Differentiated pattern of sodium channel expression in dissociated Purkinje neurons maintained in long-term culture

Cerebellar Purkinje neurons in vivo exhibit high frequency and multi-spike action potentials with transient (INaT), resurgent (INaR) and persistent (INaP) Na+ currents arising from voltage-gated Na+ channels, which play important roles in shaping the action potentials and electrical activity of these cells. However, little is known about Na+ channel expression in cultured Purkinje neurons despite the use of in vitro approaches to study these cells. Therefore, GFP-expressing Purkinje neurons isolated from transgenic mice were analysed after four weeks in culture, when, coincident with distinct axonal and dendritic morphologies, cultured Purkinje neurons exhibited dendrite-specific MAP2 expression characteristic of polarized neurons. In cell-attached patch clamp recordings, Na+ currents occurred at significantly higher frequencies and amplitudes in patches from the soma and axon than from dendrites, similar to the polarized distribution observed in vivo. INaT, INaR and INaP Na+ currents with properties similar to those observed in acutely isolated Purkinje neurons were detected in nucleated outside-out patches from cultured Purkinje cells. RT-PCR analysis detected Nav1.1, Nav1.2 and Nav1.6, but not Nav1.3, Nav1.4, Nav 1.5 or Nav1.8 Na+ channel alpha subunit gene expression in cultured Purkinje neurons, as observed in vivo. Together, the results indicate that key aspects of Na+ channel expression in mature Purkinje neurons in vivo occur in vitro.     

Early events in node of Ranvier formation during myelination and remyelination in the PNS

Action potential conduction velocity increases dramatically during early development as axons become myelinated. Integral to this process is the clustering of voltage-gated Na(+) (Nav) channels at regularly spaced gaps in the myelin sheath called nodes of Ranvier. We show here that some aspects of peripheral node of Ranvier formation are distinct from node formation in the CNS. For example, at CNS nodes, Nav1.2 channels are detected first, but are then replaced by Nav1.6. Similarly, during remyelination in the CNS, Nav1.2 channels are detected at newly forming nodes. By contrast, the earliest Nav-channel clusters detected during developmental myelination in the PNS have Nav1.6. Further, during PNS remyelination, Nav1.6 is detected at new nodes. Finally, we show that accumulation of the cell adhesion molecule neurofascin always precedes Nav channel clustering in the PNS. In most cases axonal neurofascin (NF-186) accumulates first, but occasionally paranodal neurofascin is detected first. We suggest there is heterogeneity in the events leading to Nav channel clustering, indicating that multiple mechanisms might contribute to node of Ranvier formation in the PNS.     

Dynamic compartmentalization of the voltage-gated sodium channels in axons

One of the major physiological roles of the neuronal voltage-gated sodium channel is to generate action potentials at the axon hillock/initial segment and to ensure propagation along myelinated or unmyelinated fibers to nerve terminal. These processes require a precise distribution of sodium channels accumulated at high density in discrete subdomains of the nerve membrane. In neurons, information relevant to ion channel trafficking and compartmentalization into sub-domains of the plasma membrane is far from being elucidated. Besides, whereas information on dendritic targeting is beginning to emerge, less is known about the mechanisms leading to the polarized distribution of proteins in axon. To obtain a better understanding of how neurons selectively target sodium channels to discrete subdomains of the nerve, we addressed the question as to whether any of the large intracellular regions of Nav1.2 contain axonal sorting and/or clustering signals. We first obtained evidence showing that addition of the cytoplasmic carboxy-terminal region of Nav1.2 restricted the distribution of a dendritic-axonal reporter protein to axons of hippocampal neurons. The analysis of mutants revealed that a di-leucine-based motif mediates chimera compartmentalization in axons and its elimination in soma and dendrites by endocytosis. The analysis of the others generated chimeras showed that the determinant conferring sodium channel clustering at the axonal initial segment is contained within the cytoplasmic loop connecting domains II-III of Nav1.2. Expression of a soluble Nav1.2 II-III linker protein led to the disorganization of endogenous sodium channels. The motif was sufficient to redirect a somatodendritic potassium channel to the axonal initial segment, a process involving association with ankyrin G. Thus, it is conceivable that concerted action of the two determinants is required for sodium channel compartmentalization in axons.     

Two TTX-resistant Na+ currents in mouse colonic dorsal root ganglia neurons and their role in colitis-induced hyperexcitability

The composition of Na+ currents in dorsal root ganglia (DRG) neurons depends on their neuronal phenotype and innervation target. Two TTX-resistant (TTX-R) Na+ currents [voltage-gated Na channels (Nav)] have been described in small DRG neurons; one with slow inactivation kinetics (Nav1.8) and the other with persistent kinetics (Nav1.9), and their modulation has been implicated in inflammatory pain. This has not been studied in neurons projecting to the colon. This study examined the relative importance of these currents in inflammation-induced changes in a mouse model of inflammatory bowel disease. Colonic sensory neurons were retrogradely labeled, and colitis was induced by instillation of trinitrobenzenesulfonic acid (TNBS) into the lumen of the distal colon. Seven to ten days later, immunohistochemical properties were characterized in controls, and whole cell recordings were obtained from small (<40 pF) labeled DRG neurons from control and TNBS animals. Most neurons exhibited both fast TTX-sensitive (TTX-S)- and slow TTX-R-inactivating Na+ currents, but persistent TTX-R currents were uncommon (<15%). Most labeled neurons were CGRP (79%), tyrosine kinase A (trkA) (84%) immunoreactive, but only a small minority bind IB4 (14%). TNBS-colitis caused ulceration, thickening of the colon and significantly increased neuronal excitability. The slow TTX-R-inactivating Na current density (Nav1.8) was significantly increased, but other Na currents were unaffected. Most small mouse colonic sensory neurons are CGRP, trkA immunoreactive, but not isolectin B4 reactive and exhibit fast TTX-S, slow TTX-R, but not persistent TTX-R Na+ currents. Colitis-induced hyperexcitability is associated with increased slow TTX-R (Nav1.8) Na+ current. Together, these findings suggest that colitis alters trkA-positive neurons to preferentially increase slow TTX-R Na+ (Nav1.8) currents.     

Mutations in the stumpy gene reveal intermediate targets for zebrafish motor axons

Primary motoneurons, the earliest developing spinal motoneurons in zebrafish, have highly stereotyped axon projections. Although much is known about the development of these neurons, the molecular cues guiding their axons have not been identified. In a screen designed to reveal mutations affecting motor axons, we isolated two mutations in the stumpy gene that dramatically affect pathfinding by the primary motoneuron, CaP. In stumpy mutants, CaP axons extend along the common pathway, a region shared by other primary motor axons, but stall at an intermediate target, the horizontal myoseptum, and fail to extend along their axon-specific pathway during the first day of development. Later, most CaP axons progress a short distance beyond the horizontal myoseptum, but tend to stall at another intermediate target. Mosaic analysis revealed that stumpy function is needed both autonomously in CaP and non-autonomously in other cells. stumpy function is also required for axons of other primary and secondary motoneurons to progress properly past intermediate targets and to branch. These results reveal a series of intermediate targets involved in motor axon guidance and suggest that stumpy function is required for motor axons to progress from proximally located intermediate targets to distally located ones.     

Coupled left-shift of Nav channels: modeling the Na+-loading and dysfunctional excitability of damaged axons

Injury to neural tissue renders voltage-gated Na(+) (Nav) channels leaky. Even mild axonal trauma initiates Na(+) -loading, leading to secondary Ca(2+)-loading and white matter degeneration. The nodal isoform is Nav1.6 and for Nav1.6-expressing HEK-cells, traumatic whole cell stretch causes an immediate tetrodotoxin-sensitive Na(+)-leak. In stretch-damaged oocyte patches, Nav1.6 current undergoes damage-intensity dependent hyperpolarizing- (left-) shifts, but whether left-shift underlies injured-axon Nav-leak is uncertain. Nav1.6 inactivation (availability) is kinetically limited by (coupled to) Nav activation, yielding coupled left-shift (CLS) of the two processes: CLS should move the steady-state Nav1.6 "window conductance" closer to typical firing thresholds. Here we simulated excitability and ion homeostasis in free-running nodes of Ranvier to assess if hallmark injured-axon behaviors-Na(+)-loading, ectopic excitation, propagation block-would occur with Nav-CLS. Intact/traumatized axolemma ratios were varied, and for some simulations Na/K pumps were included, with varied in/outside volumes. We simulated saltatory propagation with one mid-axon node variously traumatized. While dissipating the [Na(+)] gradient and hyperactivating the Na/K pump, Nav-CLS generated neuropathic pain-like ectopic bursts. Depending on CLS magnitude, fraction of Nav channels affected, and pump intensity, tonic or burst firing or nodal inexcitability occurred, with [Na(+)] and [K(+)] fluctuating. Severe CLS-induced inexcitability did not preclude Na(+)-loading; in fact, the steady-state Na(+)-leaks elicited large pump currents. At a mid-axon node, mild CLS perturbed normal anterograde propagation, and severe CLS blocked saltatory propagation. These results suggest that in damaged excitable cells, Nav-CLS could initiate cellular deterioration with attendant hyper- or hypo-excitability. Healthy-cell versions of Nav-CLS, however, could contribute to physiological rhythmic firing.     

Zebrafish topped is required for ventral motor axon guidance

Zebrafish primary motor axons extend along stereotyped pathways innervating distinct regions of the developing myotome. During development, these axons make stereotyped projections to ventral and dorsal myotome regions. Caudal primary motoneurons, CaPs, pioneer axon outgrowth along ventral myotomes; whereas, middle primary motoneurons, MiPs, extend axons along dorsal myotomes. Although the development and axon outgrowth of these motoneurons has been characterized, cues that determine whether axons will grow dorsally or ventrally have not been identified. The topped mutant was previously isolated in a genetic screen designed to uncover mutations that disrupt primary motor axon guidance. CaP axons in topped mutants fail to enter the ventral myotome at the proper time, stalling at the nascent horizontal myoseptum, which demarcates dorsal from ventral axial muscle. Later developing secondary motor nerves are also delayed in entering the ventral myotome whereas all other axons examined, including dorsally projecting MiP motor axons, are unaffected in topped mutants. Genetic mosaic analysis indicates that Topped function is non-cell autonomous for motoneurons, and when wild-type cells are transplanted into topped mutant embryos, ventromedial fast muscle are the only cell type able to rescue the CaP axon defect. These data suggest that Topped functions in the ventromedial fast muscle and is essential for motor axon outgrowth into the ventral myotome.     

Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments.

The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Nav1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Nav1.6, betaIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and betaIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Nav1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Nav1.6 therefore do not initiate protein assembly at initial segments. betaIV spectrin, Nav1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.     

Determinants of voltage-gated sodium channel clustering in neurons

In mammalian neurons, the generation and propagation of the action potential result from the presence of dense clusters of voltage-gated sodium channels (Nav) at the axonal initial segment (AIS) and nodes of Ranvier. In these two structures, the assembly of specific supra-molecular complexes composed of numerous partners, such as cytoskeletal scaffold proteins and signaling proteins ensures the high concentration of Nav channels. Understanding how neurons regulate the expression and discrete localization of Nav channels is critical to understanding the diversity of normal neuronal function as well as neuronal dysfunction caused by defects in these processes. Here, we review the mechanisms establishing the clustering of Nav channels at the AIS and in the node and discuss how the alterations of Nav channel clustering can lead to certain pathophysiologies.     

Control of the spatial distribution of sodium channels in giant fiber lobe neurons of the squid

Na+ channels are present at high density in squid giant axon but are absent from its somata in the giant fiber lobe (GFL) of the stellate ganglion. GFL cells dispersed in vitro maintain growing axons and develop a Na+ channel distribution similar to that in vivo. Tunicamycin, a glycosylation inhibitor, selectively disrupts the spatially appropriate, high level expression of Na+ channels in axonal membrane but has no effect on expression in cell bodies, which show low level, inappropriate expression in vitro. This effect does not appear to involve alteration in Na+ channel turnover or axon viability. K+ channel distribution is unaffected. Thus, glycosylation appears to be involved in controlling Na+ channel localization in squid neurons.     

Defective Ca2+ channel clustering in axon terminals disturbs excitability in motoneurons in spinal muscular atrophy

Proximal spinal muscular atrophy (SMA) is a motoneuron disease for which there is currently no effective treatment. In animal models of SMA, spinal motoneurons exhibit reduced axon elongation and growth cone size. These defects correlate with reduced beta-actin messenger RNA and protein levels in distal axons. We show that survival motoneuron gene (Smn)-deficient motoneurons exhibit severe defects in clustering Cav2.2 channels in axonal growth cones. These defects also correlate with a reduced frequency of local Ca2+ transients. In contrast, global spontaneous excitability measured in cell bodies and proximal axons is not reduced. Stimulation of Smn production from the transgenic SMN2 gene by cyclic adenosine monophosphate restores Cav2.2 accumulation and excitability. This may lead to the development of new therapies for SMA that are not focused on enhancing motoneuron survival but instead investigate restoration of growth cone excitability and function.     

Paraxial mesoderm specifies zebrafish primary motoneuron subtype identity

We provide the first analysis of how a segmentally reiterated pattern of neurons is specified along the anteroposterior axis of the vertebrate spinal cord by investigating how zebrafish primary motoneurons are patterned. Two identified primary motoneuron subtypes, MiP and CaP, occupy distinct locations within the ventral neural tube relative to overlying somites, express different genes and innervate different muscle territories. In all vertebrates examined so far, paraxial mesoderm-derived signals specify distinct motoneuron subpopulations in specific anteroposterior regions of the spinal cord. We show that signals from paraxial mesoderm also control the much finer-grained segmental patterning of zebrafish primary motoneurons. We examined primary motoneuron specification in several zebrafish mutants that have distinct effects on paraxial mesoderm development. Our findings suggest that in the absence of signals from paraxial mesoderm, primary motoneurons have a hybrid identity with respect to gene expression, and that under these conditions the CaP axon trajectory may be dominant.     

Voltage-gated sodium channels and pain pathways

Acute, inflammatory, and neuropathic pain can all be attenuated or abolished by local treatment with sodium channel blockers such as lidocaine. The peripheral input that drives pain perception thus depends on the presence of functional voltage-gated sodium channels. Remarkably, two voltage-gated sodium channel genes (Nav1.8 and Nav1.9) are expressed selectively in damage-sensing peripheral neurons, while a third channel (Nav1.7) is found predominantly in sensory and sympathetic neurons. An embryonic channel (Nav1.3) is also upregulated in damaged peripheral nerves and associated with increased electrical excitability in neuropathic pain states. A combination of antisense and knock-out studies support a specialized role for these sodium channels in pain pathways, and pharmacological studies with conotoxins suggest that isotype-specific antagonists should be feasible. Taken together, these data suggest that isotype-specific sodium channel blockers could be useful analgesics.     

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。     

Trigeminal-Rostral Ventromedial Medulla circuitry is involved in orofacial hyperalgesia contralateral to tissue injury

Background

Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. ?? and ??ENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear.

Results

Here we show that deleting ?? and ??ENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro.

Conclusion

Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.     

Ankyrin G restricts ion channel diffusion at the axonal initial segment before the establishment of the diffusion barrier

In mammalian neurons, the precise accumulation of sodium channels at the axonal initial segment (AIS) ensures action potential initiation. This accumulation precedes the immobilization of membrane proteins and lipids by a diffusion barrier at the AIS. Using single-particle tracking, we measured the mobility of a chimeric ion channel bearing the ankyrin-binding motif of the Nav1.2 sodium channel. We found that ankyrin G (ankG) limits membrane diffusion of ion channels when coexpressed in neuroblastoma cells. Site-directed mutants with decreased affinity for ankG exhibit increased diffusion speeds. In immature hippocampal neurons, we demonstrated that ion channel immobilization by ankG is regulated by protein kinase CK2 and occurs as soon as ankG accumulates at the AIS of elongating axons. Once the diffusion barrier is formed, ankG is still required to stabilize ion channels. In conclusion, our findings indicate that specific binding to ankG constitutes the initial step for Nav channel immobilization at the AIS membrane and precedes the establishment of the diffusion barrier.     

Polysialic acid influences specific pathfinding by avian motoneurons.

The influence of polysialic acid (PSA) on the neural cell adhesion molecule on motoneuron outgrowth and pathway formation was investigated by determining its temporal and spatial pattern of expression and by the effect that its removal had on motoneuron projection patterns. Motoneurons first expressed PSA as their growth cones began to segregate into motoneuron pool-specific groups in the plexus region; furthermore, PSA levels differed between motoneurons projecting to different targets. When PSA was removed during the period of axonal segregation in the plexus region projection errors were common. However, later removal during the process of muscle nerve formation did not result in projection errors. These results suggest that PSA modulates interactions between motoneuron axons and guidance molecules in the plexus region during axonal pathfinding.     

Inflammatory mediators increase Nav1.9 current and excitability in nociceptors through a coincident detection mechanism

Altered function of Na+ channels is responsible for increased hyperexcitability of primary afferent neurons that may underlie pathological pain states. Recent evidence suggests that the Nav1.9 subunit is implicated in inflammatory but not acute pain. However, the contribution of Nav1.9 channels to the cellular events underlying nociceptor hyperexcitability is still unknown, and there remains much uncertainty as to the biophysical properties of Nav1.9 current and its modulation by inflammatory mediators. Here, we use gene targeting strategy and computer modeling to identify Nav1.9 channel current signature and its impact on nociceptors' firing patterns. Recordings using internal fluoride in small DRG neurons from wild-type and Nav1.9-null mutant mice demonstrated that Nav1.9 subunits carry the TTX-resistant "persistent" Na+ current called NaN. Nav1.9(-/-) nociceptors showed no significant change in the properties of the slowly inactivating TTX-resistant SNS/Nav1.8 current. The loss in Nav1.9-mediated Na+ currents was associated with the inability of small DRG neurons to generate a large variety of electrophysiological behaviors, including subthreshold regenerative depolarizations, plateau potentials, active hyperpolarizing responses, oscillatory bursting discharges, and bistable membrane behaviors. We further investigated, using CsCl- and KCl-based pipette solutions, whether G-protein signaling pathways and inflammatory mediators upregulate the NaN/Nav1.9 current. Bradykinin, ATP, histamine, prostaglandin-E2, and norepinephrine, applied separately at maximal concentrations, all failed to modulate the Nav1.9 current. However, when applied conjointly as a soup of inflammatory mediators they rapidly potentiated Nav1.9 channel activity, generating subthreshold amplification and increased excitability. We conclude that Nav1.9 channel, the molecular correlate of the NaN current, is potentiated by the concerted action of inflammatory mediators that may contribute to nociceptors' hyperexcitability during peripheral inflammation.     

Use-dependent potentiation of the Nav1.6 sodium channel

Nav1.2 and Nav1.6 are two voltage-gated sodium channel isoforms that are abundant in the adult central nervous system. These channels are expressed in different cells and localized in different neuronal regions, which may reflect functional specialization. To examine this possibility, we compared the properties of Nav1.2 and Nav1.6 in response to a rapid series of repetitive depolarizations. Currents through Nav1.6 coexpressed with beta1 demonstrated use-dependent potentiation during a rapid train of depolarizations. This potentiation was in contrast to the use-dependent decrease in current for Nav1.2 with beta1. The voltage dependence of potentiation correlated with the voltage dependence of activation, and it still occurred when fast inactivation was removed by mutation. Rapid stimulation accelerated a slow phase of activation in the Nav1.6 channel that had fast inactivation removed, resulting in faster channel activation. Although the Nav1.2 channel with fast inactivation removed also demonstrated slightly faster activation, that channel showed very pronounced slow inactivation compared to Nav1.6. These results indicate that potentiation of Nav1.6 sodium currents results from faster channel activation, and that this effect is masked by slow inactivation in Nav1.2. The data suggest that Nav1.6 might be more resistant to inactivation, which might be helpful for high-frequency firing at nodes of Ranvier compared to Nav1.2.