Potassium channels in the nodal membrane of rat myelinated fibres

Following some preliminary reports, mammalian fibres from rabbit and rat have recently been successfully studied in detail by means of the voltage clamp. The early transient or sodium conductance system was found to be similar to that in frog and squid axons. However, the delayed conductance or potassium currents were found to be negligible. Only after chemical and osmotic manipulations, which were said to expose channels buried under the myelin, did Chiu and Ritchie find delayed currents in rabbit fibres. If confirmed, this would mean that the membrane conductance system of mammalian fibres is so different from that of invertebrate and amphibian axon models as to make the data base gathered from amphibian myelinated fibres (frog and toad) and invertebrate giant axons (squid and myxicola) irrelevant to human nd other mammalian fibres. However, we show here that it is possible to find in the normal nodal membrane of rat myelinated fibres potassium currents that flow through channels which are similar in many respects to those found in the frog node of squid axons.     

小鼠视神经再生研究动物模型的建立

目的总结制作小鼠视神经完全截断性动物模型作为视神经再生研究的经验和体会。方法将雄性Bcl-2高表达转基因小鼠(Bcl-2 transgenic mice)和受GFAP启动子控制表达疱疹病毒-胸苷激酶转基因雌性小鼠(GFAP-TK)交配产生的4只8~12周成年小鼠(20~30g),Bcl-2/GFAP-TK双转基因小鼠作为实验组,同周龄4只Bcl-2转基因小鼠作为对照组。其中Bcl-2/GFAP-TK双转基因小鼠皮下植入缓释泵,连续7d释放更昔洛韦(GCV)100mg.kg-1.d-1以选择性地去除视神经损伤后激活的星形胶质细胞。更昔洛韦缓释泵植入术后2d在两组动物中制作右侧单眼标准完全性视神经钳夹损伤模型,视神经钳夹10d后获取组织标本。采用免疫荧光染色特异性检测再生轴突纤维并进行定量分析;结合罗丹明的霍乱毒素B亚单位(CTB-R)或增强表达绿色荧光蛋白的复制缺陷型腺相关病毒(AAV-EGFP)用作顺行性标记物以显示再生轴突是否到达大脑靶器官。结果在Bcl-2/GFAP-TK双转基因小鼠中存在免疫荧光阳性的再生视神经轴突,再生轴突计数为71.99±24.04,并可见生长锥(growth cone)样结构,但是再生轴突纤维未能延伸达到大脑靶器官。在对照组Bcl-2转基因小鼠中未见明显再生迹象。结论小鼠视神经完全截断性动物模型可用于视神经病变的再生研究。     

Retinofugal fibres change conduction velocity and diameter between the optic nerve and tract in ferrets

In earlier studies of central nervous fibre tracts, it was tacitly assumed that individual axons are relatively uniform along their length. In the retinofugal pathway in particular, axon diameter, myelin thickness and correlated conduction properties have been treated as constant throughout the optic nerve, chiasm and tract. We report here that the conduction velocities of fibres contributing to the early components of the compound action potential are significantly greater in the optic tract than in the optic nerve of ferrets, and also that the diameters of the largest retinofugal fibres increase from nerve to tract. This observation raises significant questions about the developmental mechanisms in the central nervous system that relate the axons, their diameters, and the glia with which they are myelinated. In addition, it indicates that studies that have relied on the constancy of conduction velocity along the retinofugal course may require reappraisal.     

Dystrophic mice show age related muscle fibre and myelinated axon losses

Although many differences between age matched normal and dystrophic animals have been found, there have been few demonstrations of a time related quantitative change from normal to a characteristically dystrophic situation within the dystrophic strain itself. Here we report such a change-normal numbers of muscle fibres present in young dystrophic mice were rapidly lost, such that older animals showed the reduced number of muscle fibres characteristic of murine dystrophy. Although these losses began after a demonstrable loss of myelinated axons had occurred, it is not possible to say if the loss of muscle fibres was a result of the loss of nerve fibres.     

Regulation of axon growth in vivo by activity-based competition

The formation of functional neural networks requires precise regulation of the growth and branching of the terminal arbors of axons, processes known to be influenced by early network electrical activity. Here we show that a rule of activity-based competition between neighbouring axons appears to govern the growth and branching of retinal ganglion cell (RGC) axon arbors in the developing optic tectum of zebrafish. Mosaic expression of an exogenous potassium channel or a dominant-negative SNARE protein was used to suppress electrical or neurosecretory activity in subsets of RGC axons. Imaging in vivo showed that these forms of activity suppression strongly inhibit both net growth and the formation of new branches by individually transfected RGC axon arbors. The inhibition is relieved when the activity of nearby 'competing' RGC axons is also suppressed. These results therefore identify a new form of activity-based competition rule that might be a key regulator of axon growth and branch initiation.     

Sodium channel density in hypomyelinated brain increased by myelin basic protein gene deletion.

Trophic control over the expression and membrane distribution of voltage-dependent ion channels is one of the principal organizing events underlying the maturation of excitable cells. The myelin sheath is a major structural determinant of regional ion channel topography in central axons, but the exact molecular signals that mediate local interactions between the oligodendrocyte and axolemma are not known. We have found that large caliber fibre pathways in the brain of the mutant mouse shiverer (shi, gene on chromosome 18), whose developmental fate of myelination is averted by deletion of five exons in the myelin basic protein gene, have a striking excess of sodium channels. As cytoplasmic membranes of shiverer oligodendroglia still adhere to axons, the evidence indicates that myelin basic protein or a myelin basic protein-dependent glial transmembrane signal associated with compact myelin formation, rather than a simple glial-axon contact inhibition or an intrinsic genetic program of neuronal differentiation, could be critical in downregulating sodium channel density in axons. Here we use the shiverer mutant to show that mature central nervous system projection neurons with large caliber unmyelinated fibres sustain functional excitability by increasing sodium channel density. This axon plasticity, triggered by the absence of a single glial protein, contributes to the unexpectedly mild degree of neurological impairment in the mutant brain without myelin, and may be a potentially inducible mechanism determining the recovery of function from dysmyelinating disease.     

Nerve sheaths and motoneurone collateral sprouting

When disease or injury causes partial loss of innervation from a muscle, the remaining axons sprout and form new connections to the denervated muscle fibres. Sprouting can occur in two ways: from axon terminals (terminal sprouting) or from the intramuscular axons themselves, probably from the nodes of Ranvier (collateral sprouting). Terminal sprouting has been induced experimentally using various methods, including partial denervation, nerve conduction block and nerve transmission block. A common factor in the induction of terminal sprouting seems to be changes in the surface membrane of muscle fibres; these changes and terminal sprouting are prevented by direct stimulation of the muscle. Collateral sprouting has been induced only by partial denervation and is not prevented by direct stimulation. This has been taken as evidence for an earlier suggestion that products of nerve or axon degeneration may be a direct stimulus for collateral sprouting. We report here that axon degeneration products alone are probably not the stimulus for collateral sprouting.     

Target size regulates calibre and myelination of sympathetic axons

Axons in vertebrate peripheral nerves are ensheathed by Schwann cells. For some axons, this sheath consists of a single layer of glial cell cytoplasm and plasma membranes; for other axons, Schwann cells form multilayered myelin. Whether or not a Schwann cell makes myelin is determined by a signal from the axon, but the nature of this signal is not known. Here I show that sympathetic postganglionic axons, which are normally not myelinated, become myelinated when their calibre is increased as a result of increasing the size of the peripheral target they innervate. This result implies that axon calibre, which is known to be correlated with myelination, is in fact the crucial determinant of whether an axon becomes myelinated. Furthermore, the finding that increasing or decreasing target size causes corresponding increases or decreases in axon size indicates that axon calibre is itself regulated by retrograde signals from peripheral target tissues.     

Clustering of voltage-dependent sodium channels on axons depends on Schwann cell contact.

In myelinated nerves, segregation of voltage-dependent sodium channels to nodes of Ranvier is crucial for saltatory conduction along axons. As sodium channels associate and colocalize with ankyrin at nodes of Ranvier, one possibility is that sodium channels are recruited and immobilized at axonal sites which are specified by the subaxolemmal cytoskeleton, independent of glial cell contact. Alternatively, segregation of channels at distinct sites along the axon may depend on glial cell contact. To resolve this question, we have examined the distribution of sodium channels, ankyrin and spectrin in myelination-competent cocultures of sensory neurons and Schwann cells by immunofluorescence, using sodium channel-, ankyrin- and spectrin-specific antibodies. In the absence of Schwann cells, sodium channels, ankyrin and spectrin are homogeneously distributed on sensory axons. When Schwann cells are introduced into these cultures, the distribution of sodium channels dramatically changes so that channel clusters on axons are abundant, but ankyrin and spectrin remain homogeneously distributed. Addition of latex beads or Schwann cell membranes does not induce channel clustering. Our results suggest that segregation of sodium channels on axons is highly dependent on interactions with active Schwann cells and that continuing axon-glial interactions are necessary to organize and maintain channel distribution during differentiation of myelinated axons.     

Fluorescently labelled Na+ channels are localized and immobilized to synapses of innervated muscle fibres

Segregation of voltage-dependent sodium channels to the hillock of motoneurones and nodes of Ranvier in myelinated axons is crucial for conduction of the nerve impulse. Much less is known, however, about the distribution of voltage-dependent Na+ channels on muscle fibres. Recently, Beam et al. have shown that Na+ channels are concentrated near the neuromuscular junction. To determine the topography and mechanisms governing the distribution of voltage-dependent Na+ channels on muscle, microfluorimetry and fluorescence photobleach recovery (FPR) have now been used to measure the density and lateral mobility of fluorescently labelled Na+ channels on uninnervated and innervated muscle fibres. On uninnervated myotubes, Na+ channels are diffusely distributed and freely mobile, whereas after innervation the channels concentrate at neuronal contact sites. These channels are immobile and co-localize with acetylcholine receptors (AChRs). At extrajunctional regions the Na+ channel density is lower and the channels more mobile. The results suggest that the nerve induces Na+ channels to redistribute, immobilize and co-localize with AChRs at sites of neuronal contact.     

Experimentally induced alteration in the polarity of developing neurons

Despite the great diversity of shapes exhibited by different classes of nerve cells, nearly all neurons share one feature in that they have a single axon and several dendrites. The two types of processes differ in their morphology, in their rate of growth, in the macromolecular composition of their cytoskeletons and surface membranes, and in their synaptic polarity. When hippocampal neurons are dissociated from the embryonic brain and cultured, they reproducibly establish this basic form with a single axon and several dendrites, despite the absence of any spatially organized environmental cues, and without the need for cell to cell contact. We have cut the axons of young hippocampal neurons within a day of their development: in some cases the initial axon regenerated, but more frequently one of the other processes, which if undisturbed would have become a dendrite, instead became the axon. Frequently the stump of the original axon persisted following the transection and subsequently became a dendrite. Evidently the neuronal processes that first develop in culture have the capacity to form either axons or dendrites. The acquisition of axonal characteristics by one neuronal process apparently inhibits the others from becoming axons, so they subsequently become dendrites.     

Reformation of long axon pathways in adult rat central nervous system by human forebrain neuroblasts.

The failure of lesioned axons to regenerate over long distances in the mammalian central nervous system (CNS) is not due to an inability of central neurons to regenerate, but rather to the non-permissive nature of the CNS tissue environment. Regenerating CNS axons, which grow well within a peripheral nerve, for example, fail to penetrate mature CNS tissue by more than about 1 mm. Recent evidence indicates that this may be due to inhibitory membrane proteins associated with CNS oligodendrocytes and myelin. We report here that human telencephalic neuroblasts implanted into the excitotoxically lesioned striatum of adult rats can escape or neutralize this inhibitory influence of the adult CNS environment and extend axons along major myelinated fibre tracts for distances of up to approximately 20 mm. The axons were seen to elongate along the paths of the striato-nigral and cortico-spinal tracts to reach the substantia nigra, the pontine nuclei and the cervical spinal cord, which are the normal targets for the striatal and cortical projection neurons likely to be present in these implants.     

Common mechanisms of nerve and blood vessel wiring

Blood vessels and nerve fibres course throughout the body in an orderly pattern, often alongside one another. Although superficially distinct, the mechanisms involved in wiring neural and vascular networks seem to share some deep similarities. The discovery of key axon guidance molecules over the past decade has shown that axons are guided to their targets by finely tuned codes of attractive and repulsive cues, and recent studies reveal that these cues also help blood vessels to navigate to their targets. Parallels have also emerged between the actions of growth factors that direct angiogenic sprouting and those that regulate axon terminal arborization.     

Axoplasmic transport of muscarinic receptors

The reality of axoplasmic transport is widely accepted; various neutrotransmitters, enzymes, labelled proteins and peptides are known to move rapidly along the axons of different nerve fibres. In the terminals of sympathetic nerves, noradrenaline release is controlled by various regulatory mechanisms which imply the occurrence of presynaptic receptors. In this regard, there is considerable indirect physiological evidence for the existence of muscarinic cholinergic receptors in the sympathetic nerve endings; the stimulation by acetylcholine of such presynaptic receptors elicits an inhibitory effect on noradrenaline release. We not provide direct biochemical evidence for the occurrence in dog splenic nerve of muscarinic receptors which seem to move along the axon as suggested by their rapid accumulation on either side of a ligature.     

Synthesis of fast myosin induced by fast ectopic innervation of rat soleus muscle is restricted to the ectopic endplate region

Skeletal muscle fibres, long multinucleated cells, arise by fusion of mononucleated myoblasts to form a myotube that matures into the adult fibre. The two major types of mature fibre, fast and slow fibres, differ physiologically in their rate of isotonic shortening. At the molecular level these type-specific physiological properties are ascribed to different isoforms of myosin, a major protein involved in shortening. Differentiation of fast and slow fibres seems to be under the control of motoneurones, and mature fibres are innervated by only one motoneurone. When rat soleus muscle (SOL, a slow muscle) is dually innervated with a fast nerve, it acquires some properties of a fast muscle, that is, low sensitivity to caffeine and high glycogen content. We report here that in dually innervated soleus muscle the foreign fast nerve induces synthesis of fast isoforms of myosin, but only in the segment of the muscle fibre that is close to the foreign endplate. The localized influence of the nerve endplates suggest that factors controlling the phenotypic expression of the muscle fibre have a short range of activity.     

A central role for denervated tissues in causing nerve sprouting

One of the oldest known forms of neuronal plasticity is the ability of peripheral nerves to grow and form functional connections after damage to neighbouring axons. Yet the source of the signal which elicits this "sprouting" remains unknown. In mammalian muscles, paralysis-which gives rise to many of the changes which occur in denervated muscles-causes motor nerve terminals to sprout. Could the inactive muscle fibres (rather than nerve degeneration products, another likely source) be responsible for some of the sprouting found in partial denervation? We confirm in this paper that direct stimulation of a partially denervated muscle inhibits sprouting and show that stimulation does so by activating the denervated fibres. Consequently after partial denervation the same signal as that which causes terminal sprouting in a paralysed muscle is able to spread from the denervated muscle fibres to the nerves on the innervated fibres and initiate terminal sprouting.     

电压门控性钾离子通道对人骨肉瘤细胞增殖的影响

研究钾离子通道在人骨肉瘤细胞的表达及通道抑制剂对人骨肉瘤细胞系增殖的影响.通过RT-PCR对三株人骨肉瘤细胞系MG63、Saos-2和SOSP-9607中相关钾离子通道,包括kv1.1, kv1.3, kv1.5, kv2.1, kv3.3/3.4, kv4.1, kv5.1, kv9.3, herg, heag 和kcnq1的基因表达进行检测;用非特异性的电压门控性钾离子通道抑制剂4-AP(4-aminopyridine,4-氨基吡啶)和氯化铯(CsCl),特异性的HERG、HEAG 、和KCa通道抑制剂E-4031、丙咪嗪(imipramine) 和TEA (tetraethylammonium,四乙铵)通过MTT法检测抑制剂对三株人骨肉瘤细胞系MG63、Saos-2和SOSP-9607细胞增殖的影响.通过RT-PCR发现kv1.3, kv1.5, kv2.1, kv3.3/3.4, kv4.1, kv9.3, herg和heag通道基因在三株骨肉瘤细胞系中均有表达,而kv5.1通道基因只在MG63细胞中表达,kcnq1通道基因只在SOSP-607和MG63细胞中表达.MTT法检测发现:E-4031, imipramine 和TEA 对三株骨肉瘤细胞的增殖没有明显的影响,而4-AP药物浓度在3 mol和5 mol时对细胞增殖影响明显.CSCL药物浓度在5 mol时对细胞增殖也有影响,但影响程度较4-AP小.提示钾离子通道在RNA水平广泛地表达于实验的三株人骨肉瘤细胞中,但只有电压门控性钾离子通道参与细胞增殖过程的调节.     

Chondroitinase ABC promotes functional recovery after spinal cord injury

The inability of axons to regenerate after a spinal cord injury in the adult mammalian central nervous system (CNS) can lead to permanent paralysis. At sites of CNS injury, a glial scar develops, containing extracellular matrix molecules including chondroitin sulphate proteoglycans (CSPGs). CSPGs are inhibitory to axon growth in vitro, and regenerating axons stop at CSPG-rich regions in vivo. Removing CSPG glycosaminoglycan (GAG) chains attenuates CSPG inhibitory activity. To test the functional effects of degrading chondroitin sulphate (CS)-GAG after spinal cord injury, we delivered chondroitinase ABC (ChABC) to the lesioned dorsal columns of adult rats. We show that intrathecal treatment with ChABC degraded CS-GAG at the injury site, upregulated a regeneration-associated protein in injured neurons, and promoted regeneration of both ascending sensory projections and descending corticospinal tract axons. ChABC treatment also restored post-synaptic activity below the lesion after electrical stimulation of corticospinal neurons, and promoted functional recovery of locomotor and proprioceptive behaviours. Our results demonstrate that CSPGs are important inhibitory molecules in vivo and suggest that their manipulation will be useful for treatment of human spinal injuries.     

Assembly of microtubules at the tip of growing axons

The growth of axons in the developing nervous system depends on the elongation of the microtubules that form their principal longitudinal structural element. It is not known whether individual microtubules in the axon elongate at their proximal ends, close to the cell body, and then move forward into the lengthening axon, or whether tubulin subunits are transported to the tip of the axon and assembled there onto the free ends of microtubules. The former possibility is supported by studies of slow axonal transport in mature nerves from which it has been deduced that microtubule assembly occurs principally at the neuronal cell body. By contrast, the polarity of microtubules in axons, which have their 'plus' or 'fast-growing' ends distal to the cell body, suggests that assembly occurs at the growing tip, or growth cone, of the axon. We have addressed this question by topically applying Colcemid (N-desacetyl-N-methylcolchicine), and other drugs which alter microtubule stability, to different regions of isolated nerve cells growing in tissue culture. We find that the sensitivity to these drugs is greatest at the growth cone by at least two orders of magnitude, suggesting that this is a major site of microtubule assembly during axonal growth.