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 electron microscopy 
  • Serial electron microscopy and three-dimensional reconstructions 
  • Filled cells 
  • Lipophilic fluorescent dyes 
  • Intracellular production of fluorescent proteins 
  •  Confocal and multiphoton microscopy

 

Camillo Golgi
(1843-1924)
Santiago Ramon y Cajal
(1852-1934)


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 it 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: 

  1. 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.
  2. Dry the block shortly with filter paper. 
  3. Immerse the block into a 2% aqueous solution of silver nitrate for another 2 days. 
  4. Cut sections approximately 20-100 µm thick. 
  5. Dehydrate quickly in ethanol, clear and mount (e.g. into Depex).



Result: Black neurons on a yellow background. 

 

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.

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). 

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. (PDF)
  • 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.