Stereospecific acute neuronotoxicity of 'uncommon' plant amino acids linked to human motor-system diseases.

The L-isomer of beta-N-methylamino-L-alanine (BMAA), present in free form in seed of Cycas circinalis, elicits in spinal cord cultures a pattern of acute postsynaptic neuronal vacuolation comparable to that induced by beta-N-oxalylamino-L-alanine (BOAA), an excitotoxic amino acid of greater potency isolated from seed of Lathyrus sativus. The neuronotoxic properties of these compounds may be linked to the etiology of motor-system degenerative disorders (amyotrophic lateral sclerosis and lathyrism, respectively) found in human groups that have used these plant seeds for food.

The evolution of cellular degeneration in dorsal root ganglia exposed to doxorubicin in tissue culture.

Doxorubicin (Adriamycin) is a drug having both antibiotic and antimitotic properties. It intercalates between base pairs of DNA causing breaks in the helical strands. It is widely used as a cancer chemotherapeutic agent but is limited in use by a dose-related cardiomyopathy and necrosis of peripheral ganglia. This study of cultured mouse spinal cord and dorsal root ganglia examined the sequential pathological changes of living tissue by light and fluorescence microscopy and of fixed tissue by electron microscopy. Fluorescence microscopy showed that doxorubicin has rapid access to ganglia cells and preferentially binds to the nucleus and nucleolus. Pathological changes of the peripheral neurons include clumping of nuclear chromatin, relocation of the indented nucleus to an eccentric position, occasional loss of the nuclear envelope, dissociation of the nucleolus into the pars granulosa and the pars fibrosa, accumulation of inclusion bodies in the cytoplasm, swollen cisternae and a loss of Nissl substance. Axons display changes in localization of organelles prior to undergoing proximal Wallerian-like degeneration. Schwann cells occasionally display clumped nuclear chromatin. Some satellite cells undergo necrosis. These findings are dose- and time-related and essentially duplicate those previously reported in animal studies. Thus, the in vitro model may provide a useful tool for ascertaining the sequence of events occurring in sensory neuronopathy.

Rapid reorganization of the axonal cytoskeleton induced by a gamma diketone.

Microtubules and neurofilaments are the major components of nerve fiber axoplasm and are evenly distributed longitudinally. Rearrangement of this distribution can be induced by a number of chemicals, including 2,5-hexanedione (2,5-HD). Utilizing 2,5-HD, this study explores the earliest time points of observable rearrangement of the cytoskeletal elements. Intrafascicular injections into the sciatic nerve shows central accumulation of microtubules and peripheral relocation of neurofilaments within 1 min of injection, suggesting a direct effect of the toxin on these components.

Cation binding at the node of Ranvier: I. Localization of binding sites during development.

Cations are known to bind to the node of Ranvier and the paranodal regions of myelinated fibers. The integrity of these specialized structures is essential for normal conduction. Sites of cation binding can be microscopically identified by the electrondense histochemical reaction product formed by the precipitate of copper sulfate/potassium ferrocyanide. This technique was used to study the distribution of cation binding during normal development of myelinating fibers. Sciatic nerves of C57B1 mice, at 1, 3, 5, 6, 7, 8, 9, 13, 16, 18, 24 and 30 days of age, were prepared for electron microscopy following fixation in phosphate-buffered 2.5% glutaraldehyde and 1% osmic acid, microdissection and incubation in phosphate-buffered 0.1 M cupric sulfate followed by 0.1 M potassium ferrocyanide. Localization of reaction product was studied by light and electron microscopy. By light microscopy, no reaction product was observed prior to 9 days of age. At 13 days, a few nodes and paranodes exhibited reaction product. This increased in frequency and intensity up to 30 days when almost all nodes or paranodes exhibited reaction product. Ultrastructurally, diffuse reaction product was first observed at 3 days of age in the axoplasm of the node, in the paranodal extracellular space of the terminal loops, in the Schwann cell proper and in the terminal loops of Schwann cell cytoplasm. When myelinated axons fulfilled the criteria for mature nodes, reaction product was no longer observed in the Schwann cell cytoplasm, while the intensity of reaction product in the nodal axoplasm and paranodal extracellular space of the terminal loops increased. Reaction product in the latter site appeared to be interrupted by the transverse bands. These results suggest that cation binding accompanies nodal maturity and that the Schwann cell may play a role in production or storage of the cation binding substance during myelinogenesis and development.

Cation binding at the node of Ranvier: II. Redistribution of binding sites during electrical stimulation.

The nodal and paranodal areas of mature myelinated axons are known to bind cations. To examine whether the cation binding substance may play a role in saltatory conduction, a combined electrophysiological and histochemical study was undertaken. The sciatic nerve of anesthetized or unanesthetized adult C57B1 mice was exposed and not stimulated (control) or stimulated with constant square-wave pulses at one of the following rates: 10/sec, 30/sec, 100/sec or 500/sec. Phosphate-buffered 2.5% glutaraldehyde was either dropped onto the nerve during stimulation until cessation of the compound action potential or the nerve was fixed after discontinuing stimulation. The nerve was excised and processed for the histochemical reaction of copper sulfate/potassium ferrocyanide (which forms an electron dense precipitate at areas of cation binding), dehydrated and infiltrated with SpurrR epoxy resin. Individual nerve fibers were microdissected and counts made of the numbers of paranodal and nodal areas exhibiting the reaction product. The percentage of nodes stained, with respect to the total numbers of nodes and paranodes stained, was calculated. There was no significant difference in percent of nodes stained between the simultaneously fixed, non-stimulated, anesthetized (43.1%), the non-stimulated unanesthetized (45.3%), the animals stimulated at 10/sec (45.9%) and the animals stimulated at 30/sec (50.2%) and 100/sec(46.0%), and fixed post-stimulation. However, all values at the higher frequencies and fixed during stimulation were significantly different both from the control and from each other (30/sec-59.3%; 100/sec-70.5%; and 500/sec-76.4%). The location of cation binding appears to change in response to electrical stimulation and correlates with the increased frequency of the inward movement of sodium ions.

Quaking mouse: an ultrastructural study of the peripheral nerves.

Ultrastructural study of the peripheral nervous system of quaking mice has revealed several unusual features in the pattern of myelination in addition to hypomyelination. These are 1) the presence of 'atypical' Schmidt-Lanterman incisures, 2) irregularities of the nodal and internodal termination of Schwann cell cytoplasm and 3) the presence of non-myelinated segments without associated Schwann cell processes, but covered by basal lamina. In view of the observation of similar features during the development of the P.N.S. and also during P.N.S. remyelination, we suggest that these features are the results of modified myelination due to a defect in the control mechanisms necessary for normal myelination.

Effect of hypocholesterolemic drug AY9944 on cultured nervous tissue: morphologic and biochemical studies.

The effects of different concentrations of the hypocholesterolemic drug AY9944, an inhibitor of delta7-reductase, on organotypic cultures of fetal mouse spinal cord, were studied by light and electron microscopy. Exposure to 10(-6)M produced no observable changes. After 6 hours exposure to 10(-4)M, dense membrane-bound inclusions were occasionally observed in neurons. After 24 hours exposure to 10(-4)M, numerous cytoplasmic inclusions occurred in neurons, glia and macrophages. The form of these inclusions varied but were predominantly of two types; concentric, loosely-packed lamellae resembling membranous cytoplasmic bodies (MCB) of Tay-Sachs disease and irregular dense bodies. They were identical to those observed in our previous in vivo study. Prolonged exposure to the drug at 10(-4)M caused an increased number of inclusions in all cell types. Eventually the cultures degenerated. The number of inclusions increased for at least 38 days following a 2-5 day exposure to AY9944 at 10(-4)M. However, by 70 days, although inclusions persisted, the cultures were mostly astrocytic. In long-term cultures, in addition to these inclusions, curved or straight electron-dense paired profiles were seen in some cells, presumably macrophages. Biochemical analysis of cultures exposed to 10(-4)M revealed the continuous presence of delta7, 24-cholesta-diene-3beta-ol and 7-dehydrocholesterol even after the drug wa removed from the cultures. In our previous animal experiments, intracytoplasmic inclusions and abnormal sterols with a double bond at the 7 position disappeared quickly after discontinuation of the drug. Therefore, the results obtained in our present in vitro experiments are different in this regard from the in vivo studies of AY9944.