Neurofilament Immunoreactivity Research Articles

EXPERIMETAL MICRONEUROSURGERY OF THE CENTRAL AND PERIPHERAL NERVOUS SYSTEM IN THE STUDY OF THE NEURONAL AND GLIAL TROPHISM AND PLASTICITY

Trauma and neurodegenerative diseases commit the nervous system. After an axotomy or nerve injury in the peripheral nervous system the regeneration of the nerve fibers and reinervation of the target are seen. In central nervous system these events are restrictive, however their occurrence are related to the state of glial reaction and the synthesis of neurotrophic factors. Basic fibroblast growth factor (bFGF) has been considered an important trophic factor for neurons and astrocytes of many central nervous system regions. In this study rats were submitted to one of following neurosurgery procedures: callosotomy, pyramidectomy or complete transection of hypoglossal nerve (XII). Sham operations were made in control animals. Seven days later animals were sacrificed and their brains processed for immunohistochemistry. Coronal sections were taken from the central nervous system and incubated with antisera against the glial fibrillary acidic protein (GFAP) or neurofilament (NF), markers for astrocyte and neuronal cell body and fibers, respectively, as well as with the antiserum against the bFGF. The degree of the labelling was quantified with computer assisted stereological methods. The analysis of the NF immunoreactivity revealed a disappearance of fibers in the white matter distal to the pyramidectomy and callosotomy, however no disappearance of NF immunoreactive neurons was found in the XII nucleus following axotomy. These changes was accompanied by a massive astrocytic reaction. The reactive astrocytes synthesized increased amounts of bFGF. These findings suggest that glial reaction synthesizing neurotrophic factors may influence the wound and repair after mechanical lesions of central nervous and subsequent neuronal trophism and plasticity which may be relevant to the regenerative process of the nervous tissue

Evidence of abnormal differentiation in giant cells of tuberous sclerosis

To characterize the giant cells in tuberous sclerosis, we examined immunoreactivity for nestin, vimentin, microtubule-associated protein 111 (MAP1B), MAP2, neurorilament, and glial ribrillary acidic protein (GFAP) in cortical tubers detected in brain specimens from 6 patients with tuberous sclerosis who had undergone surgical resection for treatment of intractable epilepsy. Giant cells with a neuronal appearance, “neuron-like giant cells,” had a round centrally-placed nucleus with a single, prominent nucleolus, and Nissl substance was commonly present in cortex. These neuron-like giant cells demonstrated consistently strong immunoreactivity for neurofilament and MAP1B and occasional immunopositivity for nestin and vimentin and were rarely positive for GFAP. “Indeterminate giant cells,” characterized by abundant cytoplasm, an absence of Nissl substance, and one or more eccentric nuclei, demonstrated consistent immunoreactivity for nestin, vimentin, and MAPIB and were rarely positive for neurofilament, but more than half displayed immunoreactivity for GFAP. These observations suggest that the indeterminate giant cells exhibit limited neuronal and inconsistent astroglial characteristics, implying aberrant cellular differentiation in tuberous sclerosis.

Histological markers of neuronal, axonal and astrocytic changes after lateral rigid impact traumatic brain injury

The model of lateral, rigid impact traumatic brain injury is widely used but remains relatively poorly characterized by comparison with fluid percussion injury models. Thus, whilst the gross morphological changes that occur over the short- and long-term post-injury have been described, more subtle measures of neuronal injury and activation, and markers of axonal and glial reactions have not been investigated, complicating interpretation of data from this model. To address this issue, a variety of neurohistological markers were examined in adult male rats which had been subjected to open brain, lateral rigid impact injury. A piston device was unilaterally driven 3.0 mm into the somatosensory cortex at a speed of 3.2 m/s. Neuronal activation evidenced by Fos-like immunoreactivity showed a complex pattern at 3 h after injury which appeared to be related both to proximity to the impact site and cortical efferent connectivity. At 24 h after injury, acid fuchsin staining demonstrated dying neurons in the margin of the injury and in ipsilateral hippocampus and dorsal thalamus. Injured cells identified by heat-shock protein immunoreactivity showed a similar distribution. Axonal injury demonstrated with 68 kDa neurofilament immunoreactivity was more widely distributed. Less axonal damage was found with increasing distance from the injury site. At 7 days post-injury, glial fibrillary acidic protein immunoreactive astrocytes were prolific in the ipsilateral thalamus, hippocampus and striatum and throughout the injured cortex. In general, controlled, lateral rigid impact injury provides a more focused injury than is seen with lateral fluid percussion which may have implications for the behavioral deficits seen in this injury model.

Age-dependent changes in the immunoreactivity for neurofilaments in rabbit hippocampus

The distribution of the three subunits of neurofilaments was examined in the hippocampus of young adult rabbits (three months of age), employing a panel of six monoclonal antibodies. Thereafter, age-dependent and subunit-selective changes in neurofilament immunoreactivity in the ageing rabbit hippocampus were studied, using animals of one, three, six, 12, 24, 30, 36, 48, and 60 months. Principal cells, interneurons, axons, and various fibre systems were immunoreactive for all three subunits, although the localization and staining intensity of neurofilament immunoreactivity depended on the antibody used. Small cells immunopositive for the low subunit of neurofilament (presumably glial cells) were found abundantly in the hippocampal formation at one month, and (occasionally) at 30–36 months. Young rabbits (one to three months of age) had high numbers of interneurons stained for the high subunit of neurofilament in the stratum oriens/pyramidale. The number declined and plateaued to approximately 78% at six to 30 months, and further declined and plateaued to approximately 56% at 36–60 months. The first decline may reflect a process of maturation, while the latter decline most likely relates to ageing. Ageing pyramidal cells in 48–60 months animals revealed a slight increase for the low subunit of neurofilament, but no changes for the other subunits. Transient changes in neurofilament immunoreactivity were a striking observation in dentate gyrus granule cells during ageing. The staining intensity for the low subunit of neurofilament decreased gradually from one to 24–30 months until it was no longer detectable in these cells. The immunoreactivity then reappeared, most notably in granule cells lining the hilus, at the age of 36–48 months. By 60 months all granule cells were nearly immunonegative for this subunit. Axonal aberrations, immunoreactive for all three subunits, were found throughout the hippocampal formation. These aberrations first appeared in 24-month-old animals and increased in number and maximal size in older rabbits. The alterations in neurofilament immunoreactivity in the ageing hippocampus correlated with age-associated learning disabilities in the acquisition of a hippocampally-dependent learning task. The potential relevance of changes in the cytoskeletal profile of hippocampal neurons to age-associated learning and memory disabilities is discussed.

Pretreatment with NMDA receptor antagonist MK801 improves neurophysiological outcome after an acute spinal cord injury.

The post-traumatic release of excitatory amino acids (EAA) and their actions on N-methyl-D-aspartate (NMDA) receptors plays a major role in the spinal cord secondary injury process. The neuronal damage caused by the release of EAA may be reduced by NMDA-receptor channel blockers. To investigate the involvement of NMDA receptors in spinal cord injury (SCI), we pretreated animals with the noncompetitive NMDA antagonist MK801 (1.0 mg kg-1) before a compressive acute SCI. Pretreated animals with MK801 significantly (p = 0.038) improved the recovery of function as measured by evoked potential activities. Morphologically, specimens from rats treated with MK801 were characterized by milder and more localized hemorrhage in the gray matter. Immunohistochemical staining for glial fibrillary acidic protein (GFAP) and neurofilament (NF) histochemistry showed leakage of these antigens in traumatized cord while characteristic staining of astrocytes and neurons and their processes was observed in morphologically preserved tissue. The loss of NF immunoreactivity was reduced by MK801 treatment.

Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.

Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in the postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in developing brain. Cdk5 purified from nervous tissue phosphorylates neuronal cytoskeletal proteins including neurofilament proteins and microtubule-associated protein tau in vitro. These findings indicate that Cdk5 may have unique functions in neuronal cells, especially in the regulation of phosphorylation of cytoskeletal molecules. We report here generation of Cdk5(-/-) mice through gene targeting and their phenotypic analysis. Cdk5(-/-) mice exhibit unique lesions in the central nervous system associated with perinatal mortality. The brains of Cdk5(-/-) mice lack cortical laminar structure and cerebellar foliation. In addition, the large neurons in the brain stem and in the spinal cord show chromatolytic changes with accumulation of neurofilament immunoreactivity. These findings indicate that Cdk5 is an important molecule for brain development and neuronal differentiation and also suggest that Cdk5 may play critical roles in neuronal cytoskeleton structure and organization.

Plasticity of granule cell-mossy fiber system following kainic acid induced seizures: an immunocytochemical study on neurofilament proteins

Abnormal reestablishment of mossy fibers with the CA3 pyramidal cells and granule cells is an important aspect of postlesional plasticity in epilepsy. However, basis for the structural reorganisation and functional consequences of the event remain uncertain. Therefore we have investigated alterations of neurofilaments, major cytoskeletal components of neurons, in the rat hippocampus after the kainic acid (KA) administration, an experimental model for the temporal lobe epilepsy. The immunoreactivity for phosphorylated heavy weight neurofilament (pNFH) and non-phosphorylated heavy weight neurofilament (npNFH), in particular the pNFH, decreased in the CA1 field and inner molecular layer of the dentate gyrus during 3 and 10 days after the KA administration. After 10 days, npNFH immunoreactivity appeared in the mossy fibers, in which it is normally absent, meanwhile the pNFH staining in the mossy fibers did not decrease. From day 21, the immunoreactivity of pNFH and npNFH was normal or above normal in the CA1 stratum lacunosum-moleculare, mossy fibers, hilus and inner molecular layer of the dentate gyrus. These alterations in the later phase remained at least to day 90. The reappearance and increase of the neurofilament immunoreactivity in the inner molecular layer of the dentate gyrus probably reflects a collateral extension of the granule cell axons known as mossy fiber sprouting. The results suggest that neurofilament changes in the granule cell-mossy fiber system may be a morphological basis for the structural reconstruction of granule cell axons, and neurofilaments are involved in the plasticity after the KA induced seizures.

Immunomicroscopy of neurofilaments in chromaffin cells of the adult bovine adrenal gland.

Neurofilaments (NFs) represent a class of intermediate filaments which are highly specific for neurons. The most abundant of the native NFs is the 68 kD subunit (NF-L). Chromaffin cells of the adrenal medulla express NF subunits under culture conditions. However, NF expression in situ is questionable. It has been reported that as chromaffin cell precursors mature and begin to express catecholamine-synthesizing enzymes, their neuronal traits are extinguished and they become endocrine-like cells. This study reports that while NF-L may be lacking in the adrenal medulla of some species, NF-L immunoreactivity is clearly present in the adult bovine adrenal medulla. Immunofluorescence microscopy of bovine chromaffin cells in culture demonstrated NF immunoreactivity localized to both thin, highly ramified filaments present throughout the cell and frequently to an intensely immunofluorescent spot located near the nucleus. Double-label immunofluorescence microscopy and immunoblot analysis also demonstrated NF-L immunoreactivity in mature chromaffin cells of the bovine adrenal gland. In vitro and in situ immunofluorescence results were confirmed by immunogold and immunoperoxidase labelling, respectively. In both cases, NF-L immunoreactivity was associated with filaments in close proximity to the nucleus. Additionally, a spheroidal aggregate of immunogold-labelled NFs was seen adjacent to the nucleus in cultured cells. In conclusion, NF-L in bovine chromaffin cells demonstrates that at least one neuronal trait persists in these catecholamine-producing cells of the mature adrenal gland. In addition, this study emphasizes the fact that interspecies comparisons must be interpreted with caution, especially when attempting to formulate a generalized hypothesis.

Treatment of epilepsy with multiple subpial transections: an acute histologic analysis in human subjects.

Multiple subpial transection (MST) is a new surgical technique for treating seizures that arise from functionally critical cortical areas. It has a reported efficacy comparable to that of standard temporal lobe resections. Although the mechanism through which MST works is unknown, the carefully controlled lesions, placed 5 mm apart at the midlevel of the cortical gyri, could produce fiber damage that would prevent horizontal synchronization and spread of epileptic discharges while allowing normal cortical functions such as those related to movement or speech to be preserved. We studied the acute neuropathological features associated with MST in 8 patients with intractable temporal lobe epilepsy. Transections were made along major temporal gyri just before standard lobectomy was performed. After resection, tissue was processed by conventional histological and immunocytochemical techniques. Macroscopically, subpial transections (STs) were perpendicular to the main gyral axis and had an appropriate spacing. Microscopically, most of the lesions were perpendicular and at midlevel. However, many transections involved the lateral aspects of the small gyri, resulting in oblique or deep STs, some of which reached the gray-white matter junction due to the complex microscopic neocortical architecture, in which small gyri are superimposed on major lobar gyri, and to the variable cortical thickness. Extensive acute pyknosis and tissue edema were also evident adjacent to the transections. These changes were variable and extenDed 1-3 mm laterally as irregular columnar blocks. In the deep lesions, myelin pallor and decreased neurofilament immunoreactivity were observed in the white matter. Based on the distribution of STs and their adjacent parenchymal injury, we conclude that this technique produces block-type lesions that probably disrupt propagation of epileptogenic activity. In most instances, midlevel horizontal fibers are damaged; one third of the cases showed additional deep injury that would sever afferent and efferent axons. Therefore, in addition to horizontal desynchronization, a deafferentation mechanism involving different fiber systems may contribute to the anti-seizure effects of MST. We hypothesize that preservation of cortical function is mediated by cortex remaining in the sulcus and gyral crown and possibly by reorganization of tissue adjacent to transections.

Neurodegenerative changes including altered tau phosphorylation and neurofilament immunoreactivity in mice transgenic for the serine/threonine kinase mos

Transgenic mice expressing the oncogenic protein-Serine/threonine kinase Mos at high levels in the brain display progressive neuronal degeneration and gliosis. Gliosis developed in parallel with the onset of postnatal transgene expression and led to a dramatic increase in the number of astrocytes positive for GFAP, vimentin, and possibly tau. Interestingly, vimentin is normally expressed only in immature or neoplastic astrocytes, but appears to be induced to high levels in Mos-transgenic, mature astrocytes. Mos can activate mitogen activated protein kinase (MAPK) and MAPK has been implicated in Alzheimer-type tau phosphorylation. In the Mos-transgenic brain we found increased levels of phosphorylation at one epitope on tau containing serines 199 and 202 (numbering according to human tau), a pattern similar but not identical to that found in Alzheimer's disease. In addition, Mos-transgenic mice express a novel neurofilament-related protein that might be a proteolytic neurofilament heavy chain degradation product. These results suggest that activation of protein phosphorylation in neurons can result in changes in cytoskeletal proteins that might contribute to neuronal degeneration.

Glial cell line-derived neurotrophic factor enhances survival and growth of prenatal and postnatal spinal cord transplants.

Glial cell line-derived neurotrophic factor was first described as a trophic factor for developing dopamine neurons. However, it has been shown that glial cell line-derived neurotrophic factor messenger RNA is also expressed in several areas of the developing brain and spinal cord, suggesting that it may have additional roles in the nervous system. Intraocular transplantation of neural tissue provides a unique method to examine in vivo effects of trophic factors. We have therefore studied the effects of glial cell line-derived neurotrophic factor on spinal cord survival and development following grafting to the anterior chamber of the eye of adult rats. We used spinal cord tissue from fetal stages (embryonic days 14 and 18) and postnatal days 1 and 14 as donors. The spinal cord tissue was allotransplanted to the anterior eye chamber of Sprague-Dawley host rats after incubation in buffered saline containing 100 micrograms glial cell line-derived neurotrophic factor/ml or 100 micrograms cytochrome C/ml. One group of postnatal day 1 spinal cord grafts was also treated with concentrations of 20 and 10 micrograms glial cell line-derived neurotrophic factor/ml. In all cases, 5 microliters of the same solution was injected into the anterior eye chamber on postgrafting days 5, 10, 15 and 20 (total amounts 0.5, 0.1 and 0.05 microgram/eye/injection, respectively). We found that all glial cell line-derived neurotrophic factor-treated spinal cord grafts grew more than controls. The effect of glial cell line-derived neurotrophic factor was most prominent in grafts from newborn rats. In these grafts we found a dose-dependent effect of glial cell line-derived neurotrophic factor on growth. Moreover, grafts treated with the highest dose (0.5 microgram) grew to sizes exceeding the initial size at transplantation. In these transplants we also found greater numbers of large neurons compared to controls. Glial fibrillary acidic protein immunoreactivity, in contrast, showed increased gliosis in controls. Similar results were found with syngeneic spinal cord postnatal day 1 grafts in Fisher hosts. Spinal cord tissue grafts from two-week-old rats treated with the highest glial cell line-derived neurotrophic factor dose every fifth day, through day 35 postgrafting, responded with increased growth and less necrotic tissue compared with controls; however, we could not detect neurofilament immunoreactivity in these transplants. Taken together, these results suggest that glial cell line-derived neurotrophic factor may be a potent trophic factor for neurons in the spinal cord and in spinal cord transplants. Of particular importance is that glial cell line-derived neurotrophic factor treatment can be used to obtain survival of postnatal spinal cord tissue, that would otherwise show minimal or no survival. Thus, glial cell line-derived neurotrophic factor allows successful transplantation of more mature spinal cord tissue, which may have important implications for both basic and clinical neuroscience.

Effects of Axonal Injury on Astrocyte Proliferation and Morphologyin Vitro:Implications for Astrogliosis

Mechanisms inducing gliosis following injury in the central nervous system are poorly understood. We evaluated the effect of axonal injury on astrocyte and Schwann cell proliferation and morphologyin vitro.Purified rat dorsal root ganglion neurons grown on monolayers of rat neonatal cortical astrocytes (N-ASneonatalcultures) or sciatic nerve-derived Schwann cells (N-SC cultures) were mechanically injured. Noninjured cultures served as controls. Cell proliferation near lesions was monitored by autoradiography 1, 2, 4, and 8 days postinjury. Axonal injury caused a significant transient increase in astrocyte proliferation immediately proximal and distal to the lesion. The lesion did not induce marked changes in the intensity of glial fibrillary acidic protein (GFAP) immunoreactivity. However, processes from GFAP-positive cells usually arranged in random fashion in noninjured cultures were aligned perpendicularly to the cut distal to lesions. Ultrastructural analysis in lesioned N-ASneonatalcultures indicated that proximal to the lesion filament-filled astrocytes were intermingled with axons. Distal to the lesion astrocyte processes formed layers, between which an increased amount of collagen-like material appeared with time postlesion. Axons distal to the lesion degenerated by 2 days, coinciding with the early disappearance of neurofilament immunoreactivity. In noninjured and proximally in injured N-SC cultures, Schwann cells extended processes, engulfing some axons. Distal to the lesion, Schwann cells appeared more rounded and neurites remained until 4 days postinjury. Media conditioned by injured or noninjured N-ASneonatalcultures did not affect neuron-induced Schwann cell proliferation. These findings demonstrate that axonal injury and degeneration cause a transient increase in astrocyte proliferation and induce morphological changes in astrocytes consistent with the onset of gliosis.

In utero exposure to low-doses of ionizing radiation decelerates neuronal migration in the developing rat brain.

In order to elucidate the possible mechanisms for the effects of low doses of ionizing radiation on the developing rat cerebral cortex we studied how neuronal migration can be affected by prenatal in utero irradiation. We have demonstrated an effect of ionizing radiation on neuronal migration at doses as low as 15 cGy together with a changing pattern of expression of the neural cell adhesion molecule N-CAM. After a dose of 15 cGy or more there was a reduction in N-CAM immunoreactivity in the matrix cell zone which became apparent about 24 h after exposure and continued until 48 h after exposure. Normal reactivity occurred 3 days after exposure to radiation. However, there was no obvious change in the immunoreactivity for L1, MAP2, tau protein or neurofilament. Our findings suggest the possible role of N-CAM in neuronal migration and also suggest the presence of a threshold in terms of the effects of small radiation doses on the developing cerebral cortex.

Neurochemical markers in human cutaneous Merkel cells

Merkel cells (MCs) are specialized sensory cells widely distributed in the epithelia of vertebrates. A variable immunohistochemical pattern of neuronal and neurotransmitter markers has been demonstrated in MCs of several species including man. In the present study, we investigated the expression of neurochemical markers in a selected population of human cutaneous MCs by immunofluorescence. The structural neural proteins protein gene product 9.5 and neuron-specific enolase were found to be the most reliable markers for MC identification. Moreover, neurofilament immunoreactivity was shown in a small subset of epidermal MCs. Among the neurotransmitter markers, evidence for expression of calcitonin gene-related peptide, vasoactive intestinal polypeptide, peptide histidine isoleucine amide, neuropeptide Y, neurokinin A, galanin, substance P, somatostatin and phenylethanolamine N-methyltransferase was found. These immunoreactivities were highly variable as far as number of positive cells and staining intensity were concerned. The results indicate that a complex and heterogeneous immunophenotype can be expressed even within a homogeneous population of human MCs.

Neuronal and non-neuronal catechol-O-methyltransferase in primary cultures of rat brain cells

Previous biochemical and histochemical studies have suggested that catechol-O-methyltransferase (COMT) is a predominantly glial enzyme in the brain. The aim of this work was to study its localization and molecular forms in primary cultures, where cell types can be easily distinguished with specific markers. COMT immunoreactivity was studied in primary astrocytic cultures from newborn rat cerebral cortex, and in neuronal cultures from rat brain from 18-day-old rat embryos using antisera against rat recombinant COMT made in guinea pig. Double-staining studies with specific cell markers to distinguish astrocytes, neurons and oligodendrocytes were performed. COMT immunoreactivity colocalized with a specific oligodendrocyte marker galactocerebroside in cells displaying oligodendrocyte morphology, flat cells displaying type-1 astrocyte morphology and glial fibrillary acidic protein, in branched cells displaying type-2 astrocyte morphology and in cell bodies of neurons, the processes of which displayed neurofilament immunoreactivity. Western blots detected both soluble 24 kDa and membrane-bound 28-kDa COMT proteins in neuronal and astrocyte cultures. The results suggest that COMT is synthesized by cultured astrocytes, oligodendrocytes and neurons.

Integrin alpha 2 beta 1 mediates interactions between developing embryonic retinal cells and collagen.

In the developing nervous system, the extracellular matrix provides a source of extrinsic cues to guide determination of cell fate, neuroblast migration, axon outgrowth and synapse formation. In the neural retina, undifferentiated neuroepithelial precursor cells contact extracellular matrix that contains multiple collagen types. Collagens have been shown to support retinal cell adhesion and neurite outgrowth, but the integrin receptors mediating neuronal responses are not understood. Here we provide evidence that integrin alpha 2 beta 1 acts as a collagen receptor in the developing avian retina and examine its expression pattern. Using a recently described monoclonal antibody, MEP-17, alpha 2 protein was detected in the developing retina by immunofluorescence in tissue sections and dissociated cells, and by immunoprecipitation. At embryonic day 4 (E4), when the majority of retinal cells are undifferentiated neuroepithelial cells, alpha 2 immunoreactivity in sections was widespread and about half of cells dissociated in culture were alpha 2 positive. At E6, after the retinal ganglion cell layer had differentiated, immunoreactivity in sections decreased in the central, more developed portion of the retina and 25% of dissociated cells were alpha 2 positive. E6 retinal ganglion cells, identified by neurofilament immunoreactivity, did not express detectable alpha 2 immunoreactivity. Immunoprecipitation experiments using E6 extracts demonstrated that the alpha 2 subunit was paired with the beta 1 integrin subunit. By E12, alpha 2 immunoreactivity in sections was confined to the extreme peripheral retina, although the antigen may be masked since expression levels comparable to or slightly higher than E6 could be detected in dissociated cells and extracts. By employing function blocking antibodies, it was shown that alpha 2 beta 1 integrin is necessary for cell adhesion and process outgrowth by embryonic retinal cells on collagens I and IV. Although alpha 2 expression continued through E12, alpha 2 activity was down regulated with increasing embryonic age, since alpha 2-dependent adhesion and outgrowth declined. These data suggest a role for alpha 2 beta 1 in neuroepithelial cell interactions with collagen rather than for axon extension by retinal ganglion cells.

Brief potassium depolarization decreases levels of neurofilament proteins in CNS culture

Little is known about the effects of brief potassium depolarization that occurs concurrently with transient ischemia, epilepsy and head trauma. To investigate the effect of short-term depolarization on light (NF-L), middle (NF-M), and heavy (NF-H) neurofilament proteins and determine the role played by calcium in that effect, mixed septo-hippocampal cultures were exposed to 60 mM K + for 6 min, in the presence of 0 to 11.8 mM Ca 2+. Twenty-four hours later, neurofilament immunoreactivity in Western blots of depolarized cultures was decreased to 60% or less of control levels. Decreases were Ca 2+-dependent, not due to cell loss, and affected both phosphorylated and nonphosphorylated proteins. The phosphorylation state of NF-M and NF-H influenced the degree of loss observed. Changes in the pattern of immunolabelling of neuritic processes were also associated with depolarization. Thus, brief potassium depolarization may contribute to cytoskeletal disruption following brain injury.

Maturation of cerebellar granule cells is delayed in cultures derived from ethanol-treated chick embryos: Survival and proliferation studies

Previous studies from this laboratory have shown that ethanol administration to chick embryos during embryonic days 1–3, a critical period of neuroembryogenesis, differentially affects primordial CNS structures. In this study, chick embryos were treated in ovo with ethanol (10 mg/50 μl/day) at E1 to E3. At 14 days of embryonic age cerebellar (E14CE) granule cell cultures were prepared from both control and ethanol-treated embryos. Growth patterns were evaluated morphologically and the neuronal nature of these cultures was evaluated immunocytochemically. E14CE granule cell cultures exhibited neurofilament immunoreactivity demonstrating the neuronal-nature of these cultures. In addition E14CE granule cultures contained numerous glutamateric neurons as assessed by positive glutamate immunoreactivity and also some GABAergic neurons as assessed by positive GABA immunoreactivity. Cultures derived from both control and ethanol-treated embryos were labeled with 3H-thymidine and assessed for effects on survival and proliferation in culture. Cultures derived from ethanol-treated embryos showed a higher rate of proliferation and survival during the first 3 days in culture as compared to those derived from controls. However, after 3 days in culture, survival was lower in the cultures from ethanol-treated embryos as compared to those derived from controls. We interpret these findings to mean that (a) ethanol arrested cerebellar granule cell development at an immature state; (b) immature neurons have a higher survival capacity than differentiated neurons; and (c) ethanol accelerates normal neuronal cell death as previously reported.

Phosphorylated and non-phosphorylated neurofilament proteins: distribution in the rat hippocampus and early changes after kainic acid induced seizures

The regional distribution of neurofilament proteins in the rat hippocampus and their early changes after kainic acid induced seizures were investigated immunocytochemically with antibodies against light weight neurofilament, phosphorylated and non-phosphorylated heavy weight neurofilament. The light weight and non-phosphorylated heavy weight neurofilaments were distributed more unevenly than the phosphorylated neurofilament. The perikarya and processes of pyramidal cells in the CA3 field contained the highest light weight and non-phosphorylated heavy weight neurofilaments, while the perikarya of granule cells contained only few light weight neurofilament and the perikarya of CAI pyramidal cells were even devoid of immunoreactivity of both light and heavy weight neurofilaments. The fiber staining of the light weight and non-phosphorylated heavy weight neurofilaments, especially the former, was less in the CAI field and molecular layer of dentate gyrus. The phosphorylated neurofilament immunoreactivity was identified only in axons. Mossy fibers, the axons of granule cells, contained the light weight and phosphorylated heavy weight neurofilaments, but not the non-phosphorylated neurofilament. Seven days after the kainic acid induced seizures, the phosphorylated neurofilament staining was greatly reduced in the CAI and inner molecular layer of the dentate gyrus, probably resulting from the axonal degeneration of the Schaffer collaterals and the commissural/associational fibers. Furthermore, the non-phosphorylated neurofilament appeared in the mossy fibers of the CA3 stratum lucidum, which normally do not express such immunoreactivity. The results indicate that the neurofilaments are altered following the neuronal degeneration and post lesional plasticity caused by the kainic acid administration. Therefore, the examination of various phosphorylated neurofilaments may offer a comprehensive understanding of major hippocampul pathways, axonal plasticity and the possible roles of neurofilaments in the hippocampus following excitotoxic insults.

Choline acetyltransferase-like immunoreactivity in small diameter neurones of the rat dorsal root ganglion

In the rat choline acetyltransferase (ChAT)-like immunoreactivity (ChAT-LI) was demonstrated in the dorsal root ganglion (DRG), in the superficial spinal cord and in the subepithelial layer of the ureter using immunohistochemical techniques. In the L1 DRG, 66% of the neurones were ChAT-LI. They did not express neurofilament immunoreactivity (RT97 negative) but could also contain calcitonin gene-related peptide-like immunoreactivity (CGRP-LI). In the superficial spinal cord and in the subepithelial plexus of the ureter - both areas where high numbers of fine afferent fibres have been demonstrated - CGRP-LI and ChAT-LI were co-distributed, indicating that ChAT can be found in the peripheral and central endings of small afferents. The data provide morphological evidence that a substantial proportion of afferent fibres are cholinergic.