Rate of Movement and Composition of Rapidly Transported Proteins in Regenerating Olfactory Nerve

In a previous study, three successive groups of regenerative fibers, growing initially at 5.8, 2.1, and 0.8 mm/day, were observed in the regenerating garfish olfactory nerve. In the present study, fast axonal transport in the most rapidly regenerating axons (phase I and II) has been examined. Rapid transport in phase I fibers occurs at a velocity of 208 +/- 9 mm/day at 23 degrees, a rate identical to that measured in intact nerves. This first phase of regenerating fibers represents only 3 to 5% of the original axonal population, but each fiber appears to contain 6 to 16 times more transported radioactivity than an axon in an intact nerve. Subcellular distribution of rapidly moving material in phase I and II fibers was closely related to the distribution obtained in intact nerves. Small but significant differences indicate a shift of the transported radioactivity from a heavier to a light axonal membranous fraction. This shift might be characteristic of the immature membrane of a growing axon. The polypeptide distribution of transported radioactivity was also very similar to that of a normal nerve, with most of the radioactivity associated with high-molecular-weight polypeptides.     

Visualizing Peripheral Nerve Regeneration by Whole Mount Staining

Peripheral nerve trauma triggers a well characterised sequence of events both proximal and distal to the site of injury. Axons distal to the injury degenerate, Schwann cells convert to a repair supportive phenotype and macrophages enter the nerve to clear myelin and axonal debris. Following these events, axons must regrow through the distal part of the nerve, re-innervate and finally are re-myelinated by Schwann cells. For nerve crush injuries (axonotmesis), in which the integrity of the nerve is maintained, repair may be relatively effective whereas for nerve transection (neurotmesis) repair will likely be very poor as few axons may be able to cross between the two parts of the severed nerve, across the newly generated nerve bridge, to enter the distal stump and regenerate. Analysing axon growth and the cell-cell interactions that occur following both nerve crush and cut injuries has largely been carried out by staining sections of nerve tissue, but this has the obvious disadvantage that it is not possible to follow the paths of regenerating axons in three dimensions within the nerve trunk or nerve bridge. To try and solve this problem, we describe the development and use of a novel whole mount staining protocol that allows the analysis of axonal regeneration, Schwann cell-axon interaction and re-vascularisation of the repairing nerve following nerve cut and crush injuries.     

Increased regeneration rate in peripheral nerve axons following double lesions: Enhancement of the conditioning lesion phenomenon

The rate of regeneration of rat sciatic nerve sensory axons was measured using the pinch-reflex test method, and confirmed by studying the transport of labelled protein into the regenerating axons. For nerves receiving a single test crush lesion the rate was 4.02 ± 0.03 (SE) mm/day. For nerves with a conditioning lesion made at the knee seven days prior to the test lesion at the hip the rate was 5.73 ± 0.06 mm/day, and for nerves where both conditioning and test lesions were made at the same site (hip or knee) but separated by seven days, the rate was 6.76 ± 0.04 mm/day, a 68% increase over the normal rate, showing that pre-degeneration of the nerve distal to the site of the test lesion increases the rate of regeneration. It is concluded that the rate of axon regeneration can be influenced by the environment through which the regenerating axons grow.     

Regeneration of optic nerve fibers of adult mammals

The pathway from the retina to the brain in mammals provides a well-defined model system for investigation of not only surviving axotomy but also axonal regeneration of injured neurons. Here I introduce our recent works on axonal regeneration in the optic nerve (OpN) of adult cats. Fibers of retinal ganglion cells (RGCs) extend beyond the crush site of OpN with injections of a macrophage stimulator (oxidized galectin-1) or a Rho kinase (ROCK) inhibitor (Y-39983 or Y-27632) while axonal extension is blocked with injection of saline. Elongation of crushed optic fibers, however, is slowed after 2 weeks. Transplantation of peripheral nerve makes RGCs regenerate their transected axons into a graft but regenerated fibers extend only a few mm in the brain. Effectiveness of combination of the drugs and treatments has to be verified in future.     

Temporary colonization of the site of lesion by macrophages is a prelude to the arrival of regenerated axons in injured goldfish optic nerve

Summary. In crushed goldfish optic nerve, regenerating axons cross the site of lesion within 10 days following injury. Some 30 days later, Schwann cells accumulate at the lesion, where they myelinate the new axons. In this study, we have used immunohistochemistry and electron microscopy to examine the cellular environment of the crush site prior to the establishment of Schwann cells in order to learn more about the early events that contribute to axonal regeneration. During the first week following injury, macrophages enter the site of lesion and efficiently phagocytose the debris. The infiltration of macrophages precedes the arrival of regenerating axons that abut and surround these phagocytes. Based on EM morphology and phagocytic capacity, macrophages of the type observed at the site of lesion are not present in the degenerating distal nerve segment, where debris clearance is shared between conventional microglia and astrocytes over a period of several weeks. During this period, axon bundles emerging distally from the injury zone become enwrapped by astrocyte processes, thereby re-establishing the characteristic fascicular cytoarchitecture of the optic nerve. The process of fasciculation also leads to the displacement of myelin debris to the margins of the fiber bundles, where it is trapped by the astrocytes. Our results suggest that the early robust appearance of macrophages at the lesion, and their effectiveness as phagocytes compared with the microglia distally, may contribute to the vigorous axonal regeneration across the crush, beyond which axons<197>excepting the pioneers<197>extend through newly formed debris-free channels delineated by astrocyte processes.     

Proximodistal degeneration of C-fibers detached from their perikarya

Degeneration was followed in the garfish olfactory nerve after removal of the mucosa containing the cell bodies. Degeneration, as measured by a decrease in the weight of consecutive 3-mm nerve segments, spreads at constant velocity from the site of injury toward the synaptic area. The proximodistal degeneration is temperature dependent and progresses from 0.3 mm/d at 10 degrees C to 13.0 mm/d at 35 degrees C. Between 14 and 35 degrees C, the velocity increases linearly with temperature. At all the temperatures investigated, these proximodistal degeneration velocities are identical to the rates of slow intraaxonal flow measured in axons detached from their cell bodies, or to the rates measured in regenerating fibers, and, except at 10 degrees C, are 3.3 times faster than the rate of slow flow in intact nerves. These results were confirmed by light and electron microscopy. We hypothesize that the collapse and subsequent degeneration of the axons is the result of a proximodistal depletion of cytoskeletal elements no longer provided by the cell body to the axon by slow intraaxonal flow. A significant number of axons disappeared rapidly from the nerve before the arrival of the slow degenerative wave. From studies by other groups, this rapid degeneration may be the result of a lack of rapidly transported, mainly membranous components.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by &#110 motor neurons may explain the increase of motor axons counted proximally to the lesion.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by gamma motor neurons may explain the increase of motor axons counted proximally to the lesion.     

Synthesis, axonal transport, and turnover of the high molecular weight microtubule-associated protein MAP 1A in mouse retinal ganglion cells: tubulin and MAP 1A display distinct transport kinetics

Microtubule-associated proteins (MAPs) in neurons establish functional associations with microtubules, sometimes at considerable distances from their site of synthesis. In this study we identified MAP 1A in mouse retinal ganglion cells and characterized for the first time its in vivo dynamics in relation to axonally transported tubulin. A soluble 340-kD polypeptide was strongly radiolabeled in ganglion cells after intravitreal injection of [35S]methionine or [3H]proline. This polypeptide was identified as MAP 1A on the basis of its co-migration on SDS gels with MAP 1A from brain microtubules; its co-assembly with microtubules in the presence of taxol or during cycles of assembly-disassembly; and its cross-reaction with well-characterized antibodies against MAP 1A in immunoblotting and immunoprecipitation assays. Glial cells of the optic nerve synthesized considerably less MAP 1A than neurons. The axoplasmic transport of MAP 1A differed from that of tubulin. Using two separate methods, we observed that MAP 1A advanced along optic axons at a rate of 1.0-1.2 mm/d, a rate typical of the Group IV (SCb) phase of transport, while tubulin moved 0.1-0.2 mm/d, a group V (SCa) transport rate. At least 13% of the newly synthesized MAP 1A entering optic axons was incorporated uniformly along axons into stationary axonal structures. The half-residence time of stationary MAP 1A in axons (55-60 d) was 4.6 times longer than that of MAP 1A moving in Group IV, indicating that at least 44% of the total MAP 1A in axons is stationary. These results demonstrate that cytoskeletal proteins that become functionally associated with each other in axons may be delivered to these sites at different transport rates. Stable associations between axonal constituents moving at different velocities could develop when these elements leave the transport vector and incorporate into the stationary cytoskeleton.     

Changes in neurofilament transport coincide temporally with alterations in the caliber of axons in regenerating motor fibers

The delivery of neurofilaments via axonal transport has been proposed as an important mechanism for regulating axonal caliber. If this hypothesis is correct, alterations in axonal caliber should appear coincident with changes in the delivery of neurofilaments to the axon. The purpose of this study was to determine whether alterations in the caliber of axons in the proximal stumps of transected motor fibers precede, coincide with, or occur substantially later than changes in the delivery of neurofilaments via axonal transport. Between 3 d and 12 wk after crushing the sciatic nerves of 7-wk-old rats, lumbar motor neurons were labeled by the intraspinal injection of [35S]methionine. In neurons labeled between 3 d and 6 wk after axotomy, the relative amount of neurofilament protein in the slow component, as reflected by the ratio of the radioactivities of the 145-kD neurofilament protein to tubulin, was reduced to 30-40% of the control value. Moreover, as determined by immunoreactivity on blots, the amounts of neurofilament protein and tubulin in these nerve fibers were reduced fourfold and twofold, respectively. Thus, changes in the ratio of labeled neurofilament protein to tubulin correlated with comparable changes in the quantities of these proteins in nerve fibers. This decrease in the quantity of neurofilament proteins delivered to axons coincided temporally with reductions in axonal caliber. After regeneration occurred, the delivery of neurofilament proteins returned to pre-axotomy levels (i.e., 8 wk after axotomy), and caliber was restored with resumption of normal age-related radial growth of these axons. Thus, changes in axonal caliber coincided temporally with alterations in the delivery of neurofilament proteins. These results suggest that the majority of neurofilaments in these motor fibers continuously move in the anterograde direction as part of the slow component of axonal transport and that the transport of neurofilaments plays an important role in regulating the caliber of these axons.     

Control of axonal caliber by neurofilament transport

The role of neurofilaments, the intermediate filaments of nerve cells, has been conjectural. Previous morphological studies have suggested a close relationship between neurofilament content and axonal caliber. In this study, the regenerating neuron was used as a model system for testing the hypotheses that neurofilaments are intrinsic determinants of axonal caliber, and that neurofilament content is controlled by the axonal transport of neurofilaments. This system was chosen because previous studies had shown that, after axotomy, axonal caliber was reduced within the proximal stump of the regenerating nerve and, because the relative amount of neurofilament protein undergoing axonal transport in regenerating axons was selectively reduced. The relationship between axonal caliber and neurofilament number was examined in a systematic fashion in both regenerating and control motor axons in rat L5 ventral root. Reconstruction of the spatial and temporal sequences of axonal atrophy in the proximal stump after axotomy showed that reductions in axonal caliber were first detected in the most proximal region of the root and subsequently progressed in a proximal-to-distal direction at a rate of 1.7 mm/day, which is identical to the rate of neurofilament transport in these neurons. Quantitative ultrastructural studies showed that these reductions in caliber correlated with a proportional decrease in the number of axonal neurofilaments but not microtubules. These results support the hypotheses that neurofilament content is a major intrinsic determinant of axonal caliber and that neurofilament content is controlled by the axonal transport of neurofilaments. On this basis, we suggest a role for neurofilaments in the control of axonal volume.     

Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.     

Olfactory nerve fibers

Cross sections of olfactory nerves present a unique appearance. They indicate the presence of large numbers of very small nerve fibers, with a modal diameter of about 0.2 µ and a narrow range for their size variation. From one side of the nasal septum of a pig the yield of fibers was estimated at 6,000,000; the number arising from the turbinates would be considerably larger. The fibers are attached to the membranes of the Schwann sheaths in large bundles through mesaxons longer and more branched than those that have been seen in other nerves. Continuity of the axons between the nerves and the bipolar cells was traced in an examination of the olfactory mucous membrane; and the indication of a one-to-one relationship between cells and axons was reinforced by a comparative count. After the axons leave the bipolar cells they become incased in the central projections of the sustentacular cells. Where the latter come into contact with the basal cells the axons emerge to push back the plasma membranes of the basal cells in the first step in acquiring their nerve sheaths. Later steps are described. When the axons are delivered by the basal cells to the collecting Schwann tubes, they are already aggregated into small bundles with sheaths fundamentally the same as those they will possess until they are delivered to the glia in the olfactory bulb. Some of the aspects of the cytology of the bipolar cells and adjoining sustentacular cells are described. A survey of the physiological properties of olfactory nerve fibers was made in some experiments on the olfactory nerve of the pike. Almost all of the action potential is encompassed within a single elevation, manifesting at its front a conduction velocity of 0.2 m./sec. For a comparison, the last elevation in the C action potential in the sciatic nerve of the frog is cited as an example of conduction at the same velocity. Though expressed through long time constants, the properties of the pike olfactory fibers conform to the generalized schema for properties of vertebrate nerve fibers. This conformity signalizes that they differ from the exceptional properties of the unmedullated fibers of dorsal root origin. An afferent function for unmedullated nerve fibers does not imply that the fibers concerned are alike in their physiological properties.     

Faster nerve regeneration after sciatic nerve injury in mice over-expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF-2) is expressed in the peripheral nervous system and is up-regulated after nerve lesion. It has been demonstrated that administration of FGF-2 protects neurons from injury-induced cell death and promotes axonal regrowth. Using transgenic mice over-expressing FGF-2 (TgFGF-2), we addressed the importance of endogenously generated FGF-2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild-type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF-2. Morphometric evaluation of intact nerves from TgFGF-2 mice revealed no difference in number and size of myelinated fibers compared to wild-type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF-2 over-expression on Schwann cell proliferation during the early regeneration process, we used BrdU-labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild-types. We propose that endogenously synthesized FGF-2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination.     

Continuation of fast axonal transport in regenerating axons in vitro.

A previous study by McLean and co-workers reported that regenerating axons of the rabbit vagus nerve were unable to sustain axonal transport in vitro for several months after nerve injury. In contrast, we found that sensory axons of the rat sciatic nerve were able to transport 3H-labeled protein into their regenerating portions distal to the site of injury within a week after injury when placed in vitro. Transport in vitro was not significantly less than transport in axons maintained in vivo for the same period. Transport occurred in the medium that was used by the McLean group, but was significantly reduced in calcium-free medium. When axon regeneration was delared, only small amounts of activity were present in the nerve distal to the site of injury, showing that labeled protein normally present in that part of the nerve was associated with axons and was not a result of local precursor uptake by nonneural elements in the sciatic nerve. We were not able to explain the failure of McLean and co-workers to demonstrate transport in vitro in regenerating vagus nerve, but we conclude that there is no general peculiarity of growing axons that makes them unable to sustain transport in vitro.     

Foveate vision in deep-sea teleosts: a comparison of primary visual and olfactory inputs

The relative importance of vision in a foveate group of alepocephalid teleosts is examined in the context of a deep-sea habitat beyond the penetration limits of sunlight. The large eyes of Conocara spp. possess deep convexiclivate foveae lined with Müller cells comprising radial shafts of intermediate filaments and horizontal processes. Photoreceptor cell (171.8 x 10(3) rods mm(-2)) and retinal ganglion cell (11.9 x 10(3) cells mm(-2)) densities peak within the foveal clivus and the perifloveal slopes, respectively, with a centro-peripheral gradient between 3:1 (photoreceptors) and over 20:1 (ganglion cells). The marked increase in retinal sampling localized in temporal retina, coupled with a high summation ratio (13:1), suggest that foveal vision optimizes both spatial resolving power and sensitivity in the binocular frontal visual field. The elongated optic nerve head is comprised of over 500 optic papillae, which join at the embryonic fissure to form a thin nervous sheet behind the eye. The optic nerve is divided into two axonal bundles; one receiving input from the fovea (only unmyelinated axons) and the other from non-specialized retinal regions (25% of axons are myelinated), both of which appear to be separated as they reach the visual centres of the central nervous system. Comparison of the number of primary (first-order) axonal pathways for the visual (a total of 63.4 x 10(6) rod photoreceptors) and olfactory (a total of 15.24 x 10(3) olfactory nerve axons) inputs shows a marked visual bias (ratio of 41:1). Coupled with the relative size of the optic tecta (44.0 mm3) and olfactory bulbs (0.9 mm3), vision appears to play a major role in the survival of these deep-sea teleosts and emphasizes that ecological and behavioural strategies account for significant variation in sensory brain structure.     

Target-Dependent Regulation of Retinal Nicotinic Acetylcholine Receptor and Tubulin RNAs During Optic Nerve Regeneration in Goldfish

A fundamental issue in central nervous system development regards the effect of target tissue on the differentiation of innervating neurons. We address this issue by characterizing the role the retinal ganglion cell target, i.e., the optic tectum, plays in regulating expression of tubulin and nicotinic acetylcholine receptor genes in regenerating retinal ganglion cells. Tubulins are involved in axonal growth, whereas nicotinic acetylcholine receptors mediate communication across synapses. Retinal ganglion cell axons were induced to regenerate by crushing the optic nerve. Following crush, there was a rapid increase in alpha-tubulin RNAs (3 days), which preceded the increase in nicotinic acetylcholine receptor RNAs (10-15 days). Both classes of RNAs approached control levels by the time retinotectal synapses and functional recovery were restored (4-6 weeks). If the optic nerve was repeatedly crushed or its target ablated, tubulin RNAs remained elevated, and the increase in receptor RNAs that would otherwise be seen 2 weeks after a single nerve crush did not occur. The interaction of retinal ganglion cell axons with their targets in the optic tectum appears, then, to exert a suppressive effect on the RNA encoding a cytoskeletal protein, tubulin, and an inductive effect on RNAs encoding nicotinic acetylcholine receptors involved in synaptic communication.     

Phosphorylation of Proteins in Normal and Regenerating Goldfish Optic Nerve

Within 6 h after radiolabeled phosphate was injected into the eye of goldfish, labeled acid-soluble and acid-precipitable material began to appear in the optic nerve and subsequently also in the lobe of the optic tectum, to which the optic axons project. From the rate of appearance of the acid-precipitable material, a maximal velocity of axonal transport of 13-21 mm/day could be calculated, consistent with fast axonal transport group II. Examination of individual proteins by two-dimensional gel electrophoresis revealed that approximately 20 proteins were phosphorylated in normal and regenerating nerves. These ranged in molecular weight from approximately 18,000 to 180,000 and in pI from 4.4 to 6.9. Among them were several fast transported proteins, including protein 4, which is the equivalent of the growth-associated protein GAP-43. In addition, there was phosphorylation of some recognizable constituents of slow axonal transport, including alpha-tubulin, a neurofilament constituent (NF), and another intermediate filament protein characteristic of goldfish optic axons (ON2). At least some axonal proteins, therefore, may become phosphorylated as a result of the axonal transport of a phosphate carrier. Some of the proteins labeled by intraocular injection of 32P showed changes in phosphorylation during regeneration of the optic axons. By 3-4 weeks after an optic tract lesion, five proteins, including protein 4, showed a significant increase in labeling in the intact segment of nerve between the eye and the lesion, whereas at least four others (including ON2) showed a significant decrease. When local incorporation of radiolabeled phosphate into the nerve was examined by incubating nerve segments in 32P-containing medium, there was little or no labeling of the proteins that showed changes in phosphorylation during regeneration. Segments of either normal or regenerating nerves showed strong labeling of several other proteins, particularly a group ranging in molecular weight from 46,000 to 58,000 and in pI from 4.9 to 6.4. These proteins were presumably primarily of nonneuronal origin. Nevertheless, if degeneration of the axons had been caused by removal of the eye 1 week earlier, most of the labeling of these proteins was abolished. This suggests that phosphorylation of these proteins depends on the integrity of the optic axons.     

Axonal Transport of Choline Lipids in Normal and Regenerating Rat Sciatic Nerve

Abstract: Biochemical methods were used to study the time course of transport of choline phospholipids (labeled by the injection of [³H]choline into the ventral horn of the lumbar spinal cord) in rat sciatic nerve. Autoradiographic methods were used to localize the transported lipid within motor axons. Transported phospholipid, primarily phosphatidylcholine, present in the nerve at 6 h, continued to accumulate over the following 12 days. No discrete waves of transported lipid were observed (a small wave of radioactive phospholipid moving at the high rate would have been missed); the amounts of radioactive lipid increased uniformly along the entire sciatic nerve. In light-microscope autoradiographs, a class of large-caliber axons, presumably motor axons, retained the labeled lipid. Some lipid, even at 6 h, was seen within the myelin sheaths. Later, the labeling of the myelin relative to axon increased. The continued accumulation of choline phospholipids in the axons probably signifies their prolonged release from cell bodies and their retention in various axonal membranes, including the axolemma. The build-up of these phospholipids in myelin probably represents their transfer from the axons to the myelin sheaths surrounding them. When nerves are crushed and allowed to regenerate for 6 or 12 days, choline phospholipids transported during these times enter the regenerating nerve. In light and electron microscope autoradiographs, transported lipid was seen to be localized primarily in the regenerating axons. However, grains overlay the adjacent Schwann cell cytoplasm, indicating transported lipids were transferred from the regenerating axons to the associated Schwann cells. In addition, some cells not associated with growing axons were labeled, suggesting that phosphatidylcholine and possibly acetylcholine, carried to the regenerating axons by axonal transport, were actively metabolized in the terminal, with released choline label being used by other cells. These results demonstrate that axonal transport supplies mature and growing axons and their glial cells with choline phospholipids.