Cold Blockade of Axonal Transport Activates Premitotic Activity of Schwann Cells and Wallerian Degeneration

Between 3 and 4 days after transection of cat sciatic nerve, Schwann cell-associated premitotic activity spreads anterogradely along degenerating distal nerve stumps at a rate of approximately 200 mm/day. We investigated whether fast anterograde axonal transport contributes to the initiation of this component of Wallerian degeneration. Axonal transport was blocked in intact and transected cat sciatic nerves by focally chilling a proximal segment to temperatures below 11 degrees C for 24 hr. Incorporation of [3H]thymidine (a marker of premitotic DNA synthesis) was then measured 3 and 4 days posttransection in cold blocked- and control-degenerating nerves. Effects of cold block prior to and concomitant with nerve transection were studied. Results failed to support the hypothesis that Schwann-cell premitotic activity after axotomy is associated with entry into the axon of mitogenic substances and their anterograde fast transport along the distal stump. Instead, data suggested that progressive anterograde failure of fast anterograde transport distal to transection serves to effect the Schwann-cell premitotic response to axotomy.     

Differential time course of the response to axotomy induced by cut or crush in the leech AP cell

The time course of the reaction to axotomy in the leech AP cell was determined by measuring the duration of the spontaneous spikes at different times after the operation. The axotomy performed by section of the segmental roots containing the AP axon induced an increase of the spike duration, which persisted over 30 days. A different time course was found when the axotomy was performed by nerve crush: the changes in duration of the spontaneous spikes, which occurred during the early 2 weeks, were significantly reduced afterwards. Dye staining of some cells axotomized by crushing revealed that the reversion of the changes, which had been set up by axotomy, was in some cases concomitant with the reconnection between proximal and distal axon stumps. The section of a single axonal branch was never sufficient to affect the membrane properties of the AP cells. It is concluded that the changes observed in axotomized AP cells are not produced by simple axonal injury and that the maintainance of normal properties in the somatic membrane requires the presence of at least part of the distal axon arborization.     

Impaired function of HDAC6 slows down axonal growth and interferes with axon initial segment development

The development of morphological neuronal polarity starts by the formation and elongation of an axon. At the same time the axon initial segment (AIS) is generated and creates a diffusion barrier which differentiate axon and somatodendritic compartment. Different structural and functional proteins that contribute to the generation of neuronal action potential are concentrated at the axon initial segment. While axonal elongation is controlled by signalling pathways that regulate cytoskeleton through microtubule associated proteins and tubulin modifications, the microtubule cytoskeleton under the AIS is mostly unknown. Thus, understanding which proteins modify tubulin, where in the neuron and at which developmental stage is crucial to understanding how morphological and functional neuronal polarity is achieved. In this study performed in mice and using a well established model of murine cultured hippocampal neurons, we report that the tubulin deacetylase HDAC6 is localized at the distal region of the axon, and its inhibition with TSA or tubacin slows down axonal growth. Suppression of HDAC6 expression with HDAC6 shRNAs or expression of a non-active mutant of HDAC6 also reduces axonal length. Furthermore, HDAC6 inhibition or suppression avoids the concentration of ankyrinG and sodium channels at the axon initial segment (AIS). Moreover, treatment of mouse cultured hippocampal neurons with detergents to eliminate the soluble pool of microtubules identified a pool of detergent resistant acetylated microtubules at the AIS, not present at the rest of the axon. Inhibition or suppression of HDAC6 increases acetylation all along the axon and disrupts the specificity of AIS cytoskeleton, modifying the axonal distal gradient localization of KIF5C to a somatodendritic and axonal localization. In conclusion, our results reveal a new role of HDAC6 tubulin deacetylase as a regulator of microtubule characteristics in the axon distal region where axonal elongation takes place, and allowing the development of acetylated microtubules microdomains where HDAC6 is not concentrated, such as the axon initial segment.     

中枢神经系统内神经元信号处理(英文)

一种新颖的轴突断端(axon bleb)膜片钳记录方法大力促进了中枢神经系统轴突功能的研究。我们的工作应用这一方法揭示了大脑皮层锥体神经元的数码信号(具全或无特性的动作电位)的爆发和传播机制。在轴突始段(axon initial segment,AIS)远端高密度聚集的低阈值Na+通道亚型Nav1.6决定动作电位的爆发;而在AIS近端高密度聚集的高阈值Na+通道亚型Nav1.2促进动作电位向胞体和树突的反向传播。应用胞体和轴突的同时记录,我们发现胞体阈下膜电位的变化可以在轴突上传播较长的距离并可到达那些离胞体较近的突触前终末。进一步的研究证明了胞体膜电位的变化调控动作电位触发的突触传递,该膜电位依赖的突触传递是一种模拟式的信号传递。轴突上一类特殊K+通道(Kv1)的活动调制动作电位的波形,特别是其波宽,从而调控各种突触前膜电位水平下突触强度的变化。突触前终末的背景Ca2+浓度也可能参与模拟信号的传递。这些发现深化了我们对中枢神经系统内神经信号处理基本原理的认识,进而帮助我们理解脑如何工作。     

A little nip and tuck: axon refinement during development and axonal injury

While building the nervous system, regions of some developing axons are eliminated; this can also happen as a result of axonal injury. During development, many axon branches that are formed in excess of an organism's needs are fated for removal in a process called axon pruning. By contrast, when axons are injured the axon segment distal to the injury site is compartmentalized and eliminated. In both cases, the end result is similar -- a region of an axon is selected for removal. Recent evidence suggests that there are some similarities in the cellular and molecular mechanisms that regulate axon elimination in development and during axonal injury.     

Resealing of the proximal and distal cut ends of transected axons: Electrophysiological and ultrastructural analysis

The fates of the proximal and distal segments of transected axons differ. Whereas the proximal segment usually recovers from injury and regenerates, the distal segment degenerates. In the present report we studied the kinetics of the recovery processes of both proximal and distal axonal segment following axotomy and its temporal relations to the alterations in the cytoarchitecture of the injured neuron. The experiments were performed on primary cultured metacerebral neurons (MCn) isolated from Aplysia. We transected axons while monitoring the changes in transmembrane potential and input resistance (Rn) by inserting intracellular microelectrodes into the soma and axon. Correlation between the electrophysiological status of the injured axon and its ultrastructure was provided by rapid fixation of the neuron at selected times postaxotomy. Axotomy leads to membrane depolarization from a mean of ?55.7 S.D. 12.8 mV to ?12.7 S.D. 3.3 mV and decreased Rn from tens of MΩ to 1–3 MΩ. The transected axons remained depolarized for a period of 10–260 s for as long as the axoplasm was in direct contact with the bathing solution. Rapid repolarization and partial recovery of Rn was associated with the formation of a membrane seal over the cut ends by the constriction and subsequent fusion of the axolema. Prior to the formation of a membraneous barrier, electron-dense deposits aggregate at the tip of the cut axon and appear to form an axoplasmic “plug.” Electrophysiological analysis revealed that this “plug” does not provide resistance for current flow and that the axoplasmic resistance is homogenously distributed. The kinetics of injury and recovery processes as well as the ultrastructural changes of the proximal and distal segments are cannot be attributed to differences in the immediated response of the segments to axotomy. © 1993 John Wiley & Sons, Inc.     

Using fluorescence photoablation to study the regeneration of singly cut leech axons

The regeneration of the axons of leech Retzius cells was compared following two different methods of axonal severing: (1) a crush of the whole connective that includes the Retzius axon; and (2) photoablation of a small segment of only the Retzius axon. The photoablation was carried out after filling the Retzius cell with Lucifer Yellow (LY). Several tests were carried out to determine whether the photoablation actually severed the axon. These included (1) using the lipophilic membrane probe DiI as an indicator of membrane severance (2) electron microscopic examination of the photoablated axon after filling it with horseradish peroxidase (HRP); and (3) filling the Retzius cell first with HRP, then photoablating, and looking for the disappearance of the HRP in the photoablated region. These and other observations indicated that the photoablated axon was actually severed. Two differences were seen in the regeneration of the Retzius axon after crush versus after photoablation. First, the sprouting following crush was far more disorganized, and included significantly more lateral spread. Second, after photoablation, over 70% of the axons, upon refilling with LY after 3 days or more, showed the newly introduced LY suddenly extending far down the distal segment, indicating that the proximal and distal segments had become reconnected. This was never seen following a crush. The photoablated axons did not pass HRP into the distal segment, suggesting that the reconnection was not by fusion, but perhaps by a gap junction. The results show that axonal regeneration can take a dramatically different form than it does following a standard crush procedure if, instead, the axon is severed in a way that preserves the structural integrity of the surrounding tissue.     

Simple models of stimulation of neurones in the brain by electric fields

The excitation of pyramidal cells in the motor cortex, produced by electric fields generated by distant electrodes or by electromagnetic induction, has been modelled. Linear, steady-state models of myelinated axons capture most of the geometrical aspects of neurone activation in electric fields. Some non-linear features can be approximated. Models with a proximal sealed-end and distal infinite axon, or of finite length, are both serviceable. Surface anodal stimulation produces hyperpolarisation of the proximal axon (closest to the anode) and depolarisation in the distal axon. The point of maximum depolarisation can be influenced by the location of the cathode (greater separation of anode and cathode causes more distal depolarisation). Axon bends can produce very localised depolarisation. Cathodal stimulation may be less effective than anodal as a result of anodal block of conduction of action potentials in the distal axon. The latencies of responses to anodal stimulation, recorded in the distal axon, will decrease as the stimulus strength is increased and the point of action potential initiation moves distally node by node. Larger jumps in latency will be produced when the point of action potential initiation moves from one axon bend to another.     

Anterograde axonal transport of endopeptidase 24.15 in rat sciatic nerves

Axonal transport of endopeptidase 24.15 (EP24.15), a putative neuropeptide degrading-enzyme, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. At 48h after ligation, a significant amount of the axonal transport of EP24.15 activity was found in the proximal segment, while axonal transport of deamidase activity, a lysosomal enzyme, increased in both proximal and distal segments. Western blot analysis of EP24.15 showed that EP24.15 immunoreactivity in the proximal segment was 1.8-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in the immunoreactive EP24.15 in the proximal segment in comparison with that in the middle segment. In the distal segment, no axonal transport of EP24.15 was found in all methods examined, indicating that EP24.15 is mainly transported by an anterograde axonal flow. These observations suggest that EP24.15 may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts.     

Axonal shortening and the mechanisms of axonal motility

Axons in tissue culture retract and shorten if their tips are detached from the substrate. The shortening reaction of the axon involves contractile forces that also arise during normal axonal motility, elongation, and retraction. We studied shortening in axonal segments isolated from their parent axons by transecting the axon between the growth cone and the most distal point of adhesion to the substrate. Within 15-20 minutes after transection, an isolated axonal segment shortened and pulled its tail end toward the growth cone. During the shortening process, long sinusoidal bends arose along the axon. The identical shortening reaction occurs without transection, when the axon tip is detached from the substrate. Pharmacological studies with inhibitors of glycolysis indicate that the shortening mechanisms utilize metabolic energy, presumably ATP. The rate of sinusoidal shortening is similar to both the rate of polymer translocation in the axon by slow axonal transport and the rate of normal axonal elongation. Taxol inhibits the shortening reaction with a similar dose dependence to its inhibition of axonal growth. Together, all these observations suggest that the same basic intracellular motility mechanisms are involved in normal axonal growth, in slow axonal transport, and in the shortening reaction: the intracellular dynamic system that utilizes ATP to generate longitudinal movements of polymers within the axon may be the same mechanism underlying both the retraction and the elongation of the axon.     

Stop-flow: a new technique for measuring axonal transport, and its application to the transport of dopamine-beta-hydroxylase.

An apparatus was devised which utilizes local cooling to reversibly interrupt the axonal transport of dopamine-beta-hydroxylase (DBH) in rabbit sciatic nerves in vitro. Lowering the temperature of a short region of nerve to between 1 and 3 degrees C, while keeping the remainder at 37 degrees C, caused DBH activity to accumulate in and proximal to the cooled region. This accumulation was evident after 0.5 hr of cooling and increased in a nearly linear fashion with time for about 3 hr. The cooling-induced interruption in transport was rapidly reversed when nerves were rewarmed to 37 degrees C. Upon rewarming after local cooling for 1.5 hr, a peak of accumulated DBH activity migrated toward the distal end of the nerve at a velocity of 300 +/- 17 mm/day. This velocity was maintained for as long as the peak could be followed and was four times greater than the average velocity estimated from the rate of accumulation of DBH activity above a ligature at the distal end of these same nerves. It is concluded that ligation experiments grossly underestimate the true velocity of axonal transport of DBH and that the present technique offers great advantages in permitting direct study of the migration of separate axonal compartments of transported materials.     

Development of the axon cap neuropil of the Mauthner cell in the goldfish

Summary Development of the axon cap neuropil of the Mauthner neuron in post-hatching larval goldfish brains was observed electron-microscopically. The axonal initial segment of newly hatched (day-4) larvae is completely covered with synaptic terminals containing clear spherical synaptic vesicles. Profiles of thin terminal axons, the spiral fibers, containing similar synaptic vesicles, rapidly increase in number around the initial segment and form glomerular neuropil similar to the central core of the adult axon cap by day 7. Three types of synapses are formed in the core neuropil. Bouton-type synapses contacting the initial segment are most abundant in day-4 to-14 larvae; they decrease thereafter and are rare on the distal half of the initial segment of day-40 larvae. Asymmetric axo-axonic synapses are commonly observed between spiral fibers in the core neuropil of day-7 to -19 larvae, but become fewer by day 40. Unique symmetrical axo-axonic synapses showing accumulation of synaptic vesicles on either side of apposed membrane thickenings first appear in day-14 core neuropil, gradually increase in number, and become the predominant type in day-40 core neuropil. Thick myelinated axons, which lose their myelin sheaths in the glial cap cell layer, start to penetrate into the axon cap on day 10. They gradually increase in number and form the peripheral part of the axon cap together with the cap dendrites, which finally grow into the axon cap from the axon hillock region of the Mauthner cell by day 40.     

Stop-flow: A new technique for measuring axonal transport,and its application to the transport of dopamine-β-hydroxylase

An apparatus was devised which utilizes local cooling to reversibly interrupt the axonal transport of dopamine-β-hydroxylase (DBH) in rabbit sciatic nerves in vitro. Lowering the temperature of a short region of nerve to between 1 and 3°C, while keeping the remainder at 37°C, caused DBH activity to accumulate in and proximal to the cooled region. This accumulation was evident after 0.5 hr of cooling and increased in a nearly linear fashion with time for about 3 hr. The cooling-induced interruption in transport was rapidly reversed when nerves were rewarmed to 37°C. Upon rewarming after local cooling for 1.5 hr, a peak of accumulated DBH activity migrated toward the distal end of the nerve at a velocity of 300 ± 17 mm/day. This velocity was maintained for as long as the peak could be followed and was four times greater than the average velocity estimated from the rate of accumulation of DBH activity above a ligature at the distal end of these same nerves. It is concluded that ligation experiments grossly underestimate the true velocity of axonal transport of DBH and that the present technique offers great advantages in permitting direct study of the migration of separate axonal compartments of transported materials.     

Microtubule stability along mammalian peripheral nerves

Microtubule (MT) number, axonal area, and MT density were examined in unmyelinated axons of rat cervical vagus nerve. Study of nerve regions proximal (1-5 mm) and distal (35-40 mm) to the nodosum ganglion in controls (incubation at 37 degrees C for 1 h) showed that the number of MT per axon is significantly less in distal than in proximal nerve regions. Cooling (incubation at 0 degree C for 1 h) caused a significant reduction in the number of MT per axon in both nerve regions. The unmyelinated axons from both nerve regions showed a comparable reduction in MT number by cooling, indicating that axonal MT stability to cold was not significantly different between these two nerve regions. In these nerves no detectable changes were found in cross-axonal area of unmyelinated axons between distal and proximal nerve regions. In another experimental series, in distal nerve regions (35-40 mm from the nodosum ganglion) the number of MT was not further reduced in nerves incubated at 0 degree C by increasing the incubation time. Similar results were obtained from colchicine treated nerves (incubation at 37 degrees C, with 10 mM colchicine for 1 and 2 h). Distal nerve regions (35-40 mm from the nodosum ganglion) showed a similar reduction in the number of MT per axon when nerves were incubated at 0 degree C or with colchicine, suggesting that this drug, as well as cold, may be affecting a similar population of axonal MT, i.e., MT susceptible to anti-MT agents. These results indicate that approximately one-half of the axonal MT are stable to cold as well as to colchicine in rat unmyelinated axons.     

Uptake of lipoproteins for axonal growth of sympathetic neurons

Lipoproteins originating from axon and myelin breakdown in injured peripheral nerves are believed to supply cholesterol to regenerating axons. We have used compartmented cultures of rat sympathetic neurons to investigate the utilization of lipids from lipoproteins for axon elongation. Lipids and proteins from human low density lipoproteins (LDL) and high density lipoproteins (HDL) were taken up by distal axons and transported to cell bodies, whereas cell bodies/proximal axons internalized these components from only LDL, not HDL. Consistent with these observations, the impairment of axonal growth, induced by inhibition of cholesterol synthesis, was reversed when LDL or HDL were added to distal axons or when LDL, but not HDL, were added to cell bodies. LDL receptors (LDLRs) and LR7/8B (apoER2) were present in cell bodies/proximal axons and distal axons, with LDLRs being more abundant in the former. Inhibition of cholesterol biosynthesis increased LDLR expression in cell bodies/proximal axons but not distal axons. LR11 (SorLA) was restricted to cell bodies/proximal axons and was undetectable in distal axons. Neither the LDL receptor-related protein nor the HDL receptor, SR-B1, was detected in sympathetic neurons. These studies demonstrate for the first time that lipids are taken up from lipoproteins by sympathetic neurons for use in axonal regeneration.     

Early ablation of target muscles modulates the arborisation pattern of an identified embryonic Drosophila motor axon.

The Drosophila RP3 motor axon establishes a stereotypic arborisation along the adjoining edges of muscles 6 and 7 by the end of embryogenesis. The present study has examined the role of the target muscles in determining this axonal arborisation pattern. Target muscles were surgically ablated prior to the arrival of the RP3 axon. Following further development of the embryo in culture medium, the morphology of target-deprived RP3 motor axons was assayed by intracellular injection with the dye Lucifer Yellow. Axonal arborisations were formed on a variety of non-target muscles when muscles 6 and 7 were removed and these contacts were maintained into stage 16. The pattern of axonal arborisations over non-target muscles varied between preparations in terms of the number of muscles contacted, and the distribution of arborisations on individual muscles. Following removal of muscle 6, the RP3 motor axon frequently contacted muscle 7, and axonal arborisations were present along the distal edge of the muscle. In the absence of muscle 7, the RP3 axon often did not contact muscle 6 and when muscle 6 was contacted, the arborisation of RP3 was poorly developed. Axonal processes were retained on non-target muscles when only one target muscle was present. Therefore, the establishment of a stereotypic arborisation by the RP3 motor axon is apparently dependent on growth cone contact with both target muscles.     

Fiber composition of the distal accessory flexor muscle in several decapod crustaceans

The fiber composition of the distal accessory flexor muscle (DAFM) and the branching pattern of its excitor axon were compared in several species of crabs, in the lobster and the crayfish. The muscle is composed exclusively of long sarcomere (> 6 μm) fibers and therefore of the slow type. In all the crab species, except one, there is a distal to proximal gradient of fibers with increasing sarcomere lengths; this gradient is reverse in lobsters and crayfish. A proximal to distal gradient of increasing fiber diameters occurs in the DAFM of all crab species but not in the lobster and crayfish, in which all the fibers are approximately equal in diameter. The single excitatory axon traverses the width of the DAFM and gives off primary branches on either side in the lobster and crayfish but on only one side in crabs. The hypothesis that the axonal branching pattern may govern the regional distribution of fibers with differing sarcomere lengths in proposed.     

Alternative splicing regulates kv3.1 polarized targeting to adjust maximal spiking frequency

Synaptic inputs received at dendrites are converted into digital outputs encoded by action potentials generated at the axon initial segment in most neurons. Here, we report that alternative splicing regulates polarized targeting of Kv3.1 voltage-gated potassium (Kv) channels to adjust the input-output relationship. The spiking frequency of cultured hippocampal neurons correlated with the level of endogenous Kv3 channels. Expression of axonal Kv3.1b, the longer form of Kv3.1 splice variants, effectively converted slow-spiking young neurons to fast-spiking ones; this was not the case for Kv1.2 or Kv4.2 channel constructs. Despite having identical biophysical properties as Kv3.1b, dendritic Kv3.1a was significantly less effective at increasing the maximal firing frequency. This suggests a possible role of channel targeting in regulating spiking frequency. Mutagenesis studies suggest the electrostatic repulsion between the Kv3.1b N/C termini, created by its C-terminal splice domain, unmasks the Kv3.1b axonal targeting motif. Kv3.1b axonal targeting increased the maximal spiking frequency in response to prolonged depolarization. This finding was further supported by the results of local application of channel blockers and computer simulations. Taken together, our studies have demonstrated that alternative splicing controls neuronal firing rates by regulating the polarized targeting of Kv3.1 channels.     

Protein Transport in Intact and Severed (Anucleate) Crayfish Giant Axons

Abstract: Using video-enhanced microscopy and a pulse-radiolabeling paradigm, we show that proteins synthesized in the medial giant axon cell body of the crayfish ( Procambarus clarkii ) are delivered to the axon via fast (∼62 mm/day) and slow (∼0.8 mm/day) transport components. These data confirm that the medial giant axon cell body provides protein to the axon in a manner similar to that reported for mammalian axons. Unlike mammalian axons, the distal (anucleate) portion of a medial giant axon remains intact and functional for >7 months after severance. This axonal viability persists long after fast transport has ceased and after the slow wave front of radiolabeled protein has reached the terminals. These data are consistent with the hypothesis that another source (i.e., local glial cells) provides a significant amount of protein to supplement that delivered to the medial giant axon by its cell body.     

Dynactin is required for transport initiation from the distal axon

Dynactin is a required cofactor for the minus-end-directed microtubule motor cytoplasmic dynein. Mutations within the highly conserved CAP-Gly domain of dynactin cause neurodegenerative disease. Here, we show that the CAP-Gly domain is necessary to enrich dynactin at the distal end of primary neurons. While the CAP-Gly domain is not required for sustained transport along the axon, we find that the distal accumulation facilitates the efficient initiation of retrograde vesicular transport from the neurite tip. Neurodegenerative disease mutations in the CAP-Gly domain prevent the distal enrichment of dynactin thereby inhibiting the initiation of retrograde transport. Thus, we propose a model in which distal dynactin is a key mediator in promoting the interaction among the microtubule, dynein motor, and cargo for the efficient initiation of transport. Mutations in?the CAP-Gly domain disrupt the formation of the?motor-cargo complex, highlighting the specific defects in axonal transport that may lead to neurodegeneration.