Primary Axons Research Articles

Radial glia phagocytose axonal debris from degenerating overextending axons in the developing olfactory bulb.

Axon targeting during the development of the olfactory system is not always accurate, and numerous axons overextend past the target layer into the deeper layers of the olfactory bulb. To date, the fate of the mis-targeted axons has not been determined. We hypothesized that following overextension, the axons degenerate, and cells within the deeper layers of the olfactory bulb phagocytose the axonal debris. We utilized a line of transgenic mice that expresses ZsGreen fluorescent protein in primary olfactory axons. We found that overextending axons closely followed the filaments of radial glia present in the olfactory bulb during embryonic development. Following overextension into deeper layers of the olfactory bulb, axons degenerated and radial glia responded by phagocytosing the resulting debris. We used in vitro analysis to confirm that the radial glia had phagocytosed debris from olfactory axons. We also investigated whether the fate of overextending axons was altered when the development of the olfactory bulb was perturbed. In mice that lacked Sox10, a transcription factor essential for normal olfactory bulb development, we observed a disruption to the morphology and positioning of radial glia and an accumulation of olfactory axon debris within the bulb. Our results demonstrate that during early development of the olfactory system, radial glia play an important role in removing overextended axons from the deeper layers of the olfactory bulb.

Estimation of spike initiation zone and synaptic input parameters of a Drosophila motoneuron using a morphologically reconstructed model

The fruitfly Drosophila melanogaster has recently emerged as a powerful model for studying neuronal circuit development and function through identified neurons. A major obstacle to these studies has been a lack of computational models of these neurons for generating and testing hypotheses with fast-throughput simulations. Here, we constructed a computational model of an identified Drosophila motoneuron, aCC/MN1-Ib, from its recorded biophysical and anatomical properties [1]. Constructing this model required estimating the location and properties of distal spike-generating Na+ and K+ channels. The effects of channel location were simulated by including anatomical details in the model through a morphological reconstruction. By simulating a match to the observed in vivo electrophysiological characteristics, we predict the anatomical location of the spike initiation zone (SIZ) to be distal on aCC’s primary axon [2]. The complex morphology of the neuron effectively filters fast activating ionic currents which occur at the SIZ but are recoded from the soma in vivo. In particular, the transient component of the voltage-activated sodium current is extensively filtered relative to the slower persistent component. Input-output behavior in a broader network context was also predicted [2]: about 20 miniature excitatory potentials are required for initiating an action potential and about 200 individual potentials underlie evoked spontaneous rhythmic currents (SRC) in this neuron. Our model of aCC, which is the first morphologically-realistic active model of an identified Drosophila motoneuron, provides a template for further investigation of neuronal function in health and disease.

Highly Effective Photonic Cue for Repulsive Axonal Guidance

In vivo nerve repair requires not only the ability to regenerate damaged axons, but most importantly, the ability to guide developing or regenerating axons along paths that will result in functional connections. Furthermore, basic studies in neuroscience and neuro-electronic interface design require the ability to construct in vitro neural circuitry. Both these applications require the development of a noninvasive, highly effective tool for axonal growth-cone guidance. To date, a myriad of technologies have been introduced based on chemical, electrical, mechanical, and hybrid approaches (such as electro-chemical, optofluidic flow and photo-chemical methods). These methods are either lacking in desired spatial and temporal selectivity or require the introduction of invasive external factors. Within the last fifteen years however, several attractive guidance cues have been developed using purely light based cues to achieve axonal guidance. Here, we report a novel, purely optical repulsive guidance technique that uses low power, near infrared light, and demonstrates the guidance of primary goldfish retinal ganglion cell axons through turns of up to 120 degrees and over distances of ∼90 µm.

Semaphorin 5B is a repellent cue for sensory afferents projecting into the developing spinal cord

During vertebrate development, centrally projecting sensory axons of the dorsal root ganglia neurons first reach the embryonic spinal cord at the dorsolateral margin. Instead of immediately projecting into the grey matter, they bifurcate and extend rostrally and caudally to establish the longitudinal dorsal funiculus during a stereotyped waiting period of approximately 48 h. Collateral fibres then extend concurrently across multiple spinal segments and project to their appropriate targets within the grey matter. This rostrocaudal extension of sensory afferents is crucial for the intersegmental processing of information throughout the spinal cord. However, the precise cues that prevent premature entry during the waiting period remain to be identified. Here, we show that semaphorin 5B (Sema5B), a member of the semaphorin family of guidance molecules, is expressed in the chick spinal cord during this waiting period and dorsal funiculus formation. Sema5B expression is dynamic, with a reduction of expression apparent in the spinal cord concomitant with collateral extension. We show that Sema5B inhibits the growth of NGF-dependent sensory axons and that this effect is mediated in part through the cell adhesion molecule TAG-1. Knockdown of Sema5B in the spinal cord using RNA interference leads to the premature extension of cutaneous nociceptive axons into the dorsal horn grey matter. These premature projections predominantly occur at the site of dorsal root entry. Our results suggest that Sema5B contributes to a repulsive barrier for centrally projecting primary sensory axons, forcing them to turn and establish the dorsal funiculus.

Cortical Neuron Response Properties Are Related to Lesion Extent and Behavioral Recovery after Sensory Loss from Spinal Cord Injury in Monkeys

Lesions of the dorsal columns at a mid-cervical level render the hand representation of the contralateral primary somatosensory cortex (area 3b) unresponsive. Over weeks of recovery, most of this cortex becomes responsive to touch on the hand. Determining functional properties of neurons within the hand representation is critical to understanding the neural basis of this adaptive plasticity. Here, we recorded neural activity across the hand representation of area 3b with a 100-electrode array and compared results from owl monkeys and squirrel monkeys 5-10 weeks after lesions with controls. Even after extensive lesions, performance on reach-to-grasp tasks returned to prelesion levels, and hand touches activated territories mainly within expected cortical locations. However, some digit representations were abnormal, such that receptive fields of presumably reactivated neurons were larger and more often involved discontinuous parts of the hand compared with controls. Hand stimulation evoked similar neuronal firing rates in lesion and control monkeys. By assessing the same monkeys with multiple measures, we determined that properties of neurons in area 3b were highly correlated with both the lesion severity and the impairment of hand use. We propose that the reactivation of neurons with near-normal response properties and the recovery of near-normal somatotopy likely supported the recovery of hand use. Given the near-completeness of the more extensive dorsal column lesions we studied, we suggest that alternate spinal afferents, in addition to the few spared primary axon afferents in the dorsal columns, likely have a major role in the reactivation pattern and return of function.

Time-lapse in vivo imaging of dorsal root nerve regeneration in mice.

Primary sensory axon injury is common after spinal cord and root injuries and causes patients to suffer chronic pain and persistent loss of sensation and motor coordination. The devastating consequences of such injuries are due primarily to the failure of severed axons to regenerate within the damaged CNS. Our understanding of the molecular and cellular events that play key roles in preventing or promoting functional regeneration is far from complete, in part because complex and dynamic changes associated with nerve injury have had to be deduced from comparisons of static images obtained from multiple animals after their death. Revolutionary innovations in optics and mouse transgenics now permit real-time monitoring of regenerating dorsal root axons directly in living animals. Here, we describe detailed procedures for repetitive monitoring of identified axons in a lumbar dorsal root over hours to weeks using both widefield and two-photon microscopes. We also discuss the strengths and limitations of in vivo imaging and provide suggestions based on our own experience for troubleshooting issues associated with repeated anesthetization, an extensive laminectomy, and post-op care. These techniques provide the unprecedented opportunity to obtain novel insights into why sensory axons fail to reenter the spinal cord.

Overdose of D-serine Induces Movement Disorder and Neuromuscular Changes of Zebrafish Larvae.

D-serine is a well-known activator of N-methyl-D-aspartate receptors; however, little is known about the teratogenic effects of D-serine overdose during early embryonic development. Here, we used zebrafish as a model to test toxicity and teratogenicity, since they have transparent eggs, making the organogenesis of zebrafish embryos easier to be observed. After D-serine injection (100–1000 ppm), the most evident defective phenotypes were bent trunk phenotypes, including malformed somite boundary, twisted body axis and shorter body length. As the injection dosages increased, the rates of embryos with bent trunk phenotypes decreased (0% for 0 ppm, n=573; 59.9~84.3% for 100–1000 ppm of D-serine, n=383–451). In addition, D-serine-injected embryos exhibited significantly reduced the frequencies of spontaneous in-chorion contraction (21.7 for 0 ppm vs. 18.3–0.9 for 100–1000 ppm D-serine, n=30) in comparison with mock-treated controls (0 ppm). Subtle changes are easily observed by staining with specific monoclonal antibodies F59, Znp1, Zn5 and α-bungarotoxin to detect morphological changes in muscle fibers, primary motor axons, secondary motor axon projections and neuromuscular junctions, respectively. Our data show that overdose of D-serine leads to misalignment of muscle fibers and motor neuron defects, especially secondary motor neuron axonal growth defects.

Dorsal column sensory axons degenerate due to impaired microvascular perfusion after spinal cord injury in rats

The mechanisms contributing to axon loss after spinal cord injury (SCI) are largely unknown but may involve microvascular loss as we have previously suggested. Here, we used a mild contusive injury (120kdyn IH impactor) at T9 in rats focusing on ascending primary sensory dorsal column axons, anterogradely traced from the sciatic nerves. The injury caused a rapid and progressive loss of dorsal column microvasculature and oligodendrocytes at the injury site and penumbra and an ~70% loss of the sensory axons by 24h. To model the microvascular loss, focal ischemia of the T9 dorsal columns was achieved via phototoxic activation of intravenously injected rose bengal. This caused an ~53% loss of sensory axons and an ~80% loss of dorsal column oligodendrocytes by 24h. Axon loss correlated with the extent and axial length of microvessel and oligodendrocyte loss along the dorsal column. To determine if oligodendrocyte loss contributes to axon loss, the glial toxin ethidium bromide (EB; 0.3μg/μl) was microinjected into the T9 dorsal columns, and resulted in an ~88% loss of dorsal column oligodendrocytes and an ~56% loss of sensory axons after 72h. EB also caused an ~75% loss of microvessels. Lower concentrations of EB resulted in less axon, oligodendrocyte and microvessel loss, which were highly correlated (R2=0.81). These data suggest that focal spinal cord ischemia causes both oligodendrocyte and axon degeneration, which are perhaps linked. Importantly, they highlight the need of limiting the penumbral spread of ischemia and oligodendrocyte loss after SCI in order to protect axons.

Bioenergetic deficits in peripheral nerve sensory axons during chemotherapy-induced neuropathic pain resulting from peroxynitrite-mediated post-translational nitration of mitochondrial superoxide dismutase

Many of the widely used anticancer drugs induce dose-limiting peripheral neuropathies that undermine their therapeutic efficacy. Animal models of chemotherapy-induced painful peripheral neuropathy (CIPN) evoked by a variety of drug classes, including taxanes, vinca alkaloids, platinum-complexes, and proteasome-inhibitors, suggest that the common underlying mechanism in the development of these neuropathies is mitotoxicity in primary nerve sensory axons (PNSAs) arising from reduced mitochondrial bioenergetics [eg adenosine triphosphate (ATP) production deficits due to compromised respiratory complex I and II activity]. The causative mechanisms of this mitotoxicity remain poorly defined. However, peroxynitrite, an important pro-nociceptive agent, has been linked to mitotoxicity in several disease states and may also drive the mitotoxicity associated with CIPN. Our findings reveal that the development of mechano-hypersensitivity induced by paclitaxel, oxaliplatin, and bortezomib was prevented by administration of the peroxynitrite decomposition catalyst Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTE-2-PyP5+) without interfering with their anti-tumor effects. Peak CIPN was associated with the nitration and inactivation of superoxide dismutase in the mitochondria, but not in the cytosol, as well as a significant decrease in ATP production within the PNSAs; all of these events were attenuated by MnTE-2-PyP5+. Our results provide continued support for the role of mitotoxicity in the development of CIPN across chemotherapeutic drug classes, and identify peroxynitrite as a key mediator in these processes, thereby providing the rationale towards development of “peroxynitrite-targeted” therapeutics for CIPN.

A System for Investigation of Biological Effects of Diagnostic Ultrasound on Development of Zebrafish Embryos

A system for scanning zebrafish embryos with diagnostic ultrasound was developed for research into possible biological effects during development. Two troughs for holding embryos were formed from agarose in a rectangular dish and separated by an ultrasound absorber. A 4.9 MHz linear array ultrasound probe was positioned to uniformly scan all the embryos at the bottom of one trough, with the other used for controls. Zebrafish embryos were scanned continuously from 10-24 h post fertilization (hpf) during the segmentation period and gross morphological parameters were measured at 30 hpf, including viability, length, number of visible axons, and the progression of the lateral line primordium (LLP). Our initial tests were encumbered by the thermal effects of probe self-heating, which resulted in accelerated development of the zebrafish embryos. After subsequent optimization, our test revealed a significant retardation of primary motor axons and the migration of the LLP in embryos scanned with ultrasound, which indicated a potential for nonthermal effects on neuronal development. This diagnostic ultrasound exposure system is suitable for further investigation of possible subtle bioeffects, such as perturbation of neuronal migration.

Modeling of interstitial branching of axonal networks

A mathematical model is developed to describe and investigate interstitial branching of axonal networks during nervous system development. The model under consideration describes axonal network growth in which the concentration of axon guidance molecules controls axon's growth and interstitial branching from axon shaft. Numerical simulations show that in the model framework axonal networks branch similarly to real neural networks in vitro.

Notum Homolog Plays a Novel Role in Primary Motor Innervation

To form complex neuronal networks, growth cones use intermediate targets as guideposts on the path to more distant targets. In the developing zebrafish (Danio rerio), the muscle pioneers (MPs) are intermediate targets for primary motor neurons (PMNs) that innervate the trunk musculature. The mechanisms regulating PMN axon guidance at the MPs are not fully understood. We have identified a new member of the Notum family in zebrafish, Notum 2, which is expressed exclusively in the MPs during primary motor innervation. While homologs of Notum, including zebrafish Notum 1a, negatively regulate the Wnt/β-catenin signaling pathway, we discovered a novel function of Notum 2 in regulating motor axon guidance. Knockdown of Notum 2 resulted in a failure of caudal primary (CaP) axons to migrate beyond the MPs, despite the proper specification of the intermediate target. In contrast, mosaic Notum 2 overexpression induced branching of PMN axons. This effect is specific to Notum 2, as overexpression of Notum 1a does not affect PMN axon trajectory. Ectopic expression of Notum 2 by cells contacting the growing CaP axon induced the highest frequency of branching, suggesting that localized Notum 2 expression affects axon behavior. We propose a model where Notum 2 expression at the MPs provides a cue to release CaP motor axons from their intermediate targets, allowing growth cones to proceed to secondary targets in the ventral muscle. This work demonstrates an unexpected role for a Notum homolog in regulating growth cone migration, separate from the well established functions of other Notum homologs in Wnt signaling.

Modeling transport of a pulse of radiolabeled organelles in a Drosophila unipolar motor neuron

Based on published experimental evidence, this paper develops a model for the transport of a pulse of radiolabeled organelles in a unipolar Drosophila motor neuron. In particular, since published data indicate that no microtubules (MTs) travel from the primary neurite into the dendrite, it is investigated how organelles are transported into the dendrite. Analytical solutions describing concentrations of kinesin- and dynein-driven organelles in the primary neurite, axon, and dendrite are obtained. The effects of increasing the width of the pulse and increasing the rate of organelle transition rate from the kinesin-driven to the dynein-driven state are investigated.

Adverse outcome pathways during zebrafish embryogenesis: A case study with paraoxon

Using paraoxon as a reference acetylcholinesterase (AChE) inhibitor, the objective of this study was to develop an adverse outcome pathway (AOP) that provided quantitative linkages across levels of biological organization during zebrafish embryogenesis. Within normal zebrafish embryos, we first demonstrated that ache transcripts and AChE activity increased in a stage-dependent manner following segmentation. We then showed that static exposure of embryos to paraoxon (31.2–500nM) from 5 to 96hpf resulted in significant stage- and concentration-dependent AChE inhibition, albeit these effects were fully reversible within 48h following transfer to clean water. However, even in the presence of significant AChE inhibition, exposure to non-teratogenic paraoxon concentrations (≤250nM) did not adversely impact secondary motoneuron development at 96hpf. Therefore, we investigated the potential effects of paraoxon exposure on spontaneous tail contractions at 26hpf – an early locomotor behavior that results from innervation of primary (not secondary) motoneuron axons to target axial muscles. Based on these studies, the frequency of spontaneous tail contractions at 26hpf – a developmental stage with minimal AChE expression and activity – was significantly higher following exposure to paraoxon concentrations as low as 31.2nM. Overall, our data suggest that (1) normal AChE activity is not required for secondary motoneuron development and (2) spontaneous tail contractions at 26hpf are sensitive to paraoxon exposure, an effect that may be independent of AChE inhibition. Using a well-studied reference chemical, this study highlights the potential challenges in developing quantitative AOPs to support chemical screening and prioritization strategies.

An inherited TUBB2B mutation alters a kinesin-binding site and causes polymicrogyria, CFEOM and axon dysinnervation

Microtubules are essential components of axon guidance machinery. Among β-tubulin mutations, only those in TUBB3 have been shown to cause primary errors in axon guidance. All identified mutations in TUBB2B result in polymicrogyria, but it remains unclear whether TUBB2B mutations can cause axon dysinnervation as a primary phenotype. We have identified a novel inherited heterozygous missense mutation in TUBB2B that results in an E421K amino acid substitution in a family who segregates congenital fibrosis of the extraocular muscles (CFEOM) with polymicrogyria. Diffusion tensor imaging of brains of affected family members reveals aberrations in the trajectories of commissural projection neurons, implying a paucity of homotopic connections. These observations led us to ask whether axon dysinnervation is a primary phenotype, and why the E421K, but not other, TUBB2B substitutions cause CFEOM. Expression of exogenous Tubb2b-E421K in developing callosal projection neurons is sufficient to perturb homotopic connectivity, without affecting neuronal production or migration. Using in vitro biochemical assays and yeast genetics, we find that TUBB2B-E421K αβ-heterodimers are incorporated into the microtubule network where they alter microtubule dynamics and can reduce kinesin localization. These data provide evidence that TUBB2B mutations can cause primary axon dysinnervation. Interestingly, by incorporating into microtubules and altering their dynamic properties, the E421K substitution behaves differently than previously identified TUBB2B substitutions, providing mechanistic insight into the divergence between resulting phenotypes. Together with previous studies, these findings highlight that β-tubulin isotypes function in both conserved and divergent ways to support proper human nervous system development.

Sensory Axon Regeneration: A Review from an in vivo Imaging Perspective

Injured primary sensory axons fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Re-entry is prevented at the dorsal root entry zone (DREZ), the CNS-PNS interface. Why axons stop or turn around at the DREZ has generally been attributed to growth-repellent molecules associated with astrocytes and oligodendrocytes/myelin. The available evidence challenges the contention that these inhibitory molecules are the critical determinant of regeneration failure. Recent imaging studies that directly monitored axons arriving at the DREZ in living animals raise the intriguing possibility that axons stop primarily because they are stabilized by forming presynaptic terminals on non-neuronal cells that are neither astrocytes nor oligodendrocytes. These observations revitalized the idea raised many years ago but virtually forgotten, that axons stop by forming synapses at the DREZ.

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo(1). These mice have been used extensively for in vivo imaging of muscle(2-4) and brain(5-7), and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy(8,9). Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished(10). Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord(11). In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

Image-based Screening Identifies Novel Roles for IκB Kinase and Glycogen Synthase Kinase 3 in Axonal Degeneration

Axon degeneration is an active, evolutionarily conserved self-destruction program by which compromised axons fragment in response to varied insults. Unlike programmed cell death, axon degeneration is poorly understood. We have combined robotic liquid handling with automated microscopy and image analysis to create a robust screening platform to measure axon degeneration in mammalian primary neuronal cultures. Using this assay, we performed an unbiased screen of 480 bioactive compounds, identifying 11 that reproducibly delay fragmentation of severed axons in vitro, including two inhibitors of glycogen synthase kinase 3 and two inhibitors of IκB kinase. Knockdown of each of these targets by shRNA lentivirus also delays axon degeneration in vitro, further supporting their role in the axon degeneration program.

Notum 2 is a novel regulator of primary motor axon guidance

Notum 2 is a novel regulator of primary motor axon guidance

Mechanistic Logic Underlying the Axonal Transport of Cytosolic Proteins

Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivatable GFP (PAGFP) move with a slow motor-dependent anterograde bias distinct from both vesicular trafficking and diffusion of untagged PAGFP. The overall bias is likely generated by an intricate particle kinetics involving transient assembly and short-range vectorial spurts. In vivo biochemical studies reveal that cytosolic proteins are organized into higher order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supramolecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multiprotein complexes that are directly/indirectly conveyed by motors.