Antibody against mouse liver 5'-nucleotidase immunostains white matter in the adult mouse central nervous system

An antiserum against rat liver 5'-nucleotidase has been shown to immunostain myelinated fibers and oligodendrocytes in the rat CNS, consistent with evidence for 5'-nucleotidase activity in rat brain myelin and oligodendrocytes (Cammer, Sacchi and Kahn, Devel. Brain Res., 1985, 20: 89-96). However, in the mouse CNS, in which myelin also has 5'-nucleotidase activity, that antiserum stained only blood vessels. To obtain an antibody against the mouse enzyme, 5'-nucleotidase was partly purified from mouse liver membranes by detergent extraction, heat treatment, affinity chromatography, acidification, and ammonium sulfate fractionation. The preparation, which was enriched about 110-fold in 5'-nucleotidase specific activity, compared to the starting extract, was electrophoresed on a preparative slab gel containing Triton X-100, a strip was stained histochemically for 5'-nucleotidase, and the material corresponding to the stained band was used to immunize a rabbit. The immune IgG fraction, but not the preimmune IgG, reacted with mouse brain homogenates. The immune serum gave consistently greater inhibition of 5'-nucleotidase activity in mouse liver homogenates, mouse brain myelin and mouse brain homogenates, but not rat brain or liver homogenates, compared to the preimmune serum. The immune serum, but not the preimmune serum, immunostained white matter in the normal adult mouse brain and spinal cord. The findings suggest that the mouse may have one isozyme of 5'-nucleotidase similar to that in rat with respect to subunit sizes but differing in primary structure at one or more antigenic sites and support previous observations of 5'-nucleotidase activity in myelin from mouse brains and spinal cords.     

Characterization of a novel monoclonal anti-myelin basic protein antibody: use in immunoblotting and immunohistochemical studies

Monoclonal antibodies (MAbs) to the myelin basic protein (MBP) were produced in CAF1 (BALB/c x A/J) mice immunized with intact bovine MBP. A number of MAbs were obtained, one of which was characterized in detail with respect to its isotype, antigenic determinant on the MBP, the spectrum of antigens with which it reacted in mouse brain, and its immunohistochemical staining characteristics. This monoclonal, GB-1 (an IgG1), recognized an epitope within residues 30-51 of bovine MBP. It also reacted with a family of MBP-related proteins present in brain homogenates of mice from 7-35 days. Immunohistochemically, GB-1 stained myelinated fibers and oligodendrocytes in the rodent CNS. A second monoclonal (GB-2, and IgM) was partially characterized. It reacted with intact MBP when it was immobilized to plastic or nitrocellulose, but it was not found to be useful for immunoblots or immunohistochemistry.     

Comparison of dopamine beta-hydroxylase activity in normal rat adrenal medulla and rat pheochromocytoma cells

Dopamine beta-hydroxylase activity is reportedly negligible in malignant rat adrenal cell lines. However, in two pheochromocytoma cell lines, PC12 and PCG2, considerable amounts of this catecholamine enzyme exist but its characteristics differ from the enzyme found in normal rat tissue in two ways. First, in normal adrenal medullary tissue, dopamine beta-hydroxylase activity increases linearly with increasing protein concentration. Second, there is a stringent requirement for copper. Concentrations of copper above or below the optimum inhibit enzymatic activity. In contrast, in pheochromocytoma cells, dopamine beta-hydroxylase exhibits a sigmoidal response with increasing tissue content. At dilute protein concentrations where considerable dopamine beta-hydroxylase activity is observed in normal adrenal medullary extracts, enzymatic activity is negligible in the pheochromocytoma cell lines. As the protein concentration is increased, activation of the enzyme occurs, and enzymatic activity increases linearly with further increases in protein concentration. At very high concentrations of protein, enzymatic activity plateaus. For both PC12 and PCG2 cells, dopamine beta-hydroxylase activity shows minimal copper dependency, suggesting that endogenous inhibitors present in normal tissue are absent in these malignant cell lines. Under conditions of maximum activation, the activity of the enzyme in PC12 cells becomes equivalent to that in normal rat adrenal medulla but remains 45-fold greater than that in PCG2 cells.     

Connexin32 in oligodendrocytes and association with myelinated fibers in mouse and rat brain

The distribution and cellular localization of connexin32 (Cx32) in the brain and spinal cord of the mouse and rat was investigated by light microscope (LM) and electron microscope (EM) immunohistochemistry by using several different antibodies against Cx32. By double immunofluorescence staining for Cx32 and either the oligodendrocyte markers cyclic nucleotide phosphodiesterase (CNPase) or Rip, Cx32 was consistently found in oligodendrocyte cell bodies and proximal processes. Cx32 immunoreactivity was also clearly visualized along CNPase- and Rip-positive myelinated fibers. Both immunopositive cells and fibers were heterogeneously distributed and were often more intensely labeled when dispersed in or associated with regions of gray matter than when concentrated in major white matter tracts. Labeling of myelin sheaths along fibers was restricted to subpopulations of myelinated axons. In the cerebellar cortex, for example, it was selectively localized to sheaths around Purkinje cell axons. Punctate staining, distinct from that corresponding to cells or fibers, was evident in the olfactory bulb and hippocampus. By EM, oligodendrocytes exhibited cytoplasmic labeling associated with rough endoplasmic reticulum and Golgi apparatus. Their processes were intermittently stained, most intensely when surrounding myelinated fibers and occasionally in paranodal loops. Cx32-immunoreactive gap junctions with symmetric labeling (staining on both junctional membranes) were observed between oligodendrocytic somata and processes as well as between presumptive oligodendrocytic processes. Unidentifiable elements forming asymmetrically labeled gap junctions (staining on only one side of junctional membranes) were less frequently encountered. Western blot analysis confirmed anti-Cx32 antibody detection of Cx32 in whole brain homogenates and an enrichment of the protein in isolated myelin fractions. These results are consistent with earlier ultrastructural studies showing the occurrence of inter-oligodendrocytic gap junctions, but indicate that these may be more prevalent than previously thought. Furthermore, the results suggest a specialized role of gap junctions composed of Cx32 along myelinated fibers belonging to subpopulations of neurons. J. Comp. Neurol. 379:571–591, 1997. © 1997 Wiley-Liss, Inc.     

The Cholesterol Side-Chain Cleavage Complex in Human Brain White Matter

Specific antibodies obtained in rabbits after injection of bovine cholesterol side-chain cleavage enzymes cytochrome P-450scc, adrenodoxin and adrenodoxin-reductase were used for immunohistochemical studies in human brain. The three enzymes were co-localized in the white matter of the cerebellum. This observation strongly suggests the existence of steroidogenic activity in human oligodendrocytes, as previously reported in the rat, and suggests that the concept of 'neurosteroids' can be applied to Δ5–3ß-hydroxysteroid metabolites of cholesterol that accumulate in human brain.     

Human fetal neocortical tissue grafted to rat brain cavities survives,leads to reciprocal nerve fiber growth,and accumulates host IgG

The human-to-rat xenograft approach offers possibilities to study aspects of primate cortex development and function without monkeys. Human fetal cortical tissue was grafted to prepared cortical cavities of immunosuppressed host rats. Fetal tissue fragments were collected after routine low-pressure vacuum aspiration abortions performed in the first trimester of gestation. Human derived neurons and human nerve fiber outgrowth were visualized by immunohistochemistry with antibodies against human neurofilament protein 70 kD (hNFP70). Ingrowth from rat host striatum or cortex into the grafts was analyzed by immunohistochemistry with antibodies against tyrosine hydroxylase. Astrocytes were evaluated by immunohistochemistry with antibodies against glial fibrillary acidic protein. The grafts grew into different sizes (1–10 mm in diameter) and contained large numbers of hNFP70-positive nerve fibers. All grafts gave rise to outgrowth of hNFP70-positive fibers into the host with partly a cortical layering; layers III and IV received a majority of the human fibers. In several cases, the graft-derived nerve fibers entered the host brain at restricted areas, while there was no crossing over of nerve fibers at the rest of the graft-host interface. Tyrosine hydroxylase-positive fibers were usually not abundant in the grafts. Interestingly, cases of massive ingrowth occurred from host striatum into the graft in a pattern suggesting “permissive sites” at the graft-host interface in the same way as outgrowth from graft to host was found. Additionally, tyrosine hydroxylase-immunoreactive fibers from host cortex were found to grow into the transplant. Glial fibrillary acidic protein immunoreactivity was increased at the interfaces between graft and host cortex or host striatum. Immunohistochemistry using antibodies against rat IgG indicated the presence of rat IgG within the grafts, and in bordering areas of host brain, possibly indicating a defective graft-host barrier. Taken together, these results show that human cortical-tissue pieces grafted to cortical cavities of immunosuppressed rats survive grafting and develop, and that reciprocal nerve fiber growth between grafts and hosts occur. Human cortical neurons can grow into the rat host brain in a pattern which is partly determined by host cortical architecture. © Wiley-Liss, Inc.     

Localization of immunoreactive glutamyl aminopeptidase in rat brain. I. Association with cerebral microvessels

Glutamyl aminopeptidase (aminopeptidase-A, EC 3.4.11.7) is an ectoenzyme that selectively hydrolyses N-terminal glutamyl and aspartyl residues from oligopeptides, including (Asp¹)angiotensin II. Here we sought to determine the distribution of glutamyl aminopeptidase (EAP) in rat brain. EAP was purified to homogeneity from rat kidney and polyclonal antiserum raised in rabbits. Immune serum inhibited EAP enzyme activity in kidney homogenates and labeled two major protein bands of M_r ≈ 136,000 and M_r = 101,000 in immunoblots of kidney protein. EAP-like immunoreactivity was concentrated on kidney proximal tubule brush borders. Immunocytochemical staining of rat brain indicated that EAP-like immunoreactivity was primarily associated with cerebral microvessels. Positive staining was detected in microvessels ranging in size from capillaries up to vessels approximately 50 μm in diameter. Isolated cerebral microvessels had a 23-fold enrichment in EAP enzyme activity (193.1 ± 40.4 nmol/mg protein/h) compared to brain homogenate. Finally, immunoblots of isolated cerebral microvessels resulted in a pattern of labeling similar to that seen with kidney homogenates. These results indicate that EAP activity in brain is primarily associated with cerebral microvessels, and suggest that EAP may be involved in the metabolism of circulating or locally formed peptides.     

Xenobiotic metabolism in human brain--presence of cytochrome P-450 and associated mono-oxygenases

The cytochromes P-450, a family of heme proteins, play an important role in the oxidation of drugs and carcinogens, as well as endogenous substrates. We report the presence of cytochrome P-450 and associated mono-oxygenase activity in human brain regions and their selective enrichment in the brainstem. Immunocytochemical studies on human medulla with antibodies raised to phenobarbital-inducible rat liver cytochrome P-450 indicate that the enzyme is primarily localized in the neuronal cell bodies and to a lesser extent in the axons. These observations indicate that the human brain could be involved in metabolism of xenobiotics and endogenous compounds, mediated through cytochrome P-450.     

Brain enzyme adaptation to mild normobaric intermittent hypoxia

The adaptation to repeated periods of intermittent normobaric hypoxia (oxygen:nitrogen = 10:90, 12 hr daily for 5 days) of some specific enzymatic activities related to energy metabolism has been observed in different rat brain areas (cerebral cortex, hippocampus, corpus striatum, hypothalamus, cerebellum, and medulla oblongata). The evaluation of the maximum rate (Vmax) of the enzymes was carried out on: the homogenate "in toto," the nonsynaptic mitochondrial fraction, and the crude synaptosomal fraction. The adaptation to intermittent normobaric hypoxic exposure was characterized by significant modifications of some enzyme activities in the homogenate "in toto" (decrease of hexokinase activity in cerebellum), in the nonsynaptic mitochondrial fraction (increase of succinate dehydrogenase activity in corpus striatum and decrease of cytochrome oxidase activity in cerebral cortex), and, particularly, in the synaptosomal fraction (decrease of cytochrome oxidase activity in cerebral cortex, hippocampus, corpus striatum, and cerebellum, and decrease of malate dehydrogenase and lactate dehydrogenase activity in cerebellum). The adaptation to normobaric intermittent hypoxia differs according to the brain area, subcellular fraction, and enzyme activity tested.     

Quantitation of myelin carbonic anhydrase-development and subfractionation of rat brain myelin and comparison with myelin from other species.

A number of related studies have been performed to characterize further the carbonic anhydrase activity of myelin. Recent assertions that carbonic anhydrase activity is intrinsic to the myelin sheath were subjected to the additional test of isolation of rat brain myelin in the presence of purified carbonic anhydrase. This procedure did not increase the carbonic anhydrase activity in myelin above the endogenous level, indicating that this enzyme does not stick to myelin membranes. A developmental study of rat brain carbonic anhydrase showed that the enzyme activity increased in whole brain homogenates and in myelin, with the greatest increments in enzyme activity occurring before the animals were 60 days old. When myelin from adult rat brains was fractionated on a density gradient, carbonic anhydrase activity was relatively enriched in the heavy subfraction but was present in all three layers. This finding suggested that the activity in myelin preparations was not due to contamination with a carbonic anhydrase-rich membrane fragment. Carbonic anhydrase in myelin was not confined to the rat. Beef brain homogenates and myelin had low activities of the enzyme, but myelin from rabbit, cat, monkey and mouse had carbonic anhydrase activities comparable to that of the rat, accounting for 6.3--13.6% of the respective homogenate activities.     

Brain cytochrome oxidase: purification, antibody production, and immunohistochemical/histochemical correlations in the CNS

Cytochrome oxidase (CO) is a mitochondrial energy-generating enzyme used in brain studies as a marker of neural functional activity. The activity of CO in different brain regions, revealed histochemically, is distributed nonhomogeneously but in distinct patterns. Localized differences in CO activity could arise from localized differences in enzyme amount or from localized regulation of enzyme turnover number (molecular activity). To distinguish between these alternatives, we used antibodies against purified calf brain CO to assess the immunohistochemical distribution of CO amount (protein immunoreactivity) in several brain regions. Calf brain mitochondria (synaptic and nonsynaptic populations) were isolated from gray matter homogenates by differential centrifugation. CO was purified from detergent extracts of the mitochondria by cytochrome c-Sepharose 4B affinity chromatography. Antisera against the purified CO were raised in rabbits. The antibodies reacted specifically with CO, predominantly subunit IV, in SDS immunoblots. The antibodies did not react in SDS immunoblots with any other proteins solubilized from mitochondria or caudate nucleus but did cross-react with brain CO from other mammalian species and with bovine heart CO. The immunohistochemical distribution of CO amount matched the histochemical distribution of CO activity in all regions tested, including the monkey hippocampus and the mouse olfactory bulb, somatosensory (barrel) cortex, and cerebellum. Thus, the amount of CO in neural tissue is distributed in the same nonhomogeneous pattern as the histochemical activity of CO. The results suggest that mechanisms exist by which CO molecules are selectively distributed within neurons to meet local metabolic demands posed by neural functional activity.     

Immunohistochemical localization of aspartoacylase in the rat central nervous system

Aspartoacylase (ASPA; EC 3.5.1.15) catalyzes deacetylation of N-acetylaspartate (NAA) to generate free acetate in the central nervous system (CNS). Mutations in the gene coding ASPA cause Canavan disease (CD), an autosomal recessive neurodegenerative disease that results in death before 10 years of age. The pathogenesis of CD remains unclear. Our working hypothesis is that deficiency in the supply of the NAA-derived acetate leads to inadequate lipid/myelin synthesis during development, resulting in CD. To explore the localization of ASPA in the CNS, we used double-label immunohistochemistry for ASPA and several cell-specific markers. A polyclonal antibody was generated in rabbit against mouse recombinant ASPA, which reacted with a single band (approximately 37 kD) on Western blots of rat brain homogenate. ASPA colocalized throughout the brain with CC1, a marker for oligodendrocytes, with 92-98% of CC1-positive cells also reactive with the ASPA antibody. Many cells were labeled with ASPA antibodies in white matter, including cells in the corpus callosum and cerebellar white matter. Relatively fewer cells were labeled in gray matter, including cerebral cortex. No astrocytes were labeled for ASPA. Neurons were unstained in the forebrain, although small numbers of large reticular and motor neurons were faintly to moderately stained in the brainstem and spinal cord. Many ascending and descending neuronal fibers were moderately stained for ASPA in the medulla and spinal cord. Microglial-like cells showed faint to moderate staining with the ASPA antibodies throughout the brain by the avidin/biotin-peroxidase detection method, and colocalization studies with labeled lectins confirmed their identity as microglia. The predominant immunoreactivity in oligodendrocytes is consistent with the proposed role of ASPA in myelination, supporting the case for acetate supplementation as an immediate and inexpensive therapy for infants diagnosed with CD.     

Differences in myelination between spinal cord and corticular tissue transplanted intraocularly in rats

This paper compares the myelination in rat cortex and spinal cord transplanted on the embryonic day (E) 12, 14, 16, 18, 20 and right after birth (P0) into the anterior eye chamber of adult rats. Myelinated fibers were not observed in either cortical or spinal cord transplants of E12. When transplanted on E14, abundant myelinated fibers developed in the spinal cord and gathered at the periphery of the transplants as a ‘‘white matter’’. In the grafted cortex myelinated fibers were found when transplantation occurred on E16 or later. The myelinated fibers, however, remained scarce or formed only narrow bundles. The number and distribution of myelinated fibers did not depend on the donor’s age between E16 and P0 : even in the latter case transplanted cortex were found without myelination. The differences could be attributed to the different development of the cortical and spinal cord oligodendrocytes and to the different intrinsic organization of the grafted samples.     

Apoptotic mitochondrial pathway in neurones and astrocytes after neonatal hypoxia-ischaemia in the rat brain

Neuronal apoptosis plays an essential role in early brain development and contributes to secondary neuronal loss after acute ischaemia. Recent studies have provided evidence that caspase-3 is an important downstream event after hypoxia-ischaemia in the immature brain, but a minor event in the adult brain. Our investigations have focused on cell populations that expressed apoptotic effectors in the enzymatic death pathway including cytochrome c, caspase-9 and caspase-3. Expression, activation and cellular localization of these proteins were studied using cleavage of fluorogenic substrate and immunohistochemistry in neonatal rat brain after unilateral focal ischaemia. Caspase-3 enzyme activity was elevated in brain homogenate between 6 and 48 h after reperfusion. This activation was preceded by that of caspase-9, between 3 and 24 h. Apoptotic cell death was finally accomplished by poly-ADP-ribose polymerase cleavage, an endogenous caspase-3 substrate. In addition, immunodetection demonstrated that cytochrome c and activated caspase-9 and caspase-3 were expressed not only in the neurones, the primarily affected cells, but also within the astrocytes, which constituted a dense network delineating the infarct. These results suggested that glial injury may promote the formation of cystic lesions such as those observed clinically in the newborn brain.     

In vitro biotransformation of the selective serotonin reuptake inhibitor citalopram, its enantiomers and demethylated metabolites by monoamine oxidase in rat and human brain preparations

This study was conducted to identify enzyme systems eventually catalysing a local cerebral metabolism of citalopram, a widely used antidepressant of the selective serotonin reuptake inhibitor type. The metabolism of citalopram, of its enantiomers and demethylated metabolites was investigated in rat brain microsomes and in rat and human brain mitochondria. No cytochrome P-450 mediated transformation was observed in rat brain. By analysing H2O2 formation, monoamine oxidase A activity in rat brain mitochondria could be measured. In rat whole brain and in human frontal cortex, putamen, cerebellum and white matter of five brains monoamine oxidase activity was determined by the stereoselective measurement of the production of citalopram propionate. All substrates were metabolised by both forms of MAO, except in rat brain, where monoamine oxidase B activity could not be detected. Apparent Km and Vmax of S-citalopram biotransformation in human frontal cortex by monoamine oxidase B were found to be 266 microM and 6.0 pmol min(-1) mg(-1) protein and by monoamine oxidase A 856 microM and 6.4 pmol min(-1) mg(-1) protein, respectively. These Km values are in the same range as those for serotonin and dopamine metabolism by monoamine oxidases. Thus, the biotransformation of citalopram in the rat and human brain occurs mainly through monoamine oxidases and not, as in the liver, through cytochrome P-450.     

Cysteine dioxygenase: regional localisation of protein and mRNA in rat brain.

Cysteine dioxygenase (CDO) converts cysteine to cysteinesulphinic acid and is the rate-limiting step in sulphate production. Most studies have centred upon the hepatic form of the enzyme, but several studies have investigated brain CDO using activity assays and western blotting. The aim of this study was to investigate the expression of CDO in the rat brain using a combination of immunohistochemistry and in situ hybridisation. Affinity-purified anti-R and anti-H CDO antibodies were immunoprecipitated using rat brain homogenate to determine whether the antibodies could remove enzyme activity. Immunohistochemistry and in situ hybridisation were then used to determine the cellular and regional expression of both CDO protein and mRNA. Immunoprecipitation of rat brain homogenate removed up to 98% and 70% (anti-R and anti-H, respectively) of enzyme activity. Nonimmune sheep serum had no effect upon enzyme activity. CDO protein and mRNA was localised solely to the neurones of the brain, including the pyramidal cells of the hippocampus and the Purkinje cells of the cerebellum. Regional localisation varied, with high levels of expression in the hippocampus, the dentate gyrus, the outer cortices of the brain, and the substantia nigra. The relative expression of CDO activity and protein in these regions is most probably a result of the relative abundance of neurones in these regions. CDO expression in the brain may have several possibilities functions, the most likely being the prevention of free radical production by the autoxidation of cysteine and dopamine.     

Production, characterization, and immunohistochemical application of monoclonal antibodies to glutaminase purified from rat brain

Monoclonal antibodies were produced against phosphate-activated glutaminase (EC 3.5.1.2) as a marker for glutamatergic neurons: The enzyme was purified 1000-fold from rat brain mitochondria with a recovery of 27%. Upon SDS-PAGE the purified enzyme showed a single band up to 1.7 micrograms after the silver staining at molecular weight 62,000. Two monoclonal antibodies (IgMs) were produced; these absorbed more than 90% of glutaminase activity in rat brain homogenate. In immunoblotting after PAGE of the homogenate, the antibodies recognized only 1 protein band at the same position as that of the purified enzyme. Thus, the antibodies are specific and sufficient markers for glutaminase. Many neuronal cells in the rat brain were labeled immunohistochemically with these antibodies, but non-neuronal elements such as glial cells and vessels were not. Intense labeling was consistently observed in putative glutamatergic neurons such as pyramidal cells of layers V and VI in the cerebral neocortex. Intense staining was also seen in possible mossy fiber endings in the granular layer of the cerebellar cortex and in neurons giving off mossy fibers such as those in the pontine nuclei, pontine tegmental reticular nucleus of Bechterew, lateral reticular nucleus of the medulla oblongata, and external cuneate nucleus.     

Acute conduction block in vitro following exposure to antiganglioside sera

We studied the role of antiganglioside antibodies in the pathogenesis of autoimmune neuropathies using an in vitro preparation of a rat sciatic nerve. Human and rabbit sera with high titers of the antibodies were applied to a restricted segment of the sciatic nerve mounted in a recording chamber, and the compound nerve action potentials of the myelinated and unmyelinated fibers were observed. Myelinated fiber conduction became blocked at the segment within a few hours, whereas the unmyelinated fiber conduction remained unchanged. These results suggest that antiganglioside sera directly produce an acute conduction block only in myelinated nerve fibers and that this in vitro model is useful for studying the ionic mechanism by which the acute conduction block occurs. © 1993 John Wiley & Sons, Inc.     

GABA uptake by rabbit restiform body homogenates

Restiform body (inferior cerebellar peduncle) preparations were obtained from rabbit brain stem slices and homogenized. When challenged with labelled GABA, these homogenates took it up briskly. We have characterized pharmacologically this uptake which resulted almost equally neuronal and glial. The neuronal component of the GABA uptake might be due to the adjacent cochlear nuclei coming along in the preparations, whereas the glial component probably belongs to the restiform body proper. Another possibility is that actually both components are due to the myelinated fibers and glia which make up the restiform body.     

Distribution of a novel 57 kDa intermediate filament (IF) protein in the nervous system

A 57 kDa protein, that is not vimentin, is the major component of intermediate filaments (IF) obtained after 2 cycles of in vitro assembly from PC12 cells (Parysek and Goldman, 1987). By use of an antiserum to the 57 kDa protein, a cross-reacting antigen (of identical molecular weight) was detected on immunoblots of IF preparations and by immunofluorescence of various rat tissues. Immunolocalization studies on 3-4 micron frozen sections of tongue, small intestine, and adrenal gland showed bright labeling of nerve bundles and fine-caliber nerve fibers. The chromaffin cells and ganglion cells of the adrenal medulla also were labeled. In the nervous system, intense labeling was seen in small-caliber nerve fibers in sciatic nerve and spinal cord dorsal roots, as well as in the dorsal white columns and dorsal root ganglia. Of the ganglion cells, preferential labeling was seen in small-sized ganglion cells, whereas a monoclonal antibody to the 150 and 200 kDa neurofilament triplet (NFT) components labeled the large-sized ganglion cells. In the areas of the brain thus far examined with 57 kDa antiserum, there was labeling of components of cranial nerves and labeling of thin fibers in several areas, including the granular layer of the cerebellum and the corticospinal tract in the brain stem. For each tissue, adjacent sections treated with vimentin or glial fibrillary acidic protein antibody revealed labeling patterns distinct from that seen with either the 57 kDa or NFT antibodies. These results indicate that the 57 kDa IF protein is a neural IF component. Furthermore, this protein is distributed in only certain neuronal elements; these elements may be unified by an as yet unrecognized pattern of function in the nervous system.