Flybrain

Golgi Impregnations

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* Introduction (see below)
-completed- Mushroom Body Complex
-completed- Central Body Complex
-completed- Antennal Lobe
-completed- Projection Neurons
-completed- Intrinsic Neurons
-completed- Multiglomerular Neurons
-disconnected- Receptor Terminals in progress
-disconnected- Mechanosensory Neuropils in progress
-completed- Antennal Nerve Endings
-completed- Optic Lobes
-completed- Protocerebral Neuropils (except Central and Mushroom Body Complexes)
-development- Descending Neurons
-disconnected- Suboesophageal Ganglia
-disconnected- Thoracic-Abdominal Ganglia in progress
* Staining Method (see below)

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Introduction

The Golgi technique selectively impregnates single neurons with silver chromate. Modifications of the technique can be used to show up many independent neurons in each brain. It has provided indispensable information about the way in which sets of neuronal elements contribute to the gross structure of neuropils and tracts. Impregnations show up as black, purple or reddish brown against a pale yellow background. The Golgi method is essentially a stochastic technique, the exact chemical mechanism of which remains unclear.


Staining method

Golgi impregnations were carried out as follows. Up to six cold immobilised flies were threaded by their necks, and in the same orientation, between two parallel leaves of gelatin (substituting steel) fixed to a teflon block as diagrammed for paraffin histology. The block was immersed in 2.5% potassium dichromate. Cuticle between the compound eyes and at the frons, between the antennae, was removed taking care to duplicate this as exactly as possible between individuals. The block was then transferred to a bath containing five parts 2.5% potassium dichromate, one part 25% electron microscopy grade glutaraldehyde (Polysciences), for five days, in the dark, at 4C°. Blocks were subsequently treated by one of two methods. One involved direct immersion in 0.75% silver nitrate for 48h (the Golgi Colonnier method; Strausfeld, 1980). In the other, blocks were next washed in 2.5% potassium dichromate for 1hr, followed by four days incubation in 100 parts 2.5% potassium dichromate, one part 1% osmium tetroxide (mixed Golgi rapid/Colonnier method; Strausfeld, 1980) and then immersed in 0.75% silver nitrate. Finally the mounted flies were dehydrated through propylene oxide, and embedded in fresh Durcupan (Fluka Chemicals). Serial 30µm sections were cut on a sliding microtome.

Images were collected using a Leitz Diaplan microscope equipped with a Sony DKC-5000 CCD with image-capture software, running as a plug-in through Adobe Photoshop. Each image was scanned at 200 dpi, using a 40x or 100x oil-immersion planapochromat objective with either 1x or 0.6x intermediate magnification. Many of the Golgi and cobalt pictures are composite images. They have been reconstructed from serial optical sections taken at every 2 µm of depth through 1-4 consecutive 12-14 µm thick plastic sections. Initial alignment was manual: outlines of the last optical section of the first plastic section were drawn on a transparent overlay on the monitor, and the first optical section of the second section was aligned to this rendition using a rotating stage and X Y controls. Final alignment was accomplished using software. Each captured image was defined as a separate layer, and successively overlaid with the next captured image using the software compositing controls on 'DARKEN'. Due to the high contrast of the histological material, namely, dark staining on a light background, the most highly defined structures at each depth of focus are additively displayed. The images were then saved and labeled in CorelDraw (approx. 25MB), and reduced to JPEG format (approx. 150KB). Examples of this technique can be seen by clicking here or here.

Other images, such as those submitted by contributors, were digitized from transparencies using a Hewlitt Packard Scanjet 3C/T and scanning hardware. The resulting images were labeled using CorelDraw. Compression reduced the file size from TIFF (approx. 5MB) to JPEG (approx. 150 KB).

Reference:
Strausfeld, N.J. (1980) The Golgi method: Its application to the insect nervous system and the phenomenon of stochastic impregnation. In: Strausfeld, N.J., Miller, T.A. (Eds.) Neuroanatomical Techniques. Insect Nervous System. Springer. New York. pp 132-205.


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