The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (CaV). Different classes of sodium and potassium channels are scaffolded at specific sites within the AIS, conferring unique functions, but how calcium channels are functionally distributed within the AIS is unclear. Here, we utilize conventional 2-photon laser scanning and diffraction-limited, high-speed spot 2-photon imaging to resolve action potential-evoked calcium dynamics in the AIS with high spatiotemporal resolution. In mouse layer 5 prefrontal pyramidal neurons, calcium influx was mediated by a mix of CaV2 and CaV3 channels that differentially localized to discrete regions. CaV3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-sensitive stores, whereas CaV2 localized to non-hotspot regions. Thus, different pools of CaVs appe...

To assemble the nervous system’s functional circuits, the neuronal axonal growth cones must be precisely guided to their proper targets, which can be achieved through cell-surface guidance receptor activation by ligand binding in the periphery. We investigated the function of paxillin, a focal adhesion protein, as an essential growth cone guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory selection in the limb mesenchyme. Using in situ mRNA detection, we first show paxillin expression in LMC neurons of chick and mouse embryos at the time of spinal motor axon extension into the limb. Paxillin loss- and gain-of-function using in-ovo electroporation in chick LMC neurons, of either sex, perturbed LMC axon trajectory selection demonstrating an essential role of paxillin in motor axon guidance. In addition, a neuron-specific paxillin deletion in mice led to LMC axon trajectory selection errors. We also show that knocking down paxillin attenuates the growth preference ...

The neuronal CAM L1 is important in cell migration, axon growth and guidance. L1KO mice have defects in brain development, including hydrocephalus, abnormal corticospinal tract (CST) and hypoplasia of the cerebellar vermis. We studied mice in which the 6th Ig domain of L1 was deleted (6DKO), eliminating L1 homophilic binding. We studied the CST and the thalamocortical tract by injecting DiI crystals into the motor cortex or ventral thalamic nuclei. Both tracts showed normal development and axonal fasciculation. Histological studies and immunohistochemical analyses of neurofilament, MAP2, calbindin, and L1 cytoplasmic domain were performed. The brains from 6DKO mice showed normal anatomy without hydrocephalus or midline malformations. Other pathways including corpus callosum, anterior and hippocampal communicating fibers, striosome of the basal ganglia, and the spinal cord appeared normal. The cerebral cortex, basal ganglia, thalamus, brain stem and spinal cord showed normal cytoarchitecture. The results sh...

In this study of the efferent projections of the centre median thalamic nucleus (CM) in squirrel monkeys (Saimiri sciureus), microiontophoretic injections of BDA combined with electrophysiological recording led to the Golgi-like labeling of 7 large CM perikarya from which emerged several long, poorly branched and sparsely spined dendrites. Entire reconstruction of the axon of these neurons revealed a similar pattern of distribution. Each axon coursed through the lateral thalamic nuclei, emitted a few collaterals in the reticular thalamic nucleus, and bifurcated within the internal capsule. One branch ascended via the corona radiata to the motor cortex, where it arborized moderately in layers V and VI, and much less profusely in layer I. The other branch ran through the pallidum and entered the dorsolateral sector of the putamen (sensorimotor territory), where it broke out into 3-5 smaller collaterals. These thin branches divided into numerous shorter collaterals that displayed pedunculated varicosities org...

Thrombospondin (TSP) is a secreted glycoprotein that exists either in a soluble form or in a bound form associated with the extracellular matrix. This raises the hypothesis that each form exerts distinct effects on axon guidance. Immunostaining of whole mount Xenopus embryos reveals that TSP is associated with axon tracts in the spinal cord during early stages of neuronal development. Since TSP is localized to areas of axon outgrowth, we examined the effects of TSP on neurons developing in vitro. Neurons grown on TSP adsorbed to the culture dish have axons ~60% longer than those grown on tissue culture plastic, and exhibit a prominent increase in filopodia along their length. To assess the effects TSP on axon guidance, we monitored growth cone responses to both a gradient of TSP in solution and when encountering a substrate border of TSP adsorbed to the culture dish. Interestingly, growth cones displayed contrasting behaviors when encountering TSP in these different forms. A soluble gradient of TSP stimula...

Chemical communication controls a wide range of behaviors via conserved signaling networks. Axon regeneration in response to injury is determined by the interaction between the extracellular environment and intrinsic growth potential. In this study, we investigated the role of chemical signaling in axon regeneration in Caenorhabditis elegans . We find that the enzymes involved in ascaroside pheromone biosynthesis, ACOX-1.1, ACOX-1.2, and DAF-22, participate in axon regeneration by producing a dauer-inducing ascaroside, ascr#5. We demonstrate that the chemoreceptor genes, srg-36 and srg-37 , which encode G protein-coupled receptors (GPCRs) for ascr#5, are required for adult-specific axon regeneration. Furthermore, the activating mutation in egl-30 encoding Gqα suppresses axon regeneration defective phenotype in acox-1.1 and srg-36 srg-37 mutants. Therefore, the ascaroside signaling system provides a unique example of a signaling molecule that regulates the regenerative pathway in the nervous system. SIGNIF...

The Eph family of receptor tyrosine kinases has been implicated in a number of developmental processes, including axonal pathfinding. Previously, we demonstrated that retinal axon guidance to the optic disc in mouse involves EphB receptors. However, in addition to their roles as receptors, evidence from biochemical and cell mixing experiments show that Eph molecules may serve as ligands by a reverse signaling mechanism. This mechanism may be acting in the retina since animals lacking both EphB3 and EphB2 proteins have increased axon guidance errors to the disc compared to animals lacking EphB3 but expressing the EphB2 extracellular domain without the kinase domain. To test directly whether retinal axon growth cones respond to EphB extracellular domains, we employed a substrate choice assay using fusion proteins with the extracellular domain of EphB1, B2, or B3 fused to the IgG Fc domain. Results showed that embryonic mouse retinal axons preferred to grow on laminin compared to laminin plus EphB extracellul...

Developing neurons exhibit spontaneous fluctuations in intracellular calcium which regulate several aspects of their differentiation, including axon outgrowth. In previous studies we found that branching of cortical axons was preceded by pausing and enlargement of the primary growth cone. To test whether calcium transients play a role in slowing the advance of cortical growth cones, we measured intracellular calcium dynamics in postnatal cortical growth cones using the sensitive calcium indicator Fluo-4. Time lapse fluorescence imaging for periods up to 1 hour revealed that cortical neurons exhibit calcium oscillations, that apparently occur simultaneously throughout the neuron. These oscillations appeared in bursts at frequencies that varied from 10 per hour up to several per minute. We measured amplitude relative to baseline in growth cones of different sizes and growth states. Preliminary analysis suggests that the large paused growth cones exhibit the highest frequency (average 1 per minute) and amplit...

Synapses can be independently modified by neural activity. Such synaptic plasticity may contribute to neural development, learning and responses to injury. Synaptic protein synthesis could account in part for rapid but persistent selective synaptic modification. Research into synaptic protein synthesis has focused on postsynaptic mechanisms. Here we report evidence for presynaptic protein synthesis. Purified axon terminals (synaptosomes) were isolated from adult rat cortex. Immunoblots demonstrated that synaptosomes were enriched for synapsin I, a synaptic marker, and negative for histone protein, a nuclear marker. Transmission electron microscopy showed the synaptosome fraction was enriched for axon terminals and contained few axons with attached dendrite. We found using RT-PCR that the synaptosomes contain beta-actin, alpha-CaMKII and elongation factor-2 mRNA but not transcription factor zif/268 mRNA. Synaptosomes were shown to undergo extramitochondrial protein synthesis by incorporation of 35S-methioni...

About 15% of retinal ganglion cells survive diffuse axonal crush of the optic nerve (ONC) in adult rats. Initially rats are blind, but vision recovers within 3 weeks. To study the basis of this recovery, we investigated recovery of anterograde and retrograde axonal transport, the restitution of the cytoskeletal organization, the time course of the calcium-influx into the retinal ganglion cells (RGCs), and the expression of axon-skeletal elements. In axons surviving injury the anterograde transport of MiniRuby recovers distal to the crush site and derrangements of heavy neurofilaments are restored after 3 weeks. Using the method of in vivo confocal neuroimaging we also visualized retrograde transport of RGCs in the living rat and found, following injections of the fluorescence marker Yellow Green into the superior colliculus, recovery 3 weeks after ONC. Using the calcium marker Oregon Green BAPTA we showed that high amounts of intracellular calcium in dying RGCs correlated in time with increased levels of m...