Golgi-like demonstration of ?dark? neurons with an argyrophil III method for experimental neuropathology

Abstract

A silver method is proposed for the selective, well-contrasted and reproducible demonstration of "dark" neurons in frozen, vibratome and paraffin sections cut at a thickness of 5 to 200 microns from aldehyde-fixed brains. The Golgi-like staining of the dendrites enables assorting of "dark" neurons according to characteristic neuron classifications. The staining procedure includes an esterification with 1-propanol, a treatment with diluted acetic acid and development. The esterification strongly increases the argyrophilia of both "dark" neurons and mitochondria. Unwanted co-staining of mitochondria is suppressed by the acetic acid treatment, while a special developer is used to render the staining controllable. The applicability of the method to experimental neuropathology is demonstrated by Golgi-like staining of "dark" neurons in rat brains exposed, before transcardial perfusion-fixation and delayed autopsy, to various pathological conditions including ischemia, hypoglycemia, trauma, status epilepticus, deafferentation and poisoning with kainic acid, colchicine and sodium azide, respectively.

Instantaneous Perturbation of Dentate Interneuronal Networks by a Pressure Wave-Transient Delivered to the Neocortex

“…For the silver stain, the procedure was similar to that described earlier (Gallyas et al, 1990(Gallyas et al, , 1992avan den Pol and Gallyas, 1990). Briefly, the animals were anesthetized with sodium pentobarbital and perf used with a fixative containing 4% paraformaldehyde and 2.5% glutaraldehyde.…”

“…Briefly, the animals were anesthetized with sodium pentobarbital and perf used with a fixative containing 4% paraformaldehyde and 2.5% glutaraldehyde. The brains were left in the skull overnight (Gallyas et al, 1990;van den Pol and Gallyas, 1990), and the following day they were removed and post-fixed in the same fixative as described above. Sections were cut with a vibratome and placed in 50%, 75%, and 100% 1-propanol for 5 min each, and then in 1-propanol containing 0.8% sulf uric acid for 16 hr at 56°C.…”

“…The sections were then rehydrated in 50% and 25% 1-propanol, washed in distilled water, and treated with 3% acetic acid for 5 min. Subsequently, the sections were placed in a silicotungstate physical developer (for the composition and preparation of the developer, see Gallyas et al, 1990; van den Pol and Gallyas, 1990) for 10 min, dehydrated, mounted on slides, and coverslipped.…”

“…It was not feasible to test this hypothesis using either electrophysiological or immunocytochemical methods; e.g., it takes time to cut slices and establish whole-cell recordings, and protein levels in the injured cells are unlikely to change in an immediate manner after the passing through of the pressure wave. However, the Gallyas silver stain (Gallyas et al, 1990(Gallyas et al, , 1992avan den Pol and Gallyas, 1990) can be used to detect injury to neuronal processes that occur quickly after the insult. The principle of the stain is thought to be related to neuronal polymeric cytoskeletal elements breaking during the insult, exposing charges from the monomers that may trigger the process of silver accumulation, in a manner reminiscent of the intensification process during photography (Gallyas et al, 1992c); obviously, although this silver stain is ideal for the identification of injured or stressed neurons in a rapid manner, it is not designed to determine whether the labeled neuron will or will not survive the initial injury (see below).…”

“…The principle of the stain is thought to be related to neuronal polymeric cytoskeletal elements breaking during the insult, exposing charges from the monomers that may trigger the process of silver accumulation, in a manner reminiscent of the intensification process during photography (Gallyas et al, 1992c); obviously, although this silver stain is ideal for the identification of injured or stressed neurons in a rapid manner, it is not designed to determine whether the labeled neuron will or will not survive the initial injury (see below). Indeed, this method has been shown to reveal injured neurons immediately after direct physical damage to neurons (van den Pol and Gallyas, 1990;Gallyas et al, 1992a). Therefore, we used the Gallyas silver stain to determine whether the hilar cells are injured in an immediate manner after fluid percussion head trauma and whether the immediate injury is selective to hilar neurons compared with the granule cells, which are smaller and tightly packed in a cell layer.…”