Long-term maintenance of Na+ channels at nodes of Ranvier depends on glial contact mediated by gliomedin and NrCAM.

Clustering of Na(+) channels at the nodes of Ranvier is coordinated by myelinating glia. In the peripheral nervous system, axoglial contact at the nodes is mediated by the binding of gliomedin and glial NrCAM to axonal neurofascin 186 (NF186). This interaction is crucial for the initial clustering of Na(+) channels at heminodes. As a result, it is not clear whether continued axon-glial contact at nodes of Ranvier is required to maintain these channels at the nodal axolemma. Here, we report that, in contrast to mice that lack either gliomedin or NrCAM, absence of both molecules (and hence the glial clustering signal) resulted in a gradual loss of Na(+) channels and other axonal components from the nodes, the formation of binary nodes, and dysregulation of nodal gap length. Therefore, these mice exhibit neurological abnormalities and slower nerve conduction. Disintegration of the nodes occurred in an orderly manner, starting with the disappearance of neurofascin 186, followed by the loss of Na(+) channels and ankyrin G, and then ßIV spectrin, a sequence that reflects the assembly of nodes during development. Finally, the absence of gliomedin and NrCAM led to the invasion of the outermost layer of the Schwann cell membrane beyond the nodal area and the formation of paranodal-like junctions at the nodal gap. Our results reveal that axon-glial contact mediated by gliomedin, NrCAM, and NF186 not only plays a role in Na(+) channel clustering during development, but also contributes to the long-term maintenance of Na(+) channels at nodes of Ranvier.

Caspr and caspr2 are required for both radial and longitudinal organization of myelinated axons.

In myelinated peripheral axons, Kv1 potassium channels are clustered at the juxtaparanodal region and at an internodal line located along the mesaxon and below the Schmidt-Lanterman incisures. This polarized distribution is controlled by Schwann cells and requires specific cell adhesion molecules (CAMs). The accumulation of Kv1 channels at the juxtaparanodal region depends on the presence of Caspr2 at this site, as well as on the presence of Caspr at the adjacent paranodal junction. However, the localization of these channels along the mesaxonal internodal line still persists in the absence of each one of these CAMs. By generating mice lacking both Caspr and Caspr2 (caspr(-/-)/caspr2(-/-)), we now reveal compensatory functions of the two proteins in the organization of the axolemma. Although Kv1 channels are clustered along the inner mesaxon and in a circumferential ring below the incisures in the single mutants, in sciatic nerves of caspr(-/-)/caspr2(-/-) mice, these channels formed large aggregates that were dispersed along the axolemma, demonstrating that internodal localization of Kv1 channels requires either Caspr or Caspr2. Furthermore, deletion of both Caspr and Caspr2 also resulted in widening of the nodes of Ranvier, suggesting that Caspr2 (which is present at paranodes in the absence of Caspr) can partially compensate for the barrier function of Caspr at this site even without the formation of a distinct paranodal junction. Our results indicate that Caspr and Caspr2 are required for the organization of the axolemma both radially, manifested as the mesaxonal line, and longitudinally, demarcated by the nodal domains.

Paranodal dysmyelination in peripheral nerves of Trembler mice.

Subtle defects in paranodes of myelinated nerve fibers can cause significant physiological malfunction. We have investigated myelinated fibers in the peripheral nervous system (PNS) of the Trembler mouse, a model of CMT-1A neuropathy, for evidence of such defects. Ultrastructural analysis shows that the "transverse bands," which attach the myelin sheath to the axon at the paranodal axoglial junction, are grossly diminished in number in Trembler nerve fibers. Although paranodes often appear to be greatly elongated, it is only a short region immediately adjacent to the node of Ranvier that displays transverse bands. Where transverse bands are missing, the junctional gap widens, thus reducing resistance to short circuiting of nodal action currents during saltatory conduction and increasing the likelihood that axonal K(+) channels under the myelin sheath will be activated. In addition, we find evidence that structural domains in Trembler axons are incompletely differentiated, consistent with diminution in nodal Na channel density, which could further compromise conduction. Deficiency of transverse bands may also increase susceptibility to disruption of the paranodal junction and retraction of the myelin sheath. We conclude that Trembler PNS myelinated fibers display subtle defects in paranodal and nodal regions that could contribute significantly to conduction defects and increased risk of myelin detachment.

A critical role for the protein tyrosine phosphatase receptor type Z in functional recovery from demyelinating lesions.

Several lines of evidence suggest that tyrosine phosphorylation is a key element in myelin formation, differentiation of oligodendrocytes and Schwann cells, and recovery from demyelinating lesions. Multiple sclerosis is a demyelinating disease of the human central nervous system, and studies of experimental demyelination indicate that remyelination in vivo requires the local generation, migration or maturation of new oligodendrocytes, or some combination of these. Failure of remyelination in multiple sclerosis could result from the failure of any of these processes or from the death of oligodendrocytes. Ptprz encodes protein tyrosine phosphatase receptor type Z (Ptpz, also designated Rptpbeta), which is expressed primarily in the nervous system but also in oligodendrocytes, astrocytes and neurons. Here we examine the susceptibility of mice deficient in Ptprz to experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We observe that mice deficient in Ptprz show impaired recovery from EAE induced by myelin oligodendrocyte glycoprotein (MOG) peptide. This sustained paralysis is associated with increased apoptosis of mature oligodendrocytes in the spinal cords of mutant mice at the peak of inflammation. We further demonstrate that expression of PTPRZ1, the human homolog of Ptprz, is induced in multiple sclerosis lesions and that the gene is specifically expressed in remyelinating oligodendrocytes in these lesions. These results support a role for Ptprz in oligodendrocyte survival and in recovery from demyelinating disease.

Permeability of the paranodal junction of myelinated nerve fibers.

We have used fluorescent dextran tracers to test the tightness of the paranodal junction of living or fixed myelinated fibers in mouse sciatic nerve. Both 3 and 70 kDa tracers are able to penetrate from the perinodal space symmetrically into the paranodes on either side of the node of Ranvier at a rate consistent with diffusion through an elongated helical pathway between the paranodal terminal loops of the myelin sheath. This pathway thus provides an access route for movement of water soluble nutrients and metabolites to and from the internodal axon and constitutes a pathway through which juxtaparanodal potassium channels may be activated and may in turn affect nodal excitability. This pathway may also allow access of antibodies and toxic molecules to the internodal axon in paraneoplastic syndromes and demyelinating diseases.

Paranodal permeability in "myelin mutants".

Fluorescent dextran tracers of varying sizes have been used to assess paranodal permeability in myelinated sciatic nerve fibers from control and three "myelin mutant" mice, Caspr-null, cst-null, and shaking. We demonstrate that in all of these the paranode is permeable to small tracers (3 kDa and 10 kDa), which penetrate most fibers, and to larger tracers (40 kDa and 70 kDa), which penetrate far fewer fibers and move shorter distances over longer periods of time. Despite gross diminution in transverse bands (TBs) in the Caspr-null and cst-null mice, the permeability of their paranodal junctions is equivalent to that in controls. Thus, deficiency of TBs in these mutants does not increase the permeability of their paranodal junctions to the dextrans we used, moving from the perinodal space through the paranode to the internodal periaxonal space. In addition, we show that the shaking mice, which have thinner myelin and shorter paranodes, show increased permeability to the same tracers despite the presence of TBs. We conclude that the extent of penetration of these tracers does not depend on the presence or absence of TBs but does depend on the length of the paranode and, in turn, on the length of "pathway 3," the helical extracellular pathway that passes through the paranode parallel to the lateral edge of the myelin sheath.

A novel path for rapid transverse communication of vestibular signals in turtle cerebellum.

Voltage-sensitive dye activity within the thin, unfoliated turtle cerebellar cortex (Cb) was recorded in vitro during eighth cranial nerve (nVIII) stimulation. Short latency responses were localized to the middle of the lateral edges of both ipsilateral and contralateral Cb [vestibulocerebellum (vCb)]. Even with a severed contralateral Cb peduncle, stimulation of the nVIII ipsilateral to the intact peduncle evoked contralateral vCb responses with a mean latency of only 0.25 ms after the ipsilateral responses, even though the distance between them was ∼ 5 mm. We investigated whether a rapidly conducting commissure exists between each vCb by stimulating one of them directly. Responses in both vCb spread sagittally, but, surprisingly, there was no sequential activation along a transverse Cb beam between them. In contrast, stimulation medial to either vCb evoked transverse beams that required ∼ 20 ms to cross the Cb. Therefore, the rapid commissural connection between each vCb is not mediated by slowly conducting parallel fibers. Also, the vCb was not strongly activated by climbing fiber stimulation, suggesting that inputs to vCb involve distinct cerebellar circuits. Responses between the two vCb remained following knife cuts through the rostral and caudal Cb along the midline, through both peduncles, and even shallow midline cuts to the middle Cb through its white matter and granule cell layer. Commissural responses were still observed only with a narrow transverse bridge between each vCb or in thick transverse Cb slices. Horseradish peroxidase transport from one vCb labeled transverse axons traveling within the Purkinje cell layer that were larger than parallel fibers and lacked varicosities. In sagittal sections, cross-section profiles of myelinated axons were observed around Purkinje cells midway between the rostral and caudal Cb. This novel pathway for transverse communication between lateral edges of turtle Cb suggests that afferents may directly conduct vestibular information rapidly across the Cb to coordinate vestibulomotor reflex behaviors.

Myelin protein composition is altered in mice lacking either sulfated or both sulfated and non-sulfated galactolipids.

Myelin is highly enriched in galactocerebroside (GalCer) and its sulfated form sulfatide. Mice, unable to synthesize GalCer and sulfatide (CGT(null)) or sulfatide alone (CST(null)), exhibit disorganized paranodal structures and progressive dysmyelination. To obtain insights into the molecular mechanisms underlying these defects, we examined myelin composition of these mutants by two-dimensional differential fluorescence intensity gel electrophoresis proteomic approach and immunoblotting. We identified several proteins whose expressions were significantly altered in these mutants. These proteins are known to regulate cytoskeletal dynamics, energy metabolism, vesicular trafficking or adhesion, suggesting a disruption in these physiological processes in the absence of myelin galactolipids. Further analysis of one of these proteins, nucleotide diphosphate kinase (NDK)/Nm23, showed that it was reduced in myelin of CGT(null) and increased in CST(null), but not in whole brain homogenate. Immunostaining showed an increase in its expression in the cell bodies of CGT(null)- and a decrease in CST(null)-oligodenrocytes, together leading to the hypothesis that transport of NDK/Nm23 from oligodenrocyte cell bodies into myelin may be differentially dysregulated in the absence of these galactolipids. This study provides new insights into the changes that occur in the composition/distribution of myelin proteins in mice lacking either unsulfated and/or sulfated galactolipids and reinforces the role of these lipids in intracellular trafficking.

The ultrastructure and contractile properties of a fast-acting, obliquely striated, myosin-regulated muscle: the funnel retractor of squids.

We investigated the ultrastructure, contractile properties, and in vivo length changes of the fast-acting funnel retractor muscle of the long-finned squid Doryteuthis pealeii. This muscle is composed of obliquely striated, spindle-shaped fibers ~3 mum across that have an abundant sarcoplasmic reticulum, consisting primarily of membranous sacs that form 'dyads' along the surface of each cell. The contractile apparatus consists of 'myofibrils' approximately 0.25-0.5 microm wide in cross section arrayed around the periphery of each cell, surrounding a central core that contains the nucleus and large mitochondria. Thick myofilaments are approximately 25 nm in diameter and approximately 2.8 microm long. 'Dense bodies' are narrow, resembling Z lines, but are discontinuous and are not associated with the cytoskeletal fibrillar elements that are so prominent in slower obliquely striated muscles. The cells approximate each other closely with minimal intervening intercellular connective tissue. Our physiological experiments, conducted at 17 degrees C, showed that the longitudinal muscle fibers of the funnel retractor were activated rapidly (8 ms latent period following stimulation) and generated force rapidly (peak twitch force occurred within 50 ms). The longitudinal fibers had low V(max) (2.15 +/-0.26 L(0) s(-1), where L(0) was the length that generated peak isometric force) but generated relatively high isometric stress (270+/-20 mN mm(-2) physiological cross section). The fibers exhibited a moderate maximum power output (49.9 W kg(-1)), compared with vertebrate and arthropod cross striated fibers, at a V/V(max) of 0.33+/-0.044. During ventilation of the mantle cavity and locomotion, the funnel retractor muscle operated in vivo over a limited range of strains (+0.075 to -0.15 relative to resting length, L(R)) and at low strain rates (from 0.16 to 0.91 L(R) s(-1) ), corresponding to a range of V/V(max) from 0.073 to 0.42. During the exhalant phase of the jet the range of strains was even narrower: maximum range less than +/-0.04, with the muscle operating nearly isometrically during ventilation and slow, arms-first swimming. The limited length operating range of the funnel retractor muscles, especially during ventilation and slow jetting, suggests that they may act as muscular struts.

Neuregulin-1 type III determines the ensheathment fate of axons.

The signals that determine whether axons are ensheathed or myelinated by Schwann cells have long been elusive. We now report that threshold levels of neuregulin-1 (NRG1) type III on axons determine their ensheathment fate. Ensheathed axons express low levels whereas myelinated fibers express high levels of NRG1 type III. Sensory neurons from NRG1 type III deficient mice are poorly ensheathed and fail to myelinate; lentiviral-mediated expression of NRG1 type III rescues these defects. Expression also converts the normally unmyelinated axons of sympathetic neurons to myelination. Nerve fibers of mice haploinsufficient for NRG1 type III are disproportionately unmyelinated, aberrantly ensheathed, and hypomyelinated, with reduced conduction velocities. Type III is the sole NRG1 isoform retained at the axon surface and activates PI 3-kinase, which is required for Schwann cell myelination. These results indicate that levels of NRG1 type III, independent of axon diameter, provide a key instructive signal that determines the ensheathment fate of axons.

Spongiform pathology in mouse CNS lacking 'neuropathy target esterase' and cellular prion protein.

Conditional inactivation of the 'neuropathy target esterase' (NTE) gene in mouse nerve cells was previously shown to result in CNS pathology comparable to the spongiform encephalopathy characteristic of prion diseases. To determine whether cellular prion protein (PrPc) is essential for development of this pathology we examined hippocampi of mice lacking NTE alone, PrPc alone or both NTE and PrPc. Light microscopic survey showed clear-cut spongiform changes in a majority of NTE-/- and NTE/PrP-/- double knockout mice but in only one PrP-/- mouse. EM analysis of spongiform lesions from NTE-/- and NTE/PrP-/- mice, and from the one affected PrP-/- mouse, revealed patches of branching tubular inclusions, comparable to the 'tubulovesicular inclusions' described previously in prion diseases. We conclude that spongiform pathology in conditional NTE knockout mice is not mediated by PrPc, and that tubulovesicular inclusions can be seen in spongiform encephalopathy of other etiologies and are not pathognomonic of prion disease.

Multiple functions of the paranodal junction of myelinated nerve fibers.

Myelin sheaths include an extraordinary structure, the "paranodal axoglial junction" (PNJ), which attaches the sheath to the axon at each end of each myelin segment. Its size is enormous and its structure unique. Here we review past and current studies showing that this junction can serve multiple functions in maintaining reliable saltatory conduction. The present evidence points to three functions in particular. 1) It seals the myelin sheath to the axon to prevent major shunting of nodal action currents beneath the myelin sheath while still leaving a narrow channel interconnecting the internodal periaxonal space with the perinodal space. This pathway represents a potential route through which juxtaparanodal and internodal channels can influence nodal activity and through which nutrients, such as glucose, and other metabolites can diffuse to and from the internodal periaxonal space. 2) It serves as a mechanism for maintaining discrete, differentiated axolemmal domains at and around the node of Ranvier by acting as a barrier to the lateral movement of ion channel complexes within the axolemma, thus concentrating voltage-gated sodium channels at the node and segregating fast voltage-gated potassium channels to the juxtaparanode under the myelin sheath. 3) It attaches the myelin sheath to the axon on either side of the node and can thus maintain nodal dimensions in the face of mechanical stresses associated with stretch or other local factors that might cause disjunction. It is therefore the likely means for maintaining constancy of nodal surface area and electrical parameters essential for consistency in conduction.

Paranodal interactions regulate expression of sodium channel subtypes and provide a diffusion barrier for the node of Ranvier.

The node of Ranvier is a distinct domain of myelinated axons that is highly enriched in sodium channels and is critical for impulse propagation. During development, the channel subtypes expressed at the node undergo a transition from Nav1.2 to Nav1.6. Specialized junctions that form between the paranodal glial membranes and axon flank the nodes and are candidates to regulate their maturation and delineate their boundaries. To investigate these roles, we characterized node development in mice deficient in contactin-associated protein (Caspr), an integral junctional component. Paranodes in these mice lack transverse bands, a hallmark of the mature junction, and exhibit progressive disruption of axon-paranodal loop interactions in the CNS. Caspr mutant mice display significant abnormalities at central nodes; components of the nodes progressively disperse along axons, and many nodes fail to mature properly, persistently expressing Nav1.2 rather than Nav1.6. In contrast, PNS nodes are only modestly longer and, although maturation is delayed, eventually all express Nav1.6. Potassium channels are aberrantly clustered in the paranodes; these clusters are lost over time in the CNS, whereas they persist in the PNS. These findings indicate that interactions of the paranodal loops with the axon promote the transition in sodium channel subtypes at CNS nodes and provide a lateral diffusion barrier that, even in the absence of transverse bands, maintains a high concentration of components at the node and the integrity of voltage-gated channel domains.

Dysmyelination with preservation of transverse bands in a long-lived allele of the quaking mouse.

The new mutant mouse shaking (shk) differs from other "myelin mutants" in having a more stable neurological impairment and a much longer lifespan. We have shown that transverse bands (TBs), the component of the paranodal junction (PNJ) that attaches the myelin sheath to the axon, are present in the shk central nervous system (CNS), in contrast to more severely affected mutants, in which TBs are absent or rare. We have proposed that TBs are the major determinant underlying shk neurological stability and longevity. Here we report that TBs are abundant not only in the shk CNS but also in its peripheral nervous system (PNS), which, as in other "myelin mutants", is not as severely dysmyelinated as the CNS but does display structural abnormalities likely to affect impulse propagation. In particular, myelin sheaths are thinner than normal, and some axonal segments lack myelin sheaths entirely. In addition, we establish that the shk mutation, previously localized to chromosome 17, is a quaking (qk) allele consisting of a 105-nucleotide insertion in the qk regulatory region that decreases qk transcription but does not extend to the Parkin and Parkin coregulated genes, which are affected in the qk allele. We conclude that: 1) dysmyelination is less severe in the shk PNS than in the CNS, but TBs, which are present in both locations, stabilize the PNJs and prevent the progressive neurological deficits seen in mutants lacking TBs; and 2) the insertional mutation in shk mice is sufficient to produce the characteristic neurological phenotype without involvement of the Parkin and Parkin coregulated genes.

Distribution and morphology of transgenic mouse oligodendroglial-lineage cells following transplantation into normal and myelin-deficient rat CNS.

Glial cells from neonatal MbetaP5 transgenic mice, which express bacterial beta-galactosidase (lacZ) under control of the myelin basic protein (MBP) promoter (Gow et al, 1992), were transplanted into the spinal cord or cerebral hemisphere of immunosuppressed normal and myelin-deficient (md) rats in order to assess the ability of the donor cells to survive, migrate, and differentiate within normal compared with myelin-deficient central nervous system (CNS). LacZ+ cells were detected as early as 6-7 days after transplantation into the low thoracic cord and by 10 days had spread rostrally to the brainstem and caudally to the sacral spinal cord. Initially, compact lacZ+ cells, lacking processes, were found associated with small blood vessels and with the glia limitans. Cells of this type persisted throughout the experiment. Later, lacZ+ cells with processes were seen along fiber tracts in the dorsal columns and, after intracerebral injection, subjacent to ventricular ependyma, as well as scattered in cerebral white and gray parenchyma. The extent of spread was comparable in md and normal rats, but in the md group, the success rate was higher, and more cells differentiated into process-bearing oligodendrocytes. Acceptance of xenografts in immunosuppressed recipients equaled that of allografts. The overall spread of grafted cells exceeded that of injected charcoal, indicating active migration. In contrast to earlier studies that identified oligodendrocytes based on morphology alone, this study has allowed us to identify and track oligodendrocytes based on myelin gene expression. We show some oligodendrocytes whose morphology is consistent with classical morphological descriptions, some that resemble astrocytes, and a class of compact perivascular oligodendrocyte-lineage cells that we suggest are migratory.

Molecular architecture of myelinated nerve fibers: leaky paranodal junctions and paranodal dysmyelination.

Myelinated nerve fibers have evolved to optimize signal propagation. Each myelin segment is attached to the axon by the unique paranodal axoglial junction (PNJ), a highly complex structure that serves to define axonal ion channel domains and to direct nodal action currents through adjacent nodes. Surprisingly, this junction does not entirely seal the paranodal myelin sheath to the axon and thus does not entirely isolate the perinodal space from the internodal periaxonal space. Rather the paranode is penetrated by extracellular pathways between the myelin sheath and the axolemma for movement of molecules and the flow of current to and from the internodal axon. This review summarizes past and current studies demonstrating these pathways and considers what functional roles they subserve. In addition, modern genetic engineering methods permit modification of individual PNJ constituents, which provides an opportunity to define their specific functions. One component in particular, the transverse bands, plays a key role in maintaining the structure and function of the PNJ. Loss of transverse bands results not in frank demyelination but rather in subtle dysmyelination, which causes significant functional impairment. The consequences of such subtle defects in the PNJ are considered along with the relevance of these studies to human diseases of myelin.

Dependence of paranodal junctional gap width on transverse bands.

Mouse mutants with paranodal junctional (PNJ) defects display variable degrees of neurological impairment. In this study we compare control paranodes with those from three mouse mutants that differ with respect to a conspicuous PNJ component, the transverse bands (TBs). We hypothesize that TBs link the apposed junctional membranes together at a fixed distance and thereby determine the width of the junctional gap, which may in turn determine the extent to which nodal action currents can be short-circuited underneath the myelin sheath. Electron micrographs of aldehyde-fixed control PNJs, in which TBs are abundant, show a consistent junctional gap of ∼3.5 nm. In Caspr-null PNJs, which lack TBs entirely, the gap is wider (∼6-7 nm) and more variable. In CST-null PNJs, which have only occasional TBs, the mean PNJ gap width is comparable to that in Caspr-null mice. In the shaking mutant, in contrast, which has approximately 60% of the normal complement of TBs, mean PNJ gap width is not significantly different from that in controls. Correspondingly, shaking mice are much less impaired neurologically than either Caspr-null or CST-null mice. We conclude that in the absence or gross diminution of TBs, mean PNJ gap width increases significantly and suggest that this difference could underlie some of the neurological impairment seen in those mutants. Surprisingly, even in the absence of TBs, paranodes are to some extent maintained in their usual form, implying that in addition to TBs, other factors govern the formation and maintenance of overall paranodal structure.

The cytoskeletal adapter protein 4.1G organizes the internodes in peripheral myelinated nerves.

Myelinating Schwann cells regulate the localization of ion channels on the surface of the axons they ensheath. This function depends on adhesion complexes that are positioned at specific membrane domains along the myelin unit. Here we show that the precise localization of internodal proteins depends on the expression of the cytoskeletal adapter protein 4.1G in Schwann cells. Deletion of 4.1G in mice resulted in aberrant distribution of both glial adhesion molecules and axonal proteins that were present along the internodes. In wild-type nerves, juxtaparanodal proteins (i.e., Kv1 channels, Caspr2, and TAG-1) were concentrated throughout the internodes in a double strand that flanked paranodal junction components (i.e., Caspr, contactin, and NF155), and apposes the inner mesaxon of the myelin sheath. In contrast, in 4.1G(-/-) mice, these proteins "piled up" at the juxtaparanodal region or aggregated along the internodes. These findings suggest that protein 4.1G contributes to the organization of the internodal axolemma by targeting and/or maintaining glial transmembrane proteins along the axoglial interface.

Role of transverse bands in maintaining paranodal structure and axolemmal domain organization in myelinated nerve fibers: effect on longevity in dysmyelinated mutant mice.

The consequences of dysmyelination are poorly understood and vary widely in severity. The shaking mouse, a quaking allele, is characterized by severe central nervous system (CNS) dysmyelination and demyelination, a conspicuous action tremor, and seizures in approximately 25% of animals, but with normal muscle strength and a normal lifespan. In this study we compare this mutant with other dysmyelinated mutants including the ceramide sulfotransferase deficient (CST-/-) mouse, which are more severely affected behaviorally, to determine what might underlie the differences between them with respect to behavior and longevity. Examination of the paranodal junctional region of CNS myelinated fibers shows that "transverse bands," a component of the junction, are present in nearly all shaking paranodes but in only a minority of CST-/- paranodes. The number of terminal loops that have transverse bands within a paranode and the number of transverse bands per unit length are only moderately reduced in the shaking mutant, compared with controls, but markedly reduced in CST-/- mice. Immunofluorescence studies also show that although the nodes of the shaking mutant are somewhat longer than normal, Na(+) and K(+) channels remain separated, distinguishing this mutant from CST-/- mice and others that lack transverse bands. We conclude that the essential difference between the shaking mutant and others more severely affected is the presence of transverse bands, which serve to stabilize paranodal structure over time as well as the organization of the axolemmal domains, and that differences in the prevalence of transverse bands underlie the marked differences in progressive neurological impairment and longevity among dysmyelinated mouse mutants.

Effects of osmolality on PLP-null myelin structure: implications re axon damage.

In order to test the adhesiveness of PLP-null compact myelin lamellae we soaked aldehyde-fixed CNS specimens from PLP-null and control mice overnight in distilled water, in Ringer's solution or in Ringer's solution with added 1 M sucrose. Subsequent examination of the tissue by EM showed that both PLP-null and control white matter soaked in Ringer remained largely compact. After the distilled water soak, control myelin was virtually unchanged, but PLP-null myelin showed some decompaction, i.e., separation of myelin lamellae from one another. After the sucrose/Ringer soak, normal myelin developed foci of decompaction, but the great majority of lamellae remained compact. In the PLP-null specimens, in contrast, many of the myelin sheaths became almost completely decompacted. Such sheaths became thicker overall and were comprised of lamellae widely separated from one another by irregular spaces. Thus, in normal animals, fixed CNS myelin lamellae are firmly adherent and resist separation; PLP-null myelin lamellae, in contrast, are poorly adherent and more readily separated. Mechanisms by which impaired adhesiveness of PLP-null myelin lamellae and fluctuations in osmolality in vivo might underlie slowing of conduction and axon damage are discussed.