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Visualization of Dendritic Spines
by Josef Spacek and John Fiala
Numerous methods have been used to observe the small spines that extend from neuronal dendrities and make synaptic
connections with passing axons. These methods include:
- Golgi impregnation and light microscopy
- Transmission electron microscopy
- Freeze-fracture and electron microscopy
- Serial electron microscopy and three-dimensional
reconstructions
- Filled cells
- Lipophilic fluorescent dyes
- Intracellular production of fluorescent proteins
- Confocal and multiphoton microscopy
Golgi Impregnation and Light Microscopy
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Camillo Golgi
1843-1924
Golgi method 1873
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Nobel prize 1906 |
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Santiago Ramon y Cajal
1852-1934
Dendritic spines 1891
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Although Santiago Ramon y Cajal made the first observations of dendritic spines (1891) in brain tissue
stained with methylene blue according to Ehrlich, his favorite method was
the silver impregnation method based on Golgi's original technique. In the
hands of innumerable neurohistologists this method has been admirably fruitful since its
introduction by Camillo Golgi (1873) and continues to be widely used. Golgi and
Ramon y Cajal were jointly awarded the Nobel prize for medicine in 1906, for
their contributions to understanding the cellular structure of the brain using this
method.
The classical Golgi impregnation method is very elegant in its simplicity:
- Immerse a block (approx. 10x5 mm) of formol-fixed (or paraformaldehyde-
glutaraldehyde-pefused) brain tissue into a 2% aqueous solution of
potassium dichromate for 2 days
- Dry the block shortly with filter paper.
- Immerse the block into a 2% aqueous solution of silver nitrate for
another 2 days.
- Cut sections approx. 20-100 µm thick.
- Dehydrate quickly in ethanol, clear and mount (e.g., into Depex).
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Result:
Black neurons on a yellow background. |
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| Click on image to animate |
The principle of the method is based on the crystallization of silver chromate:
the microcrystalline precipitate either grows directly from the surface of the
tissue block into transected neuronal processes or spreads from nucleation
centers inside the block into nerve cell processes like in preformed channels -
until the neuron has been completely filled. The frequency of
nucleation centers is responsible for the unique selectivity of this method,
staining only 1-10% of neurons. Without this selectivity, no individual neurons
would be seen but only a non-informative overcrowded jungle of cells and
processes.
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| Click on image to stop animation |
More information about the dynamics of Golgi impregnation and its methodical
expansions can be found in Harris et al. (1980), Spacek (1989) and Spacek (1992).
See also the website of Valverde
for a gallery of Golgi preparations.
References
- Golgi C (1873) Sulla struttura della sostanza grigia dell cervello. Gazz. Med.
Lombarda 33:244-246.
- Ramon y Cajal S (1995) Histology of the Nervous System of Man and Vertebrates.
Oxford University Press: New York, English translation by N Swanson and LM Swanson.
- Harris KM, Cruce WRL, Greenough WT, Teyler TJ (1980) A Golgi
impregnation technique for thin brain slices maintained in vitro. J Neurosci
Methods 2:363-371.
- Spacek J (1989) Dynamics of Golgi method: A time-lapse study of the early
stages of impregnation in single sections. J Neurocytol 18:27-38.
- Spacek J (1992) Dynamics of the Golgi impregnation in neurons. Microscopy
Res Tech 23:264-274.
Next: Transmission Electron Microscopy