Fenestration in the myelin sheath of nerve fibers of the shrimp: A novel node of excitation for saltatory conduction

Giant nerve fibers of the shrimp family Penaeidae conduct impulses at the velocity highest among all animal species (∼210 m/s; highest in mammals = 120 m/s). We examined these giant and other small nerve fibers morphologically using a differential interference contrast microscope as well as an electron microscope, and found a very specialized form of excitable membrane that functions as a node for saltatory conduction of the impulse. This node appeared under the light microscope as a characteristic pattern of concentrically aligned rings in a very small spot of the myelin sheath. The diameter of the innermost ring of the node was about 5 μm, and the distance between these nodes was as long as 12 mm. Via an electron microscope, these nodes were characterized by a complete lack of the myelin sheath, forming a fenestration that has a tight junction with an axonal membrane. Voltage clamp measurements by a sucrose gap technique demonstrated that the axonal membrane at these fenestration nodes is exclusively excitable and that the large submyelinic space is a unique conductive pathway for loop currents for saltatory conduction through such fenestration nodes. © 1996 John Wiley & Sons, Inc.     

Aspects of the protein and the lipid composition of myelinoid marchi-positive bodies from mammalian spinal cord

The fraction floating on 0.32 M sucrose was isolated from normal mammalian spinal cord and analyzed with regard to protein and lipid composition. Comparisons were made with the myelin fraction isolated from the same spinal cord. A close relationship between the two fractions was indicated by a similar protein banding on SDS-polyacrylamide gel electrophoresis. The relative amounts of various proteins however were different and some high molecular weight proteins appeared unique to the floating fraction. The phospho- and galactolipid patterns, as revealed by thin-layer chromatography, were similar in the floating and the myelin fractions. The proportion of hydrophobic lipids, such as sterols and isoprenyl derivatives, was higher in the floating fraction. Bands co-migrating with cholesterol esters were detected only in the floating fraction from guinea pigs. Marchi-positive material of possible paranodal origin is enriched in the floating fraction. The present findings of a biochemical composition of the floating fraction closely resembling that of myelin is in line with the view that myelin turnover includes a step of degradation localized to the paranodal regions.     

Soluble and bound acid protease activity of myelin from bovine cerebral white matter and spinal cord

Isolated myelin of bovine spinal cord was found to degrade exogenous myelin basic protein (MBP) at pH 4.4. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Some of the proteolytic activity was soluble at increased ionic strength, some remained bound, withstanding extraction at 37°C for up to 12 hr. While being measurable with exogenous MBP, bound protease degraded neither bound MBP nor any other major intrinsic myelin protein. Both soluble and bound protease activity was completely inhibited by pepstatin A. The patterns of limited proteolysis of MBP they produced were identical. Myelin of cerebral white matter also exhibited soluble and bound acid protease activity which was likewise inhibited by pepstatin A. Protease activity of spinal cord and cerebral myelin is therefore suggested to be due to a cathepsin D-like endopeptidase, present in a loosely and tightly bound form. Both forms increased by 50 to 80% in activity when myelin was isolated from mixtures of white and cortical gray matter. While increased soluble activity of myelin is consistent with binding of cathepsin D of lysosomal origin during the isolation of myelin the tightly bound form might point to a principal mechanism through which exogenous proteins may become attached to the myelin sheath in vivo.     

Specific vulnerability of mouse spinal cord motoneurons to membrane depolarization

Intracellular calcium (Ca²⁺) concentration determines neuronal dependence on neurotrophic factors (NTFs) and susceptibility to cell death. Ca²⁺ overload induces neuronal death and the consequences are thought to be a probable cause of motoneuron (MN) degeneration in neurodegenerative diseases. In the present study, we show that membrane depolarization with elevated extracellular potassium (K⁺) was toxic to cultured embryonic mouse spinal cord MNs even in the presence of NTFs. Membrane depolarization induced an intracellular Ca²⁺ increase. Depolarization-induced toxicity and increased intracellular Ca²⁺ were blocked by treatment with antagonists to some of the voltage-gated Ca²⁺ channels (VGCCs), indicating that Ca²⁺ influx through these channels contributed to the toxic effect of depolarization. Ca²⁺ activates the calpains, cysteine proteases that degrade a variety of substrates, causing cell death. We investigated the functional involvement of calpain using a calpain inhibitor and calpain gene silencing. Pre-treatment of MNs with calpeptin (a cell-permeable calpain inhibitor) rescued MNs survival; calpain RNA interference had the same protective effect, indicating that endogenous calpain contributes to the cell death caused by membrane depolarization. These findings suggest that MNs are especially vulnerable to extracellular K⁺ concentration, which induces cell death by causing both intracellular Ca²⁺ increase and calpain activation.     

Kir4.1 channels regulate swelling of astroglial processes in experimental spinal cord edema

In glial cells, inwardly rectifying K⁺ channels (Kir) control extracellular [K⁺]_o homeostasis by uptake of K⁺ from the extracellular space and release of K⁺ into the microvasculature. Kir channels were also recently implicated in K⁺-associated water influx and cell swelling. We studied the time-dependent expression and functional implication of the glial Kir4.1 channel for astroglial swelling in a spinal cord edema model. In this CNS region, Kir4.1 is expressed on astrocytes from the second postnatal week on and co-localizes with aquaporin 4 (AQP4). Swelling of individual astrocytes in response to osmotic stress and to pharmacological Kir blockade were analyzed by time-lapse-two-photon laser-scanning microscopy in situ . Application of 30% hypotonic solution induced astroglial soma swelling whereas no swelling was observed on astroglial processes or endfeet. Co-application of hypotonic solution and Ba²⁺, a Kir channel blocker, induced prominent swelling of astroglial processes. In Kir4.1−/− mice, however, somatic as well as process swelling was observed upon application of 30% hypotonic solutions. No additional effect was provoked upon co-application with Ba²⁺. Our experiments show that Kir channels prevent glial process swelling under osmotic stress. The underlying Kir channel subunit that controls glial process swelling is Kir4.1, whereas changes of the glial soma are not substantially related to Kir4.1.     

Role of peroxynitrite in secondary oxidative damage after spinal cord injury

Peroxynitrite (PON, ONOO(-)), formed by nitric oxide synthase-generated nitric oxide radical ( NO) and superoxide radical (O(2) (-)), is a crucial player in post-traumatic oxidative damage. In the present study, we determined the spatial and temporal characteristics of PON-derived oxidative damage after a moderate contusion injury in rats. Our results showed that 3-nitrotyrosine (3-NT), a specific marker for PON, rapidly accumulated at early time points (1 and 3 h) and a significant increase compared with sham rats was sustained to 1 week after injury. Additionally, there was a coincident and maintained increase in the levels of protein oxidation-related protein carbonyl and lipid peroxidation-derived 4-hydroxynonenal (4-HNE). The peak increases of 3-NT and 4-HNE were observed at 24 h post-injury. In our immunohistochemical results, the co-localization of 3-NT and 4-HNE results indicates that PON is involved in lipid peroxidative as well as protein nitrative damage. One of the consequences of oxidative damage is an exacerbation of intracellular calcium overload, which activates the cysteine protease calpain leading to the degradation of several cellular targets including cytoskeletal protein (alpha-spectrin). Western blot analysis of alpha-spectrin breakdown products showed that the 145-kDa fragments of alpha-spectrin, which are specifically generated by calpain, were significantly increased as soon as 1 h following injury although the peak increase did not occur until 72 h post-injury. The later activation of calpain is most likely linked to PON-mediated secondary oxidative impairment of calcium homeostasis. Scavengers of PON, or its derived free radical species, may provide an improved antioxidant neuroprotective approach for the treatment of post-traumatic oxidative damage in the injured spinal cord.     

Acrolein induces axolemmal disruption, oxidative stress, and mitochondrial impairment in spinal cord tissue

Acrolein, a byproduct of oxidative stress and lipid peroxidation, has been implicated in neurodegenerative disorders such as Alzheimer's disease, but not in spinal cord trauma, as a possible key factor in neuronal degeneration. Using an isolated guinea pig spinal cord model, we have found that acrolein, in a dose- and time-dependent manner, inflicts severe membrane disruption, a factor thought to be critical in triggering axonal deterioration and cell death. The concentration threshold of such detrimental effect is shown to be around 1 microM when acrolein was exposed for 4 h. The membrane damage is likely mediated in part by reactive oxygen species and lipid peroxidation, which were elevated in response to acrolein exposure. Antioxidants were able to significantly reduce acrolein-mediated membrane disruption which further supports the role of reactive oxygen species in the loss of membrane integrity. Mitochondrial function was also impaired after acrolein exposure which not only implicates but emphasizes the role of this organelle in reactive oxygen species generation. In summary, our data strongly suggest that at a clinically relevant concentration, acrolein can severely compromise membrane integrity and may further serve as an initiating toxin triggering secondary injury cascades following the initial physical insult to the spinal cord.     

Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase

Despite its growing use as a radiological indicator of neuronal viability, the biological function of N-acetylaspartate (NAA) has remained elusive. This is due in part to its unusual metabolic compartmentalization wherein the synthetic enzyme occurs in neuronal mitochondria whereas the principal metabolizing enzyme, N-acetyl-L-aspartate amidohydrolase (aspartoacylase), is located primarily in white matter elements. This study demonstrates that within white matter, aspartoacylase is an integral component of the myelin sheath where it is ideally situated to produce acetyl groups for synthesis of myelin lipids. That it functions in this manner is suggested by the fact that myelin lipids of the rat optic system are well labeled following intraocular injection of [14C-acetyl]NAA. This is attributed to uptake of radiolabeled NAA by retinal ganglion cells followed by axonal transport and transaxonal transfer of NAA into myelin, a membrane previously shown to contain many lipid synthesizing enzymes. This study identifies a group of myelin lipids that are so labeled by neuronal [14C]NAA, and demonstrates a different labeling pattern from that produced by neuronal [14C]acetate. High performance liquid chromatographic analysis of the deproteinated soluble materials from the optic system following intraocular injection of [14C]NAA revealed only the latter substance and no radiolabeled acetate, suggesting little or no hydrolysis of NAA within mature neurons of the optic system. These results suggest a rationale for the unusual compartmentalization of NAA metabolism and point to NAA as a neuronal constituent that is essential for the formation and/or maintenance of myelin. The relevance of these findings to Canavan disease is discussed.     

Tamoxifen attenuates inflammatory-mediated damage and improves functional outcome after spinal cord injury in rats

Tamoxifen has been found to be neuroprotective in both transient and permanent experimental ischemic stroke. However, it remains unknown whether this agent shows a similar beneficial effect after spinal cord injury (SCI), and what are its underlying mechanisms. In this study, we investigated the efficacy of tamoxifen treatment in attenuating SCI-induced pathology. Blood–spinal cord barrier (BSCB) permeability, tissue edema formation, microglial activation, neuronal cell death and myelin loss were determined in rats subjected to spinal cord contusion. The results showed that tamoxifen, administered at 30 min post-injury, significantly decreased interleukin-1β (IL-1β) production induced by microglial activation, alleviated the amount of Evans blue leakage and edema formation. In addition, tamoxifen treatment clearly reduced the number of apoptotic neurons post-SCI. The myelin loss and the increase in production of myelin-associated axonal growth inhibitors were also found to be significantly attenuated at day 3 post-injury. Furthermore, rats treated with tamoxifen scored much higher on the locomotor rating scale after SCI than did vehicle-treated rats, suggesting improved functional outcome after SCI. Together, these results demonstrate that tamoxifen provides neuroprotective effects for treatment of SCI-related pathology and disability, and is therefore a potential neuroprotectant for human spinal cord injury therapy.     

Relationship between myelin production and dopamine synthesis in the PKU mouse brain

Phenylketonuria is caused by specific mutations in the phenylalanine hydroxylase gene and is characterized by elevated blood phenylalanine levels, hypomyelination in forebrain structures, reduced dopamine levels, and cognitive difficulties. To determine whether brain tyrosine levels and/or myelination play a role in the up-regulation of dopamine, phenylketonuric mice were placed on a low phenylalanine diet for 4 weeks and as blood phenylalanine levels dropped to normal, the relationships between phenylalanine, tyrosine, dopamine, myelin proteins, and axonal proteins in frontal cortex and striatum were determined using gas chromatography mass spectrometry, histology, and western blotting techniques. Blood phenylalanine rapidly decreased from an eight-fold elevation to near control levels, and blood tyrosine gradually rose from about 50% to near normal values. In frontal cortex and striatum, phenylalanine levels dropped to 2- and 1.5-fold elevations above control, respectively, and tyrosine levels increased but remained less than 70% of control in both structures. In frontal cortex, increases in dopamine and myelin basic protein occurred in a similar biphasic pattern, reaching near normal levels by week 4. In striatum, dopamine and MBP dramatically increased to near normal levels in the first week. Myelination was confirmed histologically and by western blot quantification of phosphorylated neurofilaments. In summary, our results showed: (i) an increase in dopamine despite low brain tyrosine levels and (ii) similar recovery patterns for myelination and dopamine. Since myelin/axonal interactions trigger signaling pathways that result in axonal maturation, we speculate that this interaction also may trigger signals that up-regulate neurotransmitter synthesis.     

Acute spinal cord injury induces genetic damage in multiple organs of rats

Spinal cord injury (SCI) is a devastating condition with important functional and psychological consequences. However, the underlying mechanisms by which these alterations occur are still not fully understood. The aim of this study was to analyze genomic instability in multiple organs in the acute phase of SCI by means of single cell gel (comet) assay. Rats were randomly distributed into two groups (n = 5): a SHAM and a SCI group killed 24 h after cord transection surgery. The results pointed out genetic damage in blood cells as depicted by the tail moment results. DNA breakage was also detected in liver and kidney cells after SCI. Taken together, our results suggest that SCI induces genomic damage in multiple organs of Wistar rats.     

Role of calpain in spinal cord injury: Increased calpain immunoreactivity in rat spinal cord after impact trauma

Impact spinal cord injury (20 g-cm) was induced in rat by weight drop. The immunoreactivity of mcalpain was examined in the lesion and adjacent areas of the cord following trauma. Increased calpain immunoreactivity was evident in the lesion compared to control and the immunostaining intensity progressively increased after injury. The calpain immunoreactivity was also increased in tissue adjacent to the lesion. mCalpain immunoreactivity was significantly stronger in glial and endothelial cells, motor neurons and nerve fibers in the lesion. The calpain immunoreactivity also increased in astrocytes and microglial cells in the adjacent areas. Proliferation of microglia and astrocytes identified by GSA histochemical staining and GFAP immunostaining, respectively, was seen at one and three days after injury. Many motor neurons in the ventral horn showed increased calpain immunoreactivity and were shrunken in the lesion. These studies indicate a pivotal role for calpain and the involvement of glial cells in the tissue destruction in spinal cord injury. Special issue dedicated to Dr. Marion E. Smith.     

Study of myelin purity in relation to axonal contaminants

Axonal remnants are considered a probable source of contamination of isolated myelin in view of the relatively tight axon-glial intercellular junction. Using the rabbit optic system to label specifically axonal components, we have found the levels of such contaminants to depend on the myelin isolation procedure, the tissue source, and the nature of the contaminant. A procedure employing repetitive treatments with EGTA was found to be highly effective in removing proline-labeled axonal proteins, the estimated upper limit of such contamination being approximately 0.6–1.2% of the myelin protein. The standard isolation procedure of Norton and Poduslo, supplemented with an additional discontinuous gradient step, proved equally effective in removing rapidly transported proteins from myelin isolated from the superior colliculus or lateral geniculate body. When the optic tract was the source, however, the EGTA procedure proved more effective in removing both rapidly and slowly transported proteins. Axonal gangliosides labeled with N-[ ³ H]acetylmannosamine were efficiently removed by both procedures, adding support to the proposition that gangliosides detected in isolated myelin are intrinsic to that membrane.     

Internodal sodium channels ensure active processes under myelin manifesting in depolarizing afterpotentials

The current opinion about processes in myelinated axon is that action potential saltatorially propagates between nodes of Ranvier and passively charges internodal axolemma thus causing depolarizing afterpotentials (DAP). Demyelination blocks the conduction that gives additional argument in favor of hypothesis that internode is not able to be activated by the existing internodal sodium channels. The results of our modeling study shows that, when periaxonal space is sufficiently narrow, saltatorial action potential is able to activate internodes. Low density of internodal sodium channels is sufficient to generate active internodal waves that slowly propagate from nodes towards corresponding midinternodes where they collide. The periaxonal width that stops internodal wave propagation (about 400 nm) is significantly larger than the highest value of the physiological range for this parameter (30 nm). Internodal activation is directly manifested as transmembrane internodal potential or as a full-sized action potential in periaxonal space where it can hardly be detected, and only as a small deflection in intracellular space. However, changes in the periaxonal potential cause transmyelin currents that lead to significant DAP. The shape and amplitude of DAP depends on myelin parameters and densities of internodal channels. Several technical parameters affect the results of calculations. Internodal spatial segmentation has to be sufficiently fine (at most 20 microm) for the model to be able to simulate internodal activation. We employ 338 internodal segments as compared with up to 21 used in previous models. Ionic accumulation together with related diffusive and electrical processes alter the calculated DAP amplitude. Inclusion of these processes in calculations demands such increase in the total number of segments that the numerical methods used up to now become unapplicable. To overcome the problem, an iterative implicit approach is proposed. It reduces a matrix of general type in multi-cable models to tridiagonal one and accelerates calculations considerably.     

Potassium currents in the giant axon of the crabCarcinus maenas

Summary Measurements were made of the kinetic and steadystate characteristics of the potassium conductance in the giant axon of the crabCarcinus maenas. These measurements were made in the presence of tetrodotoxin, using the feedback amplifier concept introduced by Dodge and Frankenhaeuser (J. Physiol. (London) 143:76–90). The conductance increase during depolarizing voltage-clamp pulses was analyzed assuming that two separate potassium channels exist in these axons. The first potassium channel exhibited activation and fast inactivation gating which could be fitted using them ³ h, Hodgkin-Huxley formalism. The second potassium channel exhibited the standardn ⁴ Hodgkin-Huxley kinetics. These two postulated channels are blocked by internal application of caesium, tetraethylammonium and sodium ions. External application of 4 amino-pyridine also blocks these channels.     

Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1

In myelinated axons, K+ channels are concealed under the myelin sheath in the juxtaparanodal region, where they are associated with Caspr2, a member of the neurexin superfamily. Deletion of Caspr2 in mice by gene targeting revealed that it is required to maintain K+ channels at this location. Furthermore, we show that the localization of Caspr2 and clustering of K+ channels at the juxtaparanodal region depends on the presence of TAG-1, an immunoglobulin-like cell adhesion molecule that binds Caspr2. These results demonstrate that Caspr2 and TAG-1 form a scaffold that is necessary to maintain K+ channels at the juxtaparanodal region, suggesting that axon-glia interactions mediated by these proteins allow myelinating glial cells to organize ion channels in the underlying axonal membrane.     

Systemic administration of acromelic acid induces selective neuron damage in the rat spinal cord

A single systemic administration of acromelic acid A (ACRO), a novel kainate analogue (kainoid), induces a series of characteristic behavioral changes in association with selective damage of interneurons in the caudal spinal cord in adult rats. When ACRO (5 mg/kg) was systemically administered, rats displayed forced extension of hindlimbs followed by frequent cramps and generalized convulsion. Most rats died during the convulsions without neuropathological change. Two rats developed long-lasting spastic paraparesis which persisted at least 3 months. Neuropathological changes were observed only in the rats with persistent paraparesis, in which neuron damage was identified selectively in small interneurons in the lumbosacral cord. The regional difference between kainate- and ACRO-induced neuron damage suggests the existence of plural kinds of kainate receptor subtypes.     

Electrotonic synapses in the mammalian spinal cord

By means of light and electron microscopy methods structural peculiarities of motor nuclei have been studied in the rat spinal cord (17 animals) on the 1st-3d and on the 10th-18th days of postnatal ontogenesis. Synaptic junctions of the gap type are revealed; they are considered as electrotonic synapses. Dendro-somatic and dendrodendritic synaptic junctions of the gap type are found. Together with the electrotonic synapses, morphologically mixed synapses of axo-somatic and axo-axonal types are disclosed; they contain, besides organells, specific for chemical synapses, close opposition areas of pre- and postsynaptic membranes of the gap junction type. Morphologically mixed synapses occur in neuropil of the motor nuclei of the spinal cord in young rats of all age groups studied. Homologous synapses are detected in the motor nuclei of the white mouse spinal cord. Synaptic junctions of the gap type in the mammalian spinal cord could be a substrate of electrical interaction between its motor neurons.     

KB-2796, a calcium channel blocker,ameliorates ischemic spinal cord damage in rabbits

The effect of the calcium channel blocker (KB-2796) on metabolic and functional recovery in rabbit spinal cord after 20, 30, and 40 min ischemia and 4 days of recovery was investigated. The drug was given intraperitoneally in three different doses, 10, 20, or 50 mg/kg pre-or post-ischemia of 20, 30, or 40 min duration. Both higher doses 20 and 30 mg/kg completely recovered energy state and significantly improved neurological functions in the spinal cord following 20 and 30 min ischemia. Partial protection was observed even after 40 min ischemia. The protective effect of KB-2796 exceeds the effect of calcium blockers previously used in experimental spinal cord ischemia.