Positive injury signals induce growth and prolong survival in Aplysia neurons

Injury to a peripheral nerve initiates changes that can lead to regeneration of the damaged axons. How information about a distant injury is communicated to the cell body is not clear. Using the nervous system of Aplysia californica, we tested the idea that some of this information is conveyed via positive injury signals-axoplasmic proteins that are activated at the injury site and transported to the cell soma. We collected these proteins by crushing pedal nerves and then placing a ligation proximal to the ligation. The contralateral nerves were ligated as controls. Twenty h later, axoplasm was extruded from the nerve segment just distal to the ligation on the crushed nerves (cr/lig) and on the control nerves (lig). The total proteins were rhodaminated and injected into the cytoplasm of neurons in vitro to look for nuclear import. Punctate fluorescence was detected in the nucleus of all seven neurons injected with the cr/lig axoplasm. Only two of five neurons injected with lig axoplasm had any fluorescence. Equal amounts of cr/lig and lig axoplasm were then injected directly into the cell bodies of neurons maintained in vitro. The cells injected with cr/lig axoplasm exhibited renewed growth and significantly longer survival: 25.9 +/- 2.1 days (mean +/- SEM: n = 22) relative to the cells injected with lig axoplasm (15.3 +/- 1.2 days; n = 14) and to those that were not injected (12.2 +/- 1.7 days; n = 24). Fractionation of the cr/lig axoplasm indicated that different factors are responsible for growth and survival.     

Ontogenesis of the myenteric plexus in the cat gastro-intestinal sphincters. II. Axonal differentiation

The differentiation of the axons in the cat myenteric ganglia of the gastro-intestinal sphincters has been examined during pre- and postnatal development. The quantitative analysis has been also used. The differentiation of the axons was a prolonged process that advanced parallel to the maturation of the myenteric nerve perikarya and dendrites. The early fetal period was marked by axonal growth cones. Regardless of the fact that during the development their frequency decreased at the expense of axon varicosities, growth cones were also observed in the first postnatal month. The formation of the axon varicosities was intensive in the late fetal period and in the first weeks after birth. This was judged from the changes in the volume fraction of the varicosities to total neuropil and the number of the varicosities per 100 sp x micrometer of neuropil. The maturation of the varicosities exhibited a longer course which was evident from the changes in the number of the vesicles and in the varicosity area. The cholinergic varicosities differentiated first and most quickly. The so-called p-type varicosities appeared as early as the fetal period, but their number continued to increase after birth. The adrenergic varicosities developed most slowly, which was confirmed by the experiments with 6-OHDA. The axons differentiated with a different speed in the three sphincters examined.     

Cytoplasmic structure in rapid-frozen axons

Turtle optic nerves were rapid-frozen from the living state, fractured, etched, and rotary shadowed. Stereo views of fractured axons show that axoplasm consists of three types of longitudinally oriented domains. One type consists of neurofilament bundles in which individual filaments are interconnected by a cross-bridging network. Contiguous to neurofilament domains are domains containing microtubules suspended in a loose, granular matrix. A third domain is confined to a zone, 80-100 nm wide, next to the axonal membrane and consists of a dense filamentous network connecting the longitudinal elements of the axonal cytoskeleton to particles on the inner surface of the axolemma. Three classes of membrane-limited organelles are distinguished: axoplasmic reticulum, mitochondria, and discrete vesicular organelles. The vesicular organelles must include lysosomes, multivesicular bodies, and vesicles which are retrogradely transported in axons, though some vesicular organelles may be components of the axoplasmic reticulum. Organelles in each class have a characteristic relationship to the axonal cytoskeleton. The axoplasmic reticulum enters all three domains of axoplasm, but mitochondria and vesicular organelles are excluded from the neurofilament bundles, a distribution confirmed in thin sections of cryoembedded axons. Vesicular organelles differ from mitochondria in at least three ways with respect to their relationships to adjacent axoplasm: (a) one, or sometimes both, of their ends are associated with a gap in the surrounding granular axoplasm; (b) an appendage is typically associated with one of their ends; and (c) they are not attached or closely apposed to microtubules. Mitochondria, on the other hand, are only rarely associated with gaps in the axoplasm, do not have an appendage, and are virtually always attached to one or more microtubules by an irregular array of side-arms. We propose that the longitudinally oriented microtubule domains are channels within which organelles are transported. We also propose that the granular material in these channels may constitute the myriad enzymes and other nonfibrous components that slowly move down the axon.     

The effects of nitroprusside and putative agonists on guanylate cyclase activity in squid giant axons

cGMP content of axoplasm from the giant axon of Loligo forbesi was investigated after subjecting the axon to various treatments. Repetitive electrical stimulation or depolarisation by high K+ caused no change in cGMP content. Glutamate and serotonin were also without effect. The nicotinic agonist carbachol (100 microM) increased cGMP levels by 90% (n = 5). A large transient elevation of cGMP content was evoked by external nitroprusside (10 nM-20 microM in intact axons. Nitroprusside injected into both extruded axoplasm and intact axons also increased cGMP content, the stimulation being considerably higher in intact axons where the axolemma was also present. Nitroprusside was also active in axons where the soluble cytoplasmic components were washed out by internal perfusion.     

Transfer of molecules from glia to axon in the squid may be mediated by glial vesicles

Although the transfer of glial proteins into the squid giant axon is well documented, the mechanism of the transfer remains unknown. We examined the possibility that the transfer involved membrane-bound vesicles, by taking advantage of the fact that the fluorescent compound, 3,6-acridinediamine, N,N,N,',N'-tetramethylmonohydride [acridine orange (AO)], rapidly and selectively stains vesicular structures in glial cells surrounding the giant axon. We labeled cleaned axons (1–3 cm long) by incubation for 1 min in filtered seawater (FSW) containing AO. Because the AO was concentrated in glial vesicular organelles, these fluoresced bright orange when the axon was examined by epifluorescence microscopy. To look for vesicle transfer, axoplasm was extruded from such AO-treated axons at various times after labeling. During the initial 15 min, an increasing number of fluorescent vesicles were observed. No further increases were observed between 15 and 60 min post AO. The transfer of the fluorescent vesicles into the axoplasm seemed to be energy dependent, as it was inhibited in axons treated with 2 mM KCN. These results suggest that a special mode of exchange exists between the adaxonal glia and the axon, perhaps involving phagocytosis by the axon of small portions of the glial cells.     

Movement of Bax from the Cytosol to Mitochondria during Apoptosis

Bax, a member of the Bcl-2 protein family, accelerates apoptosis by an unknown mechanism. Bax has been recently reported to be an integral membrane protein associated with organelles or bound to organelles by Bcl-2 or a soluble protein found in the cytosol. To explore Bcl-2 family member localization in living cells, the green fluorescent protein (GFP) was fused to the NH₂ termini of Bax, Bcl-2, and Bcl-X_L. Confocal microscopy performed on living Cos-7 kidney epithelial cells and L929 fibroblasts revealed that GFP–Bcl-2 and GFP–Bcl-X_L had a punctate distribution and colocalized with a mitochondrial marker, whereas GFP–Bax was found diffusely throughout the cytosol. Photobleaching analysis confirmed that GFP–Bax is a soluble protein, in contrast to organelle-bound GFP–Bcl-2. The diffuse localization of GFP–Bax did not change with coexpression of high levels of Bcl-2 or Bcl-X_L. However, upon induction of apoptosis, GFP–Bax moved intracellularly to a punctate distribution that partially colocalized with mitochondria. Once initiated, this Bax movement was complete within 30 min, before cellular shrinkage or nuclear condensation. Removal of a COOH-terminal hydrophobic domain from GFP–Bax inhibited redistribution during apoptosis and inhibited the death-promoting activity of both Bax and GFP– Bax. These results demonstrate that in cells undergoing apoptosis, an early, dramatic change occurs in the intracellular localization of Bax, and this redistribution of soluble Bax to organelles appears important for Bax to promote cell death.     

Development of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder

Postnatal development of the distribution of different isoforms of purinergic (P2X) receptors on smooth muscle cells in relation to the development of the innervation of the cells by nerve varicosities in the rat urinary bladder has been determined with immunofluorescence and confocal microscopy. Antibodies against the extracellular domains of the P2X₁ to P2X₆ receptors were used to detect the receptors in the bladder. Several other antibodies were used to identify sympathetic varicosities and Schwann cells. At one day postnatal (D1) there were few strings of varicosities denoting isolated axons, with most axons confined to large nerve trunks. Small size clusters of P2X₁ to P2X₆ receptor subtypes (about 0.4 µm diameter) were observed in the muscle which were independent of each other, and sometimes juxtaposed to the rare isolated varicosity strings. At D4 large numbers of strings of varicosities could be discerned throughout the detrusor. Most of these clouds of small P2X₁ to P2X₆ receptor clusters in their immediate vicinity. Some of these were colocalised with the varicosities, which were of parasympathetic origin as they failed to counter-stain with antibodies to tyrosine hydroxylase. Up to D14 there was a gradual coalescence of many of the isolated P2X_1–6 small receptor clusters so that they became colocalised, often at varicosities. Most of the varicosities in isolated strings possessed receptor clusters at this time. By D21 it was rare to find varicosity strings in the detrusor that were not either in close juxtaposition with P2X small receptor clusters or possessing such clusters in colocalisation. However, large numbers of small P2X receptor clusters, many of which consisted of a mixture of isoforms, could be found spatially unrelated to nerve varicosities throughout the detrusor muscle. In the adult, single axons were either coextensive with one or more isoforms of P2X receptor clusters or these were immediately juxtaposed to the axons so that is was rare to find a varicosity that did not possess a receptor cluster. However, different combinations of colocalised P2X receptor isoforms could still be discerned in small clusters unrelated to varicosities. These observations are discussed in relation to the mechanism of formation of the receptor clusters and their migration beneath parasympathetic varicosities during development.     

Intracellular control of axial shape in non-uniform neurites: a serial electron microscopic analysis of organelles and microtubules in AI and AII retinal amacrine neurites

AI and AII cat retinal amacrine cells have highly varicose non-uniform, neuritic processes. Processes of both types were reconstructed via a computer system using serial electron micrographs. These reconstructions were analyzed for (a) varicosity volume, surface area, and length, (b) "neck" volume, surface area, and length, (c) number of microtubules within the varicosity, (d) number of microtubules within the "neck," and (e) volume and surface area of mitochondria and smooth endoplasmic reticulum and large smooth vesicular bodies within the processes. Correlation of these parameters revealed a linear relationship between the number of microtubules in the necks and mean neck cross-sectional area (rs = 0.780, P less than 0.001), while microtubule number within the varicosities showed no correlation with varicosity volume (rs = 0.239, P greater than 0.2). Varicosity volume did, however, correlate strongly with the summed volume of mitochondria and smooth vesicular bodies contained within the varicosity for both cell types examined. The ratio between membranous organelle volume and varicosity volume for AI amacrine processes of 1:6.97 (rs = 0.927), differed from the ratio of 1:1.80 for the AII amacrine processes (rs = 0.987). Similar relationships were observed in other nonvaricose neurites such as optic tract axons. Membranous organelles appear to contribute an additional obligatory volume to the cytosol that can be as much as seven times the organelles' direct volume. These observations suggest that both the cytoskeletal components, and the membrane organelles play a direct role in determining neurite shape.     

Tetanic stimulation and cyclic adenosine monophosphate regulate segregation of presynaptic inputs on a common postsynaptic target neuron in vitro

Previous studies indicated that Aplysia sensory neurons (SNs) compete when reestablishing synapses with a motor cell target (1.7) in vitro. The competition is characterized by a cell number-dependent decrease in the efficacy of each connection, an increase in the elimination of SN varicosities, a reduction in the formation of new SN varicosities, and the segregation of varicosities of each SN to restricted portions of the target axons. The changes do not require spike activity, since both the SNs and L7 do not fire spontaneously. Here, we examined whether adding activity to SNs during the early stages of synapse formation with stimuli known to evoke facilitatory responses in stable SN-L7 connections—tetanic stimulation or increase in intracellular cyclic adenosine monophosphate (cAMP)—would modulate the intrinsic segregatory process. Tetanic stimulation to one SN increased synapse efficacy and the number of varicosities of the stimulated SNs while reducing the functional changes by the nonstimulated SNs in the same cultures. An increase in the stability of preexisting varicosities contributed to the overall increase in varicosities evoked by tetanus. The functional changes evoked by tetanus were not expressed when the same tetanic stimulation was also given to the other SN, or when L7 was hyperpolarized during the tetanus to the SN. Raising cAMP levels in one SN increased synapse efficacy and the rate of new varicosity formation by the injected SNs without affecting the development of the connections formed by the noninjected SNs. These results suggest that different forms of presynaptic and postsynaptic activities in neurons can regulate specific aspects of the competitive process associated with the fine-tuning of connections formed by converging presynaptic inputs. © 1996 John Wiley & Sons, Inc.     

Disrupted axo-glial junctions result in accumulation of abnormal mitochondria at nodes of ranvier

Mitochondria and other membranous organelles are frequently enriched in the nodes and paranodes of peripheral myelinated axons, particularly those of large caliber. The physiologic role(s) of this organelle enrichment and the rheologic factors that regulate it are not well understood. Previous studies suggest that axonal transport of organelles across the nodal/paranodal region is locally regulated. In this study, we have examined the ultrastructure of myelinated axons in the sciatic nerves of mice deficient in the contactin-associated protein (Caspr), an integral junctional component. These mice, which lack the normal septate-like junctions that promote attachment of the glial (paranodal) loops to the axon, contain aberrant mitochondria in their nodal/paranodal regions. These mitochondria are typically large and swollen and occupy prominent varicosities of the nodal axolemma. In contrast, mitochondria located outside the nodal/paranodal regions of the myelinated axons appear normal. These findings suggest that paranodal junctions regulate mitochondrial transport and function in the axoplasm of the nodal/paranodal region of myelinated axons of peripheral nerves. They further implicate the paranodal junctions in playing a role, either directly or indirectly, in the local regulation of energy metabolism in the nodal region.     

The presence of transfer RNA in the axoplasm of the squid giant axon

Previous work has revealed that 4S RNA is the primary species of RNA in the axoplasm from the giant axons of the squid and Myxicola. This study shows that axoplasmic 4S RNA from the squid giant axon has the functional properties of tRNA. Axoplasmic RNA was charged with amino acids by aminoacyl-tRNA synthetases prepared from squid brain. The aminoacylation was prevented by incubating the RNA with RNase prior to running the reaction. The amino acid-RNA complex was labile at pH 9, which is characteristic of the acyl linkage between an amino acid and its tRNA. Aminoacyl-tRNA synthetase activity was also present in the axoplasm, primarily in the soluble fraction.     

K+-selective microelectrode study of internally dialyzed squid giant axons.

Intracellular potassium activity, (aK)i, and axoplasmic K+ concentration, [K+]i, were measured by means of K+-selective microelectrodes and atomic absorption spectroscopy, respectively, in squid giant axons dialyzed with K+-free dialysis solution and bathed in K+-free artificial sea water. (aK)i measurements indicated that axoplasmic free K+ could be depleted by dialysis, whereas [K+]i measurements on axoplasm extruded from these axons suggest substantial retention of K+ (15.5 +/- 1.7 mmol/kg axoplasm K+; n = 9). In comparison, [K+]i in axoplasm extruded from freshly dissected axons was 330 +/- 16 mmol/kg axoplasm (n = 6). These data suggest that approximately 5% of the axoplasmic K+ ions are not easily removed by dialysis and that these ions are either bound to macromolecular sites or sequestered into membrane-enclosed organelles.     

A novel pathway regulates thyroid hormone availability in rat and human hypothalamic neurosecretory neurons

Hypothalamic neurosecretory systems are fundamental regulatory circuits influenced by thyroid hormone. Monocarboxylate-transporter-8 (MCT8)-mediated uptake of thyroid hormone followed by type 3 deiodinase (D3)-catalyzed inactivation represent limiting regulatory factors of neuronal T3 availability. In the present study we addressed the localization and subcellular distribution of D3 and MCT8 in neurosecretory neurons and addressed D3 function in their axons. Intense D3-immunoreactivity was observed in axon varicosities in the external zone of the rat median eminence and the neurohaemal zone of the human infundibulum containing axon terminals of hypophysiotropic parvocellular neurons. Immuno-electronmicroscopy localized D3 to dense-core vesicles in hypophysiotropic axon varicosities. N-STORM-superresolution-microscopy detected the active center containing C-terminus of D3 at the outer surface of these organelles. Double-labeling immunofluorescent confocal microscopy revealed that D3 is present in the majority of GnRH, CRH and GHRH axons but only in a minority of TRH axons, while absent from somatostatin-containing neurons. Bimolecular-Fluorescence-Complementation identified D3 homodimers, a prerequisite for D3 activity, in processes of GT1-7 cells. Furthermore, T3-inducible D3 catalytic activity was detected in the rat median eminence. Triple-labeling immunofluorescence and immuno-electronmicroscopy revealed the presence of MCT8 on the surface of the vast majority of all types of hypophysiotropic terminals. The presence of MCT8 was also demonstrated on the axon terminals in the neurohaemal zone of the human infundibulum. The unexpected role of hypophysiotropic axons in fine-tuned regulation of T3 availability in these cells via MCT8-mediated transport and D3-catalyzed inactivation may represent a novel regulatory core mechanism for metabolism, growth, stress and reproduction in rodents and humans.     

Visualization and trafficking of the vesicular acetylcholine transporter in living cholinergic cells

The present experiments investigated the trafficking of the vesicular acetylcholine transporter (VAChT) tagged with the enhanced green fluorescent protein (EGFP) in living cholinergic cells (SN56). The EGFP-VAChT chimera was located in endosomal-like compartments in the soma of SN56 cells, and it was also targeted to varicosities of neurites. In contrast, EGFP alone in cells was soluble in the cytoplasm. The C-terminal cytoplasmic tail of VAChT has been implicated in targeting of VAChT to synaptic vesicles; thus, we have examined the role of the C-terminal region in the trafficking to varicosities. A C-terminal fragment tagged with EGFP appeared to be selectively accumulated in varicosities when expressed in SN56 cells. Interestingly, the protein was not freely soluble in the cytosol, and it presented a punctate pattern of expression. However, EGFP-C terminus did not present this peculiar pattern of expression in a nonneuronal cell line (HEK 293). Moreover, the C-terminal region of VAChT did not seem to be essential for VAChT trafficking, as a construct that lacks the C-terminal tail was, similar to EGFP-VAChT, partially targeted to endocytic organelles in the soma and sorted to varicosities. These experiments visualize VAChT for the first time in living cells and suggest that there might be multiple signals that participate in trafficking of VAChT to sites of synaptic vesicle accumulation.     

THE FINE STRUCTURE OF THE AXON AND GROWTH CONE OF THE DORSAL ROOT NEUROBLAST OF THE RABBIT EMBRYO

The centrally directed neurite of the dorsal root neuroblast has been described from the period of its initial entrance into the neural tube until a well-defined dorsal root is formed. Large numbers of microtubules, channels of agranular reticulum, and clusters of ribosomes are found throughout the length of the early axons. The filopodia of the growth cone appear as long thin processes or as broad flanges of cytoplasm having a finely filamentous matrix material and occasionally small ovoid or elongate vesicles. At first the varicosity is a small expansion of cytoplasm, usually containing channels of agranular reticulum and a few other organelles. The widely dilated cisternae of agranular reticulum frequently found within the growth cone probably correspond to the pinocytotic vacuoles seen in neurites in tissue culture. The varicosities enlarge to form bulbous masses of cytoplasm, which may measure up to 5 µ in width and 13 µ in length. They contain channels of agranular reticulum, microtubules, neurofilaments, mitochondria, heterogeneous dense bodies, and a few clusters of ribosomes. Large ovoid mitochondria having ribonucleoprotein particles in their matrix are common. Dense membrane specializations are found at the basal surface of the neuro-epithelial cell close to the area where the early neurites first enter the neural tube.     

Trafficking of green fluorescent protein tagged-vesicular acetylcholine transporter to varicosities in a cholinergic cell line

Synaptic vesicle proteins are suggested to travel from the trans-Golgi network to active zones via tubulovesicular organelles, but the participation of different populations of endosomes in trafficking remains a matter of debate. Therefore, we generated a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) and studied the localization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP-VAChT is distributed to both early and recycling endosomes in the cell body and is also observed to accumulate in endocytic organelles within varicosities of SN56 cells. GFP-VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with FM4-64 and GFP-Rab5, markers of endocytic vesicles and early endosomes, respectively. A GFP-VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasma membrane in SN56 cells. This endocytosis-defective GFP-VAChT mutant is localized primarily at the somal plasma membrane and exhibits reduced neuritic targeting. Furthermore, the VAChT mutant did not accumulate in varicosities, as did VAChT. Our data suggest that clathrin-mediated internalization of VAChT to endosomes at the cell body might be involved in proper sorting and trafficking of VAChT to varicosities. We conclude that genesis of competent cholinergic secretory vesicles depends on multiple interactions of VAChT with endocytic proteins.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> cyclin-dependent kinase inhibitors are nuclear-localized and show different localization patterns within the nucleoplasm

The Arabidopsis genome contains seven cyclin-dependent kinase (CDK) inhibitors (ICK for inhibitor/interactor with cyclin-dependent kinase) which share a small conserved C-terminal domain responsible for the CDK-inhibition activity by these proteins. Different ICK/KRPs have been shown to have unique expression patterns within tissues, organs and during the cell cycle. Previous studies have shown that overexpressing one of the ICK/KRPs inhibits CDK activity, cell division, and profoundly affects plant growth and development. In this study, we investigated the subcellular localization of the seven Arabidopsis ICK proteins and domains responsible for this localization. Using transgenic expression in Arabidopsis plants and transient expression in tobacco leaf cells, all ICK/KRPs fused to green fluorescent protein (GFP) were localized to the nucleus, suggesting that the nucleus is the cellular compartment for the plant CDK inhibitors to function. While ICK2/KRP2, ICK4/KRP6, and ICK5/KRP7 were localized to the nucleoplasm in a homogeneous manner, ICK1/KRP1, ICK3/KRP5, ICK6/KRP3, and ICK7/KRP4 showed a punctate pattern of localization. A small motif conserved amongst the latter group of ICK/KRPs is required to confer this subcellular pattern as deletion of this motif from ICK7/KRP4 resulted in a shift from a punctate to a homogeneous pattern of localization. While a single nuclear localization signal (NLS) is responsible for the nuclear localization of ICK2/KRP2, multiple mechanisms for nuclear localization are suggested to exist for the other six ICK/KRPs since deletion mutants lacking predicted NLS motifs and the conserved C-terminal domain are still localized in the nucleus.     

Subcellular Localization of Functionally Differentiated Microtubules in Squid Neurons: Regional Distribution of Microtubule-Associated Proteins and β-Tubulin Isotypes

The subcellular localization of microtubule proteins in the neurons of squid (Doryteuthis bleekeri) was immunologically studied using monoclonal antibodies against the microtubule proteins. We found that (1) the squid neurons contained three kinds of high-molecular-weight microtubule-associated proteins [MAP A of approximately 300 kilodaltons (kD), MAP B of 260 kD, and axolinin of 260 kD] and two kinds of beta-tubulin isotypes (beta 1 and beta 2); (2) the cell body of the squid giant neuron contained MAP A, MAP B, and the two beta-tubulin isotypes (beta 1 and beta 2); (3) axolinin and the beta 1 isotype were present exclusively in the peripheral axoplasm of the giant axon; and (4) a small amount of axolinin, MAP A, and the beta 1 isotype was found in the insoluble aspect of the central axoplasm, whereas the soluble aspect of the central axoplasm contained an abundant amount of MAP A along with the modified form of the beta 1 isotype. The regional difference of the distribution of the microtubule protein components may explain the differences in stability among axonal microtubules. Microtubules in the soluble aspect of the central axoplasm are sensitive to any treatment with colchicine, cold temperature, and high ionic strength but those both in the insoluble aspect of the central axoplasm and in the peripheral axoplasm are highly insensitive to the treatment.     

The Fine Structure of Degenerating Nerve Fibers, their Sheaths, and their Terminations in the Central Nerve Cord of the Cockroach (Periplaneta americana)

The connectives above and below the second thoracic ganglion and nerves to and from the mesothoracic leg were severed in Periplaneta americana. Isolated ganglia and severed nerve cord were examined in the electron microscope. In the connectives, sheaths of degenerating fibers remain continuous but become thicker and more dense. There is increase in number and more haphazard disposition of the neuroglial processes which ensheath the axons. The cytoplasm contains vacuoles. Dense droplets normally intercalated between the layers of neuroglial processes ensheathing the axons are strikingly increased in number. The axoplasm with its organelles forms dense clumps. Mitochondria in axons are enlarged, the intramitochondrial matrix is more dense, and the internal folds are disorganized. In ganglia, mitochondrial changes in terminal parts of the axons appear similar to those described in the parent axons in the connective. The synaptic portions of nerve fibers appear very dense. Alterations of the sheath are minimal. Synaptic particles in the degenerating axoplasmic coagulum undergo only slight morphological changes and are still present up to 6 days after severance of their nerve fibers. It is difficult to assess whether there are any alterations in the total number of synaptic particles during degeneration.     

Three-dimensional distribution of TrkA neurotrophin receptors in neurite varicosities of differentiated PC12 cells treated with NGF determined by immunoelectron tomography

Nerve growth factor (NGF) initiates the activation of TrkA tyrosine kinase receptors and numerous subsequent signaling cascades. However, the dynamics of the process including the translocation of TrkA is still unclear. In this study, the effect of NGF or membrane depolarization on the endocytic process and TrkA localization in the neuronal cell line PC12 was analyzed by live-cell imaging and immunoelectron tomography using an ultra-high voltage electron microscope (UHVEM). Both NGF re-stimulation and high potassium-induced depolarization enhanced the endocytic uptake of the fluorescent indicator into acidic organelles within varicosities as well as cell bodies. However, the transition of uptake differed completely. NGF also significantly increased the number of TrkA-containing varicosities. Immunoelectron tomography in whole-mounted cells showed that NGF induced the recruitment of TrkA to the surface membrane of neurite varicosities as well as the multivesicular bodies (MVBs) and lysosomal complexes inside the varicosities. Three-dimensional analysis revealed that invagination pits and intralumenal vesicles of MVBs contained TrkA immunoreactivity. In addition, TrkA immunoreactivity was scattered in the lysosomal matrices after NGF treatment. These results suggest that the neurite varicosities are intensely active in intracellular membrane trafficking, and play an important role in the degradation and accumulation of the NGF receptor, TrkA, after ligand stimulation.