Dendrite regeneration in C. elegans is controlled by the RAC GTPase CED-10 and the RhoGEF TIAM-1.

Neurons are vulnerable to physical insults, which compromise the integrity of both dendrites and axons. Although several molecular pathways of axon regeneration are identified, our knowledge of dendrite regeneration is limited. To understand the mechanisms of dendrite regeneration, we used the PVD neurons in C. elegans with stereotyped branched dendrites. Using femtosecond laser, we severed the primary dendrites and axon of this neuron. After severing the primary dendrites near the cell body, we observed sprouting of new branches from the proximal site within 6 hours, which regrew further with time in an unstereotyped manner. This was accompanied by reconnection between the proximal and distal dendrites, and fusion among the higher-order branches as reported before. We quantified the regeneration pattern into three aspects-territory length, number of branches, and fusion phenomena. Axonal injury causes a retraction of the severed end followed by a Dual leucine zipper kinase-1 (DLK-1) dependent regrowth from the severed end. We tested the roles of the major axon regeneration signalling hubs such as DLK-1-RPM-1, cAMP elevation, let-7 miRNA, AKT-1, Phosphatidylserine (PS) exposure/PS in dendrite regeneration. We found that neither dendrite regrowth nor fusion was affected by the axon injury pathway molecules. Surprisingly, we found that the RAC GTPase, CED-10 and its upstream GEF, TIAM-1 play a cell-autonomous role in dendrite regeneration. Additionally, the function of CED-10 in epidermal cell is critical for post-dendrotomy fusion phenomena. This work describes a novel regulatory mechanism of dendrite regeneration and provides a framework for understanding the cellular mechanism of dendrite regeneration using PVD neuron as a model system.

Regulation of UNC-40/DCC and UNC-6/Netrin by DAF-16 promotes functional rewiring of the injured axon.

The adult nervous system has a limited capacity to regenerate after accidental damage. Post-injury functional restoration requires proper targeting of the injured axon to its postsynaptic cell. Although the initial response to axonal injury has been studied in great detail, it is rather unclear what controls the re-establishment of a functional connection. Using the posterior lateral microtubule neuron in Caenorhabditis elegans, we found that after axotomy, the regrowth from the proximal stump towards the ventral side and accumulation of presynaptic machinery along the ventral nerve cord correlated to the functional recovery. We found that the loss of insulin receptor DAF-2 promoted 'ventral targeting' in a DAF-16-dependent manner. We further showed that coordinated activities of DAF-16 in neuron and muscle promoted 'ventral targeting'. In response to axotomy, expression of the Netrin receptor UNC-40 was upregulated in the injured neuron in a DAF-16-dependent manner. In contrast, the DAF-2-DAF-16 axis contributed to the age-related decline in Netrin expression in muscle. Therefore, our study revealed an important role for insulin signaling in regulating the axon guidance molecules during the functional rewiring process.

Swimming Exercise Promotes Post-injury Axon Regeneration and Functional Restoration through AMPK.

Restoration of lost function following a nervous system injury is limited in adulthood as the regenerative capacity of nervous system declines with age. Pharmacological approaches have not been very successful in alleviating the consequences of nervous system injury. On the contrary, physical activity and rehabilitation interventions are often beneficial to improve the health conditions in the patients with neuronal injuries. Using touch neuron circuit of Caenorhabditis elegans, we investigated the role of physical exercise in the improvement of functional restoration after axotomy. We found that a swimming session of 90 min following the axotomy of posterior lateral microtubule (PLM) neuron can improve functional recovery in larval and adult stage animals. In older age, multiple exercise sessions were required to enhance the functional recovery. Genetic analysis of axon regeneration mutants showed that exercise-mediated enhancement of functional recovery depends on the ability of axon to regenerate. Exercise promotes early initiation of regrowth, self-fusion of proximal and distal ends, as well as postregrowth enhancement of function. We further found that the swimming exercise promotes axon regeneration through the activity of cellular energy sensor AAK-2/AMPK in both muscle and neuron. Our study established a paradigm where systemic effects of exercise on functional regeneration could be addressed at the single neuron level.

let-7 miRNA controls CED-7 homotypic adhesion and EFF-1-mediated axonal self-fusion to restore touch sensation following injury.

Neuronal injury often leads to devastating consequences such as loss of senses or locomotion. Restoration of function after injury relies on whether the injured axons can find their target cells. Although fusion between injured proximal axon and distal fragment has been observed in many organisms, its functional significance is not clear. Here, using Caenorhabditis elegans mechanosensory neurons, we address this question. Using two femtosecond lasers simultaneously, we could scan and sever posterior lateral microtubule neurons [posterior lateral microtubules (PLMs)] on both sides of the worm. We showed that axotomy of both PLMs leads to a dramatic loss of posterior touch sensation. During the regenerative phase, only axons that fuse to their distal counterparts contribute to functional recovery. Loss of let-7 miRNA promotes functional restoration in both larval and adult stages. In the L4 stage, loss of let-7 increases fusion events by increasing the mRNA level of one of the cell-recognition molecules, CED-7. The ability to establish cytoplasmic continuity between the proximal and distal ends declines with age. Loss of let-7 overcomes this barrier by promoting axonal transport and enrichment of the EFF-1 fusogen at the growing tip of cut processes. Our data reveal the functional property of a regenerating neuron.

Nonlinear femtosecond laser induced scanning tunneling microscopy.

We demonstrate ultrafast laser driven nonlinear scanning tunneling microscopy (STM), under ambient conditions. The design is an adaptation of the recently introduced cross-polarized double beat method, whereby z-polarized phase modulated fields are tightly focused at a tunneling junction consisting of a sharp tungsten tip and an optically transparent gold film as substrate. We demonstrate the prerequisites for ultrafast time-resolved STM through an operative mechanism of nonlinear laser field-driven tunneling. The spatial resolution of the nonlinear laser driven STM is determined by the local field intensity. Resolution of 0.3 nm-10 nm is demonstrated for the intensity dependent, exponential tunneling range. The demonstration is carried out on a junction consisting of tungsten tip and gold substrate. Nano-structured gold is used for imaging purposes, to highlight junction plasmon controlled tunneling in the conductivity limit.

Electron transport in dodecylamine capped gold nanocluster films using current sensing atomic force microscope (C-AFM).

Electron transport across cataphoretically deposited dodecylamine capped gold nanocluster rough films on Si(111) substrate is investigated using current sensing atomic force microscopy. Contact mode images depict uniform deposition of agglomerates of gold nanoparticles. The current images display strong correlation with topographic images. The I-V measurement on a single agglomerate of approximately = 250 nm size at different forces exhibits force dependent threshold voltage. The electron transport from tip to sample is found to be ohmic in contrast to that from sample to tip which, exhibits Fowler-Nordheim behavior up to 35 nN force. At higher forces, the I-V behavior could be attributed to other electron transfer processes such as Schottky/Poole-Frenkel or trapping/detrapping, although no exact mechanism could be identified. The results are discussed in the light of models based on Coulomb blockaded collective charge transport in nanoparticle arrays duly accounting for the potential role of the capping layer.