendrites and cell bodies of neurons but not in axons.
• Tau: A microtubule-associated protein concentrated in axons; with MAP2 it distinguishes the axon from dendrites.
• Transfection: Putting DNA, such as a plasmid encoding GFP, into cells; in neurons it usually reaches only a small fraction of cells.
• Vehicle control: Cells that receive the same solvent (for example DMSO) as the treated cells, but without the compound.
• Sholl analysis: Counting how many times the dendrites cross circles of increasing radius around the cell body, to describe branching.
• Independent culture: A culture prepared from different animals (or a separate differentiation) on a different day; the unit of replication.