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Nearest-neighbor distance of intermediate filaments in axons and Schwann cells. Distinction between axons and schwann cell processes in the denervated and reinnervated peripheral nerves.

To distinguish axons from Schwann cell processes in the denervated (Büngner's bands) and reinnervated peripheral nerves, the nearest-neighbor distance of intermediate filaments (NND) was measured in axons and Schwann cells from denervated and subsequent regenerating peripheral nerves. It was revealed that the NND was much larger in regenerating axons (41.9 +/- 14.1 nm) than in Schwann cell processes (23.1 +/- 7.1 nm in regeneration and 19.7 +/- 5.8 nm in denervation). In addition, the NND was also measured in the normal adult and developing peripheral nerves, and it became clear that in all cases the NND in axons (29.0-41.9 nm) was larger than in Schwann cells (19.7-23.1 nm). Thus, it can be generally considered that the NND is larger in axons than in Schwann cells. This fact can be used for the distinction between axons and Schwann cell processes, when the latter have a profile similar to that of the former as in Büngner's bands and in the regenerating nerves.

Animals↗

Occurrence of long non-myelinated axonal segments intercalated in myelinated, presumably sensory axons: electron microscopic observations in the dog atrial endocardium.

Electron microscopy of serial sections revealed the occurrence of long non-myelinated segments in myelinated, presumably sensory axons running in the left atrial endocardium of normal adult dogs. Four such non-myelinated segments were analysed in three myelinated axons. They varied from 20 to 150 microns in length, and differed from nodes of Ranvier in being invested by Schwann cells in the manner of unmyelinated nerve fibers. Short non-myelinated portions (20-25 microns long) were associated with a single Schwann cell, whereas the longest such segment (150 microns) had five. The non-myelinated axonal segments were non-varicose and similar in diameter (1.2-3.0 microns) to adjacent myelinated segments, which had myelin sheaths 6-25 lamellae thick. The cytoplasm of the non-myelinated axonal segments contained numerous neurofilaments and microtubules, some mitochondria and smooth endoplasmic reticulum. The short non-myelinated segments were enclosed by perineurium, whereas the long non-myelinated segment was devoid of perineurium at its mid-portion; instead fibroblast-like cells made a loose boundary around the axon at this level. The significance of these non-myelinated segments was discussed with special emphasis on the question of whether they result from focal degeneration of the myelin sheath (demyelination) or are generally present in the preterminal regions of some axons.

Animals↗

The paratrigeminal nucleus. II. Identification and inter-relations of catecholamine axons, indoleamine axons, and substance P immunoreactive cells in the neuropil.

The localization of three different putative neurotransmitters -- indoleamine, catecholamine, and Substance P -- was studied in the paratrigeminal nucleus of the rat and rhesus monkey at the light and electron microscope level by autoradiography following administration of [3H]5-hydroxytryptamine, or [3H]norepinephrine, and by immunocytochemistry using the unlabelled anti-Substance P antiserum peroxidase--antiperoxidase technique. The paratrigeminal neurons are not monoaminergic but certain cells exhibit Substance P-like immunoreactivity. These cells receive a rich plexus of indoleamine afferents, a sparse catecholamine input, and a rich plexus of fibres with Substance P-like immunoreactivity. Of the entire monoaminergic population of labelled axons, more than 60% are synaptic and less then 40% nonsynaptic, and this proportion is the same for indoleamines as for catecholamines. Indoleamine axons form a heterogeneous population with at least four different morphological types that are synaptic and three that are nonsynaptic. They bear distinctive collections of small, clear, tubular or large granular vesicles, which distinguish one category of axon from another. These axons engage in numerous axo--somatic, axo--spinous, axo--dendritic, and possibly axo--axonic relations with paratrigeminal neurons. The catecholamine axons are also heterogeneous in axoplasmic morphology but their terminal contacts are distributed to more peripheral portions of dendrites. The significance of the inter-relations between the monaminergic and peptidergic elements in the paratrigeminal nucleus is discussed in relation to the possible functions of this nucleus as a nociceptive, chemosensitive, or pressure-sensitive centre on the lateral medullary surface.

Animals↗

Different kinds of axon terminals forming symmetric synapses with the cell bodies and initial axon segments of layer II/III pyramidal cells. I. Morphometric analysis.

An examination of material prepared for conventional electron microscopy has indicated that there are at least four different types of axon terminals forming symmetric synapses with the cell bodies and initial axon segments of layer II/III pyramidal cells in the rat visual cortex. One type of terminal synapses with the initial axon segment and it is derived from the chandelier cell. Because the location and features of these terminals allow them to be readily recognized, chandelier cell terminals were used to determine the extent of morphometric variability that can exist among terminals originating from one cell type. It was found that there is a wide range of mean synaptic vesicle size among chandelier terminals, so that calculated mean vesicle profile diameters for individual terminals can be between 32 and 39 nm. Similar ranges of mean synaptic vesicle sizes also exist among populations of the other three axon terminal types. These terminal types are referred to as 'large', 'medium-sized', and 'dense' terminals. The large terminals synapse with the cell bodies of layer II/III pyramids and their profiles often measure 1.5 X 0.8 microns. The large terminals contain rather loosely packed pleomorphic vesicles and they frequently synapse with a second neuronal element. The medium-sized terminals are smaller, being 1.0 X 0.6-0.8 microns in size, and their synaptic vesicles are usually more closely packed than those within the large terminals. The medium-sized terminals are the ones encountered most frequently on the cell bodies of pyramidal cells and they can also occur on the axon hillock and initial axon segment. The dense terminals are usually flattened against the cell body, and they contain rather rounded and closely packed synaptic vesicles, which often seem to be enmeshed in a rather dark cytoplasmic matrix. This matrix and the close packing of the vesicles makes these terminals appear to be more dense than the others. It is now necessary to determine the origins of the large, medium and dense terminals, and to ascertain if they all use GABA as their neurotransmitter.

Animals↗

Retrograde transport of horseradish peroxidase in transected axons. 3. Entry into injured axons and subsequent localization in perikaryon.

Horseradish peroxidase (HRP) applied to crushed mouse sciatic nerves diffused through the damaged perineurium into the endoneurium. In the injured area, HRP passed into damaged myelinated and unmyelinated axons forming columns of reaction product, which extended for several millimeters proximally to the lesion. Ultrastructurally, HRP adhered to the inner surface of the axoplasm and to the surfaces of neurotubules and neurofilaments in such columns. At more proximal levels axons contained HRP in vesicular and tubular organelles and, later, nerve cell bodies of the corresponding spinal ganglia showed HRP, accumulation in cytoplasmic vesicles, cup-shaped bodies, multivesicular bodies and tubules of agranular endoplasmic reticulum. Markedly less HRP reached neurons in the spinal ganglia when applied to the nerve 30 or 60 min after the crush. After such time intervals solid HRP containing axons were also less frequently observed. Conceivably, HRP enters crushed axons momentarily after a crush as an injured cell reaction. Subsequently it is incorporated into organelles higher up in the axons, from where retrograde transport to the perikaryon will fellow. This phenomenon of a sudden non-specific influx of exogenous macromolecules into axotomized neurons and their subsequent transport to the perikaryon might be relevant for development of certain biochemical and morphological responses, e.g. lysosomal alterations, of the neuron to an axonal injury.

Animals↗

Axonal transport of taurine along neonatal and young adult rat optic axons.

Studies in this laboratory have indicated that taurine is axonally transported along goldfish optic nerves. In the present experiments the axonal transport of taurine was examined in neonatal and young adult rat optic axons. [35S]taurine was injected into the vitreous humor of right eyes of developing (1--15-day-old) or young adult (40-day-old) rats. At various times after injection ranging from 3 h to 7 days, right retinae and left and right geniculates were removed and assayed for radioactivity, left minus right lateral geniculate (L-RLG) radioactivity being used as an index of axonally transported [35S]taurine. Results indicated that taurine was rapidly transported along both neonatal and young optic axons, in contrast to other amino acids (i.e., leucine and proline) which are not axonally transported in this system. Significant developmental variations were seen in both L-RLG and right retinal [35S]taurine activity 24 h after injection. The amounts of L-RLG [35S]taurine corrected for retinal ganglion cell uptake in animals injected at 1,4,7 and 11 days after birth (prior to and during the major period of synaptogenesis in the geniculates) were 4.5, 3.1, 2.3 and 2.6 times higher, respectively, than those in the young adults. In contrast, the amount of corrected L-RLG [35S]taurine in animals injected at 15 days after birth (after synaptogenesis) were not significantly different from that in the young adult.

Age Factors↗

Morphological evidence that regenerating axons can fuse with severed axon segments.

Regenerating axons of sensory neurons in the leech nerve cord usually reconnect with their normal targets by growing the entire distance from the site of lesion to the target. However, in less than 1% to nearly 10% of cases a rapid restoration of the normal arborization occurs when the regenerating axon connects with the severed distal segment of the same cell or another cell of the same modality. The passage of horseradish peroxidase (mol. wt approximately 40,000 daltons) from the regenerating axon selectively into the axon or cell with which it has connected indicates that the two have joined or fused, rather than become linked by an electrical synapse, as sometimes occurs for other neurons in the leech. These results support the conclusions, based largely on physiological data from regenerating motor axons in crayfish, that unusually rapid and complete regeneration can occur when a growing axon fuses with its severed distal segment.

Animals↗

Assessment of axonal dysfunction in an in vitro model of acute compressive injury to adult rat spinal cord axons.

An in vitro model of spinal cord injury was developed to study the pathophysiology of posttraumatic axonal dysfunction. A 25 mm length of thoracic spinal cord was removed from the adult male rat (n = 27). A dorsal column segment was isolated and pinned in a recording chamber and superfused with oxygenated (95% O2/5% CO2) Ringer. The cord was stimulated with a bipolar electrode, while two point responses were recorded extracellularly. Injury was accomplished by compression with a modified aneurysm clip which applied a 2 g force for 15 s. With injury the compound action potential (CAP) amplitude decreased to 53.7 +/- 5.4% (P < 0.001), while the latency increased to 115.6 +/- 3.1% (P < 0.0025) of control values. The absolute refractory period increased with injury from 1.7 +/- 0.1 ms to 2.1 +/- 0.1 ms (P < 0.05). The infusion of 5 mM 4-aminopyridine (4-AP), a blocker of voltage-sensitive 'fast' K channels confined to internodal regions, resulted in broadening of the CAP of injured axons to 114.9 +/- 3.1% of control (P < 0.05). Ultrastructural analysis of the injured dorsal column segments revealed marked axonal and myelin pathology, including considerable myelin disruption. In conclusion, we have developed and characterized an in vitro model of mammalian spinal cord injury which simulates many of the features of in vivo trauma. Injured axons display characteristic changes in physiological function including a shift in refractory period and high frequency conduction failure. The ultrastructural data and response of injured axons to 4-AP suggest that myelin disruption with exposure of 'fast' K+ channels contributes to posttraumatic axonal dysfunction.

4-Aminopyridine↗

Axon growth from limb motorneurons in the locust embryo: the effect of target limb removal on the pattern of axon branching in the periphery.

Metathoracic limb buds have been unilaterally ablated from locust embryos at 25 to 30% of embryonic development and the effect of this operation on the axon morphology of the motorneuron fast extensor tibiae (FETi) observed at later embryonic stages. In control embryos this neuron sends a single axon out the main leg nerve, nerve 5, to the extensor tibiae muscle in the femur. In limb ablated embryos the axon of FETi is found in a wide variety of aberrant peripheral nerve pathways and projects to a wide range of foreign muscles. There is a degree of apparent selectivity, but no rigid hierarchy, in the choice of pathway and muscle made by FETi. A high degree of variability is found between one embryo and another in the extent and pattern of axon branching. The axon of FETi is generally found in pathways that correspond to nerves in control embryos but on occasion grows along novel routes. An anteriorly directed dendritic branch, seldom seen in control FETi neurons, is frequently seen in experimental FETis. These findings are discussed in terms of the rules for specific axon growth in normal development.

Animals↗

Dimensions and branching patterns of triceps surae alpha-motor axons and their recurrent axon collaterals in the spinal cord during the postnatal development of the cat.

Triceps surae alpha motoneurons in the cat were stained intracellularly with horseradish peroxidase (HRP) at different postnatal ages from birth to the adult stage. The motor axons and axon collaterals were studied with regard to length, diameter and branching pattern. The postnatal increase of internodal length, measured as the distance between two subsequent axon collateral origins, was about 100% which paralleled the total length increase of the main axon in the grey matter. The axon collaterals were unmyelinated at birth and branched exclusively dichotomously until after 3 weeks of age when a substantial fraction of the branching points gave off 3-5 daughter branches. This was interpreted as signs of a fusion between neighboring branching points during the period of myelination of the axon collaterals. The length analysis of the collaterals indicated that the postnatal elimination of collateral branches described previously is preferentially located in the distal parts of the collateral tree.

Animals↗

Axonal maturation in development--II. Immunofluorescence study of rat spinal cord and cerebellum with axon-specific neurofilament antibodies.

Neurofilament monoclonal antibodies derived from mice immunized with chicken brain antigen or purified bovine NF 150K and NF 200K either stained only axons or they stained neuronal perikarya, dendrites and axons. Antibodies in the second group were called conventional because they decorated tissue sections like the neurofibrillary methods of traditional histology. Axon-specific antibodies either reacted with phosphorylated epitopes or they were phosphate/phosphatase insensitive thus suggesting reactivity with post-translational modifications other than phosphorylation. Another possibility was reactivity with phosphorylated epitopes inaccessible to exogenous phosphatases. Conventional neurofilament antibodies stained motor and sensory neurons in day 12 and day 13 rat embryos, respectively, as previously reported with neurofilament antisera. Immunoreactivity with axon-specific antibodies first appeared in motor and sensory axons at different times in development: day 13-14 (3 monoclonals); day 17 (6 monoclonals); day 21 (1 monoclonal); postnatal day 2 (1 monoclonal). There were no major differences between conventional and axon-specific antibodies as to the time of appearance of Purkinje cell baskets in postnatal rat cerebellum. With two exceptions all monoclonals first stained thin baskets on day 11. Immunoreactivity of Purkinje cell baskets with two monoclonals reacting with phosphorylated NF 200K first appeared on days 14 and 20. It is suggested that post-translational modifications may stabilize the neurofilaments, thus accounting for their late appearance by electron microscopy in development.

Animals↗

Bcl-x(L) increases axonal numbers but not axonal elongation from rat retinal explants.

The Bcl-2 family of proteins has been characterized as a key regulator of cell death programs. In addition, these proteins also play important roles in cellular differentiation, such as axonal growth. The role of Bcl-2 family members on axonal regeneration and neurite extension has been controversial so far. Here, we examine the influence of Bcl-x(L) on axonal regeneration from adult retina explants in vitro. We delivered recombinant Bcl-x(L) into retinal tissue, mediated by the Tat-protein transduction domain, and observed its effect on retinal axon extension. We found that Bcl-x(L) increased the number of regenerating neurites, but did not increase their length. Our results indicate that Bcl-x(L) stimulates axonal initiation but not axonal elongation after crush injury to retinal explants, without altering the number of surviving neurons.

Animals↗

Sodium channel blockade with phenytoin protects spinal cord axons, enhances axonal conduction, and improves functional motor recovery after contusion SCI.

Accumulation of intracellular sodium through voltage-gated sodium channels (VGSCs) is an important event in the cascade leading to anatomic degeneration of spinal cord axons and poor functional outcome following traumatic spinal cord injury (SCI). In this study, we hypothesized that phenytoin, a sodium channel blocker, would result in protection of axons with concomitant improvement of functional recovery after SCI. Adult male Sprague-Dawley rats underwent T9 contusion SCI after being fed normal chow or chow containing phenytoin; serum levels of phenytoin were within therapeutic range at the time of injury. At various timepoints after injury, quantitative assessment of lesion volumes, axonal degeneration, axonal conduction, and functional locomotor recovery were performed. When compared to controls, phenytoin-treated animals demonstrated reductions in the degree of destruction of gray and white matter surrounding the lesion epicenter, sparing of axons within the dorsal corticospinal tract (dCST) and dorsal column (DC) system rostral to the lesion site, and within the dorsolateral funiculus (DLF) caudal to the lesion site, and enhanced axonal conduction across the lesion site. Improved performance in measures of skilled locomotor function was observed in phenytoin-treated animals. Based on these results, we conclude that phenytoin provides neuroprotection and improves functional outcome after experimental SCI, and that it merits further examination as a potential treatment strategy in human SCI.

Animals↗

Subsurface cisterna-lined axonal invaginations and double-walled vesicles at the axonal-myelin sheath interface.

The axonal-myelin sheath interface of vertebrate myelinated axons possesses special structural complexities, and there may be an intercellular macromolecular traffic transversing the periaxonal cleft that spans the internodal axon. By conventional electron microscopy and serial sectioning, we observed a category of double-walled vesicles at the axonal-myelin sheath interface, which often contained ribosome-like particles or endoplasmic reticulum. Some of them were demonstrated to continue with the subjacent axon with a thin stalk. In addition, we described a special category of axonal invaginations, probably mediated by subsurface cisternae. The functional implications of these specialized structures were discussed.

Action Potentials↗

The normal human optic nerve. Axon count and axon diameter distribution.

Computerized image analysis was used to determine the normal axonal count and axon diameter distribution in 12 normal human eyes. Mean axon count per nerve was 969,279 +/- 239,740 and mean axon diameter was 0.72 +/- 0.07 micron. Multiple linear regression disclosed 4909 axons lost yearly (P = 0.08). Statistical analysis did not show a relationship between axon diameter and age or time to fixation. The inferotemporal sector of the nerve had the highest fiber density (P = 0.02). The superonasal nerve had higher mean diameters (P = 0.02). This study may provide a baseline for future pathologic studies.

Adolescent↗

Intrinsic properties of the developing motor cortex in the rat: in vitro axons from the medial somatomotor cortex grow faster than axons from the lateral somatomotor cortex.

The axons that originate in the medial somatomotor cortex of the rat depart, during development, after those from the lateral somatomotor cortex, yet they arrive in the cervical spinal cord first. Either the medially originating axons elongate faster, or the laterally originating ones pause along the descent pathway. To investigate the presence of an intrinsic difference of the axonal elongation velocity between the lateral and medial somatomotor cortical areas, we cultured explants taken from these areas for 2 days, and measured the length of the outgrowth. After 2 days the explants were surrounded by a radiate corona of axons of which the longest measured 1.95 mm. A significant difference was detected between the medial and lateral somatomotor cortical areas in vitro. Axons originating from explants taken from the medial somatomotor cortical area are, after 2 days in culture, on average 0.16 mm longer than those from the lateral somatomotor cortical area. Though the observed difference is not large enough to allow for the overtaking observed in vivo, it does indicate that intrinsic differences exist within the developing rat somatomotor cortex. This in turn indicates that intrinsic cortical traits not only influence regionalization and targeting behavior of cortical projection neurons, but also their axonal elongation speed.

Animals↗

Peripheral nerve injury fails to induce growth of lesioned ascending dorsal column axons into spinal cord scar tissue expressing the axon repellent Semaphorin3A.

We have investigated the hypothesis that the chemorepellent Semaphorin3A may be involved in the failure of axonal regeneration after injury to the ascending dorsal columns of adult rats. Following transection of the thoracic dorsal columns, fibroblasts in the dorsolateral parts of the lesion site showed robust expression of Semaphorin3A mRNA. In addition, dorsal root ganglion (DRG) neurons with projections through the dorsal columns to the injury site persistently expressed both Semaphorin3A receptor components, neuropilin-1 and plexin-A1. These ascending DRG collaterals failed to invade scar regions occupied by Semaphorin3A-positive fibroblasts, even in animals which had received conditioning lesions of the sciatic nerve to enhance regeneration. Other axon populations in the dorsal spinal cord were similarly unable to penetrate Semaphorin3A-positive scar tissue. These data suggest that Semaphorin3A may create an exclusion zone for regenerating dorsal column fibres and that enhancing the intrinsic regenerative response of DRG neurons has only limited effects on axonal regrowth. Tenascin-C and chondroitin sulphate proteoglycans were also detected at the injury site, which was largely devoid of central nervous system (CNS) myelin, showing that several classes of inhibitory factors, including semaphorins, with only partially overlapping spatial and temporal patterns of expression are in a position to participate in preventing regenerative axonal growth in the injured dorsal columns. Interestingly, conditioning nerve injuries enabled numerous ascending DRG axons to regrow across areas of strong tenascin-C and chondroitin sulphate proteoglycan expression, while areas containing Semaphorin3A and CNS myelin were selectively avoided by (pre)primed axonal sprouts.

Animals↗

Heparan sulfate proteoglycan-dependent induction of axon branching and axon misrouting by the Kallmann syndrome gene kal-1.

Kallmann syndrome is a neurological disorder characterized by various behavioral and neuroanatomical defects. The X-linked form of this disease is caused by mutations in the KAL-1 gene, which codes for a secreted molecule that is expressed in restricted regions of the brain. Its molecular mechanism of action has thus far remained largely elusive. We show here that expression of the Caenorhabditis elegans homolog of KAL-1 in selected sensory and interneuron classes causes a highly penetrant, dosage-dependent, and cell autonomous axon-branching phenotype. In a different cellular context, heterologous C. elegans kal-1 expression causes a highly penetrant axon-misrouting phenotype. The axon-branching and -misrouting activities require different domains of the KAL-1 protein. In a genetic modifier screen we isolated several loci that either suppress or enhance the kal-1-induced axonal defects, one of which codes for an enzyme that modifies specific residues in heparan sulfate proteoglycans, namely heparan-6O-sulfotransferase. We hypothesize that KAL-1 binds by means of a heparan sulfate proteoglycan to its cognate receptor or other extracellular cues to induce axonal branching and axon misrouting.

Amino Acid Sequence↗