Further studies on the functional properties of spinal axons in vivo

Mammalian spinal tracts in situ demonstrate a phase of marked hyperexcitability during hypoxia or on the application of an excess of potassium or citrate ion. This is in keeping with the fact that they also show post-spike supernormality as well as hyperexcitability under cathodal polarization (17). Behavior of this kind indicates that central axons carry a well developed L fraction of membrane properties. The rhythmic state in central axons in situ, unlike peripheral nerve or spinal root, is not induced by the action of excess potassium ion. This appears to be related to the absence of a positive after-potential in dorsal columns (17). However, sodium citrate can elicit autonomous firing in central axons. When synchronized by an applied stimulus the resulting periodic oscillations have a fundamental frequency (340 to 400 C.P.S.) which is significantly greater than that of peripheral nerve.     

After-potential of spinal axons in vivo

A method is evolved whereby the after-potential sequence intrinsic to longitudinal dorsal column myelinated fibers may be studied in isolation from those events occurring in intact spinal cord subjected to an afferent volley. The recovery sequence intrinsic to dorsal column fibers, after refractoriness is over, is characterized by supernormality approximately four to five times greater than that seen in peripheral nerve. This supemormality averages 15.7 ± 4 per cent (current calibration) at peak and decays exponentially with a half-time of 7.5 msecs. It is not followed by subnormality unless conditioning is repeated more than three times at frequencies greater than 100/sec. Characterization of the recovery curve of dorsal column fibers permits by exclusion, the allocation of the origin of DRV to structures more centrally located. DCV (the dorsal column counterpart of DRV) is seen to exist equally developed in active and passive dorsal column fibers.     

An isoform of ankyrin is localized at nodes of Ranvier in myelinated axons of central and peripheral nerves

Two variants of ankyrin have been distinguished in rat brain tissue using antibodies: a broadly distributed isoform (ankyrinB) that represents the major form of ankyrin in brain and another isoform with a restricted distribution (ankyrinR) that shares epitopes with erythrocyte ankyrin. The ankyrinR isoform was localized by immunofluorescence in cryosections of rat spinal cord gray matter and myelinated central and peripheral nerves to: (a) perikarya and initial axonal segments of neuron cells, (b) nodes of Ranvier of myelinated nerve with no detectable labeling in other areas of the myelinated axons, and (c) the axolemma of unmyelinated axons. Immunogold EM on ultrathin cryosections of myelinated nerve showed that ankyrinR was localized on the cytoplasmic face of the axolemma and was restricted to the nodal and, in some cases, paranodal area. The major isoform of ankyrin in brain (ankyrinB) displayed a broad distribution on glial and neuronal cells of the gray matter and a mainly glial distribution in central myelinated axons with no significant labeling on the axolemma. These results show that (a) ankyrin isoforms display a differential distribution on glial and neuronal cells of the nervous tissue; (b) an isoform of ankyrin codistributes with the voltage-dependent sodium channel in both myelinated and unmyelinated nerve fibers. Ankyrin interacts in vitro with the voltage-dependent sodium channel (Srinivasan, Y., L. Elmer, J. Davis, V. Bennett, and K. Angelides. 1988. Nature (Lond.). 333:177-180). A specific interaction of an isoform of ankyrin with the sodium channel thus may play an important role in the morphogenesis and/or maintenance of the node of Ranvier.     

Regenerating mammalian nerve fibres: changes in action potential waveform and firing characteristics following blockage of potassium conductance

Extracellular application of potassium channel blocking agents is known to increase the amplitude and duration of the compound action potential in non-myelinated and demyelinated axons, but not in mature mammalian myelinated fibres. In the present study we used intra-axonal and whole nerve recording techniques to study the effects of the potassium channel blocking agent 4-aminopyridine (4-AP) on regenerating rat nerve fibres. Our results indicate that early regenerating (premyelinated) axons show considerable broadening of the action potential after 4-AP application and late regenerating (myelinated) axons give rise to burst activity following a single stimulus after 4-AP application. 4-AP did not affect spike waveform or firing properties of normal mature sciatic nerve fibres. These results demonstrate the importance of potassium conductance in stabilizing firing properties of myelinated regenerating axons.     

The Axonal Membrane Protein Caspr, a Homologue of Neurexin IV, Is a Component of the Septate-like Paranodal Junctions That Assemble during Myelination

We have investigated the potential role of contactin and contactin-associated protein (Caspr) in the axonal–glial interactions of myelination. In the nervous system, contactin is expressed by neurons, oligodendrocytes, and their progenitors, but not by Schwann cells. Expression of Caspr, a homologue of Neurexin IV, is restricted to neurons. Both contactin and Caspr are uniformly expressed at high levels on the surface of unensheathed neurites and are downregulated during myelination in vitro and in vivo. Contactin is downregulated along the entire myelinated nerve fiber. In contrast, Caspr expression initially remains elevated along segments of neurites associated with nascent myelin sheaths. With further maturation, Caspr is downregulated in the internode and becomes strikingly concentrated in the paranodal regions of the axon, suggesting that it redistributes from the internode to these sites. Caspr expression is similarly restricted to the paranodes of mature myelinated axons in the peripheral and central nervous systems; it is more diffusely and persistently expressed in gray matter and on unmyelinated axons. Immunoelectron microscopy demonstrated that Caspr is localized to the septate-like junctions that form between axons and the paranodal loops of myelinating cells. Caspr is poorly extracted by nonionic detergents, suggesting that it is associated with the axon cytoskeleton at these junctions. These results indicate that contactin and Caspr function independently during myelination and that their expression is regulated by glial ensheathment. They strongly implicate Caspr as a major transmembrane component of the paranodal junctions, whose molecular composition has previously been unknown, and suggest its role in the reciprocal signaling between axons and glia.     

Effects of calcium lack on action potential of motor axons of the lobster limb

The effects of external calcium deprivation on certain characteristics of the action potential of the lobster motor axon have been studied. Upon exposure to calcium-free solution the spike amplitude is rapidly decreased within a few minutes and is followed by a slow linear decline. The rates of spike rise and fall are proportionally reduced more than the spike but follow similar time courses during calcium lack. Associated with these phenomena are the loss in the normal slow spike repolarization process, the development of a large and lengthy undershoot, and the appearance of a high degree of refractoriness. The mean increase in the refractory period is 525 per cent upon 10 minutes' exposure to calcium-free solution. These effects are completely reversible upon returning the axons to normal solution. These results are compared to similar effects of calcium deprivation on frog myelinated axons and squid and lobster giant axons recently observed by other workers.     

Properties of action potentials in Drosera tentacles

Summary Action potentials of Drosera tentacles resemble those of vertebrate peripheral nerves in that they appear to be comprised of relatively uniform spikes, variable shoulders or negative after-potentials, and variable positive after-potentials. The peaking of the spike corresponds to a period of great refractoriness, while action potentials of low amplitude may be fired readily during the negative after-potential. The action potentials fired during the negative after-potential appear to be unlike those of peripheral nerves in that they are of abnormally brief duration. Also apparently different from the case in peripheral nerves is the dependence of the duration of an action potential on the interval separating it from the preceding action potential.Action potentials propagate from the neck of the stalk to its base at about 5 mm s^-1 at room temperature. Propagation may be reversed artificially, consistent with the possibility that the neuroid cells are electrically coupled.     

The Nodes of Ranvier: Molecular Assembly and Maintenance

Action potential (AP) propagation in myelinated nerves requires clustered voltage gated sodium and potassium channels. These channels must be specifically localized to nodes of Ranvier where the AP is regenerated. Several mechanisms have evolved to facilitate and ensure the correct assembly and stabilization of these essential axonal domains. This review highlights the current understanding of the axon intrinsic and glial extrinsic mechanisms that control the formation and maintenance of the nodes of Ranvier in both the peripheral nervous system (PNS) and central nervous system (CNS).Axons conduct electrical signals, called action potentials (APs), among neurons in a circuit in response to sensory input, and between motor neurons and muscles. In mammals and other vertebrates, many axons are myelinated. Myelin, made by Schwann cells and oligodendrocytes in the peripheral nervous system (PNS) and central nervous system (CNS), respectively, is a multilamellar sheet of glial membrane that wraps around axons to increase transmembrane resistance and decrease membrane capacitance. Although myelin is traditionally viewed as a passive contributor to nervous system function, it is now recognized that myelinating glia also play many active roles including regulation of axon diameter, axonal energy metabolism, and the clustering of ion channels at gaps in the myelin sheath called nodes of Ranvier. Together, the active and passive properties conferred on axons by myelin, result in axons with high AP conduction velocities, low metabolic demands, and reduced space requirements as compared with unmyelinated axons. Thus, myelin and the clustering of ion channels in axons permitted the evolution of the complex nervous systems found in vertebrates. This review highlights the current understanding of the axonal intrinsic and glial extrinsic mechanisms that control the formation and maintenance of the nodes of Ranvier in both the PNS and CNS.     

Conserved roles for Slit and Robo proteins in midline commissural axon guidance

In Drosophila, Slit at the midline activates Robo receptors on commissural axons, thereby repelling them out of the midline into distinct longitudinal tracts on the contralateral side of the central nervous system. In the vertebrate spinal cord, Robo1 and Robo2 are expressed by commissural neurons, whereas all three Slit homologs are expressed at the ventral midline. Previous analysis of Slit1;Slit2 double mutant spinal cords failed to reveal a defect in commissural axon guidance. We report here that when all six Slit alleles are removed, many commissural axons fail to leave the midline, while others recross it. In addition, Robo1 and Robo2 single mutants show guidance defects that reveal a role for these two receptors in guiding commissural axons to different positions within the ventral and lateral funiculi. These results demonstrate a key role for Slit/Robo signaling in midline commissural axon guidance in vertebrates.     

Nerve growth factor-induced growth of sympathetic axons into the optic tract of mature mice is enhanced by an absence of p75NTR expression

Postganglionic sympathetic axons display a remarkable ability for new collateral growth in response to local increases in nerve growth factor (NGF). Elevating NGF levels within the brain also induces the directional growth of sympathetic axons, but not within myelinated pathways of adult mammals. In this investigation, we provide in vivo evidence that sympathetic axons are capable of NGF-induced collateral growth through the microenvironment of mature myelinated pathways, especially in the absence of the p75 neurotrophin receptor (NTR). In transgenic mice overexpressing NGF centrally and expressing p75NTR, only a few varicose sympathetic axons invade the optic tract after the first month of postnatal life. In other transgenic mice overexpressing NGF centrally but lacking p75NTR expression, the incidence of sympathetic axons within this myelinated tract substantially increases. Moreover, numerous unmyelinated sympathetic axons cluster together to form large processes extending through the optic tract; such structures are first seen 8 weeks after birth. Only these large axon bundles display prominent immunostaining for GAP-43, which is preferentially localized to the sympathetic fibers, since nonmyelinating Schwann cells are not associated with these axon bundles. These data provide the first direct evidence that sympathetic axons are indeed capable of NGF-induced collateral growth into myelinated tracts of mature mammals, and that their continued growth through this microenvironment is markedly enhanced by the absence of p75NTR expression. We propose that p75NTR among sympathetic axons may either directly or indirectly limit collateral branching of these fibers in response to increased levels of NGF.     

Conduction along myelinated and demyelinated nerve fibres with a reorganized axonal membrane during the recovery cycle: model investigations

The changes in the excitability of the reorganized axonal membrane in myelinated and demyelinated nerve fibres as well as the causes conditioning such changes have been investigated by paired stimulation during the first 30 ms of the recovery cycle. The variations of the action potential parameters (amplitude and velocity) are traced also. The simulation of the conduction along the normal fiber is based on the Frankenhaeuser and Huxley (1964) and Goldman and Albus (1968) equations, while the demyelination is considered to be an elongation of the nodes of Ranvier. The axonal membrane reorganization is achieved by means of potassium channel blocking and increase of the sodium-channel permeability. It is shown that potassium channels block decreases membrane excitability for the myelinated and demyelinated fibres in the cases of initial and paired stimulation. With increasing sodium-channel permeability on the background of the blocked potassium channels, the membrane excitability is increased. For the fibres with a reorganized membrane, a supernormality of the membrane excitability is obtained, the latter remaining unrecovered during the 30 ms cycle under investigation. The supernormality of the excitability grows from the demyelinated fibre without reorganized membrane to the demyelinated fibre with reorganized one. For short interstimulus intervals, the second action potential propagates along the fibres with a reduced velocity and a decreased amplitude. No supernormality of the potential parameters (amplitude, velocity) is observed during the cycle up to 30 ms. The membrane properties of the myelinated and demyelinated fibres with blocked potassium channels recover in the interval from 15 to 20 ms depending on whether the sodium channels' increase of the permeability is added on the background of the blocked potassium channel or not. In the recovery cycle, the axonal membrane reorganization leads to an improvement of the conduction along most severely demyelinated fibres.     

Individual axons regulate the myelinating potential of single oligodendrocytes in vivo

The majority of axons in the central nervous system (CNS) are eventually myelinated by oligodendrocytes, but whether the timing and extent of myelination in vivo reflect intrinsic properties of oligodendrocytes, or are regulated by axons, remains undetermined. Here, we use zebrafish to study CNS myelination at single-cell resolution in vivo. We show that the large caliber Mauthner axon is the first to be myelinated (shortly before axons of smaller caliber) and that the presence of supernumerary large caliber Mauthner axons can profoundly affect myelination by single oligodendrocytes. Oligodendrocytes that typically myelinate just one Mauthner axon in wild type can myelinate multiple supernumerary Mauthner axons. Furthermore, oligodendrocytes that exclusively myelinate numerous smaller caliber axons in wild type can readily myelinate small caliber axons in addition to the much larger caliber supernumerary Mauthner axons. These data indicate that single oligodendrocytes can myelinate diverse axons and that their myelinating potential is actively regulated by individual axons.     

Nerve growth factor–induced growth of sympathetic axons into the optic tract of mature mice is enhanced by an absence of p75NTR expression

Postganglionic sympathetic axons display a remarkable ability for new collateral growth in response to local increases in nerve growth factor (NGF). Elevating NGF levels within the brain also induces the directional growth of sympathetic axons, but not within myelinated pathways of adult mammals. In this investigation, we provide in vivo evidence that sympathetic axons are capable of NGF‐induced collateral growth through the microenvironment of mature myelinated pathways, especially in the absence of the p75 neurotrophin receptor (NTR). In transgenic mice overexpressing NGF centrally and expressing p75^NTR, only a few varicose sympathetic axons invade the optic tract after the first month of postnatal life. In other transgenic mice overexpressing NGF centrally but lacking p75^NTR expression, the incidence of sympathetic axons within this myelinated tract substantially increases. Moreover, numerous unmyelinated sympathetic axons cluster together to form large processes extending through the optic tract; such structures are first seen 8 weeks after birth. Only these large axon bundles display prominent immunostaining for GAP‐43, which is preferentially localized to the sympathetic fibers, since nonmyelinating Schwann cells are not associated with these axon bundles. These data provide the first direct evidence that sympathetic axons are indeed capable of NGF‐induced collateral growth into myelinated tracts of mature mammals, and that their continued growth through this microenvironment is markedly enhanced by the absence of p75^NTR expression. We propose that p75^NTR among sympathetic axons may either directly or indirectly limit collateral branching of these fibers in response to increased levels of NGF. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 51–66, 1999     

Development of cutaneous innervation in the chick: ultrastructural and quantitative analysis (author's transl)

In the chick, at the thoracic level, the dorsal branches of spinal nerves form at 4 days of incubation (stage 25) and reach the skin between 5 1/2 and 6 days (stages 28-29). At 6 days, the growing nervous peripheral processes ("axons") form large bundles (200-1,000 fibers). At 10 days, young Schwann cells divide the bundles into groups of axons. The perineurium and endoneurium differentiate between 10 and 14 days (but epineurium is formed after hatching). At 14 days of incubation, the adult pattern of cutaneous innervation is established. At this same stage, myelogenesis begins but develops mainly after hatching : 1% of the axons is myelinated at 16 days of incubation, 4% at hatching, 40% in 6-week old chickens and 60% in adults. Thus, less than 10% of myelinated axons of the adult are already myelinated at hatching. Two modes of myelogenesis were observed: 1) early myelination, starting in the embryo around axons measuring about 1 micrometer in diameter: 2) delayed myelination, occurring in the older chickens after an increase in axon diameter. These observations suggest that there is, in the development of chick skin innervation, a critical stage (14-15 days of incubation) apparently corresponding to the stabilization of cutaneous nerve supply.     

Nodes of Ranvier act as barriers to restrict invasion of flanking paranodal domains in myelinated axons

Accumulation of voltage-gated sodium (Na(v)) channels at nodes of Ranvier is paramount for action potential propagation along myelinated fibers, yet the mechanisms governing nodal development, organization, and stabilization remain unresolved. Here, we report that genetic ablation of the neuron-specific isoform of Neurofascin (Nfasc(NF1??)) in vivo results in nodal disorganization, including loss of Na(v) channel and ankyrin-G (AnkG) enrichment at nodes in the peripheral nervous system (PNS) and central nervous system (CNS). Interestingly, the presence of paranodal domains failed to rescue nodal organization in the PNS and the CNS. Most importantly, using ultrastructural analysis, we demonstrate that the paranodal domains invade the nodal space in Nfasc(NF1??) mutant axons and occlude node formation. Our results suggest that Nfasc(NF1??)-dependent assembly of the nodal complex acts as a molecular boundary to restrict the movement of flanking paranodal domains into the nodal area, thereby facilitating the stereotypic axonal domain organization and saltatory conduction along myelinated axons.     

Morphometry and ultrastructure of the squirrel monkey (Saimiri sciureus) vestibular nerve

Vestibular nerves of squirrel monkeys (Saimiri sciureus) embedded in plastics and epoxies were examined with light microscopy (LM) and transmission electron microscopy (TEM), and computerized measures were obtained and analyzed statistically. An average of 12,412 perikarya and 12,005 myelinated nerve fibers was obtained. Approximately 0.7% of the perikarya appeared unmyelinated under LM. About 500 unmyelinated fibers were counted. The cross-sectional area of 1,864 perikarya was 200-650 micron 2. The cross-sectional area of 1,346 nerve fibers was 3-11 micron 2 for the axoplasm and 11-12 micron 2 for the myelin sheath of the same fiber. Myelin thickness was directly proportional to the axoplasm cross-sectional area of the nerve fibers. The cross-sectional area of central axons and peripheral dendrites differed significantly (p less than 0.001). The initial segments of peripheral dendrites were usually smaller, but longer than the initial segments of the central axons. Both initial segments increased in diameter after the first node of Ranvier. Schmidt-Lantermann incisures were more abundant in thick and heavily myelinated fibers than in thin and lightly myelinated fibers. Larger perikarya usually had larger fibers and vice versa, within the first 100-200 micron from the first node of Ranvier. No major ultrastructural differences were found between myelinated and unmyelinated perikarya, except at the hillock region. The Nissl substance was preferentially located in the peripheral cytoplasm.     

The optic nerve in the cat. II. Qualitative aspects of maturation in the adult cat

54 healthy cats were studied. In almost all cases, two types of degenerative signs could be seen: in the primary optic pathways, but also in the posterior funiculi and the spinal-cerebellar tracts, lesions of wide-diameter axons seen by their initial demyelination, accompanied by intense neuroglial reactions, and, on the other hand, a Wallerian degeneration of smaller axons situated in the axial part of the nerve. The percentage of these abnormalities must still be evaluated. Their eventual consequences and nature are discussed.     

Dolphin peripheral visual pathway in chronic unilateral ocular atrophy: Complete decussation apparent

Components of the peripheral visual pathway were examined in two bottlenose dolphins, Tursiops truncatus, each with unilateral ocular degeneration and scarring of 3 or more years' duration. In both animals, the optic nerve associated with the blind eye right eye in Tg419 and left eye in Tt038 had a translucent, gel-like appearance upon gross examination. This translucency was also evident in the optic tract contralateral to the affected eye. In Tg419, myelinated axons of varying diameters were apparent in the left optic nerve, whereas the right optic nerve, serving the blind eye, appeared to be devoid of axons. In Tt038, myelinated axons were associated with the right optic nerve (serving the functional eye) and left optic tract but were essentially absent in the left optic nerve and right optic tract. Examined by light microscopy in serial horizontal sections, the optic chiasm of Tt038 was arranged along its central plane in segregated, alternating pathways for the decussation of right and left optic nerve fibers. Ventral to this plane, the chiasm was comprised of fibers from the left optic nerve, whereas dorsal to the central plane, fibers derived from the right optic nerve. Because of this architectural arrangement, the right and left optic nerves grossly appeared to overlap as they crossed the optic chiasm with the right optic nerve coursing dorsally to the left optic nerve. At the light and electron microscopic levels, the optic nerves and tracts lacking axons were well vascularized and dominated by glial cell bodies and glial processes, an expression of the marked glial scarring associated with postinjury axonal degeneration. The apparent absence of axons in one of the optic tract pairs (right in Tt038 and left in Tg419) supports the concept of complete decussation of right and left optic nerve fibers at the optic chiasm in the bottlenose dolphin. © 1994 Wiley-Liss, Inc.     

Low level impedance changes following the spike in the squid giant axon before and after treatment with "veratrine" alkaloids

The increase in conductance, which accompanies the spike in the presence of sea water, is followed by a decrease to below the resting level, here designated as the "initial after-impedance," which lasts 3 msec. and is 3 per cent as great as the increase. Treatment with cevadine usually obliterates the latter but leaves the former essentially unaltered. In addition, the alkaloid gives rise to periodic conductance increases followed by a prolonged, exponentially decaying elevated conductance (the "negativity after-impedance") which correspond closely to potential oscillations and to the negative after-potential. These are also only a few per cent of the major conductance change. Veratridine causes a conductance increase which lasts longer and which also conforms closely with earlier after-potential results. Preliminary calculations indicate that the negativity after-impedance and the negative after-potential may be due to the subsidence of an elevated chloride permeability. However, no satisfactory explanation is available for the initial after-impedance or for the temporal course of the conductance changes associated with oscillations in membrane potential.