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Cobalt Fills

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* Introduction (see below)
-completed- Ascending Terminals in the Supraeosophageal Ganglion
-completed- Horizontal and Vertical Cells
-completed- Cajal Block Stain of Lobula Complex
* Staining Method (see below)


Introduction

Cobalt chloride can be used to fill neurons intracellularly or through their cut axons. For Drosophila neuroanatomy, the method has proven useful for screening the projections of neurons that are coupled via gap junctions, permitting the transsynaptic passage of cobalt ions. Examples of this procedure are shown for the giant fibre projections. Implantation of electrodes in the thoracic ganglia allow backfills anterogradely into ascending neurons or retrogradely into descending neurons, such as the DNOVS. The cobalt method is especially useful for following the route of sensory cells from the periphery into target neuropils of the thoracic ganglia and brain.


Staining method

Filling

Dilute (2-4%) solutions of cobalt chloride or cobalt nitrate in distilled water are used for three primary filling methods.

1. The solution is applied passively (no current, simple diffusion) through an electrode implanted into the brain or ganglia. Care must be taken to prevent desiccation of the tissue, one of the main drawbacks of this method used on the small CNS of Drosophila.

2. Mass fills can be accomplished by exposing the thoracic ganglia and isolating it on a sliver of thin black plastic, such as sued for trash bags. A wall of Vaseline is built around the ganglion which is then cut with sharpened needle. The cobalt solution is placed on the ganglion (which is, of course, connected to the brain via the cervical connective). Filling times at room temperature are 30-60 minutes, or at 4oC for twice this time.

3. A field of peripheral sensilla is isolated using Vaseline. Distilled water is placed in the isolated area and one or several sensilla are then broken or scratched with a sharpened minuten pin. The water is withdrawn and immediately replaced with the cobalt solution. For mass fills, an appendage may be cut and placed directly into a pool of cobalt.

At the end of the filling period, if using an electrode, withdraw the electrode and open up the cuticle over other areas of the CNS before proceeding to the next step. If filling from the periphery, remove the remaining cobalt solution with a twist of tissue paper, and clean the filling site carefully with distilled water. Then open cuticle over the CNS . If filling from a pool of cobalt, carefully suck away excess cobalt solution with a twist of tissue. Cut off the head (if filling from the thorax), open cuticle over the brain.

Cobalt is precipitated as cobalt sulphide.

Bubble hydrogen sulphide through ringer or 70% ethanol. Then immerse the preparation in the sulphide solution for about a minute. Wash the preparation in ringer (or 70%) alcohol and then fix tissue in Gregory's artificial Bouin fluid for an hour. Afterwards, the specimen is washed in 70% ethanol and cleaned of cuticle, fat bodies, trachea, etc.

Cobalt sulphide is intensified with silver.

Cobalt sulfide is a powerful catalyst for silver reduction. Cobalt sulphide-filled neurons are like latent images which can be intensified by a process similar to photographic development. The procedure of J. P. Bacon and J. S. Altman (Brain Res. 138:259-363, 1977) is the simplest one to use and is applicable to the widest range of insect species.

Tissue is hydrated and then placed in a solution of silver nitrate and a developer such as hydroquinone. To prevent immediate reaction between these, a colloid solution is used to carry the hydroquinone, with silver nitrate then being added to it. The procedure is simple. 4-10% gum arabic, and 6% sucrose (by weight) in distilled water receives 0.17 g hydroquinone. The pH of this mixture is adjusted to pH 3.0-3.2 with concentrated citric acid. This is now the stock "reducer" which is maintained at 50-60oC. A 1% solution of silver nitrate (in distilled water) is also maintained at this temperature. Next, the tissue is placed in a small amount of the reducer and maintained in it for about 30-60 minutes. Then, the tissue is placed in a freshly mixed solution of 20 volumes reducer and 1 volume silver nitrate solution. Tissue is again maintained at 50-60oC. This part of the procedure must be in the dark as light catalyzes silver reduction outside the tissue. Brief examination in red light will indicate whether the reaction has proceeded far enough. This is gauged by the darkening of the tissue to a pale to golden brown. Once this is achieved, the tissue is placed in a warm colloid solution containing only citric acid so as to stop the reaction. After cooling this solution, the tissue is washed in distilled water, dehydrated and embedded in plastic for serial sectioning.

References pertaining to this method are described in "Neuroanatomical Techniques: Insect Nervous System." (Eds: N. J. Strausfeld and T. A. Miller).Springer. Heidelberg, New York. See G.E. Gregory's "Bodian Protargol Technique" (Chapter 3) for preparing artificial Bouin. Cobalt filling and intensification methods are described in Chapters 18 - 21.

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