Axonal maturation in development--II. Immunofluorescence study of rat spinal cord and cerebellum with axon-specific neurofilament antibodies

Neurofilament monoclonal antibodies derived from mice immunized with chicken brain antigen or purified bovine NF 150K and NF 200K either stained only axons or they stained neuronal perikarya, dendrites and axons. Antibodies in the second group were called conventional because they decorated tissue sections like the neurofibrillary methods of traditional histology. Axon-specific antibodies either reacted with phosphorylated epitopes or they were phosphate/phosphatase insensitive thus suggesting reactivity with post-translational modifications other than phosphorylation. Another possibility was reactivity with phosphorylated epitopes inaccessible to exogenous phosphatases. Conventional neurofilament antibodies stained motor and sensory neurons in day 12 and day 13 rat embryos, respectively, as previously reported with neurofilament antisera. Immunoreactivity with axon-specific antibodies first appeared in motor and sensory axons at different times in development: day 13-14 (3 monoclonals); day 17 (6 monoclonals); day 21 (1 monoclonal); postnatal day 2 (1 monoclonal). There were no major differences between conventional and axon-specific antibodies as to the time of appearance of Purkinje cell baskets in postnatal rat cerebellum. With two exceptions all monoclonals first stained thin baskets on day 11. Immunoreactivity of Purkinje cell baskets with two monoclonals reacting with phosphorylated NF 200K first appeared on days 14 and 20. It is suggested that post-translational modifications may stabilize the neurofilaments, thus accounting for their late appearance by electron microscopy in development.     

Neurofilament phosphorylation in axons and perikarya: immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies

Rat dorsal root ganglia and spinal cord were stained with 12 monoclonal antibodies reacting with phosphorylated epitopes of two neurofilament proteins (NF 150K and NF 200K). Three monoclonal antibodies were axon-specific in both locations; neuronal perikarya were not stained. Nine monoclonal antibodies stained a subpopulation of neurofilament-positive sensory neurons, as indicated by double labeling experiments with polyclonal antibodies reacting with phosphorylated and dephosphorylated forms of the neurofilament protein triplet. Of these nine antibodies, two stained motor neuron perikarya in the spinal cord, while the remaining seven antibodies were axon-specific in this location. Subpopulations of stained and unstained motor neurons were not observed. With all 12 antibodies, the staining pattern in the lumbar dorsal root ganglia and spinal cord remained unchanged following sciatic nerve crush and ligature. The findings suggest that, in the neurofilament, some phosphorylated epitopes are axon specific, while other phosphorylated epitopes are present in both axons and perikarya. Furthermore, they suggest that differences exist between neuronal populations as to the presence of phosphorylated epitopes in perikaryal neurofilaments. It remains to be seen whether phosphorylation events in perikarya and axons have similar or different effects on neurofilament structure and function.     

Neurofilament phosphorylation in peripheral nerve regeneration

A monoclonal antibody to the 200 kdalton neurofilament (NF) polypeptide selectively decorated axons in tissue sections. Neuronal perikarya and dendrites, including motor and sensory neurons reacting to axotomy, were not stained. Axonal staining was abolished by dilution of the monoclonal supernatants with phosphate buffer and by digestion of tissue sections with phosphatase, thus suggesting that the antibody reacted with a phosphorylated epitope. Conventional monoclonal and polyclonal antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites) were not affected by these procedures. Compared to conventional NF antibodies, staining with the axon-specific monoclonal antibody was a late event in peripheral nerve regeneration. One week after operation, the whole distal stump of crushed rat sciatic nerve was invaded by bundles of axons strongly reacting with conventional NF antibodies. Axon-specific NF immunoreactivity was confined to the proximal segment of the stump at this time and progressively extended distally in the following week. Furthermore, NF phosphorylation appeared to coincide with the return of a normal nerve structure as evidenced by the distribution of laminin immunoreactivity. Bundles of axons growing within columns of laminin-positive Schwann cells did not stain with the axon-specific NF antibody. Immunoreactivity with this antibody coincided with the return of a normal laminin pattern, i.e. selective decoration of the endoneurial basal membranes surrounding the axons.     

Delayed phosphorylation of the largest neurofilament protein in rat optic nerve development

Monoclonal antibodies selectively reacting with the high molecular weight neurofilament proteins (NF 150K and NF 200K) on immunoblots of bovine spinal cord extracts were obtained upon immunization of mice with chicken brain antigen and with highly purified NF 150K or NF 200K isolated from bovine spinal cord by anion exchange chromatography. Antibodies reacting with NF 200K or with both NF 150K and NF 200K were selected for this study. The antibodies were screened on immunoblots for reactivity with phosphorylated epitopes by dilution of the supernatants in sodium potassium phosphate as well as by treatment of nitrocellulose transfers with alkaline phosphatase. Abolishment of staining under these conditions was taken as evidence of reactivity with phosphorylated epitopes. With phosphate/phosphatase-sensitive antibodies, NF 200K immunoreactivity was a late event in rat optic nerve development. It was first observed at day 18 on immunoblots of sodium dodecyl sulfate extracts analyzed by gel electrophoresis. Conversely, with phosphate/phosphatase-insensitive antibodies, NF 200K immunoreactivity was already present on day 10, the earliest age in this study. With one monoclonal reacting with phosphorylated NF 150K and NF 200K, NF 150K immunoreactivity was already present on day 10. It is proposed that NF 200K expression precedes NF 200K phosphorylation in development.     

Neurofilament phosphorylation in the axonless horizontal cells of rat retina

Axonless horizontal cells in the outer plexiform layer of rat retina were studied with 19 monoclonal antibodies reacting with phosphorylated and non-phosphorylated epitopes of the two high molecular weight neurofilament proteins (NF 150K and NF 200K). With 6 antibodies, immunoreactivity was confined to the nerve fiber layer on the inner surface of the retina. Horizontal cells were not stained. Four antibodies in this group were axon-specific, while the remaining two stained motor and sensory neuron perikarya in rat spinal cord and dorsal root ganglia, respectively. Of the 13 antibodies which stained horizontal cells, 11 reacted with phosphorylated epitopes and failed to decorate motor neuron perikarya in the spinal cord, while in dorsal root ganglia, they stained a subpopulation of sensory neurons.     

Early and late appearance of neurofilament phosphorylation events in nerve regeneration

Neurofilament phosphorylation in regenerating rat sciatic nerve was studied by indirect immunofluorescence with monoclonal antibodies reacting with phosphorylated epitopes of the 2 large polypeptides of the neurofilament protein triplet (NF 150K, NF 200K). One group of antibodies decorated axons early in the process. In fact, no differences were seen in double labeled sections between these antibodies and polyclonal neurofilament antibodies as to their reactivity with the distal stump of transected sciatic nerves. Another group stained axons after they had completed their elongation, i.e., after they had reached the distal part of the denervated sciatic nerve. In general, the epitopes recognized by antibodies in this group appeared more sensitive to phosphatase digestion as compared to the first group. Furthermore, there was a good correlation between the thickness of the regenerated axons and staining with these monoclonal antibodies. Thick axons (like those observed in normal nerves) were stained, while bundles of thin axons remained unstained. Monoclonal II32 stained regenerated axons in a remarkable segmental pattern. With this antibody, continuous decoration of the axons was still not observed 7 weeks after transection, the longest follow-up period in this study. We suggest that some neurofilament phosphorylation events may contribute to the stabilization of the axonal cytoskeleton and that abnormalities persist in regenerated axons as to the extent of neurofilament phosphorylation.     

Maturation of a large neurofilament protein (NF 150K) in rat postnatal development

The mammalian neurofilament is made of three neuron-specific proteins with approximate molecular weights of 70 kilodaltons (kDa) (NF 70K), 150 kDa (NF 150K), and 200 kDa (NF 200K) by SDS-PAGE. As previously reported in the rat by Strocchi et al (J Neurochem 39:1132-1141, 1982) and Nixon et al (J Cell Biol 94:150-58, 1982), NF 150K comprises three molecular weight variants with the same isoelectric point. A fourth lower molecular weight and slightly less acidic variant was identified by monoclonal and polyclonal antibodies reacting with the alpha-helical middle domain of NF 150K. With few exceptions, this lower molecular weight variant did not stain with monoclonal antibodies reacting with the peripheral carboxy-terminal domain. Staining with these antibodies was abolished or markedly reduced following neurofilament dephosphorylation. The distribution of the NF 150K variants varied in different regions of the nervous system. The higher molecular weight variant (component a) was less prominent in brain compared to spinal cord, optic nerve, and sciatic nerve. Furthermore, the lower molecular weight variant (component d) was not identified in optic nerve and sciatic nerve. All four variants were identified in brain and spinal cord extracts of newborn rats with monoclonal and polyclonal antibodies reacting with the alpha-helical middle domain of NF 150K. As a general (see Results for exceptions) monoclonal antibodies reacting with the carboxy-terminal region of NF 150K did not stain the variants in newborn rat brain extracts until day 10 when immunoreactivity of component a first appeared. The adult pattern was first observed on postnatal day 15.(ABSTRACT TRUNCATED AT 250 WORDS)     

Axonal regeneration in old multiple sclerosis plaques

Summary Cryostat sections of two old plaques removed at autopsy from the spinal cord of a 62-year-old man with multiple sclerosis of 24-year duration were studied by indirect immunofluorescence with antibodies to neurofilament proteins, glial fibrillary acidic protein (GFAP), glial hyaluronate-binding protein (GHAP), vimentin and laminin. The neurofilament monoclonal antibodies used in this study reacted with phosphorylated epitopes of the two large polypeptides of the neurofilament triplet (NF 150K, NF 200K). As previously reported [Dahl D, Labkovsky B, Bignami A (1989) Brain Res Bull 22:225–232], the neurofilament antibodies either stained axons in the distal stump of transected sciatic nerve in the early stages of regeneration or late in the process, i.e., after regenerating axons had reached the distal stump of the transected sciatic nerve. Both multiple sclerosis plaques were positive for GFAP and vimentin, but negative for GHAP, while astrocytes in myelinated spinal cord white matter stained with both GFAP and GHAP antibodies. Laminin immunoreactivity in the plaques and normal spinal cord was confined to blood vessels. One plaque was almost devoid of axons as evidenced by indirect immunofluorescence with neurofilament antibodies. Another plaque was packed with bundles of thin axons running an irregular course in the densely gliosed tissue. Axons in the plaque only stained with neurofilament antibodies reacting with sciatic nerve in the early stages of regeneration while axons in the surrounding myelinated white matter were decorated by all neurofilament antibodies, regardless of the time of appearance of immunoreactivity in crushed sciatic nerve. It is concluded that reactive astrocytes forming glial scars do not constitute a non-permissible substrate for axonal growth.Supported by NIH grant NS 13034 and by the Veterans Administration     

Novel monoclonal antibodies provide evidence for the in situ existence of a nonphosphorylated form of the largest neurofilament subunit

We have obtained five monoclonal antibodies to the Mr 200,000 neurofilament component (NF200) after immunization with polypeptides purified from enzymatically dephosphorylated bovine neurofilaments. In immunoblots of untreated neurofilament protein and protein from filaments exposed to phosphatase, these antibodies recognize nonphosphorylated or dephosphorylated, but not phosphorylated, forms of NF200. The epitopes recognized by these new monoclonal antibodies reside in the carboxyterminal domain of the NF200 polypeptide as defined by immunoreaction with limited chymotryptic fragments. Immunohistochemical studies of bovine cerebellum, spinal cord, trigeminal ganglion, and trigeminal nerve with these new monoclonal antibodies demonstrate immunoreactivity primarily in neuronal perikarya; axons and dendrites are weakly or infrequently immunostained. After enzymatic dephosphorylation of these tissues, a more extensive distribution of immunoreactivity is seen, especially in axons and dendrites. Immunostaining of cultured rat sympathetic neurons is restricted to cell bodies. These data provide evidence for the in situ existence of NF200 epitopes that are not phosphorylated in some classes of neurons or regions of a neuron, but are modified by phosphorylation in other neurons or neuronal domains. These new monoclonal antibodies are distinctly different from those in a large library (over 100) raised to, and specific for, phosphorylated neurofilament proteins. They are novel tools for probing neurofilament distribution, metabolism, structure, and possibly function.     

Neurofilament proteins in fish: a study with monoclonal antibodies reacting with mammalian NF 150K and NF 200K

Monoclonal antibodies were obtained upon immunization of mice with chicken brain antigen and with the two high molecular weight neurofilament proteins (NF 150K and NF 200K) isolated from bovine spinal cord by anion exchange chromatography. By the immunoblotting procedure, the antibodies selected for this study reacted with bovine NF 150K and NF 200K. By the same procedure the antibodies reacted with sea raven, goldfish, sea bass, shark, and trout spinal cord extracts. In goldfish and sea raven the antibodies stained a single band at approximately 150 kDa and 200 kDa, respectively. Two bands were stained in the shark, sea bass, and trout. In the shark and sea bass these bands were in the molecular weight range of mammalian NF 150K and NF 200K. In the trout the upper band was approximately 150 kDa and the lower band 130 kDa. Our findings suggest an early origin of NF 150K and NF 200K in vertebrate phylogeny as well as considerable divergence in several species.     

Immunohistochemical localization of the 150K neurofilament protein in the rat and the rabbit

Antisera to the 150K-dalton polypeptide of the bovine neurofilament triplet and chicken neurofilament antisera reacting with the 70K protein in isolated bovine brain filaments stained the same structures in rat cerebellum by immunofluorescence and peroxidase-antiperoxidase methods, that is Purkinje cell baskets, thin nerve fibers in the lower half of the molecular layer and myelinated axons. The 150K bovine neurofilament antisera did not stain large motor neurons in the anterior horns of the spinal cord in rat and rabbit, nor aluminum-induced neurofibrillary tangles in the rabbit. All these structures were demonstrated by the chicken neurofilament antisera and by silver neurofibrillary methods. IDPN-induced axonal balloons containing accumulations of neurofilaments were equally well stained by bovine 150K and chicken neurofilament antisera. These data suggest that the 150K polypeptide of the neurofilament triplet is not a subunit of the neurofilament core and probably plays a role in axonal transport.     

Temporal and topographic relationships between the phosphorylated and nonphosphorylated epitopes of the 200 kDa neurofilament protein during development in vitro

The ontogeny of the triplet of neurofilament proteins (NF), and the phosphorylated and nonphosphorylated derivatives of the 200 kDa neurofilament subunit (NF200P, NF200D) have been investigated in dissociated cultures prepared from gestational day 13 mouse spinal cord and dorsal root ganglia (DRG), using immunocytochemical methods. Neurofilament-like immunoreactivity (NF-LI), as detected with antiserum, occurred in the somata and processes of all neurons from day 1 in culture, and reached a maximum density and intensity at days 16-20. The first labeling of neurons by NF200D antibodies occurred at day 3, and was confined to DRG cells. Only a small, proximal portion of the axons from these cells exhibited NF200D-LI. At later stages, however, this immunoreactive region extended to include progressively more distal parts. Spinal cord neurons first became NF200D-positive at day 9; however, many NF200D-negative neurons still remained in mature cultures. Also at these later stages, some axons were stained for less than their full length with the NF200D antibody. NF200P-LI was first apparent at day 17, in smooth and varicose axons and only where NF-LI was also present. In contrast, NF200P- and NF200D-LI were usually localized in mutually exclusive populations of axons and other fibers. In some, predominantly thick axons, however, the proximal segment was NF200D-positive, whereas the distal part exhibited solely NF200P-LI. In contrast to NF70 and NF150, the 200 kDa neurofilament is dilatory in its appearance in most neurons in culture. The development of the nonphosphoderivative precedes that of the phosphoderivative, and the respective ontogenies are specific for different neuronal types. Posttranslational phosphorylation of NF200 seems therefore to occur at a later stage of development than the induction of NF200 itself, while there is a wide variation in its rates of phosphorylation during passage down different axons.     

Phosphorylation of neurofilaments is altered in aluminium intoxication

Summary A series of monoclonal antibodies that distinguish phosphorylated and nonphosphorylated neurofilament (NF) epitopes was used to immunostain brain stem neurons from control rabbits and from rabbits chronically intoxicated with Aluminium (Al). In controls, none of the monoclonal antibodies to phosphorylated NF stained the perikaryon of neurons. In contrast, in animals treated with Al, all neuronal perikarya containing Al-induced neurofilament bundles (NB) and some lacking well-formed NB immunoreacted with two of the five antibodies to phosphorylated NF. Axons were stained by all five antibodies to phosphorylated NF in both control and Al-treated animals. A broadly reacting monoclonal antibody to a nonphosphorylated NF epitopes immunoreacted with neuronal cell bodies, dendrites and axons in control and Al-intoxicated animals regardless of the presence of Al-induced NB. Staining of Al-induced NB with one of the antibodies to phosphorylated NF was greatly diminished after treatment of sections with trypsin and phosphatase. It is concluded that NF which compose the Al-induced NB have different immunocytochemical characteristics from those of the NF present in the perkaryon of normal neurons. It is likely that, contrary to normal perikaryal NF, NF of Al-induced NB are phosphorylated. Moreover, phosphorylation of NF of Al-induced NB is probably abnormal, since NF of Al-induced NB have immunostaining characteristics different from NF of normal axons. Al-induced NB may result from abnormal phosphorylation of NF in the perikaryon, preventing normal axonal transport of these structures.Supported by grants from the National Institutes of Health NS 14509 and AG 00795     

Effect of the substrate on neurofilament phosphorylation in mixed cultures of rat embryo spinal cord and dorsal root ganglia.

The effect of the substrate on neurofilament phosphorylation was studied in primary cultures of spinal cord and dorsal root ganglia dissociated from 15-day-old rat embryos. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites. (Primaria dishes have a modified plastic surface with a net positive charge). On both polylysine and Primaria substrates, spinal cord neurons were stained with neurofilament monoclonal antibodies reacting with phosphorylated epitopes appearing early in rat embryo development, i.e. soon after neurofilament expression. Conversely, immunoreactivity with antibodies recognizing late phosphorylation events was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, only occurred after several days in culture, at a time when non-neuronal cells (mainly astrocytes) had formed a confluent monolayer.     

Identities, antigenic determinants, and topographic distributions of neurofilament proteins in the nervous systems of adult frogs and tadpoles of Xenopus laevis

Three proteins with nominal molecular weights of 73 kDa (XNF-L), 175 kDa (XNF-M), and 205 kDa (XNF-H) were identified as putative neurofilament proteins in the nervous system of the frog, Xenopus laevis. These conclusions were based on four criteria: (1) these proteins were enriched in cytoskeletal preparations; (2) they reacted with a monoclonal antibody (anti-IFA) that cross-reacts with an epitope found in all intermediate filament proteins; (3) they cross-reacted with monoclonal antibodies directed against specific mammalian neurofilaments; and (4) antibodies that reacted with these proteins on Western blots specifically stained neurons in immunohistochemical analyses. The neurofilament proteins in Xenopus were antigenically similar, but not identical to mammalian neurofilament proteins. The principal difference was that four antibodies that reacted on Western blots with rat NF-H reacted with XNF-M in Xenopus. However, similarly to mammals, antibodies against phosphorylated XNF-M specifically labeled axons, whereas an antibody that reacted only with dephosphorylated epitopes on XNF-M specifically labeled neuronal cell bodies in immunohistochemistry. Three other antibodies that reacted equally well with untreated or alkaline-phosphatase-treated XNF-M or XNF-H proteins also showed axonally restricted staining in the adult Xenopus nervous system. An XNF-L (XC5D10) antibody was produced which stained axons and cell bodies equivalently throughout the adult Xenopus nervous system. By 3 days of development (stage 42; Xenopus tadpoles), antibodies to all three molecular weight forms of the frog neurofilament proteins detected specific neurons in the brainstem and spinal cord; and antibodies to phosphorylated and dephosphorylated epitopes on XNF-M could discriminate between axons and cell bodies in the rhombencephalon. The immunoreactivities of four antibodies directed at XNF-L, -M, or -H, which were unaffected by alkaline phosphatase treatment, differed significantly in their immunohistochemical staining patterns in adult vs. premetamorphic frogs.     

Neurofilament phosphorylation in neuronal perikarya following axotomy: a study of rat spinal cord with ventral and dorsal root transection

Rat spinal cord was stained by indirect immunofluorescence with 11 neurofilament monoclonal antibodies that recognize phosphorylated epitopes. All monoclonals were axon-specific in this location. The large motoneurons containing bundles of neurofilaments did not stain and the pattern remained unchanged after transection of the sciatic nerve in the thigh. With nine monoclonals, stained motoneurons were observed in the ventral horns 3 days, 5 days, 1 week, and 2 weeks after transection of the ventral roots close to the spinal cord. The abnormal motoneurons were typically scattered among normal (i.e., nonstained) cells. Even in animals showing the most severe reaction, the whole motoneuron population at the site of rhizotomy was not affected, stained and nonstained perikarya often coexisting side by side. Stained motoneurons were no longer observed 3 weeks after ventral root transection. Changes in neuronal immunoreactivity were also observed after dorsal root transection. However, a different population was affected, i.e., middle-sized neurons in dorsal horns and at the base of ventral horns. With two monoclonals (A9 and D21), cell bodies remained negative following all operations. It is concluded that axotomy in proximity of the cell body may induce certain neurofilament phosphorylation events in motor neuron perikarya, whereas other phosphorylation events remain confined to the axons under these experimental conditions. The absence of changes after transection of the sciatic nerve in the thigh suggests that neurofilament phosphorylation is a reaction to cell injury rather than a cellular event related to nerve regeneration.     

Varying degrees of phosphorylation determine microheterogeneity of the heavy neurofilament polypeptide (Nf-H)

Two-dimensional immunoblots revealed a spectrum of 200 kDa neurofilament polypeptides (Nf-H) of apparent molecular weights ranging from 200 to 170 kDa. The entire spectrum was stained immunocytochemically by three monoclonal antibodies specific for nonphosphorylated neurofilaments, while more restricted staining was revealed by four monoclonal antibodies specific for phosphorylated neurofilament epitopes. Treatment with increasing amounts of phosphatase suggested the existence of various forms of partially phosphorylated neurofilaments that possess phosphoepitopes that differ in their ease of dephosphorylation. Immunoprecipitation in low detergent concentration confirmed the existence of microheterogeneous forms of Nf-H that differed in extent of phosphorylation or in distribution of phosphorylated sites.     

Differential binding of antibodies against the neurofilament triplet proteins in different avian neurons

Using monospecific antisera against each of the three chicken neurofilament (NF) proteins, NF70, NF160 and NF180, the distribution of each of these proteins in several types of neurons was examined by immunohistochemistry. Striking differences were observed in the relative staining by the three antibodies when the soma of different types of neurons were compared, and also when the soma of some neurons were compared with their axons. Both the soma and axons of dorsal root sensory neurons were brightly stained by each of the antisera. The soma of spinal cord ventral horn neurons, however, were stained only by A-NF70 and A-NF180, not by A-NF160. The axons of these neurons were uniformly stained by A-NF70 and A-NF180, while only gradually becoming NF160-positive over the first several hundred microns. The lack of staining by A-NF160 was also observed in many neuronal soma in cultures of dissociated spinal cord cells. The soma and dendrites of adult cerebellar Purkinje cells were weakly stained by A-NF70 and A-NF180 and not at all by A-NF160, but both A-NF70 and A-NF180 yielded prominent staining of immature Purkinje cells and dendrites. These results suggest that the three NF proteins may be unequally distributed within the soma and processes of different types of neurons and/or may be subject to regionally selective modification.     

Brain filament proteins in primary cultures derived from chick embryos early in development

Brain filament expression and neurofilament post-translational modifications (phosphorylations) were studied in primary cultures derived from whole 3–4 day chick embryos. After 2–3 days in culture, neurofilament-positive cells formed neuronal aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. Aggregates and neuritic bundles were stained with several monoclonal antibodies reacting with phosphorylated neurofilament epitopes. With two monoclonal antibodies reacting with phosphorylated forms of the high molecular weight neurofilament subunit, staining was only observed after 8 and 10 days in vitro. There was a major difference between rat and chicken with respect to astrocyte differentiation in culture. In chicken, the flat cells surrounding the neuronal aggregates remained constantly GFAP-negative throughout the whole experimental period (10 days). GFAP-positive cells were first observed within the neuronal aggregates on day 8 in vitro.     

Monoclonal antibodies distinguish several differentially phosphorylated states of the two largest rat neurofilament subunits (NF-H and NF-M) and demonstrate their existence in the normal nervous system of adult rats

A new panel of greater than 300 monoclonal antibodies (mAbs) was prepared to the high, middle, and low Mr rat neurofilament (NF) subunits (NF-H, NF-M and NF-L, respectively). NF proteins were purified both from native, i.e., phosphorylated rat NFs and from enzymatically dephosphorylated rat NFs. The resulting mAbs were used to biochemically and immunochemically distinguish and characterize distinct and differentially phosphorylated isoforms of NF subunits. By immunoblot, all mAbs specific for NF-L and some mAbs specific for NF-M detected their specific NF subunit regardless of whether or not the NFs had been treated with alkaline phosphatase, and such antibodies were termed "phosphate-independent" or P[ind] mAbs. The other mAbs were specific for NF-M, NF-H, or for both NF-M and NF-H, and they recognized epitopes in the COOH termini of these subunits. Significantly, the latter mAbs could discriminate different isoforms of NF-M and NF-H, depending on the phosphorylation state of each variant. Such mAbs were assigned to one of 4 distinct categories on the basis of their performance in immunoblots of progressively dephosphorylated rat NF samples and by immunohistochemistry of various adult rat nervous tissues: (1) P[-] mAbs preferentially stained neuronal perikarya and dendrites, and they recognized only extensively dephosphorylated (and nonphosphorylated) NF-H; (2) P[+] mAbs stained axons more strongly than perikarya, and primarily blotted phosphorylated, but not nonphosphorylated, forms of NF-H and NF-M; (3) P[++] mAbs stained axons almost to the exclusion of perikarya, and in blots recognized only the extensively phosphorylated forms of NF-H and NF-M (i.e., subunits subjected to limited enzymatic dephosphorylation); (4) P[ ] mAbs also predominantly stained axons, but the briefest alkaline phosphatase treatment abolished the NF-M and NF-H immunobands produced by these mAbs. Two-dimensional gel analysis and immunoblotting of total proteins from adult rat dorsal root ganglion verified mAb specificity in situ, and showed that differentially phosphorylated isoforms of NF-M and NF-H occur in vivo. This provided additional evidence that mAbs can detect all 4 phosphorylation-dependent endogenous isoelectric variants of NF-H and NF-M.(ABSTRACT TRUNCATED AT 400 WORDS)