A Brief Review of Techniques used in the Study of Neuroanatomy
1. The earliest observations on the brain and peripheral nerves were made on unfixed animal material. Discovery of methods of fixation, and the development of simple microscopes enabled much more detailed studies to be made, but substantial advances became possible only after methods for preparing tissue sections and for staining them became available. The technique of silver impregnation was discovered in the later part of the eighteenth century by workers including Ranvier and Golgi.
2. Early discoveries that paved the way for techniques allowing the tracing of neural pathways included the staining of ‘chromatin granules’ by Nissl, who also discovered the phenomenon of chromatolysis about a hundred years ago. About the same time Weigert discovered a method for staining normal myelin, and Marchi discovered his method for degenerating myelin.
3. Changes taking place in degenerating neurons were first described by Waller in 1850 (hence the term Wallerian degeneration). After the discovery of Marchi’s method for staining degenerating myelin the way was opened for tracing of neural pathways after placing of experimental lesions in animals. However, methods for the demonstration of degenerating axons, and of axon terminals became available only in the middle of the present century (Glees, 1946; Nauta and Gygax, 1951). With the relatively recent development of stereotactic methods it has become possible to create accurate and controlled neural damage even deep within the brain.
4. While the various neuron-tracing methods developed over a century have added considerably to our knowledge of the structure of the nervous system, information thus obtained has been confirmed and greatly amplified by neuro-physiological methods. These involve controlled stimulation at one point with recording of evoked potentials at other sites. Neurophysiological methods have greatly increased in sophistication with the development of stereotaxis, of advanced electronic stimulating and recording devices, and of microelectrodes that can record potential changes even in individual neurons or from individual nerve fibres.
5. More recent advances in the study of neuroanatomy have gone hand in hand with technical developments. The capabilities of light microscopes have increased with the development of refined optics, and with innovations like dark field illumination, use of polarised light, phase contrast, and microscopy with ultraviolet light. One recent advance is the confocal microscope that provides three dimensional views of microscopic structures (without the need for preparation of thin sections and reconstructions).
Histochemical techniques have added greatly to understanding of neurology, specially in association with the results of physiological experiments. In this connection the development of immuno-histochemistry has been of great importance. This technique has made possible precise localisation of various neurotransmitters, neuromediators neuro-modulators and other substances, and has revolutionised concepts about the variety of nerve cells and nerve fibres.
Immuno-histochemical localisation has greatly improved following availability of monoclonal antibodies, and use of molecular genetics for producing antigens of great purity.
A revolutionary explosion of knowledge of the finer details of the structure of neurons, of nerve fibres, and of synapses has taken place with the development of suitable methods for preparation and examination of tissues using the electron microscope. In turn the availability of detailed information has led to the possibility of useful correlations with the results of biochemical and physiological advances. The utility of electronmicroscopy has been greatly increased by combining it with techniques like autoradiography , histochemistry, and immunological methods.
When amino acids or sugars normally present in the body are labelled with radioactive substances and are injected into an animal, they become incorporated at specific sites. These sites can be located in tissue sections by covering a section with a photographic emulsion and allowing the radioactive material to act on it for some days. When the emulsion is developed granules of silver are seen over the sites where the incorporated material is present. This technique is called autoradiography. It has been found that if labelled amino acids or sugars are injected into a part of the brain, these substances are absorbed by neurons and are transported along their axons. These neurons can be revealed by autoradiography. The preparations can be studied both by light microscopy and electronmicroscopy.
It has been found that if axons are exposed to the enzyme horse radish peroxidase (HRP), the enzyme is taken up into the axons and transported in a retrograde direction to reach the cell bodies concerned. Alternatively if HRP is injected into neuronal cell bodies it is transported down the axons. The presence of enzyme can be seen using appropriate methods. This has provided a new technique for tracing neuronal pathways. Some fluorochrome dyes act in a manner similar to HRP. Some fluorescent dyes injected into embryonic cells pass into their daughter cells thus enabling the origin of the latter to be traced.
It has been shown that embryonic neural tissue implanted into the brain can survive and can be functionally integrated with the host tissue. Such implantation may offer a method for replacing dead or deficient neurons at particular sites (e.g., the corpus striatum). They may also be used to encourage regeneration of axons after injury.