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.

Based on recent findings described in accompanying reports as well as on relevant observations in the literature we hypothesize that: (1) the fundamental elements in the mechanism of the formation of "dark" (argyrophilic) neurons are independent of the causative conditions including post-mortem or in vivo mechanical injuries and various in vivo pathometabolic processes such as blood recirculation following ischemia; (2) the causative conditions, each in its own mechanical or metabolic way, induce the same morphopathological damage at one point only within each affected neuron; (3) this damage spreads throughout the respective somato-dendritic or axonal domain and entails type III argyrophilia; (4) the intraneuronal spread of the morphopathological damage consumes mechanical energy stored by the neurofilaments in the form of a metastable inner structure, and (5) is propagated by a process working, in certain structural and energetical respects, on the domino principle; and (6) the primary neuronal damage caused in the above manner might be secondarily modified in different directions by different postcausation conditions.

Thirty-four rats were killed by transcardial perfusion fixation 1 min after a contusing concussive head injury, and 17 rats 1 day later. From the results obtained with a new silver method demonstrating traumatically damaged neuronal somata, dendrites and axons the following conclusions were drawn: (1) outside the contused territories all features of traumatically induced neuronal argyrophilia are similar to those found in non-contusing concussive head injury, as reported in an accompanying paper; (2) within contused territories the neuronal argyrophilia is abolished by some substance released either from damaged blood vessels or damage parenchymal cells, while the neuronal damage otherwise underlying the induction of argyrophilia is present; (3) different phenotypes of neurons are vulnerable to different values of the parameters of the intracranial pressure wave generated by the trauma; (4) some of the neurons may recover from the traumatically induced argyrophilic damage; (5) traumatically induced inundation of neurons with extracellular tracers, as reported by other authors, and somato-dendritic argyrophilia may be different manifestations of one and the same phenomenon; and (6) diffuse primary traumatic axonal injury in human neuropathology may be closely correlated to axonal argyrophilia.

We have investigated the possibility that, regardless of the involvement of a second messenger system, the ultimate effect of presynaptic, receptor-activated inhibitory modulation is the opening of a K channel. With the consequent hyperpolarization of the terminal, less Ca2+ would enter and this would result in the observed diminished release of a neurotransmitter. This possibility was explored utilizing rat cortical synaptosomes that were prelabeled with either 86Rb or [3H]acetylcholine, depolarizing with either K+ or veratridine, and measuring either efflux of 86Rb or release of [3H]acetylcholine in the presence or absence of inhibitory presynaptic modulators. The modulating agents used were 2-chloroadenosine, carbamylcholine, clonidine, and morphine. In all instances, these agents promoted an increased efflux of 86Rb, indicating hyperpolarization, and decreased release of acetylcholine. These results are compatible with our suggestion that an increase in K conductance may be responsible for presynaptic inhibition of the release of neurotransmitters.

Forty anesthetized rats were cooled below 3 degrees C by 30-min transcardial perfusion of chilled physiological saline before a concussive head injury. The animals were then perfusion-fixed with a buffered formaldehyde-glutaraldehyde solution. Another forty rats were fixed by 30-min transcardial perfusion of the same fixative before a similar concussive head injury. In brain sections of both groups of animals a new silver method stained, in a Golgi-like fashion, a number of neurons and long axonal segments scattered among unstained ones. The similarity between these findings and those obtained following in vivo concussive head injuries described in accompanying papers suggests that the formation of traumatically induced argyrophilic neuronal damage is independent of metabolic processes, i.e., it may be a primary morphopathological process.