Rapid arrest of axon elongation by brefeldin A: a role for the small GTP-binding protein ARF in neuronal growth cones.

Members of the ADP-ribosylation factor (ARF) family of small guanosine triphosphate-binding proteins play an essential role in membrane trafficking which subserves constitutive protein transport along exocytic and endocytic pathways within eukaryotic cell bodies. In growing neurons, membrane trafficking within motile growth cones distant from the cell body underlies the rapid plasmalemmal expansion which subserves axon elongation. We report here that ARF is a constituent of axonal growth cones, and that application of brefeldin A to neurons in culture produces a rapid arrest of axon extension that can be ascribed to inhibition of ARF function in growth cones. Our findings demonstrate a role for ARF in growth cones that is coupled tightly to the rapid growth of neuronal processes characteristic of developmental and regenerative axon elongation, and indicate that ARF participates not only in constitutive membrane traffic within the cell body, but also in membrane dynamics within growing axon endings.

S100B, a neurotropic protein that modulates neuronal protein phosphorylation, is upregulated during lesion-induced collateral sprouting and reactive synaptogenesis.

Using light and electron microscopic immunocytochemistry, we examined the expression of the Ca2+-binding protein S100B in the dentate gyrus of adult rats during lesion-induced sprouting and reactive synaptogenesis. Nine days following unilateral lesioning of the entorhinal cortex, S100B was upregulated in cells primarily in the outer part of the molecular layer of the ipsilateral dentate gyrus. When examined with electron microscopy, numerous astrocytes and synapses containing S100B were identified. These data show that during lesion-induced sprouting and reactive synaptogenesis, S100B is upregulated in astrocytes and can be found in pre- and post-synaptic compartments where it might influence neuronal protein phosphorylation.

Up-regulation of fast-axonally transported proteins in retinal ganglion cells of adult rats with optic-peroneal nerve grafts.

Metabolic labeling and quantitative 2D gel fluorography were used to assess changes in the synthesis and transport of five fast-axonally transported and developmentally regulated proteins (GAP-43, SNAP-25, and proteins of 18, 22, and 23/24 kDa) after grafting of a peroneal nerve segment onto a transected optic nerve in adult rats. After optic nerve transection alone, only GAP-43 was up-regulated significantly compared to normal adult controls. The other proteins showed little change or were down-regulated following axotomy. By 4 weeks following optic nerve transection and peroneal nerve grafting, however, GAP-43, proteins 22 and 23/24 kDa showed a sustained up-regulation in synthesis and transport compared to normal controls; SNAP-25 and protein 18 kDa showed levels of expression similar to or slightly greater than normal controls. Increased expression of GAP-43 in retinal ganglion cells was also examined with immunocytochemistry. While a transient up-regulation of GAP-43 in retinal ganglion cells was observed following optic nerve transection, a sustained increase in GAP-43 immunoreactivity was present only in animals with nerve grafts. Backfilling of retinal ganglion cells from the grafts with horseradish peroxidase combined with GAP-43 immunocytochemistry revealed that all retinal ganglion cells with axons growing into the grafts were positive for GAP-43, but not all retinal ganglion cells showing GAP-43 immunoreactivity were extending axons into the grafts. We conclude that the presence of a nerve graft sustains the up-regulation of a number of proteins including GAP-43, and that this up-regulation is correlated with an increased potential for nerve growth, but other as yet unknown factors or conditions appear to play a role in determining if this growth potential will be realized.

Modification of cysteine residues within G(o) and other neuronal proteins by exposure to nitric oxide.

Nitric oxide (NO), a free-radical gas produced endogenously by some neurons, functions as a diffusible intercellular messenger and appears to play a role in activity-dependent modification of synaptic efficacy in the mammalian CNS. The molecular targets and mechanisms of action of NO in neurons remain largely uncharacterized. Employing in vitro brain slices and isolated synaptosomes, we show here that exposure to exogenous or endogenously generated NO results in the modification of cysteine residues within neuronal proteins, as revealed by reduced binding of agents which react with cysteine sulfhydryls. In particular, exposure of synaptosomes to NO inhibits subsequent thiol-linked ADP-ribosylation of the heterotrimeric G-protein, G(o), by pertussis toxin. Our results demonstrate directly that NO may exert its neuronal effects through modification of protein cysteine thiols, and identify G(o) as a potential synaptic target of NO.

Dynamics of retinotectal synaptogenesis in normal and 3-eyed frogs: evidence for the postsynaptic regulation of synapse number.

Quantitative stereological methods were used to determine if the number, density, and types of synaptic connections formed during development are regulated by presynaptic input or by postsynaptic target cells in the optic tectum of normal and 3-eyed Rana pipiens tadpoles and frogs. Our analysis indicates that the number and size of synapses is approximately the same in both tecta of 3-eyed tadpoles and frogs, even though one tectal lobe is receiving input from twice the normal complement of retinal ganglion cells. Moreover, the number and size of synapses in the tectal lobes of 3-eyed animals did not differ significantly from values determined for normal tadpoles and frogs of the same developmental stage. These data suggest strongly that developing tectal cells regulate the number of synaptic contacts they will form. Differences in several morphological features between singly and doubly innervated tecta, however, including synapse density, distribution and complexity, amount of extracellular space, and number of myelin figures, suggest that the presence of supernumerary input retards tectal maturation. We propose that the noncorrelated activity of retinal ganglion cell terminals in the doubly innervated tectum results in fewer stabilized synapses per unit volume of neuropil and in the delayed maturation of the tectal neuropil. Taken together, our data suggest a complex dynamic interaction between retina and tectum during development.

Inhibition of protein kinase C- and casein kinase II-mediated phosphorylation of GAP-43 by S100 beta.

The effect of the glial-derived protein, S100 beta, on the in vitro phosphorylation of the growth-associated protein GAP-43 was investigated. S100 beta inhibited in a dose dependent manner the phosphorylation of GAP-43 by protein kinase C (PKC) or by casein kinase II (CKII). S100 beta appeared to slow down the rate and the degree to which GAP-43 can be phosphorylated by either kinase. The specificity of the inhibition was demonstrated by the observation that the phosphorylation of two other CKII substrates, casein and a selective peptide substrate, was not inhibited by S100 beta. The marked inhibitory effect of S100 beta required the presence of calcium in the phosphorylation reactions. In addition, S100 beta inhibition of GAP-43 phosphorylation was seen with GAP-43 purified under a variety of conditions that alter acylation, suggesting that the acylation state of GAP-43 does not affect the ability of S100 beta to modulate CKII- or PKC-mediated phosphorylation of GAP-43.

Isthmotectal axons make ectopic synapses in monocular regions of the tectum in developing Xenopus laevis frogs.

During the development of binocular maps in the tectum of Xenopus laevis, axons that relay input from the ipsilateral eye via the nucleus isthmi undergo a prolonged period of shifting connections. This shifting accompanies the dramatic change in eye position that takes place as the laterally placed eyes of the tadpole move dorsofrontally. There is a concomitant expansion of the proportion of tectum that receives contralateral retinotectal input corresponding to the binocular portion of the visual field. Electrophysiological recording demonstrates that ipsilateral units are present in those rostral tectal zones, and anatomical methods show that the isthmotectal axons arborize densely in the rostral region but also extend sparser branches into the caudal zone, which is occupied by contralateral inputs with receptive fields in the monocular zone of the visual field. A mechanism that aligns the ipsilateral and contralateral maps is activity-dependent stabilization of isthmotectal axons that exhibit firing patterns correlated with those of nearby retinotectal axons. In order for activity patterns to function in stabilizing correct connections and promoting the withdrawal of incorrect connections, synaptic communication of some sort is hypothesized to be essential. We have investigated whether isthmotectal axons make morphologically identifiable synapses during development and where such synapses are located. We find evidence for morphologically identifiable synapses in all regions of the tectum, along with many growth cones and structures that are probably immature synapses. As in the adult, the synapses contain round, clear vesicles, have asymmetric specializations, and terminate on structures that appear to be dendrites. In both adult and tadpole, the rarity of serial synapses involving isthmotectal terminals suggests that the interactions between retinotectal and isthmotectal inputs are mediated by postsynaptic dendrites.

Synthesis and transport of GAP-43 in entorhinal cortex neurons and perforant pathway during lesion-induced sprouting and reactive synaptogenesis.

Metabolic labeling and quantitative 2D gel autoradiography were used to assess changes in the synthesis and transport of GAP-43 in entorhinal cortex (EC) neurons and perforant pathway during lesion-induced sprouting and reactive synaptogenesis. In normal adult rats, there is a high constitutive level of GAP-43 synthesis and transport in EC neurons projecting to the hippocampus. Following unilateral EC lesions, there is a 2-fold (100%) increase in the transport of newly synthesized GAP-43 to the contralateral or 'sprouting' hippocampus. The timing of this upregulation (between 6 and 15 days) suggests that changes in GAP-43 expression occur in response to the growth of presynaptic terminals during sprouting.

Ultrastructure of the crossed isthmotectal projection in Xenopus frogs.

The nucleus isthmi (NI) of frogs is a relay for input from the eye to the ipsilateral tectum; each NI receives retinotopic input from one tectum and sends retinotopic output to both tecta. The crossed isthmotectal projection in Xenopus displays tremendous plasticity during development. Physiological and anatomical studies have suggested that the location at which a developing isthmotectal axon will terminate is determined by the correlation of its visually evoked activity with the activity of nearby retinotectal terminals. What structures could mediate such communication? We have examined quantitatively the ultrastructural characteristics of crossed isthmotectal axons and synapses in order to determine whether retinotectal axons communicate directly with isthmotectal axons via axo-axonic synapses or whether the communication is indirect, e.g., via common postsynaptic dendrites. Our results support the conclusion that isthmotectal axons interact with retinotectal axons indirectly and that tectal cell dendrites are the critical site of interaction.

Light-microscopic immunolocalization of the growth- and plasticity-associated protein GAP-43 in the developing rat brain.

Growth-associated protein-43 (GAP-43) is a developmentally regulated, fast-axonally transported phosphoprotein whose synthesis and transport are enhanced during periods of growth and synaptic terminal formation. GAP-43 is a substrate of protein kinase C and is identical to protein F1, a phosphoprotein which is regulated during long-term potentiation in the hippocampus. In order to characterize the cellular localization of GAP-43, we have raised a specific antiserum against it, and used this as a probe to show that GAP-43 is neuron-specific, and is localized to growing neuronal processes in developing rat brain, and to presynaptic terminals in both the peripheral and central nervous system. In the mature CNS, GAP-43 immunoreactivity is present in most neuropil areas, but is especially dense in the molecular layers of the cerebellum, neocortex, and the hippocampus, structures known to exhibit synaptic plasticity. Its localization, together with biochemical data concerning the dynamics of its synthesis and its identity as protein F1, suggest that GAP-43 may be involved in axon growth in the developing nervous system, and in some aspect of synaptic plasticity in the mature CNS. These data also suggest that axon growth and synaptic plasticity in the brain may be regulated by a common mechanism, both involving the protein kinase C-mediated phosphorylation of GAP-43.

Evidence for the coidentification of GAP-43, a growth-associated protein, and F1, a plasticity-associated protein.

GAP-43 is a fast-axonally transported protein whose expression correlates with periods of axon growth both during development and during regeneration. Similarities in molecular weight (43-47 kDa), pI (4.3-4.5), and aberrant behavior in acrylamide gels suggested that GAP-43 might be related or identical to protein F1, a protein kinase C substrate that has been shown to undergo a change in phosphorylation state during long-term potentiation in the hippocampus. Here we show that GAP-43 and protein F1 comigrate by two-dimensional PAGE and that antiserum raised against GAP-43 specifically immunoprecipitates protein F1. More direct evidence that GAP-43 and protein F1 are identical proteins was obtained by performing S. aureus V8 protease digests of a mixture of purified 32P-labeled protein F1 and purified GAP-43. Under these conditions, 2 phosphorylated peptide fragments of protein F1 corresponded exactly to 2 Coomassie-stainable bands from purified GAP-43. We conclude on the basis of these data that GAP-43 and protein F1 are identical proteins. Using light-microscopic immunocytochemistry, we also show that GAP-43/protein F1 immunoreactivity is localized to neuropil areas of the hippocampus consistent with its roles as a protein kinase C substrate in vivo and in long-term potentiation. These findings suggest that nerve growth during development and regeneration, and synaptic plasticity in the adult mammalian brain, may be mediated by a common mechanism involving the phosphorylation of GAP-43/protein F1.

A protein induced during nerve growth (GAP-43) is a major component of growth-cone membranes.

Growth cones are specialized structures that form the distal tips of growing axons. During both normal development of the nervous system and regeneration of injured nerves, growth cones are essential for elongation and guidance of growing axons. Developmental and regenerative axon growth is frequently accompanied by elevated synthesis of a protein designated GAP-43. GAP-43 has now been found to be a major component of growth-cone membranes in developing rat brains. Relative to total protein, GAP-43 is approximately 12 times as abundant in growth-cone membranes as in synaptic membranes from adult brains. Immunohistochemical localization of GAP-43 in frozen sections of developing brain indicates that the protein is specifically associated with neuropil areas containing growth cones and immature synaptic terminals. The results support the proposal that GAP-43 plays a role in axon growth.

Nerve injury stimulates the secretion of apolipoprotein E by nonneuronal cells.

Nerve trauma initiates significant changes in the composition of proteins secreted by nonneuronal cells. The most prominent of these proteins is a 37-kDa protein, whose expression correlates with the time course of nerve development, degeneration, and regeneration. We now report that the 37-kDa protein is apolipoprotein E (apoE). We produced a specific antiserum against the 37-kDa protein isolated from previously crushed nerves. This antiserum recognizes a 36-kDa protein in rat serum that we have purified and identified as apoE. The anti-37-kDa antiserum also recognizes apoE on electrophoretic transfer blots of authentic samples of high and very low density lipoproteins. The nerve 37-kDa protein comigrates with apoE by two-dimensional electrophoresis, shares a similar amino acid composition, and reacts with an antiserum against authentic apoE. The purified apoE specifically blocks the immunoprecipitation of [35S]methionine-labeled 37-kDa protein synthesized by nonneuronal cells. Thus, on the basis of its molecular mass, isoelectric point, amino acid composition, and immunological properties, we conclude that the 37-kDa protein is apoE. We also used light microscopic immunohistochemistry to localize apoE following nerve injury. In rats with optic nerve lesions, the 37-kDa antiserum bound specifically to the degenerating optic tracts and to the retino-recipient layers of the lateral geniculate nucleus and the superior colliculus. We propose that apoE is synthesized by phagocytic cells in response to nerve injury for the purpose of mobilizing lipids produced as a consequence of axon degeneration.

Retinotectal synapses formed by ipsilaterally projecting fibers in the doubly innervated goldfish tectum.

When one tectum of an adult goldfish is removed, the severed retinal fibers regenerate ipsilaterally into the remaining tectal lobe. Initially fibers from the two eyes overlap in the tectum but EM-HRP data suggest that few mature retinal synapses are formed between the ipsilateral eye and tectum at this time. At longer time periods, when some fibers appear to segregate into eye-specific termination bands, our data suggest that a significant number of synapses from the ipsilateral eye are present. These findings have important implications for how eye-specific termination bands are formed in doubly innervated tecta.

Localization of alpha-bungarotoxin binding sites to the goldfish retinotectal projection.

The optic tectum of the goldfish Carassius auratus is a rich source of alpha-bungarotoxin (alpha-Btx) binding protein. In order to determine whether some fraction of these receptors is present at retinotectal synapses, we have compared the histological distribution of receptors revealed by the use of [125I]alpha-Btx radioautography to the distribution of optic nerve terminals revealed by the use of cobalt and horseradish peroxidase (HRP) techniques. The majority of alpha-Btx binding is concentrated in those tectal layers containing primary retinotectal synapses. The same layers contain high concentrations of acetylcholinesterase (AChE), revealed histochemically. Following enucleation of one eye, there is a loss of alpha-Btx binding in the contralateral tectum, observed both by radioautography and by a quantitative binding assay of alpha-Btx binding. Approximately 40% of the alpha-Btx binding sites are lost within two weeks following enucleation. By contrast, no significant change in AChE activity could be demonstrated up to 6 months following enucleation. These results are discussed in light of recent studies which show that the alpha-Btx binding protein and the nicotinic acetylcholine receptor are probably identical in goldfish tectum. We conclude that the 3 main classes of retinal ganglion cells projecting to the goldfish tectum are nicotinic cholinergic and that little or no postdenervation hypersensitivity due to receptor proliferation occurs in tectal neurons following denervation of the retinal input.

Some aspects of the organization of the lateral geniculate nucleus in Galago senegalensis revealed by using horseradish peroxidase to label relay neurons.

Following injection of horseradish peroxidase into area 17 of the prosimian Galago senegalensis, columns of labeled neurons are seen in the dorsal lateral geniculate nucleus extending through all cell layers. Individual counts of the number of labeled and unlabeled neurons reveal that from 91 to 98% of all neurons within these densely labeled columns are labeled. These results indicate that most of the neurons within the LGN of the bushbaby project to striate cortex. Average diameter measurements of labeled and unlabeled cells within the labeled columns were used to determine whether cell layers could be separated into two types (parvocellular and magnocellular) or three types (small, medium, and large) on the basis of cell body size. These measurements indicate that the LGN of the bushbaby is composed of two layers each of small (layers 4 and 5), medium (layers 3 and 6), and large (layers 1 and 2) relay neurons. These observations are consistent with the conclusion that layers 1 and 2 in the LGN of Galago are homologous with the magnocellular layers, and layers 3 and 6 homologous with the parvocellular layers, identified in the LGN of New and Old World monkeys.

The identification of relay neurons in the dorsal lateral geniculate nucleus of monkeys using horseradish peroxidase.

Intraaxonal retrograde transport of the protein horseradish peroxidase (HRP) was used to identify relay neurons in the dorsal lateral geniculate nucleus (LGN) of owl (Aotus trivirgatus) and rhesus (Macaca mulatta) monkeys. In both species, from 94.1-98.6% of the neurons within columns extending through both parvocellular and magnocellular layers were labeled following injection of HRP into striate cortex. Labeled neurons were also identified in the thin ventral-most S(0) Layers. Although most of the cells within the thin interlaminar regions in the LGN of both species were labeled following injections of HRP, many unlabeled neurons were identified within the large cell-rich interlaminar region (IL) between the internal parvocellular and internal magnocellular layers in the LGN of the owl monkey, suggesting that IL may be a specialized region containing a large number of intrinsic neurons. Finally, measurement of the cell diameters of neurons within the densely labeled areas in relay layers revealed that labeled and unlabeled neurons could not be distinguished on the basis of cell body size alone and that some of the smallest cells of the LGN project to striate cortex. These findings indicate that nearly all of the neurons of the main relay layers of the LGN in these two primates are relay cells and that the organization of the LGN in primates may differ significantly from that of other mammals with respect to the percentage of interneurons.