Filling axons of interneurons with Cl2Co 

Objective

          To learn a very simple technique used in the different levels of analysis: 1)To selectively dye neurons, which allows us to locate the groups of cells in the  central nervous system, providing the necessary anatomic base for the position of electrodes in the physiology experiments; 2)For the study of ganglions and underlining tracts; 3)For the detailed study of the morphology and connections with the different centers of integration of the central nervous system of neurons individually identified. 

Introduction

          The study of the nervous tissue could be performed through different techniques: 1)Techniques of general coloration by using Hematoxilin-eosin for example, 2)Specific techniques for the nervous system such as the intravital injection of Methelyne blue trihydrate and the impregnations with silver, and 3)The intraneural injection of fluorescent dyes (yellow and orange) enzymes and heavy metals.

          With the use of the intraneural injection of dyes (developed in the 70's), a new era for the neurophysiology begins, now that it is possible to combine the use of intracellular electrodes with the simultaneous tinting, registering the electric activity of an individual cell and studying its anatomy in detail with an optical microscope or an electron microscope.  The dying method using cobalt is used very often due to the fact that it requires less technical demand as opposed to other methods; and the obtained sections can be analyzed in either microscope.  Also, cobalt penetrates the cell without the need of electricity; and it was also demonstrated that it could be introduced in the neurons through the cut borders of its axons.  The fact that cobalt could be applied to cut axons allows us, by carefully selecting the nerve, it is possible to analyze the projections of the central nervous system of all the neurons that compose it; and to localize the cellular bodies inside different ganglions.  This way, we can achieve the mapping of the central projections of, for example, peripheral receptors to take on an electrophysiological study.

          The basic technique finds the end of the axon cut in a pool of diluted solution of  Cobalt Chloride and it is enclosed to exile the rest of the nervous system and tissues that surround it.  After enough time has passed for the migration of the cobalt ions to the inside of the neurons, it is precipitated inside the tissue as Cobalt Sulfide (which is brown in color and soluble), with Ammonium Sulfide.

          The cells that took the Co appear like dark silhouettes against a clear background.

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