Neurofilament Protein Immunoreactivity Research Articles

Patterns of differentiation in central neurocytoma

Central neurocytoma has been characterised by its intraventricular localisation, predominant occurrence in young adults, oligodendroglioma-like histology, benign course and ultrastructural evidence for neuronal differentiation. Eleven intraventricular central neurocytomas were studied histopathologically, employing cell type-specific immunocytochemical markers and electron microscopic analysis. In the past, these lesions have caused diagnostic problems since central neurocytomas share basic histopathological features with other periventricular neoplasms. Accordingly, several tumours of this series had previously been classified as ependymomas of the foramen of Monro or oligodendrogliomas. Although generally regarded as benign lesions, two central neurocytomas of this series showed histopathological evidence of anaplasia, with focal necrosis, mitotic activity and vascular proliferation. All central neurocytomas exhibited immunoreactivity for neuron-specific enolase and synaptophysin, indicating consistent neuronal differentiation. Three tumours were studied by electron microscopy and contained synaptic vesicles, neuritic processes and neurosecretory granules. In addition, one tumour contained ganglioid cells and this was associated with focal immunoreactivity for neurofilament protein, suggesting that some central neurocytomas may, at least focally, continue to differentiate towards the formation of mature neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurofilament protein-triplet immunoreactivity in distinct subpopulations of peptide-containing neurons in the guinea-pig coeliac ganglion

A battery of polyclonal and monoclonal antibodies raised against the triplet of identified neurofilament protein subunits was used to investigate neurofilament protein immunoreactivity in neurons of the guinea-pig coeliac ganglion. Using optimal conditions of fixation and tissue processing for each antibody we found that only 20% of the postganglionic sympathetic neurons in the guinea-pig coeliac ganglion contain neurofilament protein-triplet immunoreactivity. Double labelling with neurofilament protein-triplet antibodies raised in different species demonstrated that all of these antibodies labelled the same population of neurons. Double labelling using mouse monoclonal antibodies against neurofilament proteins in combination with rabbit polyclonals to neuronal markers showed that neurofilament protein-triplet immunoreactivity is restricted to specific chemically coded subpopulations of noradrenergic neurons. Approximately 52% of neurons in the ganglion contain neuropeptide Y and are presumed vasomotor neurons projecting to blood vessels in the submucosa of the small intestine. Virtually none of the neuropeptide Y-containing neurons were labelled with neurofilament protein-triplet antibodies. Neurons that contain somatostatin (21%) project to the submucous ganglia of the small intestine. Approximately two-thirds of neurons containing somatostatin are immunoreactive for the neurofilament protein-triplet. The other postganglionic neurons in the ganglion (27%) project to the myenteric plexus of the small intestine and do not contain either neuropeptide Y or somatostatin. Approximately a quarter of these neurons were labelled with neurofilament protein-triplet antibodies. These results suggest that the neurofilament protein-triplet may not be an instrinsic component of the cytoskeleton of all neurons. Furthermore the idea of a chemical coding of neurons should be extended to cytoskeletal proteins. The finding that these neurofilament proteins are confined to specific neuronal subpopulations has important implications for the search for a role of the neurofilament protein-triplet in neurons, for the interpretation of classical neurohistological silver impregnation techniques which appear to stain only neurofilament protein-triplet-containing neurons, as well as for neuropathological conditions that may involve these proteins in disease processes.

Immunohistochemical Analysis of the Olfactory Mucosa by Use of Antibodies to Brain Proteins and Cytokeratin

The present study deals with the immunohistochemical detection of four brain-derived proteins and cytokeratin in the normal olfactory mucosa of humans and guinea pigs. Neurofilament protein immunoreactivity was found in the olfactory vesicles, dendrites, and perikaryon of receptor cells, and in thick nerve bundles located deep in the lamina propria. The antiserum to neuron-specific enolase (NSE) selectively stained olfactory receptor cells throughout the length of the bundles. The NSE immunoreactivity also was recognized in nerve bundles of various sizes throughout the lamina propria. Glia-specific S-100 protein immunoreactivity was present in Bowman's glands as well as in all nerve bundles in the lamina propria, but not in any cellular elements constituting the olfactory epithelium. Immunoreactivity for spot-35 protein, which was considered to be specific for cerebellar Purkinje cells, was found in flasklike cells (microvillar cells) occurring near the free surface of the epithelium. The basal cells in the olfactory and respiratory epithelium were stained positively with a cytokeratin antiserum.

Olfactory receptor cells: Immunocytochemistry for nervous system-specific proteins and Re-evaluation of their precursor cells.

The immunohistochemical localization of nervous system-specific proteins and cytokeratin in the olfactory mucosa is described mainly in humans and guinea pigs. Immunoreactivity for neuron-specific enolase (NSE) was demonstrated in olfactory receptor cells including their dendrite and axon. Immunoreactivity for neurofilament protein (NFP) was contained in the dendrite and perikaryon of the olfactory cells, but not in the axon. Immunoreactivity for spot-35 protein, a kind of neuron-specific proteins was selectively localized in several flask-shaped cells of the olfactory epithelium only in the guinea pig. These spot-35 protein-positive cells were suspected of being microvillar cells, considered the second type of receptor cells dispersed in the olfactory epithelium. Immunoreactivity for glia-specific S-100 protein was demonstrated in the Bowman's glands as well as Schwann cells associated with the olfactory nerves in the lamina propria. Supporting cells were not immunolabelled with any antisera against nervous system-specific proteins. Cytokeratin, a useful marker for epithelial cells, was expressed exclusively in basal cells. Axotomy of olfactory nerves induced the disappearance of the olfactory receptor cells with NSE immunoreactivity 3 days after the operation. Regenerating cells showing NSE immunoreactivity were first recognized in the lower portion of the epithelium within 7 days. The epithelium was completely repaired 3-4 weeks later and came to contain many NSE-reactive mature receptor cells. Bromodeoxyuridine (BrdU), administered during regeneration of the olfactory epithelium, was immunohistochemically detected in the cells of the layer above the basal cell layer, but not in the latter itself. This result does not support the general contention that the receptor cells originate from the basal cells.

Prenatal low-dose gamma irradiation of the inner ear induces changes in the expression of intermediate filaments.

The expression of intermediate filaments (1F) was analysed in the inner ear in normally developed adult CBA/CBA mice and in mice of the same age which had been gamma irradiated in utero with a low dose 1-2 Gy single exposure. Well characterized monoclonal antibodies (mAbs) against all classes of intermediate filament proteins (cytokeratins-Cks, vimentin, neurofilaments, desmin and glial fibrillar acidic protein) were used. With the exception of neurofilament proteins, the expression of intermediate filament proteins was the same in adult normal and irradiated inner ears, irrespective of gestational age at exposure. A complex Ck pattern occurred in the various cell types comprising the membranous labyrinth. In spite of the differences in cell shape and internal organization of organelles, epithelia actively involved in inner ear fluid homeostasis (stria vascularis, dark cell epithelium, endolymphatic duct and sac) revealed, according to our mAbs, the same expression of Cks, except for the mouse counterpart of human Ck 7, which was found exclusively in the stria vascularis and the endolymphatic duct and sac. The pattern of intermediate filament composition in the labyrinth was the same in the mouse as in man. Irradiation on gestational days 12 or 13 (the otocyst stage)--but not at more advanced embryonic age--induced immunoreactivity for neurofilament proteins in vestibular hair cells (HC) and to a minor extent also in cochlear HC. No such positivity was found in the control material.

Toluene exposure during maturation of intraocular brain tissue transplants: Alterations of host and graft cerebellar Purkinje neuron function and sensitivity to norepinephrine

The effects of chronic toluene exposure on central neurons were examined using syngeneic grafts into the anterior chamber of the eye. Young adult albino rats with intraocular brain transplants inhaled toluene (1000 ppm) for 9 weeks starting at the time of transplantation, or from Week 8 to 17 after the graft was placed in oculo. Control animals were exposed to room air during the same intervals. Toluene treatment during development did not affect general growth or morphology of any of the brain areas examined. The distribution of neurofilament or glial fibrillary acidic protein immunoreactivity was similar in the experimental group and control group as well. Extracellular recordings of cerebellar Purkinje neurons showed a significantly reduced spontaneous firing rate, of 15–25%, both in intraocular transplants and in cerebellum in situ in toluene exposed animals. Postsynaptic sensitivity of intraocular and in situ Purkinje neurons to norepinephrine (NE) was evaluated. Purkinje neurons in transplants exposed to toluene during development were markedly supersensitive to superfused NE as compared to controls, while neither Purkinje neurons in mature cerebellar grafts nor cerebellum in situ showed any effects of the toluene treatment on NE sensitivity. The tissue content of NE in transplants exposed to toluene during maturation, evaluated with high-performance liquid chromatography coupled with electrochemical detection, was greater than that in the control grafts. Moreover, the content of free (3-methoxy-4-hydroxyphenyl) ethylene glycol (MHPG) was increased in both transplant and host cerebellum after toluene exposure. Taken together, these data indicate that toluene exposure during development of cerebellar grafts in oculo causes changes in postsynaptic noradrenergic sensitivity as well as decreased spontaneous activity of Purkinje neurons. Toluene exposure of adult cerebellum in situ or in oculo appears to decrease the Purkinje neuron discharge rate and increase NE turnover, but has no marked effect on postsynaptic NE sensitivity.

Immunocytochemical identification of non-neuronal intermediate filament proteins in the developing Xenopus laevis nervous system

Intermediate filament proteins in the postmetamorphic Xenopus laevis nervous system were identified by their crossreactivities on Western blots with a pan-specific intermediate filament antibody (anti-IFA). These intermediate filament protein bands on Western blots were characterized as 3 cytokeratin-like proteins (49, 55, and 58 kDa), one vimentin-like protein (53 kDa), two distinct glial fibrillary acidic protein (GFAP)-like proteins (60 and 67 kDa), and 3 neurofilament proteins (73, 175, and 200 kDa) by evaluation of their crossreactivities with specific antibodies directed against the mammalian forms of these proteins. This panel of antibodies to mammalian proteins, and two additional antibodies directed against a Xenopus GFAP-like protein and a Xenopus neurofilament (NF-M) protein, were used in immunocytochemical studies to determine the developmental expression of these proteins in the Xenopus nervous system. The first antigen to be detected during development was cytokeratin immunoreactivity, which was located in the inner lining of the embryonic neural tube as early as stage 19, and which in immunocytochemical studies in postmetamorphic frogs was abundant in meninges and processes forming the ventricular lining of the ependymal zone. Vimentin immunoreactivity was found in numerous neuroepithelial cell processes in the rhombencephalon and anterior spinal cord by stage 22, in the prosencephalon by stage 33/34, and in the retina by stage 29/30. In the postmetamorphic frog, vimentin immunoreactivity was found to be abundant in radial processes throughout the brain and spinal cord. NF-M protein immunoreactivity was first detected in neurons in the developing neural tube between stages 22 and 24, in the retina by stages 29/30, and continued to increase throughout development. GFAP-like immunoreactivity was detected very early in radial cells in the neural tube (stage 24), and by stage 42 was found throughout the nervous system. This early appearance of GFAP-like immunoreactivity implies that the onset of glial cell differentiation is a relatively early event in Xenopus.

Differential distribution of vimentin and neurofilament protein immunoreactivity in NB2a/d1 neuroblastoma cells following neurite retraction distinguishes two separate intermediate filament systems

Mouse NB2a/d1 cells assemble all 3 neurofilament protein subunits (NFPs) into the detergent-insoluble cytoskeleton and segregate phosphorylated forms of the 200-kDa subunit (NFP-H) within neurites when differentiation is induced with dibutyryl cyclic AMP (dbcAMP). Before and after differentiation, these cells also incorporate vimentin into both the perikaryal and neuritic cytoskeleton (Shea et al., 1988, Dev. Brain Res., submitted). To determine whether NFPs and vimentin constitute separate intermediate filament systems or exist as heteropolymers, we perturbed cytoskeletal architecture by inducing the retraction of neurites with colchicine. After cells were exposed to colchicine, vimentin immunoreactivity partitioned into perikarya in the form of fibrous whorls that did not cross-react with antisera to NFPs. By contrast, NFP immunoreactivity remained dispersed throughout the cell body following neurite retraction. We interpret these different responses to colchicine to indicate that NFPs and vimentin are assembled into separate intermediate filaments in NB2a/d1 cells.

Trigeminal ganglion cells cocultured with gut express vasoactive intestinal peptide

The plasticity of neural crest cells for the expression of adrenergic and cholinergic transmitter phenotypes has been well studied. The object of this study was to determine if cells of a sensory ganglion are capable of neuropeptide transmitter plasticity. We studied whether cells of the trigeminal ganglion, which do not express the neuropeptide vasoactive intestinal peptide (VIP) in vivo, would express this peptide when grown with a tissue, the gut, that contains large numbers of VIP neurons. Embryonic aneural chick rectum was explanted with the embryonic quail trigeminal ganglion on the chorioallantoic membrane of chick hosts for 7–8 days. The explants were fixed, sectioned, and stained for VIP immunoreactivity (IR), for neurofilament protein immunoreactivity, and for the quail nucleolar marker. In sections of the explants we observed two populations of quail neurons: small (10–13 μm) VIP-IR cells and large (25–32 μm) cells lacking VIP-IR and resembling native trigeminal neurons. Trigeminal ganglia explanted with embryonic heart or trigeminal ganglia explanted alone lacked small VIP-IR cells but contained large VIP-negative neurons. These results show that cells of the trigeminal ganglion grown with the gut can express a neuropeptide they do not express in the absence of the gut or in vivo. Thus the embryonic trigeminal ganglion contains cells that are plastic with respect to neuropeptide expression.

Neuronal properties of clonal hybrid cell lines derived from central cholinergic neurons.

Clonal cell lines derived from specific types of central neurons can be used to identify and characterize properties specific to those neurons. With somatic cell fusion techniques, nine clonal hybrid cell lines have been developed from the septal region of the mouse basal forebrain. Two lines express characteristics typical of cholinergic neurons--choline acetyltransferase activity and immunoreactivity, neurite formation with neurofilament protein immunoreactivity, and aggregation in rotation-mediated cell culture. These cell lines may be useful for studying the trophic interactions that support the development and maintenance of central cholinergic connections.

Developmental change in expression of neurofilament protein-immunoreactivity in the cerebellar Purkinje cells during the postnatal development of rats

Developmental change in expression of neurofilament protein-immunoreactivity in the cerebellar Purkinje cells during the postnatal development of rats

Neurofilament breakdown products in degenerating rat and human peripheral nerves.

The fate of neurofilament (NF) proteins was examined in sections of nerves from rats that survived for 3 to 90 days following nerve transection, using anti-NF monoclonal antibodies and immunohistochemical techniques. The same methods were also applied to twenty-three human nerve biopsy specimens. Granular debris with NF immunoreactivity was observed in the rat nerves for up to 21 days after transection. Similar granular debris with NF protein immunoreactivity was observed in nerve biopsy specimens from patients with axonal degeneration. This change was most evident in specimens that demonstrated abundant ovoids in teased fiber preparations and in a patient with a recent traumatic nerve transection. Similar granular debris was not observed in normal rat or human nerves. We conclude that degenerating NF proteins have a distinctive granular appearance and that NF immunoreactivity can be demonstrated in NF breakdown products with immunohistochemical techniques for prolonged periods of time in rat and human nerves undergoing axonal degeneration. Immunohistochemical studies with anti-NF monoclonal antibodies offer a novel approach to the evaluation of human nerve biopsy specimens.