Changes in the structure of neuromuscular junctions caused by variations in osmotic pressure.

Neuromuscular junctions of the frog, Rana pipiens, were examined for structural modifications produced by exposure to increased and reduced osmotic pressure (pi). Preparations exposed to increased pi for varying lengths of time were fixed with either OSO4-Veronal with and without calcium, glutaraldehyde-phosphate, or glutaraldehyde-formaldehyde-phosphate as primary fixatives. The greatest difference between the fixatives was seen in preparations exposed to increased pi for 5 min, corresponding to the time when miniature endplate potential frequency is highest. The 5-min OSO4 calcium-free preparations appeared comparatively normal, while those fixed with OSO4 and 2 mM CaCl2 or aldehyde-phosphate had wide infoldings of the presynaptic membrane and a reduced number of synaptic vesicles. Aldehyde-phosphate had the same effect on mouse diaphragm. Another series of frog preparations were conditioned to elevated pi and then returned to normal Ringer's for varying times before fixation in OSO4-phosphate. Preparations fixed 2 min after their return to normal Ringer's showed marked disruption of the presynaptic membrane as well as apparently rupturing vesicles. If fixed after 10 min, terminals were depleted of vesicles although the presynaptic membrane had returned to its normal position and appearance.

Changes in the fine structure of the neuromuscular junction of the frog caused by black widow spider venom.

Application of black widow spider venom to the neuromuscular junction of the frog causes an increase in the frequency of miniature end-plate potentials (min.e.p.p.) and a reduction in the number of synaptic vesicles in the nerve terminal. Shortly after the increase in min.e.p.p. frequency, the presynaptic membrane of the nerve terminal has either infolded or "lifted." Examination of these infoldings or lifts reveals synaptic vesicles in various stages of fusion with the presynaptic membrane. After the supply of synaptic vesicles has been exhausted, the presynaptic membrane returns to its original position directly opposite the end-plate membrane. The terminal contains all of its usual components with the exception of the synaptic vesicles. The only other alteration of the structures making up the neuromuscular junction occurs in the axon leading to the terminal. Instead of completely filling out its Schwann sheath, the axon has pulled away and its axoplasm appears to be denser than the control. The relation of these events to the vesicle hypothesis is discussed.

Slow axonal transport of neurofilament proteins: impairment of beta,beta'-iminodipropionitrile administration.

beta,beta'-Iminodipropionitrile (IDPN) administration prevented normal slow axonal transport of [35S]methionine- or [3H]leucine-labeled proteins in rat sciatic motor axons. Ultrastructural and electrophoretic studies showed that the neurofilament triplet proteins in particular were retained within the initial 5 millimeters of the axons, resulting in neurofilament-filled axonal swellings. Fast anterograde and retrograde axonal transport were not affected. The IDPN thus selectively impaired slow axonal transport. The neurofibrillary pathology in this model is the result of the defective slow transport of neurofilaments.

Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain.

Recent evidence indicates that the nucleus basalis of Meynert, a distinct population of basal forebrain neurons, is a major source of cholinergic innervation of the cerebral cortex. Postmortem studies have previously demonstrated profound reduction in the presynaptic markers for cholinergic neurons in the cortex of patients with Alzheimer's disease and senile dementia of the Alzheimer's type. The results of this study show that neurons of the nucleus basalis of Meynert undergo a profound (greater than 75 percent) and selective degeneration in these patients and provide a pathological substrate of the cholinergic deficiency in their brains. Demonstration of selective degeneration of such neurons represents the first documentation of a loss of a transmitter-specific neuronal population in a major disorder of higher cortical function and, as such, points to a critical subcortical lesion in Alzheimer's patients.

Structural changes in murine cancer associated with hyperthermia and lidocaine.

Hyperthermia alone and hyperthermia with lidocaine cause changes in the fine structure of the CA755 tumor cell as well as the breakdown of the tumor vasculature. The first structural change, observed immediately after termination of hyperthermia of 43.5 degrees for 1 hr, is the vesiculation of the Golgi apparatus. Other structural changes occur later but with variable times of onset. The changes appear to be unrelated to the presence of lidocaine. Vascular breakdown results in hemorrhaging within the tumor, and its onset and intensity appear to vary directly with the size of the tumor. Breakdown of the tumor cell plasmalemma and degenerative changes of the cytoplasm and nucleoplasm are seen more frequently in large tumors and in the interior of small tumors at any given time after the end of hyperthermia. The vesiculation of the Golgi persists in treated cells for as long as 30 hr. This modification may represent an intensification in the function of the Golgi apparatus; however, it closely corresponds to that found in a variety of other cells treated with a class of compounds, including lidocaine, that specifically inhibits the function of the Golgi apparatus. The effect of these compounds is rapidly reversible, unlike hyperthermia. Since the Golgi apparatus probably is crucial in repairing any deleterious effects of hyperthermia, any impairment of its normal function would place most treated tumor cells in a difficult position. The rate of tumor destruction may ultimately depend on the breakdown of the tumor vasculature following hyperthermia and lidocaine.

Differential sensitivity of AKR murine leukemia and normal bone marrow cells to hyperthermia.

To determine if there is a differential effect of hyperthermia on AKR murine leukemia and AKR normal bone marrow cells incubated in vitro, the fractional survival of leukemic and of normal cells with proliferative potential as a function of heating exposure was estimated by evaluating spleen colony formation. Normal bone marrow colony-forming units were assayed in lethally irradiated (750 centigrays) mice; leukemic colony-forming units were assayed in nonirradiated mice. Electron micrographic studies of leukemic cells treated with 41.8 degrees hyperthermia found that structural damage to the cell, i.e., changes in the Golgi apparatus, was associated with the lack of ability to form colonies. AKR leukemia cells were more sensitive than normal cells to hyperthermic killing at 41.8 degrees and at 42.5 degrees. This differential was found whether cells of each type were heated separately or when mixed together. This model system demonstrates an inherently greater sensitivity of neoplastic cells, as compared to normal syngeneic stem cells, to thermal killing. This finding may have relevance to autologous bone marrow transplantation in humans.

Regulation of purine metabolism. Adenylosuccinate synthetase from Novikoff ascites tumor cells.

Adenylosuccinate synthetase has been partially purified from Novikoff ascites tumor cells. The properties of the protein are quite different from the enzyme from rat liver in that the Km for asparate is higher and the K1 for the feedback inhibitor AMP is also higher. The antibiotic hadacidin has a preferential inhibitory effect on the tumor enzyme. These results suggest that the Novikoff ascites tumor enzyme is less sensitive to normal feedback controls but may be more sensitive to specific antitumor drugs.

Reconstitution of (Na+ + K+)-ATPase proteoliposomes having the same turnover rate as the membranous enzyme.

Membranous (Na+ + K+)-ATPase from the electric eel was solubilized with 3-[3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate (Chaps). 50 to 70% of the solubilized enzyme was reconstituted in egg phospholipid liposomes containing cholesterol by using Chaps. The obtained proteoliposomes consisted of large vesicles with a diameter of 134 +/- 24 nm as the major component, and their protein/lipid ratio was 1.25 +/- 0.07 g protein/mol phospholipid. The intravesicular volume of these proteoliposomes is too small to consistently sustain the intravesicular concentrations of ligands, especially K+, during the assay. The decrease in K+ concentration was cancelled by the addition of 20 microM valinomycin in the assay medium. The low value of the protein/lipid ratio suggests that these proteoliposomes contain one Na+/K+-pump particle with a molecular mass of 280 kDa per one vesicle as the major component. In these proteoliposomes, the specific activity of the (Na+ + K+)-ATPase reaction was 10 mumol Pi/mg protein per min, and the turnover rate of the ATP-hydrolysis was 3500 min-1, the same as the original enzyme under the same assay condition. The ratio of transported Na+ to hydrolyzed ATP was 3, the same as that in the red cell. The proteoliposomes could be disintegrated by 40-50 mM Chaps without any significant inactivation. This disintegration of proteoliposomes nearly tripled the ATPase activity compared to the original ones and doubled the specific ATPase activity compared to the membranous enzyme, but the turnover rate was the same as the original proteoliposomes and the membranous enzyme. This disintegration of proteoliposomes by Chaps suggests the selective incorporation of the (Na+ + K+)-ATPase particle into the liposomes and the asymmetric orientation of the (Na+ + K+)-ATPase particle in the vesicle.

The ventral photoreceptor cells of Limulus. I. The microanatomy.

The ventral photoreceptor cells of Limulus polyphemus resemble the retinular cells of the lateral eyes both in electrical behavior and in morphology. Because of the great size of the ventral photoreceptor cells they are easy to impale with glass capillary micropipettes. Their location along the length of the ventral eye nerve makes them easy to dissect out and fix for electron microscopy. Each cell has a large, ellipsoidal soma that tapers into an axon whose length depends upon the distance of the cell from the brain. The cell body contains a rich variety of cytoplasmic organelles with an especially abundant endoplasmic reticulum. The most prominent structural feature is the microvillous rhabdomere, a highly modified infolding of the plasmalemma. The microvilli are tightly packed together within the rhabdomere, and quintuple-layered junctions are encountered wherever microvillar membranes touch each other. Glial cells cover the surface of the photoreceptor cell and send long, sheet-like projections of their cytoplasm into the cell body of the photoreceptor cell. Some of these projections penetrate the rhabdomere deep within the cell and form quintuple-layered junctions with the microvilli. Junctions between glial cells and the photoreceptor cell and between adjacent glial cells are rarely encountered elsewhere, indicating that there is an open pathway between the intermicrovillous space and the extracellular medium. The axon has a normal morphology but it is electrically inexcitable.

The axonal pathology in chronic IDPN intoxication.

Neurofilamentous axonal swellings occur in a number of degenerative and toxic disorders of the nervous system. In one of these, experimental intoxication with beta, beta'-iminodiproprionitrile (IDPN), accumulation of neurofilaments has been shown to result from a defect in slow axonal transport. The consequence of this functional abnormality is a series of changes in axonal morphology: Neurofilaments accumulate in the proximal axon; the proximal axon becomes swollen; the distal axon loses volume (axonal atrophy). These studies indicate that axonal atrophy occurs secondary to an impairment of slow axonal transport and suggest that a similar abnormality may underlie the pathological changes in certain other degenerative and toxic diseases of the nervous system.

Neuronal and glial gene expression in neocortex of Down's syndrome and Alzheimer's disease.

The association cortex of Down's syndrome (DS) predictably and prematurely undergoes neurofibrillary degeneration of Alzheimer type. Hence studies of DS are potentially useful in defining the earliest pathogenetic events in Alzheimer's disease (AD). Previous reports have described altered expression of several mRNAs in AD cortex; but the pathogenetic stage at which expression of these mRNAs begins to deviate from the norm has not been defined. We have examined this issue in neocortex of DS. Expression of mRNAs, known to be altered in AD cortex, was studied by Northern analysis, comparing frontal cortex of DS (15-45 years) with age-matched controls and with AD. Chromosome 21- and non-21-encoded mRNAs were studied, including transcripts expressed preferentially in neurons (neurofilament light subunit and amyloid precursor transcripts) and in glia (glial fibrillary acidic protein [GFAP] and S100 beta). Chromosome 21-encoded mRNAs were increased in DS cortex as expected. Except in the DS case with extensive neurofibrillary degeneration, GFAP was expressed at levels significantly below the control, suggesting that trisomy 21 exerts a suppressive effect on GFAP gene expression. We found no instance in which AD-type changes of transcript expression preceded the appearance of neurofibrillary degeneration. The findings indicate that in trisomy 21, certain changes of mRNA prevalence previously described for AD neocortex are not a necessary antecedent to neurofibrillary degeneration.

Neurofilamentous axonal swellings as a normal finding in the spinal anterior horn of man and other primates.

To define the nature and extent of axonal swellings in the normal spinal anterior horn, we studied the spinal cords of patients five days to 83 years of age from a general autopsy population. Axonal swellings were routinely found in the anterior horn of the cervical and lumbosacral spinal cord. The swellings measure 5-50 micron in diameter and are most numerous at the anterior edge of the anterior horn. They first appear about five months of age and appear to increase in number until about 20 years of age, with no increment thereafter. Ultrastructurally, they are filled with neurofilaments and surrounded by a thin myelin sheath. Most are probably aberrant components of motor axons. Identical axonal swellings, in the same anatomical site, were found in the spinal cords of cynomolgus and rhesus monkeys. On the basis of their natural history and morphologic features, they should be distinguished from the neuroaxonal dystrophy of aging. The largest of them resemble the neurofilamentous axonal swellings of early onset motor neuron disease but occur in much smaller numbers. Moreover, location on the proximal axon could not be demonstrated for any of these swellings. An awareness of this normal phenonemon is essential for the interpretation of axonal swellings in the spinal cord.

Doxorubicin intoxication: neurofilamentous axonal changes with subacute neuronal death.

Neurofilamentous axonal swellings occur in various chronic neuronal degenerations in man and animals. The pathogenesis of these swellings in the setting of neuronal degeneration remains unclear. A toxic model of neuronal degeneration can be produced by doxorubicin. This agent impairs DNA-dependent RNA synthesis and produces subacute neuronal death. The aim of the present study was to investigate whether neurofilamentous axonal swellings occur in association with neuronal nuclear derangement and subacute neuronal death produced by doxorubicin. We investigated the evolution of changes seen in retinal ganglion cells and their axons after an intravitreous injection of doxorubicin in rats. The earliest changes were in the nuclei of the retinal ganglion cells. Later, transient axonal swellings filled with neurofilaments were prominent. These neurofilamentous swellings preceded the subacute neuronal cell death. To determine whether these changes represent a direct effect of the agent on the axon, we injected doxorubicin directly into the sciatic nerve. This local injection did not produce similar changes in the axons. These results suggest that in this model neurofilamentous axonal swellings precede neuronal cell death and may be due to a primary insult to the nerve cell body.

Cortical degeneration with swollen chromatolytic neurons: its relationship to Pick's disease.

We report two cases of dementia in which cortical degeneration with widespread swollen chromatolytic neurons (SCN) was the dominant pathologic feature. Each patient had received the diagnosis of Alzheimer's disease on the basis of clinical findings. There was no deficit of cortical choline acetyltransferase activity, assayed in one case, or lesions of the nucleus basalis of Meynert. The brains had moderate to marked frontal atrophy. Comparison of SCN with several other cerebral degenerative disorders indicates a similarity with certain features of the transmissible spongiform encephalopathies and with corticodentatonigral degeneration. The pathologic features of our cases are those of a number of other cases reported as "Pick's disease," and may represent an earlier stage in the pathogenetic process than the severe, sharply circumscribed atrophy with "nonspecific" cell loss and gliosis as the only microscopic residuals. Our findings re-emphasize the need to search for pathogenetically distinct subgroups which have been wholly or partially subsumed into the concept of Pick's disease.

The neuropathology of CAG repeat diseases: review and update of genetic and molecular features.

Classification of inherited neurodegenerative diseases is increasingly based on their genetic features, which supplement, clarify, and sometimes replace the older clinical and pathologic schemata. This change has been particularly rapid and impressive for the CAG repeat disorders. In Huntington's disease, X-linked spinobulbar muscular atrophy, dentatorubropallidoluysian atrophy, and a series of autosomal dominant cerebellar atrophies, genetic advances have resolved many nosologic issues, and opened new avenues for exploration of pathogenesis. In this review, we summarize classic and current concepts in neuropathology of these CAG repeat diseases.

Aging is associated with divergent effects on Nf-L and GFAP transcription in rat brain.

We studied the effects of advancing age on the expression of several proteins important in the structure and function of the nervous system. Brains of young (3 month), middle-aged (13 month), and old (29 month) male Fischer 344 rats were examined. Run-on transcription and Northern blot hybridizations were used to determine gene-specific transcription rates and mRNA levels, respectively. With advancing age, there was a decrement in the transcription rate and mRNA levels for neurofilament-light subunit (Nf-L), but an increment in the transcription rate and mRNA levels for glial fibrillary acidic protein (GFAP). Proteolipid protein (PLP) mRNA levels were attenuated between 3 and 13 months of age, whereas amyloid precursor protein (APP) mRNA levels were attenuated in the middle-aged but not the old animals. Transcription rates for alpha-actin and fos, and mRNA levels for alpha-actin, were unaffected. These observations indicate divergent transcriptional regulation of several genes, notably Nf-L and GFAP, in the aging mammalian forebrain.

Gene expression in Alzheimer neocortex as a function of age and pathologic severity.

Previous studies have shown a marked decline in neuronal and an increase in glial gene expression in Alzheimer's disease (AD) neocortex. Severity of pathologic changes may be greater in presenile AD (PAD) than in senile AD (SAD). We evaluated whether changes in transcript expression were altered as a function of age or pathologic severity. Northern analysis revealed a marked (> 50%) decline in expression of transcripts for the neurofilament light subunit and the major amyloid precursor protein (APP) isoforms in both PAD and SAD. Expression of these neuronal transcripts declined as a function of age in AD and control cases. Expression of the glial fibrillary acidic protein (GFAP) transcript was increased in AD, particularly in the presenile group. AD cases with larger numbers of neurofibrillary tangles had higher levels of GFAP transcript; AD cases with larger numbers of senile plaques had higher levels of APP695 transcript. We conclude that the neuronal mRNA decrements of AD are superimposed on an age-related decline. Age-related shift in expression of certain genes may account for the differences in pathologic severity of PAD and SAD.

Neurotoxic effects of cisplatin therapy.

Swelling of the optic disc and loss of tendon reflexes developed in a 3 1/2-year-old girl after 11 high-dose courses of cisplatin (cis-dichlorodiammine platinum [II]) therapy for recurrent sacrococcygeal teratoma. Postmortem examination demonstrated optic disc swelling; the spinal cord showed loss of myelinated fibers and gliosis of the dorsal columns. To our knowledge this case is the first in which the neuropathologic changes associated with cisplatin therapy have been demonstrated by postmortem examination.

Postinfectious leukoencephalitis complicating Mycoplasma pneumoniae infection.

Neuropathological findings in a patient with fatal neurological complications due to infection with Mycoplasma pneumoniae were similar to those seen in postinfectious encephalitis and acute hemorrhagic leukoencephalitis. This case supports the hypothesis that immune mechanisms play a role in the pathogenesis of neurological symptoms during mycoplasmal infections.

Neurologic complications of bone marrow transplantation.

Among 78 patients who died after bone marrow transplantation, neurologic complications were present in 55 (70%) and were the cause of death in 5 (6%). Metabolic encephalopathy occurred in 29 patients (37%). CNS infections included aspergillosis (3), herpes simplex encephalitis (2), and Listeria monocytogenes meningitis (1). Six additional patients had neuropathologic changes possibly due to cytomegalovirus infection. Cerebrovascular complications occurred in five patients (two hemorrhages and three infarcts). All infarcts were associated with endocarditis. The rate of nonbacterial thrombotic endocarditis was significantly higher (p less than 0.001) than in the general autopsy population. CNS leukemia and therapy-induced injury were rare. There was no evidence of graft-versus-host disease involving the CNS.