Osmotic dilution stimulates axonal outgrowth by making axons more sensitive to tension

Mechanical tension is a potent stimulator of axonal growth rate, which is also stimulated by osmotic dilution. We wished to determine the relationship, if any, between osmotic stimulation and tensile regulation of axonal growth. We used calibrated glass needles to apply constant force to elongate axons of cultured chick sensory neurons. We find that a neurite being pulled at a constant force will grow 50-300% faster following a 50% dilution of inorganic ions in the culture medium. That is, osmotic dilution appears to cause axons to increase their sensitivity to applied tensions. Experimental interventions suggest that this effect is not mediated by dilution of extracellular calcium, or to osmotic stimulation of adenylate cyclase, or to osmotic stimulation of mechanosensitive ion channels. Rather, experiments measuring the static tension normally borne by neurites suggest a direct mechanical effect on the cytoskeletal proteins of the neurite shaft. Our results are consistent with a formal thermodynamic model for axonal growth in which removing a compressive load on axonal microtubules promotes their assembly, thus promoting axonal elongation.     

Neurotrophins support the development of diverse sensory axon morphologies

The initial outgrowth of peripheral axons in developing embryos is thought to occur independently of neurotrophins. However, the degree to which peripheral neurons can extend axons and elaborate axonal arborizations in the absence of these molecules has not been studied directly because of exquisite survival requirements for neurotrophins at early developmental stages. We show here that embryonic sensory neurons from BAX-deficient mice survived indefinitely in the absence of neurotrophins, even in highly dissociated cultures, allowing assessment of cell autonomous axon outgrowth. At embryonic day 11 (E11)-E13, stages of rapid axon growth toward targets in vivo, Bax-/- sensory neurons cultured without neurotrophins were almost invariably unipolar and extended only a rudimentary axon. Addition of neurotrophins caused outgrowth of a second axon and a marked, dose-dependent elongation of both processes. Surprisingly, morphological responses to individual neurotrophins differed substantially. Neurotrophin-3 (NT-3) supported striking terminal arborization of subsets of Bax-/- neurons, whereas NGF produced predominantly axon elongation in a different subset. We conclude that axon growth in vitro is neurotrophin dependent from the earliest stages of sensory neuron development. Furthermore, neurotrophins support the appearance of distinct axonal morphologies that characterize different sensory neuron subpopulations.     

Distinct morphological characteristics of touch, temperature, and mechanical nociceptive neurons in the crotaline trigeminal ganglia

Intrasomal recording and horseradish peroxidase injection techniques were employed in vivo to determine the morphological characteristics of touch, temperature, and mechanical nociceptive neurons in the trigeminal ganglia of crotaline snakes. The touch neurons, with a peripheral axon conducting at the A-beta range, could be subdivided into tactile and vibrotactile neurons according to their response properties, but there were no morphological differences between them. These neurons exhibited a large and oval soma and possessed a set of large stem, peripheral, and central axons which were all myelinated and equal in diameter with a constriction at the bifurcation. The temperature neurons, which conducted peripherally at the A-delta range, were physiologically separated into thermosensitive and thermo-mechanosensitive neurons, which were also morphologically indistinguishable. The temperature neurons had a round soma of medium size and a set of medium axons with varied axonal bifurcation patterns. All axons of these neurons were myelinated, but the central axon was thinner than the stem and peripheral axons. The mechanical nociceptive neurons, which had a peripheral axon conducting at the A-delta range, were morphologically heterogeneous based on their conduction velocities. The neurons conducting at the fast A-delta range were morphologically similar to the temperature neurons in the ganglion excepting their thinner central axons, whereas those at the slow A-delta range had a thinner myelinated stem axon that gave rise to a thinner myelinated peripheral axon and an unmyelinated stem axon with a bifurcation of either a triangular expansion at the bifurcating point or a central axon arising straightforwardly from the constant stem and peripheral axons. This study revealed that distinct morphological characteristics do exist for the touch and temperature neurons and the subtypes of mechanical nociceptive neurons in the trigeminal ganglion, but not for the subfunctional types of touch neurons or temperature neurons.     

Fast axonal transport is required for growth cone advance

Growth cones are capable of advancing despite linkage to a stationary axonal cytoskeleton in chick and murine dorsal root ganglion neurites. Several lines of evidence point to the growth cone as the site of cytoskeletal elongation. Fast axonal transport is probably the means by which cytoskeletal elements or cofactors are rapidly moved through the axon. We report that direct, but reversible, inhibition of fast axonal transport with laser optical tweezers inhibits growth cone motility if cytoskeletal attachment to the cell body is maintained. Advancement ceases after a distance-dependent lag period which correlates with the rate of fast axonal transport. But severing the axonal cytoskeleton with the laser tweezers allows growth cones to advance considerably further. We suggest that axon elongation requires fast axonal transport but growth cone motility does not.     

Effects of drying methods and additives on structure and function of actin: mechanisms of dehydration-induced damage and its inhibition

Neurofilaments are essential for establishment and maintenance of axonal diameter of large myelinated axons, a property that determines the velocity of electrical signal conduction. One prominent model for how neurofilaments specify axonal growth is that the 660-amino acid, heavily phosphorylated tail domain of neurofilament heavy subunit (NF-H) is responsible for neurofilament-dependent structuring of axoplasm through intra-axonal crossbridging between adjacent neurofilaments or to other axonal structures. To test such a role, homologous recombination was used to generate NF-H-null mice. In peripheral motor and sensory axons, absence of NF-H does not significantly affect the number of neurofilaments or axonal elongation or targeting, but it does affect the efficiency of survival of motor and sensory axons. Loss of NF-H caused only a slight reduction in nearest neighbor spacing of neurofilaments and did not affect neurofilament distribution in either large- or small-diameter motor axons. Since postnatal growth of motor axon caliber continues largely unabated in the absence of NF-H, neither interactions mediated by NF-H nor the extensive phosphorylation of it within myelinated axonal segments are essential features of this growth.     

Connectivity and convergence of single corticostriatal axons

The distribution of synapses formed by corticostriatal neurons was measured to determine the average connectivity and degree of convergence of these neurons and to search for spatial inhomogeneities. Two kinds of axonal fields, focal and extended, and two striatal tissue compartments, the patch (striosome) and matrix, were analyzed separately. Electron microscopic examination revealed that both kinds of corticostriatal axons made synapses at varicosities that could be identified in the light microscope, and each varicosity made a single synapse. Thus, the distribution of varicosities was a good estimate of the spatial distribution of synapses. The distance between axonal varicosities was measured to determine the density of synaptic connections formed by one axon within the volume occupied by a striatal neuron. Intersynaptic distances were distributed exponentially, except that synapses were rarely located <4 microm apart. The mean distance between synapses was approximately 10 microm, so axons made a maximum of 40 synapses within the dendritic volume of a spiny neuron. There are approximately 2840 spiny neurons located within the volume of the dendrites of one spiny cell (Oorschot, 1996), so each axon must contact 相似文献   

Transient axonal branching in the developing corpus callosum

During development, there is a transient overproduction of axons in the corpus callosum; this overproduction of axons is due, in part, to a transient excess of neurons that send an axon through the corpus callosum. However, transient axonal branching could also contribute to the developmental overproduction of callosal axons. To investigate this possibility, we filled developing callosal axons in the Syrian hamster with the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil). Light microscopic analysis showed that, indeed, developing callosal axons branch transiently in the hamster: branching was robust on postnatal day 0 (P0) and P3 (P0 = the first 24 hr after birth), less prominent on P6 and P8, and absent by P11. Immature callosal axons branched before or after crossing the midline and at all rostral-caudal and medial-lateral levels within the corpus callosum. The majority of callosal axon collaterals that were contained within individual 100-micron-thick sections were relatively short (mean = 15.1 microns) but some collaterals extended up to approximately 135 microns from the main axon trunk before passing out of the section in which they were observed. Nearly all of the collaterals emanated from the main axon trunk; higher-order collaterals were rare. Some callosal axon trunks had multiple collaterals. Branching callosal axons originated from multiple cortical areas, including area 17. Electron microscopic observations indicated that the processes designated as axon collaterals by light microscopic criteria would have been included in electron microscopic counts of developing callosal axons. Some callosal axon trunks and branches had ultrastructural features that suggested they were degenerating. In cats, developing callosal axons branch on embryonic day 57 (E57; the first 24 hr after conception = E0) and P0. Thus, it is likely that transient branching of immature callosal axons is a generalized feature of mammalian cortical development and that it contributes to the overproduction of callosal axons, albeit perhaps to varying degrees, in multiple species.     

Surround repulsion of spinal sensory axons in higher vertebrate embryos

We have tested whether the orientation of axons sprouting from bipolar dorsal root ganglion neurons is influenced by diffusible cues from surrounding tissues. Surface ectoderm, dermomyotome, and notochord exert strong chemorepulsion on axons growing in collagen gels, operating at separations beyond those found in vivo and active in cocultures of chick and mouse tissues. Basal and alar plates of the neural tube are devoid of activity, as is the posterior-half-sclerotome, which repels in a contact-dependent manner. When ganglia are sandwiched between dermomyotome and notochord placed at a distance, axon growth is channeled in a bipolar trajectory. These results show that gradients of diffusible repulsion molecules flanking axon pathways can generate linear patterns of axon growth. We suggest that such "surround repulsion" may function generally, in concert with contact-dependent guidance mechanisms, to guide axons in the developing nervous system.     

Myelinated sensory and alpha motor axon regeneration in peripheral nerve neuromas

Histochemical staining for carbonic anhydrase and cholinesterase (CE) activities was used to analyze sensory and motor axon regeneration, respectively, during neuroma formation in transected and tube-encapsulated peripheral nerves. Median-ulnar and sciatic nerves in the rodent model permitted testing whether a 4 cm greater distance of the motor neuron soma from axotomy site or intrinsic differences between motor and sensory neurons influenced regeneration and neuroma formation 10, 30, and 90 days later. Ventral root radiculotomy confirmed that CE-stained axons were 97% alpha motor axons. Distance significantly delayed axon regeneration. When distance was negligible, sensory axons grew out sooner than motor axons, but motor axons regenerated to a greater quantity. These results indicate regeneration differences between axon subtypes and suggest more extensive branching of motor axons within the neuroma. Thus, both distance from injury site to soma and inherent motor and sensory differences should be considered in peripheral nerve repair strategies.     

Myelin does not influence the choice behaviour of entorhinal axons but strongly inhibits their outgrowth length in vitro

Myelin is crucial for the stabilization of the entorhinohippocampal projection during late development and is a non-permissive substrate for regrowing axons after lesion in the adult brain. We used two in vitro assays to analyse the impact of myelin on rat entorhinohippocampal projection neurons. A stripe assay was used to study the impact of myelin on the choice behaviour of axons from the entorhinal cortex (EC). Given a choice between alternating hippocampal membrane lanes from developmental stages ranging from early postnatal to adult, EC axons preferred to extend on early postnatal hippocampal membranes. Neither the neutralization of myelin-associated factors by a specific antibody (IN-1) nor the separation of myelin from membranes interfered with the axons' choice behaviour. The entorhinal axons showed no preference in the membrane combination of adult and myelin-free adult hippocampal membranes. These stripe assay experiments demonstrate that support for EC axon choice in the developing hippocampus is maturation-dependent and is not influenced by myelin. The application of IN-1 in the outgrowth assay and the separation of myelin from membranes, enhanced elongation of outgrowing entorhinal axons on adult hippocampal membranes, whereas a control antibody did not. This shows that myelin-associated factors have a strong inhibitory effect on the outgrowth length of entorhinal axons. In conclusion, we suggest that axonal elongation in the entorhinohippocampal system during development is strongly influenced by myelin-associated growth inhibition factors and that specific target finding of entorhinal axons is regulated by a different mechanism.     

Sprouting adult CNS cholinergic axons express NILE and associate with astrocytic surfaces expressing neural cell adhesion molecule

To assess the cellular and molecular substrates for cholinergic axon growth in the adult central nervous system (CNS), we implanted grafts of control and nerve growth factor (NGF)-producing genetically modified fibroblasts within the striatum of rats. Sprouting cholinergic axonal processes that grew into grafts of NGF-producing fibroblasts were fasciculated and followed the surface of astrocytic processes for long distances within the grafts. The close and long distance anatomical relationship between the sprouted axons and the astrocytes supported previous ultrastructural evidence that astrocytes may serve as a cellular substrate for sprouting cholinergic axons in vivo. The sprouted axon processes were associated with the expression of nerve growth factor-inducible large external (NILE) glycoprotein on their surfaces. NILE expression was not seen in control grafts where there was an absence of cholinergic ingrowth. NILE has been demonstrated to play a role in axon fasciculation in a number of other neural systems. The astrocytic processes in both control and NGF-producing fibroblast grafts expressed neural cell adhesion molecule (NCAM), suggesting that NCAM-mediated adhesion may be responsible for the close relationship between the axons and astrocytes within the grafts. NGF-induced heterotypic interactions between neuronal NILE and astroglial NCAM may also be required for adult cholinergic axonal sprouting.     

Working with avoidant children: a clinical challenge

We examined axon-target interactions in cocultures of embryonic rat trigeminal, dorsal root, nodose, superior cervical ganglia or retina with a variety of native or foreign peripheral targets such as the whisker pad, forepaw, and heart explants. Axon growth into these peripheral target tissues was analyzed by the use of lipophilic tracer DiI. Embryonic day 15 dorsal root and trigeminal axons grew into isochronic normal and foreign cutaneous targets. Both axon populations avoided the same age heart tissue, but grew profusely into younger (embryonic day 13) or older (postnatal) heart explants. In contrast, embryonic day 15 superior cervical or nodose ganglion axons grew heavily into the same age heart and forepaw explants and to a lesser extent into the whisker pad explants. Embryonic day 15 retinal axons grew into all three peripheral targets used in this study. Primary sensory and sympathetic axons, but not retinal axons, formed target-specific patterns in the whisker pad and forepaw explants. DiI-labeling and immunostaining of primary sensory neurons in coculture revealed that these neurons retain their bipolar characteristics, and express class-specific markers such as parvalbumin, calcitonin gene-related peptide and TrkA receptors. In the whisker pad explants, axons positive for all three markers were seen to form patterns around the follicles. Our results indicate that developing peripheral targets can attract and support axon growth from a variety of sources. Whereas neurotrophins play a major role in attracting and supporting survival of subpopulations of sensory neurons, other substrate-bound or locally released molecules must regulate sensory neurite growth into specific peripheral and central targets.     

Specific pathway selection by the early projections of individual peripheral sensory neurons in the embryonic medicinal leech

In leech, the central annulus of each midbody segment possesses seven pairs of sensilla, which are mixed clusters of primary peripheral sensory neurons that extend their axons into the CNS where they segregate into distinct fascicles. Pathway selection by individual afferent growth cones of sensillar neurons was examined by double labeling using intracellular dye-filling with antibody labeling in early Hirudo medicinalis embryos. The monoclonal antibody Lan3-2 was used because sensillar neuronal tracts are specifically labeled by this antibody. Examining 68 individually filled neurons we found that sensillar neuron growth cones bifurcate within the CNS, that they project long filopodia capable of sampling the local environment, and that all of them appeared to choose a single particular CNS fascicle without apparent retraction or realignment of growth cones. Furthermore, each side of the bifurcating afferent growth cones always chose the same fascicle, implying a specific choice of a distinct labeled pathway. By dye-filling individual central neurons (P-cells), we show that there are centrally projecting axons present at the time sensillar afferents enter the ganglionic primordia and select a particular fascicle, and we confirm that at least the dorsal peripheral nerve is likely to be pioneered by central neurons, not by the peripheral afferents. In the sensillum studied here, we found examples of sensory neurons extending axons into one of all the available fascicles. Thus, an individual embryonic sensillum possesses a heterogeneous population of afferents with respect to the central fascicle chosen. This is consistent with the idea that segregation into distinct axon fascicles may be based upon functional differences between individual afferent neurons. Our findings argue strongly in favor of specific pathway selection by afferents in this system and are consistent with previous suggestions that there exists a hierarchy of cues, including surface glycoconjugates that mediate navigation of the sensillar growth cones and the fasciculation of their axons.     

Impairment of fast axonal transport in the proximal axons of anterior horn neurons in amyotrophic lateral sclerosis

We studied the possible impairment of fast axonal transport in patients with amyotrophic lateral sclerosis (ALS) to gain some insight into the pathogenesis of the disease. We carried out an ultrastructural investigation of the proximal axons (axon hillock and initial segment) of the anterior horn neurons on samples from 11 ALS patients; specimens from 12 age-matched individuals who died of nonneurological diseases served as controls. Eighty-seven proximal axons that emanated directly from normal-appearing neurons were examined in each group of subjects. Increased smooth endoplasmic reticulum (SER) and the formation of bundles of fibrillary SER with a single unit membrane were not uncommonly observed in the initial segment of the patients with ALS. In some instances, there was loss of the parallel SER arrangement along the longitudinal axis. When viewed in transverse sections, the bundles had a tubular appearance. These morphologic changes of SER were exclusively demonstrated in patients with ALS. A marked increase or accumulation of mitochondria and lysosomes was more common in the proximal axons, particularly in the axon hillock, of ALS patients than of control subjects. The accumulation of these membrane-bounded cytoplasmic organelles suggests that fast axonal transport is impaired in the proximal axons of individuals with ALS. In addition, there were Lewy body-like hyaline inclusions, lipofuscin granules, and multiple membranous structures in the proximal axons. The presence of these unusual structures may also be a reflection of axonal transport dysfunction. By contrast, in the central chromatolytic neurons, there was not only a decrease in the number of neurofilaments in the axon hillock and initial segment, but also of mitochondria, lysosomes, and SER. In some instances, none of these cytoplasmic organelles was seen. These findings support the notion that the outflow of cytoplasmic constituents from the anterior horn cell body into the proximal axon may be impaired in central chromatolytic neurons.     

Suppression of IL-12 production by phosphodiesterase inhibition in murine endotoxemia is IL-10 independent

Limbic system-associated membrane protein (LAMP), a 64-kDa membrane protein, is an axon guidance adhesion molecule expressed by neurons in limbic system-related areas of the CNS. During development, LAMP is expressed on growing axons, growth cones, and their target neurons, but in adults it is restricted to membranes of somata and dendrites. In the adult spinal cord, LAMP immunoreactivity is found only on neurons of lamina II, lamina X, and the intermediolateral cell column and its ultrastructural localization is entirely postsynaptic. We studied changes in the expression of LAMP in lamina II of adult rat spinal cord after L1-S2 dorsal rhizotomy, a procedure that partially deafferents lamina II neurons and induces axonal sprouting by spared systems in lamina II. At the light microscopic level, LAMP immunoreactivity in lamina II was decreased in density at 3, 10, and 60 days postoperatively. This decrease in immunoreactivity suggests that LAMP expression by lamina II neurons may normally be regulated by specific afferent activity. Ultrastructurally, in control lamina II and after deafferentation in both control and deafferented lamina II at 3 and 60 days postoperatively, LAMP expression was restricted to postsynaptic membranes. Ten days after deafferentation, however, when axons are actively sprouting, LAMP was expressed on both axonal and postsynaptic membranes. The reexpression of LAMP on axonal profiles after deafferentation may identify axons that undergo sprouting in response to deafferentation.     

Microtubule transport from the cell body into the axons of growing neurons

The present studies test the hypothesis that microtubules (MTs) are transported from the cell body into the axons of growing neurons. Dissociated sympathetic neurons were cultured using conditions that allow us to control the initiation of axon outgrowth. Neurons were injected with biotin-labeled tubulin (Bt-tub) and then stimulated to extend axons. The newly formed axons were then examined using immunofluorescence procedures for MTs with or without Bt-tub. Because the Bt-tub is fully assembly competent, all MTs that assemble after injection will contain Bt-tub. However, MTs that exist in the neuron at the time of injection and persist during the subsequent incubation will not contain Bt-tub. Because the neurons were injected before extending axons, MTs without Bt-tub are initially localized to the cell body. We specifically determined whether these MTs appeared in the newly formed axon. Such a result can only be explained by the transport of these MTs from their initial location in the cell body into the axon. The newly formed axons of many neurons contained MTs both with and without Bt-tub. MTs without Bt-tub were detected all along the axon and in some neurons represented a substantial portion of the total polymer in the proximal and middle regions of the axon. These results show that MTs are transported from the cell body into growing axons and that this transport is robust, delivering MTs to all regions of the newly formed axon.     

Neurotransmitter secretion along growing nerve processes: comparison with synaptic vesicle exocytosis

In mature neurons, synaptic vesicles continuously recycle within the presynaptic nerve terminal. In developing axons which are free of contact with a postsynaptic target, constitutive membrane recycling is not localized to the nerve terminal; instead, plasma membrane components undergo cycles of exoendocytosis throughout the whole axonal surface (Matteoli et al., 1992; Kraszewski et al., 1995). Moreover, in growing Xenopus spinal cord neurons in culture, acetylcholine (ACh) is spontaneously secreted in the quantal fashion along the axonal shaft (Evers et al., 1989; Antonov et al., 1998). Here we demonstrate that in Xenopus neurons ACh secretion is mediated by vesicles which recycle locally within the axon. Similar to neurotransmitter release at the presynaptic nerve terminal, ACh secretion along the axon could be elicited by the action potential or by hypertonic solutions. We found that the parameters of neurotransmitter secretion at the nerve terminal and at the middle axon were strikingly similar. These results lead us to conclude that, as in the case of the presynaptic nerve terminal, synaptic vesicles involved in neurotransmitter release along the axon contain a complement of proteins for vesicle docking and Ca2+-dependent fusion. Taken together, our results support the idea that, in developing axons, the rudimentary machinery for quantal neurotransmitter secretion is distributed throughout the whole axonal surface. Maturation of this machinery in the process of synaptic development would improve the fidelity of synaptic transmission during high-frequency stimulation of the presynaptic cell.     

Ganglioside synthesis during the development of neuronal polarity. Major changes occur during axonogenesis and axon elongation, but not during dendrite growth or synaptogenesis

Changes in the levels and types of gangliosides occur during neuronal differentiation and development, but no studies have correlated these changes with defined events in neuronal morphogenesis. Here, we have analyzed the relationship between ganglioside synthesis and the development of axons and dendrites in polarized neurons, using hippocampal neurons cultured in such a way that axons and dendrites are generated by a defined sequence of events and in which there is virtually no contamination by glial cells. Neurons were labeled with [4,5-3H]dihydrosphingosine, which was rapidly incorporated into cells and metabolized to 3H-labeled glycosphingolipids. The rate of 3H-labeled glycosphingolipid synthesis was directly proportional to the initial rate of [4,5-3H]dihydrosphingosine uptake and was linear versus time for up to 9 h of incubation. The major changes in 3H-labeled ganglioside synthesis occurred during the period of axonogenesis and rapid axon growth. During axonogenesis, there was a significant increase in the synthesis of complex gangliosides (i.e. GM1, GD1a, GD1b, and GT1b) with a corresponding reduction in the synthesis of glucosylceramide and ganglioside GD3. During the stage of rapid axon growth, the ratio of a- to b-series gangliosides increased significantly. However, during dendritogenesis, dendrite growth, and synaptogenesis, there was little change in ganglioside synthesis, with a small and gradual increase in the ratio of a- to b-series gangliosides and an increase in the synthesis of gangliosides GD1a and GT1b. These results indicate that despite major changes in neuronal morphology and functionality as neurons mature, changes in ganglioside synthesis are restricted to early stages of neuronal development, namely axonogenesis and rapid axon elongation.     

Localized and transient elevations of intracellular Ca2+ induce the dedifferentiation of axonal segments into growth cones

The formation of a growth cone at the tip of a severed axon is a key step in its successful regeneration. This process involves major structural and functional alterations in the formerly differentiated axonal segment. Here we examined the hypothesis that the large, localized, and transient elevation in the free intracellular calcium concentration ([Ca2+]i) that follows axotomy provides a signal sufficient to trigger the dedifferentiation of the axonal segment into a growth cone. Ratiometric fluorescence microscopy and electron microscopy were used to study the relations among spatiotemporal changes in [Ca2+]i, growth cone formation, and ultrastructural alterations in axotomized and intact Aplysia californica neurons in culture. We report that, in neurons primed to grow, a growth cone forms within 10 min of axotomy near the tip of the transected axon. The nascent growth cone extends initially from a region in which peak intracellular Ca2+ concentrations of 300-500 microM are recorded after axotomy. Similar [Ca2+]i transients, produced in intact axons by focal applications of ionomycin, induce the formation of ectopic growth cones and subsequent neuritogenesis. Electron microscopy analysis reveals that the ultrastructural alterations associated with axotomy and ionomycin-induced growth cone formation are practically identical. In both cases, growth cones extend from regions in which sharp transitions are observed between axoplasm with major ultrastructural alterations and axoplasm in which the ultrastructure is unaltered. These findings suggest that transient elevations of [Ca2+]i to 300-500 microM, such as those caused by mechanical injury, may be sufficient to induce the transformation of differentiated axonal segments into growth cones.