Guisheng Zhong

Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons.

Axonal Actin How actin is organized in the axons and dendrites of neurons is largely unknown. Xu et al. (p. 452, published online 13 December) imaged actin in axons and dendrites using stochastic optical reconstruction microscopy. Surprisingly, while actin in dendrites formed long filaments, the actin in axons was organized into evenly spaced ringlike structures at the axon circumference. Spectrin, which is known to interact with actin to form a membrane cytoskeleton in erythrocytes, formed periodic structures that alternated with those of actin. This actin-spectrin cytoskeletal structure might give mechanical support to axons and could also organize other membrane proteins. Superresolution microscopy reveals a membrane cytoskeleton in neurons comprising rings of actin separated by spectrin. Actin and spectrin play important roles in neurons, but their organization in axons and dendrites remains unclear. We used stochastic optical reconstruction microscopy to study the organization of actin, spectrin, and associated proteins in neurons. Actin formed ringlike structures that wrapped around the circumference of axons and were evenly spaced along axonal shafts with a periodicity of ~180 to 190 nanometers. This periodic structure was not observed in dendrites, which instead contained long actin filaments running along dendritic shafts. Adducin, an actin-capping protein, colocalized with the actin rings. Spectrin exhibited periodic structures alternating with those of actin and adducin, and the distance between adjacent actin-adducin rings was comparable to the length of a spectrin tetramer. Sodium channels in axons were distributed in a periodic pattern coordinated with the underlying actin-spectrin–based cytoskeleton.

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30–60 nm spatial resolution at temporal resolutions down to 1–2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.

In mice lacking V2a interneurons, gait depends on speed of locomotion

Many animals are capable of changing gait with speed of locomotion. The neural basis of gait control and its dependence on speed are not fully understood. Mice normally use a single “trotting” gait while running at all speeds, either over ground or on a treadmill. Transgenic mouse mutants in which the trotting is replaced by hopping also lack a speed-dependent change in gait. Here we describe a transgenic mouse model in which the V2a interneurons have been ablated by targeted expression of diphtheria toxin A chain (DTA) under the control of the Chx10 gene promoter (Chx10::DTA mice). Chx10::DTA mice show normal trotting gait at slow speeds but transition to a galloping gait as speed increases. Although left–right limb coordination is altered in Chx10::DTA mice at fast speed, alternation of forelegs and hindlegs and the relative duration of swing and stance phases for individual limbs is unchanged compared with wild-type mice. The speed-dependent loss of left–right alternation is recapitulated during drug-induced fictive locomotion in spinal cords isolated from neonatal Chx10::DTA mice, and high-speed fictive locomotion evoked by caudal spinal cord stimulation also shows synchronous left–right bursting. These results show that spinal V2a interneurons are required for maintaining left–right alternation at high speeds. Whether animals that generate galloping or hopping gaits, characterized by synchronous movement of left and right forelegs and hindlegs, have lost or modified the function of V2a interneurons is an intriguing question.

Persistent Sodium Currents Participate in Fictive Locomotion Generation in Neonatal Mouse Spinal Cord

The persistent sodium current (INa(P)) has been implicated in the regulation of synaptic integration, intrinsic membrane properties, and rhythm generation in many types of neurons. We characterized INa(P) in commissural interneurons (CINs) in the neonatal (postnatal days 0–3) mouse spinal cord; it is activated at subthreshold potentials, inactivates slowly, and can be blocked by low concentrations of riluzole. The role of INa(P) in locomotor pattern generation was examined by applying riluzole during fictive locomotion induced by NMDA, serotonin, and dopamine or by stimulation of the cauda equina. Blockade of INa(P) has marginal effects on the locomotion frequency but progressively weakens the rhythmic firing and locomotor-related membrane oscillation of CINs and motoneurons (MNs) and the locomotor-like bursts in ventral roots, until the motor pattern ceases. Riluzole directly affects the intrinsic firing properties of CINs and MNs, reducing their ability to fire repetitively during tonic depolarizations and raising their spike threshold. At the same time, riluzole has little effects on the strength of spike-evoked synaptic transmission onto CINs and MNs. Our results suggest that INa(P) is essential for the generation of the locomotor pattern and acts in part by regulating the frequency of interneuron firing in the central pattern generator for locomotion.

Electrophysiological Characterization of V2a Interneurons and Their Locomotor-Related Activity in the Neonatal Mouse Spinal Cord

The V2a class of Chx10-expressing interneurons has been implicated in frequency-dependent control of left–right phase during locomotion in the mouse. We have used the Chx10::CFP mouse line to further investigate the properties and locomotion-related activity of V2a interneurons in the isolated neonatal spinal cord. V2a interneurons can be divided into three classes, based on their tonic, phasic, or delayed-onset responses to step depolarization. Electrical coupling is found only between neurons of same class and helps to synchronize neuronal activity within the class. Serotonin (5-HT) excites isolated tonic V2a interneurons by depolarizing the neurons and increasing their membrane input resistance, with no significant effects on action potential properties, a mechanism distinct from 5-HT excitation of commissural interneurons. During NMDA-/5-HT-induced locomotor-like activity, patch-clamp recordings and two-photon calcium imaging experiments show that approximately half of V2a interneurons fire rhythmically with ventral root-recorded motor activity; the rhythmic V2a interneurons fired during one half of the cycle, in phase with either the ipsilateral or the contralateral L2 ventral root bursts. The percentage of rhythmically firing V2a interneurons increases during higher-frequency fictive locomotion, and they become significantly more rhythmic in their firing during the locomotor cycle; this may help to explain the frequency-dependent shift in left–right coupling in Chx10::DTA mice, which lack these neurons. Our results together with data from the accompanying paper (Dougherty and Kiehn, 2009) reinforce earlier proposals that the V2a interneurons are components of the hindlimb central pattern generator, helping to organize left–right locomotor coordination in the neonatal mouse spinal cord.

Developmental mechanism of the periodic membrane skeleton in axons

Actin, spectrin, and associated molecules form a periodic sub-membrane lattice structure in axons. How this membrane skeleton is developed and why it preferentially forms in axons are unknown. Here, we studied the developmental mechanism of this lattice structure. We found that this structure emerged early during axon development and propagated from proximal regions to distal ends of axons. Components of the axon initial segment were recruited to the lattice late during development. Formation of the lattice was regulated by the local concentration of βII spectrin, which is higher in axons than in dendrites. Increasing the dendritic concentration of βII spectrin by overexpression or by knocking out ankyrin B induced the formation of the periodic structure in dendrites, demonstrating that the spectrin concentration is a key determinant in the preferential development of this structure in axons and that ankyrin B is critical for the polarized distribution of βII spectrin in neurites. DOI: http://dx.doi.org/10.7554/eLife.04581.001

Intrinsic and Functional Differences among Commissural Interneurons during Fictive Locomotion and Serotonergic Modulation in the Neonatal Mouse

Commissural interneurons (CINs) send their axons across the midline to innervate contralateral targets and have been implicated in the coordination of left–right limb movements during locomotion. In the neonatal mouse spinal cord, we studied the firing properties and responses to serotonin (5-HT) of two classes of CINs: those whose axons turn caudally after crossing the midline (dCINs) and those whose axons bifurcate after crossing the midline (adCINs). During NMDA and 5-HT-induced locomotor-like activity, a majority of lumbar (L2) dCINs fired rhythmically with ventral root-recorded motor activity, although their firing phase was widely distributed throughout the locomotor cycle. In contrast, none of the adCINs fired rhythmically during fictive locomotion. We studied the baseline firing and membrane properties, and responses to current injection, in dCINs and adCINs that had been partially isolated by blockade of rapid synaptic transmission (with antagonists to glutamate, GABA, and glycine). No significant baseline differences were found between the cell types. In contrast, 5-HT significantly increased the excitability of the isolated dCINs by depolarizing the membrane potential, reducing the postspike afterhyperpolarization amplitude and decreasing the action potential threshold. None of these parameters were affected by 5-HT in adCINs. These results, together with our recent study of a third class of CINs, the aCINs whose axons ascend after crossing the midline (Zhong et al., 2006), suggest that dCINs and aCINs, but not adCINs, are excited by 5-HT and are rhythmically active during fictive locomotion. Thus, they may play important roles in the coordination of left–right movements during fictive locomotion.

Neuronal activity in the isolated mouse spinal cord during spontaneous deletions in fictive locomotion: insights into locomotor central pattern generator organization

•  The organization of the spinal circuitry responsible for the generation of locomotor rhythm and control of locomotion in mammals is largely unknown, though several types of spinal interneurons involved in the rodent locomotor network have been identified. •  Ventral root recordings of spinal motoneurons during fictive locomotion in the isolated mouse spinal cord show spontaneous deletions of activity. The majority of deletions in the isolated neonatal mouse spinal cord are non‐resetting: they do not change the phase of subsequent motor cycles. Flexor and extensor motoneurons express asymmetric responses during deletions: flexor deletions are accompanied by tonic ipsilateral extensor activity, while extensor deletions do not perturb rhythmic ipsilateral flexor activity. Non‐resetting deletions on one side of the cord do not perturb rhythmic activity on the other side of the cord and can occur in isolated hemicords. •  We have characterized the activity of motoneurons and identified interneurons during spontaneous motor deletions. The motoneurons and a subset of V2a interneurons fall silent during non‐resetting motor deletions while a second subset of V2a interneurons and commissural interneurons continue unperturbed rhythmic firing. This allowed us to suggest their involvement at different levels of the locomotor network operation. •  We have developed a computational model of the central pattern generator that reproduces, and proposes a mechanistic explanation for, our experimental results. The model provides novel insights into the organization of spinal locomotor networks.

Frequency-dependent recruitment of V2a interneurons during fictive locomotion in the mouse spinal cord

The principles governing the recruitment of interneurons during acceleration in vertebrate locomotion are not known. In the mouse, the V2a spinal interneurons are dispensable for left-right coordination at low locomotor frequencies, but their function is essential for maintaining left-right coordination at high frequencies. Here we explore the mechanisms driving this frequency-dependent role, using four methods to determine how V2a interneurons are recruited at different locomotor frequencies. We show that half the V2a interneurons receive rhythmic locomotor synaptic drive which increases with cycle frequency, recruiting more of the neurons to fire at higher frequencies. The other V2a interneurons do not receive locomotion-related synaptic drive, and are not recruited into the locomotor network at any frequency. The increased role of V2a interneurons at higher locomotor frequencies arises from increased synaptic drive to recruit subthreshold oscillating V2a neurons, and not from recruitment of a second set of silent V2a interneurons.

A PIK3C3–Ankyrin-B–Dynactin pathway promotes axonal growth and multiorganelle transport

Interactions between ankyrin-B and both dynactin and phosphatidylinositol 3-phosphate lipids promote fast axonal transport of organelles.