Distribution and Subcellular Localization of High-Molecular-Weight Microtubule-Associated Protein-2 Expressing Exon 8 in Brain and Spinal Cord

Abstract: The expression of high-molecular-weight (HMW) microtubule-associated protein-2 (MAP-2) expressing exon 8 (MAP-2+8) was examined by immunoblotting during rat brain development and in sections of human CNS. In rat brain, HMW MAP-2+8 expression was detected at embryonic day 21 and increased during postnatal development. In adult rats, HMW MAP-2+8 comigrated with MAP-2a. In human adult brain, HMW MAP-2+8 was expressed in select neuronal populations, including pyramidal neurons of layers III and V of the neocortex and parahippocampal cortex, pyramidal neurons in the endplate, CA2 and subiculum of the hippocampus, and the medium-sized neurons of the basal ganglia. In the cerebellum, a subpopulation of Golgi neurons in the internal granular cell layer and most Purkinje cells were also stained. In the spinal cord staining was observed in large neurons of the anterior horn. Staining was present in cell bodies and dendrites but not in axons. At the ultra-structural level, HMW MAP-2+8 immunoreactivity was observed on mitochondrial membranes and in postsynaptic densities (PSDs) of some asymmetric synapses in the midfrontal cortex and spinal cord. Immunoblots of proteins isolated from enriched mitochondrial and PSD fractions from adult human frontal lobe and rat brains confirmed the presence of HMW MAP-2+8. The presence of HMW MAP-2+8 in dendrites and in close proximity to PSDs supports a role in structural and functional attributes of select excitatory CNS synapses.     

Microtubule-associated protein 2 within axons of spinal motor neurons: associations with microtubules and neurofilaments in normal and beta,beta'-iminodipropionitrile-treated axons

We have examined the distribution of microtubule-associated protein 2 (MAP2) in the lumbar segment of spinal cord, ventral and dorsal roots, and dorsal root ganglia of control and beta,beta'-iminodipropionitrile- treated rats. The peroxidase-antiperoxidase technique was used for light and electron microscopic immunohistochemical studies with two monoclonal antibodies directed against different epitopes of Chinese hamster brain MAP2, designated AP9 and AP13. MAP2 immunoreactivity was present in axons of spinal motor neurons, but was not detected in axons of white matter tracts of spinal cord and in the majority of axons of the dorsal root. A gradient of staining intensity among dendrites, cell bodies, and axons of spinal motor neurons was present, with dendrites staining most intensely and axons the least. While dendrites and cell bodies of all neurons in the spinal cord were intensely positive, neurons of the dorsal root ganglia were variably stained. The axons of labeled dorsal root ganglion cells were intensely labeled up to their bifurcation; beyond this point, while only occasional central processes in dorsal roots were weakly stained, the majority of peripheral processes in spinal nerves were positive. beta,beta'- Iminodipropionitrile produced segregation of microtubules and membranous organelles from neurofilaments in the peripheral nervous system portion and accumulation of neurofilaments in the central nervous system portion of spinal motor axons. While both anti-MAP2 hybridoma antibodies co-localized with microtubules in the central nervous system portion, only one co-localized with microtubules in the peripheral nervous system portion of spinal motor axons, while the other antibody co-localized with neurofilaments and did not stain the central region of the axon which contained microtubules. These findings suggest that (a) MAP2 is present in axons of spinal motor neurons, albeit in a lower concentration or in a different form than is present in dendrites, and (b) the MAP2 in axons interacts with both microtubules and neurofilaments.     

Synaptic localization and axonal targeting of agrin secreted by ventral spinal cord neurons in culture

Agrin secreted by motor neurons is a critical signal for postsynaptic differentiation at the developing neuromuscular junction. We used cultures of chick ventral spinal cord neurons with rat myotubes and immunofluorescence with species-specific antibodies to determine the distribution of agrin secreted by neurons and compare it to the distribution of agrin secreted by myotubes. In addition, we determined the distribution of agrin secreted by isolated chick ventral spinal cord neurons and rat motor neurons grown on a substrate that binds agrin. In cocultures, neuronal agrin was concentrated along axons at sites of axon-induced acetylcholine receptor (AChR) aggregation and was found at every such synaptic site, consistent with its role in synaptogenesis. Smaller amounts of agrin were found on dendrites and cell bodies and rarely were associated with AChR aggregation. Muscle agrin, recognized by an antibody against rat agrin, was found at nonsynaptic sites of AChR aggregation but was not detected at synaptic sites, in contrast to neuronal agrin. In cultures of isolated chick neurons or rat motor neurons, agrin was deposited relatively uniformly around axons and dendrites during the first 2-3 days in culture. In older cultures, agrin immunoreactivity was markedly more intense around axons than dendrites, indicating that motor neurons possess an intrinsic, developmentally regulated program to target agrin secretion to axons.     

Widespread distribution of the major polypeptide component of MAP 1 (microtubule-associated protein 1) in the nervous system

We prepared a monoclonal antibody to microtubule-associated protein 1 (MAP 1), one of the two major high molecular weight MAP found in microtubules isolated from brain tissue. We found that MAP 1 can be resolved by SDS PAGE into three electrophoretic bands, which we have designated MAP 1A, MAP 1B, and MAP 1C in order of increasing electrophoretic mobility. Our antibody recognized exclusively MAP 1A, the most abundant and largest MAP 1 polypeptide. To determine the distribution of MAP 1A in nervous system tissues and cells, we examined tissue sections from rat brain and spinal cord, as well as primary cultures of newborn rat brain by immunofluorescence microscopy. Anti-MAP 1A stained white matter and gray matter regions, while a polyclonal anti-MAP 2 antibody previously prepared in this laboratory stained only gray matter. This confirmed our earlier biochemical results, which indicated that MAP 1 is more uniformly distributed in brain tissue than MAP 2 (Vallee, R.B., 1982, J. Cell Biol., 92:435-442). To determine the identity of cells and cellular processes immunoreactive with anti-MAP 1A, we examined a variety of brain and spinal cord regions. Fibrous staining of white matter by anti-MAP 1A was generally observed. This was due in part to immunoreactivity of axons, as judged by examination of axonal fiber tracts in the cerebral cortex and of large myelinated axons in the spinal cord and in spinal nerve roots. Cells with the morphology of oligodendrocytes were brightly labeled in white matter. Intense staining of Purkinje cell dendrites in the cerebellar cortex and of the apical dendrites of pyramidal cells in the cerebral cortex was observed. By double-labeling with antibodies to MAP 1A and MAP 2, the presence of both MAP in identical dendrites and neuronal perikarya was found. In primary brain cell cultures anti-MAP 2 stained predominantly cells of neuronal morphology. In contrast, anti-MAP 1A stained nearly all cells. Included among these were neurons, oligodendrocytes and astrocytes as determined by double-labeling with anti-MAP 1A in combination with antibody to MAP 2, myelin basic protein or glial fibrillary acidic protein, respectively. These results indicate that in contrast to MAP 2, which is specifically enriched in dendrites and perikarya of neurons, MAP 1A is widely distributed in the nervous system.     

An immunofluorescence study of neurofilament protein expression by developing hippocampal neurons in tissue culture

We have studied the development of intermediate filament proteins in the neurons found in hippocampal cell cultures using single and double label immunofluorescence with both monoclonal and polyclonal antibodies. Neurons in these cultures are known to differentiate in a manner similar to their counterparts in situ: in particular they develop axonal and dendritic processes which differ from each other in form, in ultrastructure, and in synaptic polarity. During the first days in culture, developing neurons could not be stained with antibodies against any of the neurofilament proteins, although many cells reacted with anti-vimentin. Later in the first week, antibody staining revealed clearly filamentous staining for the L (68 000 daltons) and the M (145 000 daltons) neurofilament subunits, though M reactivity was much stronger at this earlier stage of development. Some neurofilament positive profiles in many cells could also be stained with vimentin, though the vimentin immunoreactivity became progressively less pronounced during further development, and disappeared after about two weeks in culture. Also at about two weeks in vitro we noted the first appearance of neurofilament H protein (200 000 daltons) immunoreactivity, which was localized to a subset of long neurites which could be identified on morphological grounds as axons. These processes lacked staining for microtubule associated protein 2 (MAP2), a dendritic marker. They tended to be close to islands of glial cells, suggesting that H induction may require complex neuron-glial interactions. These results are consistent with the suggestion that H protein immunoreactivity is a marker for axonal outgrowth. In addition to obvious filamentous staining, we were able to localize neurofilament antigens to an interesting class of small ring-like structures, found increasingly frequently as the cultures aged. We also present evidence that tyrosinated alpha-tubulin is present both within dendrites and axons of neurons in these cultures.     

Biochemical and immunological analyses of cytoskeletal domains of neurons

We have used cultured sympathetic neurons to identify microtubule proteins (tubulin and microtubule-associated proteins [MAPs]) and neurofilament (NF) proteins in pure preparations of axons and also to examine the distribution of these proteins between axons and cell bodies + dendrites. Pieces of sympathetic ganglia containing thousands of neurons were plated onto culture dishes and allowed to extend neurites. Dendrites remained confined to the ganglionic explant or cell body mass (CBM), while axons extended away from the CBM for several millimeters. Axons were separated from cell bodies and dendrites by dissecting the CBM away from cultures, and the resulting axonal and CBM preparations were analyzed using biochemical, immunoblotting, and immunoprecipitation methods. Cultures were used after 17 d in vitro, when 40-60% of total protein was in the axons. The 68,000-mol-wt NF subunit is present in both axons and CBM in roughly equal amounts. The 145,000- and 200,000-mol-wt NF subunits each consist of several variants which differ in phosphorylation state; poorly and nonphosphorylated species are present only in the CBM, whereas more heavily phosphorylated forms are present in axons and, to a lesser extent, the CBM. One 145,000-mol-wt NF variant was axon specific. Tubulin is roughly equally distributed between CBM and axon-like neurites of explant cultures. MAP-1a, MAP-1b, MAP-3, and the 60,000-mol-wt MAP are also present in the CBM and axon-like neurites and show distribution patterns similar to that of tubulin. In contrast, MAP-2 was detected only in the CBM, while tau and the 210,000-mol-wt MAP were greatly enriched in axons compared to the CBM. In immunostaining analyses, MAP-2 localized to cell bodies and dendrite-like neurites, but not to axon-like neurites, whereas antibodies to tubulin and MAP-1b localized to all regions of the neurons. The regional differences in composition of the neuronal cytoskeleton presumably generate corresponding differences in its structure, which may, in turn, contribute to the morphological differences between axons and dendrites.     

Localization of Specific Epitopes on Human Microtubule-Associated Protein 2

Abstract: Microtubule-associated protein 2 (MAP-2) is an abundant neuronal cytoskeletal protein that binds to tubulin and stabilizes microtubules. Using fusion protein constructs we have defined the epitopes of 10 monoclonal antibodies (mAbs) to discrete regions of human MAP-2. Proteins were expressed in pATH vectors. After electrophoresis, immunoblotting was performed. By western blot analysis five of the mAbs (AP-14, AP-20, AP-21, AP-23, and AP-25) share epitopes with only the high molecular weight isoforms (MAP-2a, MAP-2b); two of the mAbs (AP-18 and tau 46) recognize MAP-2a, MAP-2b, and MAP-2c. Although AP-18 immunoreactivity was detected within heat-stable protein homogenates isolated from a human neuroblastoma cell line MSN, fusion protein constructs encompassing human MAP-2 were negative, suggesting that the AP-18 epitope is phosphorylated. Furthermore, AP-18 immunoreactivity was lost after alkaline phosphatase treatment of heat-stable protein preparations from MSN cells. Four of the mAbs (322, 636, 635, and 39) recognize epitopes located within amino acids 169–219 of human MAP-2. AP-21 maps to a region between amino acids 553 and 645. AP-23 maps between amino acids 645 and 993, whereas AP-20, AP-14, and AP-25 map between amino acids 995 and 1332. Expression of the region of MAP-2 between amino acids 1787 and 1824 was positive to tau 46.     

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.     

Hooks and comets: The story of microtubule polarity orientation in the neuron

It is widely believed that signature patterns of microtubule polarity orientation within axons and dendrites underlie compositional and morphological differences that distinguish these neuronal processes from one another. Axons of vertebrate neurons display uniformly plus-end-distal microtubules, whereas their dendrites display non-uniformly oriented microtubules. Recent studies on insect neurons suggest that it is the minus-end-distal microtubules that are the critical feature of the dendritic microtubule array, whether or not they are accompanied by plus-end-distal microtubules. Discussed in this article are the history of these findings, their implications for the regulation of neuronal polarity across the animal kingdom, and potential mechanisms by which neurons establish the distinct microtubule polarity patterns that define axons and dendrites.     

Maturation of neurites in mixed cultures of spinal cord neurons and muscle cells from Xenopus laevis embryos followed with antibodies to neurofilament proteins

Dissociated cell cultures of Xeopus laevis embryonic spinal cord have proved useful for studying the differentiation of neuronal ionic channel and membrane properties and for examining the dynamics of microtubules in developing neurons. To examine their usefulness for studying neurofilaments in developing neurites, we prepared similar cultures from stage 22 embryos. Between 3 and 55 h after plating, these cultures were fixed and immunostained with antibodies directed against various epitopes of neurofilament proteins from X. Laevis. These antibodies were specific for nonphosphorylated epitopes of the two low molecular weight Xenopus neurofilament proteins (Xenopus NF-L and the Xenopus neuronal intermediate filament protein, XNIF), both phosphorylated and nonphosphorylated epitopes of the Xenopus middle molecular weight neurofilament protein (NF-M), and a nonphosphorylated epitope of the Xenopus high molecular weight neurofilament protein (NF-H). The emergence of these neurofilament proteins in culture was compared to the time course previously reported for them in Xenopus spinal cord neurons in situ. To facilitate the comparison of times in culture to developmental stages, the age of cultured neurons was converted to an equivalent Nieuwkoop and Faber normal stage using data presented here on the effect of changing temperature on developmental rates of X. laevis. With the exception of the nonphosphorylated epitope of NF-H, which is indicative of the most mature axons found in situ. the emergence of the other neurofilament protein antibody epitopes closely paralleled that previously reported for these antibodies in situ. Thus, with respect to XNIF, NF-M, and NF-L, the neurities of cultured neurons were typical of young embryonic Xenopus laevis spinal cord axons. This system should prove useful for studying both the function of these neurofilament proteins during the early stages of axonal development and the dynamics of their transport. 1994 John Wiley & Sons, Inc.     

An evaluation of myelomeres and segmentation of the chick embryo spinal cord.

We have investigated whether the neuromeres of the developing chick spinal cord (myelomeres) are manifestations of intrinsic segmentation of the CNS by studying the patterns of cell proliferation and neuronal differentiation. Treatment of 2-day embryos with colchicine does produce exaggerated myelomeres, in confirmation of K?llén (Z. Anat. Entwickl.-Gesch. 123, 309-319, 1962). However, this does not imply that myelomeres are segmental proliferation centres: the undulations caused by colchicine are irregular alongside the unsegmented mesoderm, and another mitotic inhibitor, bromodeoxyuridine, has no such effects. In contrast to lower vertebrate embryos, there is no evidence for segmental groups of primary motor neurons in the chick: the earliest motor neurons express cholinesterase, and project their axons into the adjacent sclerotome, at random positions in relation to the somite boundaries. The population of motor neurons projecting HRP-labelled axons into a single somite lies out of phase with both myelomere and somite, and is placed symmetrically about the anterior half-sclerotome. The earliest intrinsic spinal cord neurons, as stained with zinc iodide-osmium tetroxide or anti-68 x Mr neurofilament antibody, show no segmental patterns of differentiation. We conclude that, in contrast to the rhombomeres of the developing hindbrain, myelomeres are not matched by segmental groupings of differentiating nerve cells, and result from mechanical moulding of the neuroepithelium by the neighbouring somites.     

Phosphorylation of Microtubule Proteins in Rat Brain at Different Developmental Stages: Comparison with That Found in Neuronal Cultures

The phosphorylation of rat brain microtubule protein on intracranial injection of labeled phosphate has been analyzed. The major microtubule protein components phosphorylated in vivo in rat brain are the high-molecular-weight microtubule-associated proteins (MAPs) MAP-1A, MAP-1B, and MAP-2. A slight phospholabeling of beta-tubulin, which corresponds to the phosphorylation of a minor neuronal beta-tubulin isotype, is also observed. Whereas MAP-1B, MAP-2, and beta-tubulin are phosphorylated in the brain of 5-day-old rat pups, when most neurons of the CNS are extending processes, MAP-1A phosphorylation is observed only after neuronal maturation takes place. The phosphorylation of MAP-1A, MAP-1B, and beta-tubulin may be due mainly to casein kinase II or a related enzyme, whereas MAP-2 appears to be modified by other enzymes such as the cyclic AMP-dependent protein kinase (protein kinase A) and the calcium/phospholipid-dependent protein kinase (protein kinase C). Microtubule protein phosphorylation has also been studied in neuronal cultures. In differentiated neuroblastoma cells, only MAP-1B and beta-tubulin are phosphorylated in a manner coupled to neurite outgrowth. In primary cultures of fetal rat brain neurons, the pattern of microtubule protein phosphorylation resembles that found in vivo in rat pup brain. As phosphorylated MAP-1A and MAP-1B are present mainly on assembled microtubules, whereas the phosphorylation of MAP-2 decreases its interaction with microtubules, a role can be suggested for the phosphorylation of these proteins in the regulation of microtubule assembly and disassembly during neuronal development.     

Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. I. Conditions under which neurons form axons but not dendrites

We have examined the morphology of fetal rat sympathetic neurons grown in serum-free medium in the absence of nonneuronal cells. Because cell density can affect phenotypic expression in vitro, the morphological analysis was subdivided into the study of isolated neurons (neurons whose somata were at least 150 micron from their nearest neighbor) and of more highly aggregated neurons. When isolated neurons were injected with intracellular markers, it was found that most (79%) had a single process emanating from their somata and that this unipolar state persisted for at least 8 weeks in vitro. The processes of unipolar sympathetic neurons had the appearance of axons in that they were thin and long, had a constant diameter, and were relatively unbranched. Cytochemical methods revealed that such processes had other axonal characteristics: (1) they were more reactive with a monoclonal antibody against phosphorylated forms of the M and H neurofilament subunits than with an antibody to nonphosphorylated forms of these proteins; (2) they also reacted with antibodies to the tau microtubule-associated protein and to the phosphorylated forms of the H neurofilament subunit; and (3) they contained only small amounts of RNA as determined by [3H]uridine autoradiography. These data indicate that neurons which normally form dendrites in vivo need not express this capacity in vitro and that axonal and dendritic growth can be dissociated under some conditions in culture. While most isolated neurons were unipolar, neurons in regions of high neuronal cell density were usually multipolar. In addition to axons, multipolar neurons had processes with some of the characteristics expected of rudimentary dendrites: they ended locally (usually within 100 micron), were often highly branched, and reacted with an antibody to nonphosphorylated forms of the M and H neurofilament subunits. The effects of density were most prominent when neurons were within aggregates in which the somata were in close apposition. Density-dependent changes in morphology were less frequently observed when neuronal somata were separated by greater distances (30-100 micron). These data indicate that the morphology of sympathetic neurons is subject to environmental regulation and that neuron-neuron interactions can promote the extension of rudimentary dendrites in vitro.     

Selective stabilization of tau in axons and microtubule-associated protein 2C in cell bodies and dendrites contributes to polarized localization of cytoskeletal proteins in mature neurons

In mature neurons, tau is abundant in axons, whereas microtubule- associated protein 2 (MAP2) and MAP2C are specifically localized in dendrites. Known mechanisms involved in the compartmentalization of these cytoskeletal proteins include the differential localization of mRNA (MAP2 mRNA in dendrites, MAP2C mRNA in cell body, and Tau mRNA in proximal axon revealed by in situ hybridization) (Garner, C.C., R.P. Tucker, and A. Matus. 1988. Nature (Lond.). 336:674-677; Litman, P., J. Barg, L. Rindzooski, and I. Ginzburg. 1993. Neuron. 10:627-638), suppressed transit of MAP2 into axons (revealed by cDNA transfection into neurons) (Kanai, Y., and N. Hirokawa. 1995. Neuron. 14:421-432), and differential turnover of MAP2 in axons vs dendrites (Okabe, S., and N. Hirokawa. 1989. Proc. Natl. Acad. Sci. USA. 86:4127-4131). To investigate whether differential turnover of MAPs contributes to localization of other major MAPs in general, we microinjected biotinylated tau, MAP2C, or MAP2 into mature spinal cord neurons in culture (approximately 3 wk) and then analyzed their fates by antibiotin immunocytochemistry. Initially, each was detected in axons and dendrites, although tau persisted only in axons, whereas MAP2C and MAP2 were restricted to cell bodies and dendrites. Injected MAP2C and MAP2 bound to dendritic microtubules more firmly than to microtubules in axons, while injected tau bound to axonal microtubules more firmly than to microtubules in dendrites. Thus, beyond contributions from mRNA localization and selective axonal transport, compartmentalization of each of the three major MAPs occurs through local differential turnover.     

Novel dendritic kinesin sorting identified by different process targeting of two related kinesins: KIF21A and KIF21B

Neurons use kinesin and dynein microtubule-dependent motor proteins to transport essential cellular components along axonal and dendritic microtubules. In a search for new kinesin-like proteins, we identified two neuronally enriched mouse kinesins that provide insight into a unique intracellular kinesin targeting mechanism in neurons. KIF21A and KIF21B share colinear amino acid similarity to each other, but not to any previously identified kinesins outside of the motor domain. Each protein also contains a domain of seven WD-40 repeats, which may be involved in binding to cargoes. Despite the amino acid sequence similarity between KIF21A and KIF21B, these proteins localize differently to dendrites and axons. KIF21A protein is localized throughout neurons, while KIF21B protein is highly enriched in dendrites. The plus end-directed motor activity of KIF21B and its enrichment in dendrites indicate that models suggesting that minus end-directed motor activity is sufficient for dendrite specific motor localization are inadequate. We suggest that a novel kinesin sorting mechanism is used by neurons to localize KIF21B protein to dendrites since its mRNA is restricted to the cell body.     

Human Skeletal Muscle Cells Synthesise a Neuronotrophic Factor Reactive with Spinal Neurons

Retrograde trophic influences originating in the skeletal musculature have been postulated to be involved in regulating survival and differentiation of embryonic motor neurons and reactive terminal sprouting of mature motor fibres. We have previously described the use of a quantitative immunoassay for neurofilament protein to bioassay in vitro the cell-type-specific neuronotrophic activity of nerve growth factor (NGF) on sensory ganglion neurons. In the present study, the effect of media conditioned by adult human muscle cells (MCM) on the in vitro development of chicken spinal neurons has been studied using a similar approach. Significant increases in neurofilament protein levels in 7-day chicken embryonic spinal cord cultures were found with doses of MCM protein as low as 0.4 microgram/ml, with a dose-response relationship yielding maximal and half-maximal effects at 4 and 1 microgram/ml, respectively. Maximal increases in neurofilament protein levels were associated with an approximate two-fold increase in neuronal cell survival. MCM also induced increases in choline acetyltransferase activity in chick spinal cord cultures. In both the absence and presence of NGF, MCM did not increase neurofilament protein expression in primary cultures of sensory neurons.     

Widespread neuronal expression of branched-chain aminotransferase in the CNS: implications for leucine/glutamate metabolism and for signaling by amino acids

Transamination of the branched-chain amino acids produces glutamate and branched-chain alpha-ketoacids. The reaction is catalyzed by branched-chain aminotransferase (BCAT), of which there are cytosolic and mitochondrial isoforms (BCATc and BCATm). BCATc accounts for 70% of brain BCAT activity, and contributes at least 30% of the nitrogen required for glutamate synthesis. In previous work, we showed that BCATc is present in the processes of glutamatergic neurons and in cell bodies of GABAergic neurons in hippocampus and cerebellum. Here we show that this metabolic enzyme is expressed throughout the brain and spinal cord, with distinct differences in regional and intracellular patterns of expression. In the cerebral cortex, BCATc is present in GABAergic interneurons and in pyramidal cell axons and proximal dendrites. Axonal labeling for BCATc continues into the corpus callosum and internal capsule. BCATc is expressed by GABAergic neurons in the basal ganglia and by glutamatergic neurons in the hypothalamus, midbrain, brainstem, and dorsal root ganglia. BCATc is also expressed in hypothalamic peptidergic neurons, brainstem serotoninergic neurons, and spinal cord motor neurons. The results indicate that BCATc accumulates in neuronal cell bodies in some regions, while elsewhere it is exported to axons and nerve terminals. The enzyme is in a position to influence pools of glutamate in a variety of neuronal types. BCATc may also provide neurons with sensitivity to nutrient-derived BCAAs, which may be important in regions that control feeding behavior, such as the arcuate nucleus of the hypothalamus, where neurons express high levels of BCATc.