Counting dendritic spines with Golgi–Cox staining

Most excitatory synapses in the brain sit on dendritic spines, small protrusions from the dendrites of neurons; a spine typically receives input from one excitatory synapse (Nimchinsky et al., 2002). Changes in the number and shape of spines are reported in many brain disorders and after experience, so counting them is one of the commonest ways to ask whether the wiring of a brain region has changed (Penzes et al., 2011). Spines come in shapes that grade into one another; the classical categories are thin, stubby and mushroom (Peters & Kaiserman-Abramof, 1970), with long, headless filopodia as a fourth, immature form (Risher et al., 2014).

The Golgi method, developed by Camillo Golgi in 1873 and used by Santiago Ramón y Cajal to describe the nervous system cell by cell (Risher et al., 2014), fills only a small, apparently random fraction of neurons, about 1–3% (Zaqout & Kaindl, 2016), completely with a dark deposit. Because so few cells are filled, a single neuron with all its dendrites and spines stands out against an almost clear background. Golgi and Cajal shared the Nobel Prize in Physiology or Medicine in 1906. The Golgi–Cox variant, which uses a solution of potassium dichromate, mercuric chloride and potassium chromate, is considered the most reliable for showing dendrites with a low background (Zaqout & Kaindl, 2016) and is one of the most commonly used methods for spines (Risher et al., 2014), and it also works on human post-mortem brain tissue, including unfixed tissue stored frozen for years (Buell, 1982; Melendez-Ferro et al., 2009). Tissue kept in formalin for many years, by contrast, impregnates very poorly with Golgi–Cox (Melendez-Ferro et al., 2009).

Spine counts are easy to get wrong. Under a light microscope, spines in front of or behind the dendrite are hidden, so counts underestimate the true number (Feldman & Peters, 1979; Harris et al., 1992); how much depends on the objective, on focusing and on the impregnation. The data are also nested: spines on dendrites, dendrites on neurons, neurons in animals. Treating every dendrite as independent is pseudoreplication and inflates false positives (Lazic, 2010; Aarts et al., 2014); the animal, or the donor, is the unit of analysis. Dendritic complexity is measured with Sholl analysis, which counts how many times the dendrites cross circles drawn around the cell body (Ferreira et al., 2014).

Learning objectives

• Explain why the Golgi–Cox method fills only a few neurons, and why that makes whole-neuron morphology visible
• Choose the tissue, impregnation time, section thickness and development, and recognise under- and over-impregnation, cut dendrites and batch effects
• Select neurons and dendritic segments blind by rules fixed in advance (fully impregnated, isolated, not cut at the section surface, defined branch order)
• Count spines at 100× under oil by focusing through the dendrite, express spine density per 10 µm, and classify spines as thin, stubby, mushroom or filopodia from their length and head width
• Run a Sholl analysis and interpret intersections against distance from the cell body
• Analyse nested data with the animal or donor as n, and show how treating dendrites as independent misleads

Ethics and safety

Animal work needs approval 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), an ethical review that applies the 3Rs (replace, reduce, refine) and trained, authorised staff; for Golgi–Cox staining the brain is removed after humane killing by an approved method, and genetically altered lines are bred under the same approvals. Studies 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). Human brain tissue comes only from brain banks (Kretzschmar, 2009), from donors who (or whose next of kin) gave informed consent to research use, under the law and research ethics approval of the country concerned; there is no experimental manipulation, so groups are diagnoses, and donors are compared with care for age, sex, post-mortem interval and medication. The Golgi–Cox solution contains mercuric chloride and dichromate, which are toxic and carcinogenic, so it is handled in a fume hood with gloves (Zaqout & Kaindl, 2016) and eye protection, and all waste is collected as hazardous heavy-metal waste.

Key terms

• Dendritic spine: A small protrusion from a dendrite, usually with a head and a neck, that receives most excitatory synapses.
• Golgi–Cox impregnation: A staining method in which a block of brain tissue soaks in a dichromate–mercury solution for days to weeks; a small fraction of neurons become completely filled with a dark deposit.
• Impregnation: Filling of a cell with the metallic deposit of the Golgi reaction, as opposed to binding of a dye.
• Development: The step after sectioning in which an ammonia solution turns the impregnated neurons dark and stable; it is followed by dehydration, clearing and coverslipping.
• Thick section: A section of about 100–200 µm, much thicker than for most stains, so that dendrites stay within it for long distances.
• Branch order: The position of a dendritic branch in the tree: branches leaving the cell body are first order, their daughter branches second order, and so on.
• Oil-immersion objective: A high-magnification objective (here 100×) used with a drop of immersion oil between lens and coverslip, giving a numerical aperture of about 1.4 and the finest detail a light microscope can resolve.
• Z-stack: A series of images, or a sequence of focus positions, taken at small steps through the depth of the section.
• Spine density: The number of spines counted along a measured length of dendrite, here given per 10 µm.
• Thin, stubby, mushroom, filopodium: Spine shapes: thin spines have a long neck and small head; stubby spines have no clear neck; mushroom spines have a large head; filopodia are long, headless protrusions.
• Sholl analysis: Counting how many times the dendrites of a neuron cross a series of circles of increasing radius centred on the cell body.
• Nested data: Data with several levels, such as spines within dendrites within neurons within animals; observations at the lower levels are not independent.
• Post-mortem interval (PMI): The time between death and the preservation (freezing or fixation) of the tissue.
• Brain bank: An organisation that collects, characterises, stores and distributes post-mortem brains from consenting donors for research.