Cerebrospinal fluid from amyotrophic lateral sclerosis patients causes fragmentation of the Golgi apparatus in the neonatal rat spinal cord.

We have previously shown in our laboratory that cerebrospinal fluid from ALS patients (ALS-CSF) contains putative toxic factor(s). In the present study we determined the effect of ALS-CSF on the integrity of the Golgi apparatus of spinal motor neurons in the neonatal rats. CSF was injected intrathecally into three-day-old rat pups and subsequently the ultrastructural changes in the motor neurons were studied after 48 h, 1, 2 and 3 weeks. We observed that ALS-CSF causes fragmentation of the Golgi apparatus in a considerable number of motor neurons in the spinal cord. This was further confirmed when motor neurons were stained with an antibody against a medial Golgi protein (MG160). Thus, we suggest that the putative toxin(s) present in ALS-CSF may cause impairment in the protein processing leading to motor neuron death.

Nuclear localization of Munc18-1 (p67) in the adult rat brain and PC12 cells.

Munc18-1, also referred to as p67, co-purifies with Cdk5 and has an important role in neurotransmitter release. The role of Munc18-1 for functional connectivity of the nervous system was demonstrated by gene knockout experiments in mice, wherein accumulation of neurotransmitter and silencing of synaptic activity was observed. Our earlier studies have shown that both Munc18-1 and Cdk5 co-purify and co-localize with cytoskeletal components, implying that apart from having a regulatory role in vesicle docking and fusion, Munc18-1 could also affect the dynamics of neuronal cytoskeleton. In the present study we have shown the presence of Munc18-1 in nuclear rich fraction from rat brain and confirmed the nuclear localization of this protein in PC12 cells and adult rat brain neurons by immunofluorescence and immunoelectron microscopy. We also demonstrate the binding of Munc18-1 to double stranded (ds) DNA. The ability of Munc18-1 to bind dsDNA, albeit the lack of DNA binding domains, suggests that the binding may be mediated through protein-protein interaction through some other DNA-binding proteins. The presence of both nuclear import and export signals in Munc18-1 primary structure corroborates its nuclear localization and makes it a putative shuttle protein between nuclear and cytoplasmic compartments, the precise physiological relevance of which needs to be elucidated.

Co-purification and localization of Munc18-1 (p67) and Cdk5 with neuronal cytoskeletal proteins.

Munc18-1 (p67, nSec1, rbSec1), a neuron-specific 67kDa protein was independently identified as a syntaxin-binding protein, and as a component that co-purifies with, and regulates the kinase activity of cyclin dependent kinase (Cdk5). Gene knockout studies have demonstrated a role for Munc18-1 in synaptic vesicle docking and neurotransmitter release. Mice lacking Munc18-1 gene were synaptically silent, but the gene deletion did not prevent normal brain assembly, including the formation of layered structures, fiber pathways and morphologically defined synapses. Previous study has shown that Munc18-1 facilitates Cdk5 mediated phosphorylation of KSPXK domains of the neuronal cytoskeletal elements, suggesting that Munc18-1 may function in the regulation of cytoskeletal dynamics. Present study demonstrates the co-purification and co-localization of Munc18 with cytoskeletal elements and forms first step towards understanding the role for Munc18-1 in cytoskeletal dynamics. Conversely, the cytoskeletal proteins and Cdk5 co-purifies with Munc18-1 in a Munc18-1 immuno-affinity chromatography, suggesting a strong protein-protein interaction. Findings from immunofluorescence studies in PC12 cells have shown co-localization of Munc18-1 and Cdk5 with neurofilaments and microtubules. Further, immunohistochemical and immuno-electron microscopic studies of rat olfactory bulb also demonstrated co-localization of Munc18-1 and Cdk5 with cytoskeletal elements. Thus, the biochemical evidence of strong interaction between Munc18-1 with cytoskeletal proteins and morphological evidence of their (Munc18 and cytoskeletal elements) identical sub-cellular localization is suggestive of the possible role for Munc18-1 in cytoskeletal dynamics.

Dinitrophenol derivatization of proteolytic products and its application in the assay of protease(s) activity.

A spectrophotometric method based on dinitrophenol (DNP) derivatization of proteolytic products was developed for monitoring the increase in NH(2)-groups as a function of protease activity. DNP derivatization of amino acids and proteolytic products was carried out at an alkaline pH of 8.8, in presence of 2,4-dinitrofluorobenzene (DNFB), followed by the stabilization of products by adjusting the pH to approximately 2.5. Using casein as substrate, under the defined assay conditions for proteases, trichloroacetic acid soluble proteolytic products were derivatized with DNFB reagent. Though alkaline pH favored the DNP derivatization of primary amino compounds, the products formed were found to be unstable. However, upon adjusting the pH to 2.5+/-0.1, DNP derivatives of amino acids and proteolytic products were found to be stable with identical lambda(max) of 395 nm. The utility of the method was evaluated by assaying the proteolytic activities of trypsin and calcium activated neutral protease (CANP). Proteolytic activity was quantified by employing the molar extinction coefficient of DNP derivatives of an equimolar concentration of glutamate and glycine. By employing this method, CANP activity in different regions of rat brain was determined. The proposed method to monitor the increase in NH(2)-end groups as a function of proteolytic activity could be employed to assay the activity of proteases.

Synaptic development in semi-dissociated cultures of rat retina.

Cultured neurons provide a simpler and more accessible environment to study the synaptic physiology. However, it is not clear if development of synapses in culture is similar to that in the in vivo condition. We studied the developmental sequence and morphological differentiation of chemical synapses in semi-dissociated rat retinal cultures that consisted of dissociated neurons as well as undissociated retinal aggregates. Synapses were quantified by synaptophysin immunoreactive puncta. During second week of in vitro development the average number of chemical synapses on the cell body decreased while that on the neurites increased significantly. Conventional synapses appeared both in aggregate and in dissociated neurons, with the developmental profile similar to that reported for in vivo retina. In contrast, the development of ribbon synapses was adversely affected by the in vitro microenvironment as suggested by following observations. The ribbon synapses were more frequently found in aggregate than in dissociated neurons, and were not associated with dyadic or triadic synaptic arrangement. The photoreceptor ribbons did not contact a postsynaptic process while bipolar ribbons made single (monadic) synapses. Further, photoreceptor ribbons in dissociated neurons were late to form and took more time to mature as compared to those in the aggregate cultures. Most of the rod bipolar cells, identified by their immunoreactivity to protein kinase C (PKC), had three or more neurites. Unlike in the in vivo retina, the dissociated rod bipolar cells did not show any PKC immunoreactive varicosities, suggesting that they failed to develop a well-differentiated synaptic terminal. Interestingly, we did not find any parvalbumin positive AII amacrine cells that are normally postsynaptic to rod bipolar cells. These results show that the conventional synapses of retina, which are similar to chemical synapses in other parts of the brain, develop normally both in aggregate and dissociated neurons. However, the highly specialized ribbon synapses have more stringent developmental requirements, and their normal development may require the presence of postsynaptic neurons in their close vicinity.

Cytochrome oxidase activity in rat retinal ganglion cells during postnatal development.

In this study, the metabolic activity of rat retinal ganglion cells during postnatal development has been examined in vivo using cytochrome oxidase histochemistry. The intensity of staining was measured by optical densitometry. The activity of cytochrome oxidase in retinal ganglion cells progressively increased from postnatal day 0 (P0) and reached a peak during the second week of postnatal development (P10-P14) and declined thereafter. Our data show that the increased levels of cytochrome oxidase seen in developing retinal ganglion cells occur at the same time, when neuronal maturity and synaptogenesis reach their peaks.

Inclusion body myositis (IBM).

Clinical, histological, immunohistochemical and ultrastructural features of 5 cases of inclusion body myositis -4 sporadic (s-IBM) and one hereditary (h-IBM) form are described. These patients (3 men, 2 women) had chronic progressive weakness of varying severity in all 4 extremities with sparing of cranial muscles. Elevation of CPK was noted in 2 patients. Electromyography revealed features of myopathy in 4 and additional neurogenic changes in 2 subjects. Clinical diagnosis was often other than inclusion body myositis. Presence of characteristic eosinophilic inclusions within the vacuoles established the diagnosis. The inclusions were congophilic and showed positivity to ubiquitin, beta-amyloid and SMI-31 in the sporadic cases while congophila was absent in the hereditary form. Immunostaining to hyperphosphorylated-tau was negative in both s-IBM and h-IBM. Membraneous whorls were observed at ultrastructural level. None of the patients improved with steroids and trial with other immunosuppressants was unsuccessful.

Centronuclear myopathy--morphological relation to developing human skeletal muscle: a clinicopathological evaluation.

Centronuclear myopathy (CNM), an uncommon condition, is one of the congenital myopathies. It is believed to arise as a result of maturational arrest, with persistence of myotubes postnatally. However, denervation being the basic disease process and its possible influence on central nervous system causing defect in nuclear migration has also been postulated. Keeping in view these existing controversies, we have studied 17 cases of CNM (neonatal - 1, childhood - 13, adulthood - 3) during the last twelve and a half years. Diagnosis was based on histological and enzyme histochemical findings of muscle biopsy along with clinical data. Ultrastructural characterstics of muscle have been studied in 10 cases. The affected muscle fibres showed a central nucleus (40-99%) with perinuclear halo. Type I fibre predominance with hypoplasia was consistently seen. Fibre type disproportion was noticed in 7 cases. The neonatal form revealed dense oxidative enzyme reaction product in the centre. The morphological features of CNM were compared with foetal skeletal muscles obtained at gestational ages ranging from 9 weeks - 36 weeks (n = 18). In the severe neonatal form th myofibres resembled the foetal myotubes. In the less severe childhood and adult form of CNM, aberrant organization of cytoskeletal network might have played a pathogenetic role in causing the disease.

Is infantile spinal muscular atrophy a disease of maturation arrest or a dynamic neurogenic atrophy of the skeletal muscle?

Spinal muscular atrophy constitutes one of the major disease entities amongst infantile neuro-muscular disorders. On the basis of morphological evidence, it had been suggested that the infantile spinal muscular atrophy (ISMA) is due to maturation arrest of the myofibres at 20 weeks gestation. Therefore, in the present study morphological features of skeletal muscles from patients with ISMA was compared with foetal muscle obtained at different gestational ages (9-36 weeks, n=18). Of the 35 cases ofISMA, 22 were diagnosed as having SMA-I and 13 cases an SMA-II, characterised histologically by fascicles composed of groups of small, normal, intermediate sized and hypertrophic fibres. The former ones belonged to both histochemical fibre types, while the hypertrophic fibres in 21/35 cases were type I in nature. Redundant basal lamina was a predominant finding at ultrastructural level. Mature myotubes, a feature seen during foetal muscle development was not noticed in any of the cases of ISMA. Our observations suggest denervation atrophy to be the basic pathogenic mechanism rather than arrest in maturation. This was further supported by the changes seen in the spinal cord specimen of a 20 day old infant from a case of SMA I which revealed marked fallout of motor neurons in the anterior horn, chromatolysis and gliosis. Thus ISMA is a dynamic and progressive neurogenic atrophy secondary to degeneration and loss of spinal motor neurons possibly resulting in lack of trophic factors.

Developmental expression of synaptophysin, synapsin I and syntaxin in the rat retina.

Expression of synaptophysin, synapsin I and syntaxin was studied immunocytochemically in the developing rat retina using indirect immunoperoxidase technique. In the inner plexiform layer (IPL), syntaxin immunoreactivity appeared at postnatal day 1 (P1) whereas synaptophysin and synapsin I staining were first observed at P2. In the outer plexiform layer (OPL), synaptophysin appeared at P4, while synapsin I and syntaxin appeared at P8. In the case of synaptophysin, a punctate pattern of staining was observed from the time of its appearance (P4) in the OPL and from P12 onwards in the IPL. Synapsin I and syntaxin immunoreactivity in the OPL were of a low intensity throughout the development and in the adult stage. These findings are discussed in relation to synaptogenesis in the rat retina.

Variable histomorphology of muscle in congenital muscular dystrophy.

Congenital muscular dystrophy (CMD) is a relatively uncommon disease with a controversial nosological status. That collagen synthesis could be the primary abnormality has been suggested earlier (Fidzianska et al., 1982). Amongst eighteen cases of CMD diagnosed during the past twelve years, muscle biopsy in three cases revealed prominence of myofibre necrosis and phagocytosis, and serum CPK was markedly elevated suggesting a rapidly progressive form. In twelve cases, marked increase in endomysial collagen, pronounced fallout of myofibres and significant fibre diameter variation was seen. This was associated with myonecrosis and regenerative activity of mild degree resembling the classical form of CMD. In the remaining three cases, polyfocal, polyphasic necrosis was noticed. Fibre splitting was more frequently observed, better delineated in the enzyme histochemical preparations, affecting both fibre types, while endomysial fibrofatty tissue was only moderately increased. The histomorphology in the latter group resembled that of limb girdle dystrophy. Ultrastructural findings in all the eighteen cases correlated well with light microscopic observations. lmmunohistochemical studies done on three of the eighteen cases showed normal localization of dystrophin protein. Such variable histomorphology, revealing a spectrum of myopathic features, suggests that the primary change in CMD is likely to be in the myofibre rather than in collagen synthesis.

Levels of dopamine and noradrenaline in the developing of retina--effect of light deprivation.

The effect of light deprivation on the levels of dopamine and noradrenaline was studied in the developing rat retina. These transmitters were estimated in three groups of rats: (i) cycling light reared; (ii) dark reared since birth; and (iii) dark reared since birth, but exposed to cycling light for 1 day prior to the estimation of catecholamines. Our results show that (1) there is a progressive decrease in the levels of dopamine and noradrenaline in the cycling light and dark reared rats during postnatal development; (2) dark rearing further reduces the content of dopamine and noradrenaline; and (3) restoration of physiological (light) stimulus in the dark-reared rats during the early postnatal period results in the recovery of noradrenaline to a greater extent than that of dopamine. This study demonstrates a progressive decrease in the plasticity of dopaminergic system during retinal development, while such a decrease is not apparent in the noradrenergic system.

Chediak-Higashi syndrome--a case report with ultrastructural and cytogenetic study.

We report the ultrastructural abnormalities of the leukocyte granules and the cytogenetic findings in a patient of Chediak-Higashi syndrome (CHS), who presented with cutaneous melanosis as the only clinical feature. The diagnosis of CHS was established by peripheral smear and bone marrow examination. Chediak-Higashi syndrome, a rare autosomal recessive disorder is characterized by enlarged abnormal organelles in leukocytes and other cells. An interesting aspect of our patient was the absence of recurrent infections or any other clinical stigmata. Ultrastructurally, the leukocytes and their precursors in the bone marrow showed characteristic homogenous and heterogenous giant inclusions of variable sizes and shapes. These represent the primary granules which enlarge to attain the giant abnormal size by fusion with other primary or secondary granules. Cytogenic study of the bone marrow cells showed monosomy of chromosomes 8 and 17 in 20 percent of the metaphases. Neither the gene nor the chromosomal abnormalities specific for CHS have been identified as yet and thus the significance of our cytogenetic finding is presently not clear.

Intraventricular neurocytoma : a clinical and electro-microscopic study.

Intraventricular neurocytomas are rare or have been earlier misdiagnosed as Oligodendroglioma, a tumour which they closely resemble at light microscopy. Three such intraventricular tumours are described, all of which were diagnosed as Oligodendroglioma on CT scan and at routine light microscopy. The judicious use of EM for a definite diagnosis of these tumours is emphasised. Cells with multiple processes, abundant RER and dense core vesicles and profiles of axon terminals with groups of synaptic vesicles, clinches the diagnosis. Even though the prognosis of neurocytomas is generally believed to be good, it may depend on whether they have only neuronal or associated Glial differentiation.

A modified technique to process peripheral nerves for light microscopy.

A new technique is described to fix, paraffin embed and cut routine peripheral nerve biopsies for light microscopy. Buffered glutaraldehyde pH 7.4 is used as a common fixative for both light and electron microscopy. The nerve bits for LM are embedded in paraffin using the 'reverse embedding' method and blocks are trimmed to cut sections on the ultra microtome using glass knives. The uniformly thin sections can be utilised for any special stain and the good resolution of these thin sections facilitates better observation and documentation.

Ultrastructural variations in frog muscles subjected to sciatectomy.

Fine structural variations in two different types of muscles of frog (Rana cyanophlictis) subjected to sciatectomy were studied electronmicroscopically. Gastrocnemius muscle showed marked myofibrillar disarray and degeneration due to sciatectomy, while sartorius muscle was relatively less affected. The extent of sciatectomy induced fine structural variation was in proportion to the degree of denervation atrophy (as reflected by loss of wet muscle weight) in these muscles. Differences in the degree of degenerative changes in atrophying muscles may be attributed to variations in fiber type composition and stretch effects imposed during swimming movements.