THE NEURON DOCTRINE

(established primarily based on anatomical studies)

1. The Neuron is an Anatomical Unit: The nerve cells has several short free protoplasmic extensions (dendrites) and one single long fiber, the axis cylinder (axon) which may have several collaterals. The axon and their branches end freely and interact by contact with other nerve cells.(Cajal, von Lenhossek, Waldeyer, Koelliker). The name dendrite was coined by His, 1889; the axon by Kolliker, 1896; the neuron by Waldeyer, 1891. The final proof for this law came after identifying the synapse with the electron microscope (Palade and Palay, 1954; DeRobertis, 1954)

1. The Law of Dynamic Polarization: Cajal formulated this concept, by which he meant the essentially unidirectional flow of information within neurons from their receptive surface (commonly the dendrites), through or past the cell body to the axon, and hence to its terminal branches. He based this concept upon his description of the short interconnections in the major centers of the brain (spinal cord, cerebellum, hippocampus, olfactory bulb, retina)

2. The Neuron is an Embryological (Developmental) Unit: Cajal shows that the axon and dendrites of a neuron grow out from the cell body during development and ends freely at all times. He also describes the growth cone. Later Harrison (1907) shows that the growth cones are capable by amoboid movement to reach their destined target.

4. The Neuron is a Metabolic (trophic) Unit: The distal part of the nerve degenerate following nerve transection (anterograde degeneration: Waller, 1852). Following the cut the cell body shows sign of atrophy (retrograde degeneration: Gudden, 1870). However, the injury usually does not spread forward or backward to the next neuron.

5. The Neuron is a Basic Information Processing Unit: this law was added by McCulloch and Pitts (1943).

Other important discoveries supporting the NEURON DOCTRINE

CELL BIOLOGY

Describing the Axonal Transport Mechanisms (Weiss and Hiscoe, 1948). Understanding the protein synthetic machinery and the subcellular organization of cells (Palade, 1954)

PHYSIOLOGY

The physiological concept of spinal reflexes. The term synapse was coined by Sherrington, 1897

Intracellular Electrophysiology

1. Generation of impulses by sequential movements of Na and K ions through channels across the membrane in the squid giant axon (Hodgkin and Huxley, 1952).

2. First intracellular recording of end-plate potentials giving rise to muscle action (Fatt and Katz, 1950).

3. First reported intracellular recording of EPSPs giving rise to action potentials in cat spinal motorneurons (Brock, Coombs, Eccles, 1951)

4. First reported intracellular recordings of small quantal deflections (miniature end-plate potentilas) at the muscle end-plate region (Fatt and Katz, 1952)

5. First central synaptic pathway mediating recurrent inhibition of motorneurons (Eccles, Fatt and Koketsu, 1953)

PHARMACOLOGY

The chemical hypothese of synapse

Thomas Elliot, 1904; Dixon, Dale 1914; Otto Loewi, 1921

REVISIONS of the NEURON DOCTRINE

1. The presence of electrical synapses – gap junctions (Furshpan and Potter, 1959)

2. Axo-axonic synapses

3. Dendro-dendritic synapses (e.g. amacrine cells of the retina; granule cells of the olfactory bulb (Shepherd)

4. Transynaptic regulation of transmitters, enzymes; transynaptic transport of amino acids, viruses

5. Metabolic subunits within the neuron (e.g. spines as microcompartments)

6. Backpropogation of action potentials from the soma to the dendrites

HISTORY

A. van Leeuwenhook, 1718

Nerve was composed of many individual hollow tubees. Confirmation of the classical idea, first held by the ancients and elaborated in the XVIIth century by Descartes, that the nerves were tubes containing fluids (spirits) that actually moved from sensory organs in the brain to carry sensations and from the spinal cord to the muscles to bring about movement.

Jan E. Purkinje, 1837

First identified nerve cell in the nervous system; first published view of the cellular composition of the histological layers within a brain region.

Matthias Schleiden, 1838

His paper in 1838 contained the essential idea that a basic cellular structure applied to all plants.

Theodor Schwann, 1839,

Came to the conclusion that the elements in animal tissue were practically identical with those in plant tissue.

Augustus Waller, 1852

In 1850 Waller reported experiments in which he cut the nerves to the tonque, and observed decomposition of the nerve "tubers" around the taste buds in the tonque, beginning 3-4 days after the time of the cut. He realized the great importance of this method for studyinhg nerve connections. He also realized that degeneration of the part of the nerve distal to the cut indicated that it had been deprived of its normal source of sustenance.

B.A. Gudden, 1870

Waller believed that, following a cut, the cell body and central stump of the nerve remained normal, but in the 1870s Gudden found that they also showed signs of atrophy. He launched a series of investigation in which he used his "this" secondary degeneration" to trace connections between the main centers of the brain.

Albrecht von Kolliker, 1817-1905

During the 1840s, despite continuous problems with flawed tissue preparations, there was a growing consesnus that nerve cells tend to give rise to several processes, and that in many cases one of these, different in structure from the rest, gives rise to a long process that presumably becomes the nerve fiber of the long tracts within the brain or of the peripheral motor nerves. He provided authoritative pronouncements on the early work in the 1840s, and continued to play this role at virtually every step along the way over the next half century. In the 1890s. he served as the final arbitrator of the work that established the neuron doctrine. Handbuch der Gewebelehre des Menschen, 1852, english tarnslation, 1853).

Camillo Golgi, 1843-?

In 1873 Golgi discovered that some neurons filled with the black reaction ("la reazione nera"), i.e. silver chromate precipitate, if brain tissue that had been hardened in potassium dichromate was subsequently treated in a silver nitrate solution. His mani discoveries: the axis cylinder, axon collaterals, short axoned (Golgi 2nd) neuron.

The paradox of his research. Golgi ideas about the structure and function of individual neurons were in error. However, one could say in his defensew that this was not for him important question; the crucial question was , rather, how does the nervous system function as a whole? At this level what mattered was how the nerve cells provide for a system of extensive interaction so that consciousness and mental activity could emerge in a holistic manner. We know now tah tholistic functions of the mind are understandable in terms of distributed systems that unite different structures with different functions and build up from individual neurons.

Some of the network idea, in fact is expressed in the computational neural nets developed in recent years to simulate brain functions.

Santiago Ramon y Cajal (1852-1934)

He used embryonic or young animal before myelination take splace which resulted a much crispy Golgi picture. Cajal supports Golgi's conclusion that the protoplasmic prolongations (dendrites) and freely and do not anastomose, but he goes further and draws the critical conclusions that this also applies to the axon and their branches as well, in contradiction to Golgi.

Confrontation in Stockholm, 1906: Cajal was furious at Golgi for his "display of pride and self worship" for an ego 'that was hermetically sealed and impermeable to the incessant changes taking place in the intellectual environment"

Morphological, chemical and electrophysiological characterization of neuronal circuits

1. The Nissl Method

The Nissl method is based on staining with basic aniline dyes, e.g., cresyl violet, thionine, or toluidine blue. These dyes stain the components containing nucleic acids including the nucleus and the ribosomes in the cytoplasm. The Nissl method reveals the size and shape of the cell bodies, and provides a map of cell assemblies and other cytoarchitectonic landmarks.

2. The Golgi Method

In 1873 Golgi discovered that some neurons became filled with a “black reaction”, i.e. silver chromate precipitate, if brain tissue that had been hardened in potassium dichromate was subsequently treated in a silver nitrate solution. A fortunate yet unexplained quality of the Golgi method is the fact that it impregnates only a few percent of the neurons in a given area, without staining surrounding elements. With the black neurons in sharp relief against a light background, the method is especially valuable for studying the distribution of dendrites and unmyelinated axons. In some cases, e.g. in the cerebellum, this enabled the classical histologists to assemble correctly the wiring of the entire neural system, in most cases, however, the correspondence between separately impregnated pre- and postsynaptic processes is not so obvious as to predict the organization of neuronal circuits.

3. Electron microscopy, Golgi-EM

With the advent of the electron microscopy in the early fifties the last piece of evidence supporting the neuron theory came by identification of synapses (Palade and Palay, 1954). In order to understand the apparently random entanglement of axons and dendrites under the electron microscope, however, additional techniques had to be employed. Using a combination of the Golgi method and electron microscopy, Blackstad’s (1965) pioneering work paved the way to a direct analysis of identified neural circuit at the synaptic level.

4. Intra- juxtacellular labeling techniques

A further dimension was opened up in the analysis of identified circuits with the application of the electron microscopic technique to brain tissue that also contained electrophysiologically characterized and intra- or juxtacellularly injected neurons using different staining protocols (Horseradish peroxidase=HRP, biocytin, Lucifer Yellow, etc.).

5. Neurochemical characterization of neurons using immunocytochemistry

Immunocytochemistry has rapidly developed into an indispensable tool in neuroscience. It provides a means of characterizing nerve cells and fibers on the basis of the presence of specific cell constituents. One of first methods used for the immunocytochemical localization of antigen was the direct immunofluorescence technique developed by Coons and his colleagues (Coons et al., 1941, Coons and Kaplan, 1950). Following the development of the direct immunofluorescence technique, other direct and indirect methods were developed for light and electron microscopy (PAP, ABC, ferritin, gold). More recently, dual immunocytochemical labeling of two antigens in the same section prepared for electron microscopy has permitted the characterization of transmitters within pre- and postsynaptic junctions in the CNS.

6. Neurochemical characterization of neurons using in situ hybridization

7. Axon tracing methods

Although individual axons can be traced with the Golgi method or by the aid of the intracellular injection techniques, the tracing of long axons is usually not possible. Various silver methods were applied after experimental lesion (Bielschowsky, Nauta, Fink-Heimer, etc.) to stain degenerating axons and terminals using the principle of Wallerian degeneration. Since degenerating terminals can also be recognized under the electron microscope, the identification of the termination of the various pathways became possible. While silver methods had a dominant role as tract-tracing methods until more sophisticated techniques became available in the mid 1970s, we are witnessing a revival of such techniques to localize neuronal degeneration in experiments using various neurotoxins to model human neurodegenerative diseses.

8. Axonal transport methods

Since the late sixties several tracer methods were introduced which used the physiological anterograde or retrograde transport mechanism instead of artificially damaging the neurons. In additions, these methods often circumvented the problem of fibers of passage, a serious drawback of the experimental lesion technique.

Anterograde tracer methods. The PHA-L method. After iontophoretic injection of the plant lectin Phaseolus vulgaris leucoagglutinin into a specific part of the nervous system, the tracer is incorporated into the cell bodies in the area of injection and some of the lectin is transported at the slow rate (4-6 mm/day) of axonal transport to the axon terminals. The neuron that have incorporated the tracer can then be detected by an immunohistochemical method.

Retrograde tracer methods. HRP, retrograde fluorescent tracers. HRP and several fluorescent tracers are readily taken up by axon terminals and transported back to the parent cell bodies, where they could be detected either by a simple staining procedure (DAB in the case of HRP) or by direct visualization in a fluorescence microscope.

9. Combined techniques: Tracing of successive links in a neuronal circuits

10. Computer assisted 3-D reconstruction of neurons and neuronal populations

11.Cell counting using ‘unbiased’ stereological techniques

12.Intra-, extracellular recording, EEG monitoring in anesthethized rats

Further Reading

1.Boulton, AA, et al. (1990) ed. Neurophysiological Techniques. Vol. 15, Neuromethods,

Humana Press, Clifton, NJ

2. Beltramino CA, deOlmos J, Gallyas F, Heimer L, Zaborszky (1993) In: Assessing Neurotoxicity of Drugs of Abuse. L Erinoff (ed.) NIDA Monograph Series, 136:101-132.

3. Bolam, J.P. (Ed) Experimental Neuroanatomy: A Practical Approach. Oxford University

Press: Oxford, 1992.

4. Heimer, L. and L. Zaborszky (eds) Neuroanatomical Tract Tracing Methods 2. Recent

Progress. Plenum Press: New York, 1989.

6.Peters, A., Palay, S.L., and Webster, H.deF. The Fine Structure of the Nervous System. 3^rd

ed., Oxford University Press, New York, 1991

7. Pinault, D. J. Neurosci. Methods. 65: 113-136, 1996.

8. Watson, S.J. and W.E. Cullinan: In: Psychopharmacology The Fourth Generation of Progress,

ed. F.E. Bloom, D.J. Kupfer, Raven Press, New York, 1995.

9. West, M.J. (1993) New stereological methods for counting neurons. Neurobiol. Aging

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