Axonal Polypeptides Cross-Reactive with Antibodies to Neurofilament Proteins

Antibodies were prepared to mammalian CNS neurofilament proteins (NFPs) and the antibody specificities were compared using a sensitive immunoblotting method. This procedure was used to detect and characterize cross-reactive proteins and their degradation products in neurofilament preparations. NFPs were prepared by axon flotation. Rabbits were immunized with 200,000, 140,000, and 70,000 NFPs (200K, 140K, and 70K) that had been electrophoretically purified by polyacrylamide gel electrophoresis (PAGE). By immunohistofluorescence it was shown that all antisera stained similar filamentous structures in rat cerebellar neurons. By use of a horseradish peroxidase-conjugated indirect antibody procedure, however, differences were detected in the cross-reactivities of the antisera to rat NFPs, separated by PAGE and electrophoretically transferred to nitrocellulose membranes. Each antiserum exhibited strong binding to the homologous NFP and, thus, was suitable for the detection of cross-reactive polypeptides and proteolytic degradation products derived exclusively from the individual NFPs. Anti-200K, anti-140K, or anti-70K was applied to overloaded two-dimensional nitrocellulose blots of NFPs prepared by axon flotation. Each of the three sera detected a group of unique nonoverlapping polypeptides, some of which were identified as NFP degradation products. A different group of polypeptides was cross-reactive with antiserum to purified glial fibrillary acidic protein. The immunostaining of polypeptides on nitrocellulose was far more sensitive for detecting NFP degradation products than was staining polyacrylamide gels with Coomassie blue. Titers for the antisera were two to three orders of magnitude higher with the immunoblotting procedure than with immunohistologic methods. The sensitivity and the specificity of the described methods suggest their usefulness for examining proteolytic cleavage products of NFPs under a variety of conditions.     

Masking of epitopes in tissue sections

Summary Antisera to chicken brain antigen (CBA) isolated by hydroxyapatite chromatography from 8 M urea extracts following repeated extractions with phosphate buffer selectively decorate neurofilaments (NF) in neuronal perikarya, dendrites and axons. The antisera also reacted with GFA protein, the astrocyte-specific intermediate filament protein, as indicated by the adsorption of NF immunoreactivity following passage of the antisera through columns prepared with purified GFA protein. Moreover, the antisera stained the polypeptides of the NF triplet (70 kd, 150 kd, 200 kd) and GFA protein by the immunoblotting procedure. Monoclonal antibodies selectively decorating NF in tissue sections were isolated from a fusion of mouse myeloma cells with spleen cells of mice immunized with CBA. By the immunoblotting procedure the antibodies decorated the 150 kd NF polypeptide and GFA protein. No staining of glial filaments or any other structure on tissue sections was also observed with antibodies derived from another fusion strongly reacting with GFA protein on immunoblots. All antibodies (monoclonal and polyclonal) appeared to react with the same region of the GFA polypeptide as indicated by immunoblots of cleavage products.     

Neurofilament phosphorylation in development. A sign of axonal maturation?

Monoclonal antibodies to the 200K neurofilament (NF) protein selectively decorated axons in tissue sections. Dilution of the antibodies in phosphate buffer and digestion with phosphatase abolished the stain. With conventional monoclonal and polyclonal NF antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites), the staining was not affected by this treatment. With all antibodies, axon-specific and conventional, the staining was abolished by trypsin digestion. Subsequent digestion with phosphatase did not restore the staining. Compared with conventional NF antibodies, staining with axon-specific anti-NF 200K was a late phenomenon in chick embryo development. NF 200K immunoreactivity was first observed in peripheral nerves and in the anterior columns of the spinal cord on day 10. Sensory ganglia and optic nerve fibers were negative. With conventional NF antibodies these structures were stained on days 4 and 5, respectively. In the following days of development the study was confined to the retina, optic nerves, cranial peripheral nerves and sensory ganglia. Up to day 16, bundles of thin peripheral nerve fibers, strongly decorated by conventional NF antibodies, did not stain with anti-NF 200K in double labelling experiments. Nerve bundles emerging from the ganglia were also negative, although some thick nerve fibers within the ganglia were stained. NF 200K immunoreactivity was first observed on day 17 in the optic nerve and in the layer of optic nerve fibers. At this time, staining was confined to the bundle emerging from the temporal side of the retina. In newborn chicken, only few fibers stained with anti-NF 200K in the nasal bundle, while the temporal bundle was well stained. It is suggested that the NF 200K antibodies reacted with a phosphorylated epitope in the axon, and that NF phosphorylation is a late event in ontogenesis probably related to axonal maturation.     

Polyclonal Antisera to the Individual Neurofilament Triplet Protdins: A Characterization Using ELISA and Immunoblotting

In this article, the preparation and characterization of polyclonal rabbit antisera against the individual polypeptides of bovine neurofilament (68, 150, and 200 kilodaltons) is described. Selected antisera against the 68- and 150-kilodalton neurofilament polypeptides were specific for the corresponding antigen in homogenates of bovine, rat, and human brain as judged by immunoblots. The antisera against the 200-kilodalton neurofilament polypeptide cross-reacted to some extent with the 150-kilodalton neurofilament polypeptide, especially with the human antigen. The most specific antisera were used to develop an enzyme-linked immunosorbent assay (ELISA), and the cross-reactivities between the antisera and the different bovine and rat neurofilament polypeptides were determined. Contrary to the results in the immunoblots, the antiserum against the 200-kilodalton neurofilament polypeptide was subunit-specific, as was the 150-kilodalton antiserum. The 68-kilodalton antiserum displayed a minute cross-reactivity against bovine 150- and 200-kilodalton neurofilaments, but it cross-reacted somewhat more with the rat 150- and 200-kilodalton antigens. Even so, the subunit specificity of the antisera is high enough to enable the development of a quantitative ELISA for determination of the individual bovine or rat neurofilament polypeptides in a mixture. This study is the necessary preparation for such an assay.     

Changes in the amounts of cytoskeletal proteins within the perikarya and axons of regenerating frog motoneurons

Changes in the amounts of tubulin, actin, and neurofilament polypeptides were found in regenerating motoneurons of grass frogs during the period of axonal elongation. Ventral roots 9 and 10 were transected unilaterally about 7 mm from the spinal cord. 35 d later, [3H]colchicine binding had decreased in the proximal stumps to approximately one-half of contralateral control values, well before the regenerating motor axons had reinnervated skeletal muscles of the hind limb. [3H]colchicine binding did not change significantly in the operated halves of the 9th and 10th spinal cord segments over a 75-d period. The relative amounts of actin, tubulin, and neurofilament polypeptides in the operated ventral roots were measured by quantitative densitometry of stained two-dimensional electrophoretic gels. Alpha-tubulin, beta-tubulin, and the 68,000 molecular weight subunit of neurofilaments (NF68) decreased within the transected ventral roots to 78%, 57%, and less than 15% of control values, respectively. The amount of actin increased to 132% of control values within the operated ventral roots, although this change was not statistically significant. Opposite changes were found within motoneuronal cell bodies isolated from the spinal cord. The relative amounts of alpha-tubulin, beta-tubulin and NF68 within axotomized perikarya increased, respectively, to 191%, 146%, and 144% of that in control perikarya isolated from the contralateral side of the spinal cord. Thus, the changes in NF68 and tubulin did not occur uniformly throughout the injured cells. The possible structural and functional consequences of these changes are discussed.     

Immunological Reactivity of Antisera Prepared Against the Sodium Dodecyl Sulfate-Treated Structural Polypeptides of Adenovirus-Associated Virus

The preparation of antisera to the three purified sodium dodecyl sulfate (SDS)-treated polypeptide components (VP1, VP2, VP3) of adenovirus-associated virus (AAV) type 3H is described. In immunofluorescence tests (FA), these antisera stained heat-stable antigens with distinct morphologies in cells co-infected with either adenovirus or herpes simplex virus. Kinetic studies of antigen formation showed that VP1 antiserum first stained the cytoplasm (14 hr) and later (by 18 hr) stained both cytoplasmic and intranuclear areas. VP2 antiserum stained only discrete intranuclear areas, and VP3 antiserum stained nearly the entire nucleus. All three VP antigens appeared at about the 14th hr postinfection, about 2 hr prior to the appearance of whole virion antigen. The VP antisera cross-reacted in FA with AAV types 1 and 2 (all at one-eighth of the homologous titer), but did not react with other parvoviruses, i.e., rat virus, hemadsorbing enteric virus of calves, minute virus of mice, or H-1 virus. These non-neutralizing antisera reacted specifically with SDS-treated AAV virion antigens in complement fixation and immunodiffusion tests, and antiserum prepared against SDS-treated helper adenovirus structural polypeptides reacted with adenovirus polypeptide antigens. All antisera to SDS-treated polypeptides were specific for new antigens revealed on the dissociated peptides and did not react with whole virions, whereas whole-virion antisera did not cross-react with the polypeptide antigens. These findings suggest that antigens unique to the polypeptides of AAV are revealed by SDS treatment and that these antigens can be detected in cells prior to the folding of the polypeptides into the molecular configuration they possess as virion subunits. These results also indicate that at least one AAV polypeptide component is synthesized in the cell cytoplasm.     

Localization of vimentin,the nonspecific intermediate filament protein,in embryonal glia and in early differentiating neurons: In vivo and in vitro immunofluorescence study of the rat embryo with vimentin and neurofilament antisera

Antisera raised against vimentin, the protein subunit of nonspecific intermediate-sized filaments (IFs), were used in conjunction with neurofilament (NF) antisera to study the early development of neurons and glia in the rat embryo. Vimentin-positive fibers spanning the entire thickness of the neural tube including the cerebral vesicles were first observed on Day 12, concomitant with the appearance of NF protein in more confined areas (anterolateral regions of spinal cord and brain stem; motor roots emerging from the NF-positive areas). From Day 15 onwards vimentin and NF antisera selectively decorated glia and neurons, respectively, both in vivo and in vitro. Before Day 15 it appeared that NF-positive structures also stained with antivimentin in cryostat sections. In vitro experiments confirmed the presence of vimentin in early differentiating neurons. NF-positive cells were observed which also reacted with antivimentin in cultures obtained from 13- and 14-day embryos, but not later in development. Most neurons in these cultures became vimentin negative after 2–3 days in vitro.     

Degradation of neurofilament proteins by purified human brain cathepsin D

Abstract: Cathepsin D (CD) was purified to homogeneity from postmortem human cerebral cortex. Incubation of CD with human neurofilament proteins (NFPs) prepared by axonal flotation led to the rapid degradation of the 200,000, 160,000, and 70,000 NFP subunits (200K, 160K, and 70K) which had been separated by one-or two-dimensional sodium dodecyl sulfate-polyacrylámide gel electrophoresis (SDS-PAGE). Degradation was appreciable at enzyme activity-to-substrate protein ratios that were two-to threefold lower than those in unfractionated homogenates from cerebral cortex. Quantitative measurements of NFPs separated by PAGE revealed that, at early stages of digestion, the 160K NFP was somewhat more rapidly degraded than the 70K subunit while the 200K NFP had an intermediate rate of degradation. At sufficiently high enzyme concentrations, all endogenous proteins in human NF preparations were susceptible to the action of CD. Human brain CD also degraded cytoskeletal proteins in NF preparations from mouse brain with a similar specificity. To identify specific NFP breakdown products, antisera against each of the major NFPs were applied to nitrocellulose electroblots of NFPs separated by two-dimensional SDS-PAGE. In addition to detecting the 200K, 160K, and 70K NFP in human NF preparations, the antisera also detected nonoverlapping groups of polypeptides resembling those in NF preparations from fresh rat brain. When human NF preparations were incubated with CD, additional polypeptides were released in specific patterns from each NFP subunit. Some of the immuno-cross-reactive fragments generated from NFPs by CD comigrated on two-dimensional gels with polypeptides present in unincubated preparations. These results demonstrate that NFPs and other cytoskel-etal proteins are substrates for CD. The physiological significance of these findings and the possible usefulness of analyzing protein degradation products for establishing the action of proteinases in vivo are discussed.     

The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state

Treatment of freshly isolated, bovine neurofilaments with Escherichia coli alkaline phosphatase removes over 90% of the phosphate groups from serine residues of the Mr 200,000 and 150,000 polypeptide components (NF200 and NF150). Dephosphorylated NF200 and NF150 remain associated with filaments, but migrate in sodium dodecyl sulfate gels with reduced apparent molecular weights. Unusual migration appears to be due to modification at regions of these polypeptides that are peripheral to the neurofilament backbone as defined by limited chymotryptic digestion. Over 90 monoclonal antibodies recognizing epitopes located within the peripheral domain of native NF200 all show reduced affinity for dephosphorylated NF200. A single monoclonal antibody binds within the filament-associated domain of NF200 and its recognition of NF200 is unaffected upon treatment of neurofilaments with phosphatase. Around 50% of our monoclonal antibodies that bind NF150 monospecifically and at epitopes within its peripheral domain have reduced affinities for NF150 from phosphatase-treated filaments, while the remaining 50% bind native and dephosphorylated NF150 equally well. The smallest neurofilament component (NF70) contains few phosphate groups, most of which remain after treatment of neurofilaments with phosphatase. The resulting form of NF70 migrates normally in gels and its recognition by antibodies is unchanged. We conclude that phosphorylation modifies the structure of the two larger neurofilament polypeptides along domains that are peripheral to the filamentous backbone and that these effects are more pronounced for NF200 than for NF150.     

Difference in phosphorylation of neurofilament proteins in motor neurons and spinal ganglion cells

The composition of the neurofilament proteins (NFPs) in neuronal perikarya was examined by two-dimensional (2-D) gel electrophoresis of isolated perikarya of bovine spinal motor neurons. The extent of phosphorylation of the high molecular weight subunit of NFP (NFP-H) was compared between motor and sensory neuronal perikarya in spinal cord and spinal ganglion by immunocytochemistry with monoclonal antibodies (MAbs) to NFP. Of the 23 MAbs used in this study, one MAb (82E10) was specific to the highly phosphorylated component of NFP-H examined by 2-D immunoblot whereas another MAb (3A8) was specific to NFP-H irrespective of its level of phosphorylation. Immunocytochemically, 82E10 did not stain the perikarya of bovine and rabbit spinal motor neurons but 3A8 stained the perikarya in both animal species. These findings are consistent with 2-D immunoblot of neuronal perikarya of bovine motor neurons isolated in bulk. As for the spinal ganglia, 82E10 stained many, but not all, perikarya of sensory neurons of both animal species. These results indicate that the extent of phosphorylation of NFP-H in the perikarya of most spinal ganglion cells is higher than that of motor neurons. These findings suggest that the rate of phosphorylation of NFP-H in perikarya or the axonal transport of NFP from perikarya to proximal axons is uniform in spinal motor neurons but variable in spinal ganglion cells.     

Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton

Neurofilaments in the axons of mammalian spinal cord neurons are extensively cross-linked; consequently, the filaments and their cross- bridges compose a three-dimensional lattice. We have used antibody decoration in situ combined with tissue preparation by the quick- freeze, deep-etch technique to locate three neurofilament polypeptides (195, 145, and 73 Kd) within this lattice. When antibodies against each polypeptide were incubated with detergent-extracted, formaldehyde-fixed samples of rabbit spinal cord, each antibody assumed a characteristic distribution: anti-73-Kd decorated the neurofilament core uniformly, but not the cross-bridges; anti-145-Kd also decorated the core, but less uniformly; sometimes the anti-145-Kd antibodies were located over the bases of cross-bridges. In contrast, anti-195-Kd primarily decorated the cross-bridges between the neurofilaments. These observations show that the 73-Kd polypeptide is a component of the central core of neurofilaments, and that the 195-Kd polypeptide is a component of the inter-neurofilamentous cross-bridges. It is consistent with this conclusion that we found few cross-bridges between neurofilaments in the optic nerves of neonatal rabbits during a developmental period when the ratio of 195 to 73 or 145-Kd polypeptides is much lower than in adults. The ratio of 195-Kd polypeptide to the other two neurofilament polypeptides also appeared much lower in the cell bodies and dendrites than in axons of adult spinal cord neurons, when the dispositions of the three polypeptides were studied by immunofluorescence experiments. The cell bodies apparently contain neurofilaments composed primarily of 145- and 73-Kd polypeptides, because we observed antibody decoration of individual neurofilaments in the cell bodies with anti-73- and -145-Kd, but not with anti-195-Kd. We conclude that the 195-Kd polypeptide participates in a cross-linking function, and that this function is, at least in certain neurons, most prevalent in the mature axon.     

LOCALIZATION OF THE THY-1 ANTIGEN IN RAT BRAIN AND SPINAL CORD BY IMMUNOFLUORESCENCE

Abstract— The Thy-1 antigen of rat brain is a membrane glycoprotein of molecular weight 17,500. It was localized in sections of brain and spinal cord by indirect immunofluorescence using rabbit antisera raised against purified Thy-1 and fluorescein conjugated purified sheep F(ab')₂, anti-(rabbit IgG) antibody fragments. The specificity of the anti-(Thy-1) sera was tested by a quantitative indirect radioactive binding assay which is particularly useful for ascertaining the specificity of reagents used in immunohistochemical studies. Purified Thy-1 was used to absorb the anti-(Thy-1) sera for controls in the immunofluorescence experiments. Strong specific fluorescence was found throughout the gray matter of brain and spinal cord with lesser amounts in white matter. The nuclei of all neural cells and also myelin lacked fluorescence. Some of the large neurons contained weak cytoplasmic fluorescence, but the majority of the immunofluorescence was located in the neuropil of the brain and spinal cord. There was an indication that Thy-1 was associated with synaptic knobs due to its presence in synaptic glomeruli and its granular appearance around some neurons. An additional association with glial membranes could not be excluded.     

Preparation of Neurofilament Protein from Guinea Pig Peripheral Nerve and Spinal Cord

Abstract: A simple and rapid method for preparation of enriched neurofilament protein from mammalian peripheral nerve or spinal cord is described. Tissue extracts from guinea pig nerve or spinal cord are fractionated by ammonium sulfate fractionation, chromatography on Sepharose 4B, and precipitation with ethanol. Molecular exclusion chromatography on Sepharose 4B, in which the neurofilament protein elutes quantitatively in the exclusion volume of the column, with little contamination by other proteins, is found to be a highly effective purification step. The protein is found to precipitate in ammonium sulfate fractions over a wide range of salt concentration, from 20 to 80% saturation. It is found to be quantitatively precipitated in 40% v/v ethanol-water. The preparative method described yields 0.25 mg of neurofilament protein per gram of nerve or spinal cord, with a purity of approximately 50%. The three principal neurofilament polypeptides, which have molecular weights by SDS-polyacrylamide gel electrophoresis of 200K, 145K, and 68K, are found to be present in the preparation in a molar ratio of 1:2:6. A variant form of neurofilament protein occurring in approximately 20% of Hartley strain guinea pigs is described, which has the polypeptide composition: 200K, 192K, 145K, 68K.     

Immunofluorescence studies of neurofilaments in the rat and human peripheral and central nervous system

Localization of antisera to neurofilament antigens derived from rat peripheral nerve was carried out in tissues of rat and human peripheral and central nervous systems by indirect immunofluorescence. Unfixed and chloroform-methanol-fixed frozen sections of tissues were incubated in purified IgG of the experimental rabbit antisera and subsequently exposed to goat anti-rabbit IgG conjugated with fluorescein isothiocyanate. Control studies were conducted on identical tissue preparations incubated in the same concentrations of nonspecific rabbit IgG or in experimental rabbit IgG absorbed with extracts of rat peripheral nerve containing neurofilament antigen. Extensive immunofluorescence was observed in rat and human peripheral and central nervous systems. The distribution and configuration of immunofluorescence corresponded to neurofilament-rich structural components of these tissues. Prominent immunofluorescence was also noted in neuronal cell bodies of spinal sensory ganglia, especially in perikarya of the large neuronal type. Immunofluorescence of the central nervous system was located predominantly in myelinated axons of the white matter in cerebrum, cerebellum, brain stem, and spinal cord. Less intense immunofluorescence was also seen in neuronal perikarya and in short thin linear processes of grey matter.     

Immunohistochemical and in situ hybridization studies of choline acetyltransferase in large motor neurons of the human spinal cord

The localization of choline acetyltransferase (ChAT) protein and mRNA was investigated in large motor neurons of the lumbar spinal cord of 10 autopsied individuals without neurological diseases, by immunohistochemistry and in situ hybridization. In the immunohistochemistry using 20 serial tissue sections with a total thickness of 80 microm, about approximately 58-85% (average 67%) of the large motor neurons (30 microm and more in somal minimal diameter) in the ventral horn were stained with the anti-human ChAT antibody. In the positive neurons, most immunoreactive products were observed focally in the perikarya. Occasionally, the perikarya of some neurons were stained diffusely. In situ hybridization with a single 4 microm-thick tissue section showed that almost all large motor neurons had positive signals (approximately 93-100%, average 98%), which were distributed diffusely in the perikarya. The positivity rate in the in situ hybridization was higher than that in the immunohistochemistry for all 10 cases. These results indicate that ChAT mRNA is transcribed in almost all large motor neurons in the ventral horn of the human spinal cord, but ChAT protein cannot always be detected in the cytoplasm by immunohistochemistry.     

Immunocytochemical localization of neuromedin K (neurokinin B) in rat spinal ganglia and cord.

Specific antisera directed against substance P and neuromedin K (neurokinin B) have been used in double-label immunofluorescence studies to unambiguously localize these two neuropeptides of the tachykinin family in single tissue sections of rat spinal cord and dorsal root ganglia. Substance P-like immunoreactivity (SPLI) is present but neuromedin K-like immunoreactivity (NMKLI) is undetectable in dorsal root ganglia. Both peptides are present in the spinal cord, but NMKLI is largely restricted to the dorsal gray while SPLI shows a broader distribution. In the spinal gray, NMKLI coexists with SPLI in some, but not all, fibers. While substance P in the dorsal spinal cord is largely of primary afferent origin, neuromedin K appears to originate largely from intrinsic spinal neurons.     

Appearance and phosphorylation of the 210 kDalton neurofilament protein in newborn rat brain,spinal cord,and sciatic nerve

The appearance and in vivo phosphorylation of the 210 kDalton (kD) neurofilament protein (NF210K) in newborn rat brain, spinal cord, and sciatic nerve were invetigated. Electron microscopic examination of neurofilaments isolated from newborn rat brain and spinal cord demonstrated morphologically distinct filaments which contained cross-bridging side arms. Neurofilament proteins, phosphorylated in vivo, were separated by sodium dodecyl sulfate slab gel electrophoresis and were transferred from acrylamide gels to nitrocellulose sheets. The nitrocellulose sheets were treated with antiserum to the 70 kD, 145 kD and 210 kD neurofilament proteins by the immunoblot technique. The three neurofilament proteins were found to be present in newborn brain, spinal cord and sciatic nerve. The presence of NF210K in newborn rat brain was further confirmed by 2-dimensional gel electrophoresis followed by indentification of this protein by the immunoblot technique. Exposure of the immunostained nitrocellulose sheets to x-ray film revealed that the NF210K, NF145K, and NF70K proteins were phosphorylated in filaments prepared from newborn rat central and peripheral nervous systems. These results suggest that the synthesis and posttranslational modification of the neurofilament proteins may be synchronized or developmentally regulated. It is feasible that phosphorylation of the NF210K subunit may be a prerequisite for the formation of neurofilament cross-bridging elements which are necessary for radial growth of axons.     

Characterization of a novel 66 kd subunit of mammalian neurofilaments

A 66 kd protein, pl 5.4, was purified from the Triton-insoluble fraction of rat spinal cord. This protein formed 10 nm filaments in vitro. The 66 kd protein was unique, although it shared homology with the 70 kd neurofilament protein (NF-L) and vimentin. An antiserum (anti-66) specific to the 66 kd protein did not cross-react with any of the neurofilament triplet proteins. In the spinal cord, anti-66 intensely stained the axons of the anterior and lateral columns. However, afferents from dorsal root ganglia and the efferents from the motoneurons were negative. In the cerebellum, anti-66 intensely stained most axons. The 66 kd protein was readily detectable in homogenates of forebrain, cerebellum, brainstem, and spinal cord, but was found only in trace amounts in adult sciatic nerves and was not found in extraneural tissues. The 66 kd protein constituted 0.5% of total protein in the spinal cord, whereas NF-L constituted about 1.5%.     

Localization of beta-endorphin-like immunoreactivity in the nervous system of the adult newt, Notophthalmus viridescens.

Using indirect immunofluorescence methods, we have localized for the first time in the newt, Notophthalmus viridescens, beta-endorphin (beta-ep)-like immunoreactivity in the neurons of spinal ganglia (SPG), spinal cord (SPC), as well as in the hypothalamic region of the brain. An examination of serially sectioned SPG showed that the beta-ep-positive neurons, cell bodies, and nerve fibers were distributed at all levels of SPG. Peripheral regions of the perikarya of beta-ep-positive SPG neurons exhibited intense staining for beta-ep, the central nuclear region remaining nonreactive. In SPC, brightly staining fibers were seen entering the afferent nociceptive input areas, namely the Lissauer's tracts, substantia gelatinosa, and the dorsal ascending columns. Dot-fiber immunofluorescence pattern was observed throughout the gray matter of SPC representing beta-ep-positive, secondary sensory neurons as well as interneurons. Also, discrete cluster of neurons located deep in the gray matter of SPC stained positively to beta-ep antisera. This study not only demonstrates for the first time the presence of beta-ep like material in the newt, more specifically in SPG and SPC, but also raises the question of a possible link between beta-ep and newt limb regeneration as previous work has shown that SPG support limb regeneration in a denervated-amputated newt forelimb.     

Immunohistological localization of desmin, the muscle-type 100 A filament protein, in rat astrocytes and Müller glia

The distribution of glial fibrillary acidic (GFA) protein and desmin was compared in cryostat sections of rat brain, spinal cord, and eye by immunofluorescence and peroxidase-antiperoxidase (PAP) staining. Desmin antisera were raised to antigen purified from chicken gizzard. In rat brain and spinal cord, GFA protein and desmin were selectively localized in astrocytes. Neurons and axons were not stained. The only difference between GFA and desmin antisera was the staining of smooth muscle in small arteries with anti-desmin. It was only in retinal glia that a difference in the localization of the two proteins was apparent. As previously reported, only the glia limitans on the inner surface of the retina was demonstrated with GFA antisera in the normal eye. With anti-desmin Müller fibers spanning the whole thickness of the retina were stained. It is concluded that GFA and desmin form two distinct systems of 100 A filaments in astroglia, as previously reported for GFA and vimentin.