Chung, Samuel H. et al. (2013) International Worm Meeting "Lesion conditioned regeneration of C. elegans amphid sensory neurons is mediated through a reduction of sensory signaling and does not require DLK-1."

Chung, Samuel H., Gabel, Christopher V.

[International Worm Meeting, 2013]

Tragically, mammals display weak neuronal regeneration within the central nervous system following traumatic neuron damage. An exciting discovery in neurotherapeutics is that mammalian neurons can strongly regenerate their central axons into and beyond an injury site if a conditioning lesion is made on their peripheral sensory axons. This lesion conditioning effect has been studied in mammals for decades, yet is still poorly understood. Employing subcellular femtosecond laser ablation we observe a strong lesion conditioning effect in multiple types of C. elegans amphid sensory neurons, where a dendrite lesion stimulates regeneration of a transected axon. Interestingly, the effect is observed in a dlk-1(ju476) mutant background demonstrating that DLK-1 is not necessary for lesion-dependent regeneration. Moreover, pharmacologically blocking or genetic mutation of the L-type voltage-gated calcium channel mimic a dendrite lesion stimulating axon regeneration. This provides a direct link to mammalian lesion-conditioned regeneration, which is mediated by a reduction of electrical sensory activity and specifically through a reduction in L-type voltage-gated channel activity. In addition, we observe a link between C. elegans lesion-conditioned regeneration and ectopic axon outgrowth in the same amphid sensory neurons. We find that genetic mutations, previously shown to cause ectopic outgrowth during development, also stimulate lesion-conditioned axon regeneration. Our results indicate that reduction of a sensory activity dependent calcium/calmodulin pathway within the amphid neurons conditions the cell for regeneration. Taken together, we demonstrate direct genetic, molecular and cellular links between three types of axon outgrowth: C. elegans lesion conditioning, C. elegans ectopic outgrowth and mammalian lesion conditioning, establishing C. elegans as a powerful model system for the study of lesion conditioning. Our findings in C. elegans are pointing towards a conserved activity-dependent pathway that modulates the nervous system's intrinsic regenerative capabilities.

Grenache DG et al. (1993) Worm Breeder's Gazette "Differential Effects of Dauer-Defective Mutations in L1-Specific Surface Antigen Switching"

Politz SM, Grenache DG

[Worm Breeder's Gazette, 1993]

DIFFERENTIAL EFFECTS OF DAUER-DEFECTIVE MUTATIONS ON L1- SPECIFIC SURFACE ANTIGEN SWITCHING. David G. Grenache and Samuel M. Politz, Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA.

Wang, Yao et al. (2019) International Worm Meeting "High-throughput Microscopy Platform toward a Roadmap for Accelerating Vertebrate Neuroregeneration Research."

Jaklitsch, Erik, Chung, Samuel, Wang, Yao

[International Worm Meeting, 2019]

Statement of Purpose Lesion conditioning to a neuron activates cellular mechanisms to enhance neuro-regeneration. Our prior study established the roundworm C. elegans as a powerful model for the rapid discovery of genes underlying lesion conditioning, which is impractical in vertebrates (Chung, S. H., et al. (2016).) Even so, a complete search for regeneration genes requires surgery and reimaging of a fluorescent neuron in >30,000 C. elegans, which represents significant efforts for the gene screening. A high-throughput surgery and reimaging platform, or specifically, a new integrated microscope platform for rapid immobilization, imaging, and optical surgery of C. elegans, would enable this gene search. Method Used Achievement of this goal requires the acceleration of two time-consuming steps: animal immobilization and precise neuronal microscopy. This can be accomplished through two specific goals: 1. Develop a microscope stage to readily immobilize many animals for in vivo imaging. 2. Enable robust automation of microscopy by improving neuron-background contrast. For Goal 1, C. elegans will be immobilized by thermoelectrically cooling them directly on their cultivation plates, instead of mounting them to slides. For Goal 2, patterned illumination approaches will enhance contrast by reducing the excitation power that illuminates extremely bright cell bodies. This patterned illumination will reduce the bright haze around the cell body from obscuring the axon and dendrites, and exposes the axon and dendrites near the cell body. Summary of Result We finished the thermal simulation of the cooling stage and tested thermoelectric performance under experimental conditions, which confirmed the feasibility of immobilizing worms on their cultivation plates. We invented a new coarse segmentation algorithm and patterned the illumination by employing a spatial light modulator, which validates our contrast improvement approaches. The accomplishment of our goals will pave the path to the high-throughput microscope platform for C. elegans research, which can be used in numerous neuroscience labs worldwide. This platform also enables our regeneration screen. Affiliations: Funding provided by Northeastern University

Wang, Yao et al. (2021) International Worm Meeting "A simple and inexpensive add-on enables confocal imaging capacity on a widefield microscope"

Chung, Samuel, Grooms, Noa, Civale, Sabrina, Wang, Yao

[International Worm Meeting, 2021]

Widefield microscopes are commonly used in many biological laboratories. However, their inability to reject scattered or out-of-focus light often produces images with obscured thin or dim structures when there are some bright structures nearby. Scanning microscopes like confocal microscopes and multiphoton microscopes can solve this problem, but they are comparatively slower and much more expensive. We developed an inexpensive way to empower confocal imaging capacity on a widefield microscope, by inserting a spatial light modulator (SLM) into the field stop of the widefield microscope and customizing the illumination pattern and acquisition methods. We assessed the performance of this SLM-inserted setup by comparing images taken at our widefield microscope, our widefield microscope with the SLM-inserted setup, and a commercial confocal microscope. While a widefield microscope showed no sectioning capability, our SLM-inserted setup showed 0.85 ± 0.04 mum and the commercial confocal showed 0.68 ± 0.04 mum optical sectioning capability. Additionally comparing images of the FLP neuron and the tightly bundled amphid neurons in C. elegans taken by the widefield, SLM-inserted setup, and confocal microscopes, we confirmed that the SLM-inserted setup greatly reduces haze from the bright cell body, allowing visualization of dim axons and dendrites nearby. Our SLM-inserted setup identified 96% of the dim neuronal fibers seen in confocal images while the widefield microscope only identified 50% of the same fibers. Our SLM-inserted setup represents a very simple (2-component) and inexpensive (<$600) approach to enable confocal capacity on a widefield microscope. This SLM-inserted setup can be broadly employed by labs that are using widefield microscopes, with minimum expense and modification.

Mehat, Pavan et al. (2013) International Worm Meeting "Acute Laser Dissection of Mechanosensory Circuitry in C. elegans."

Sun, Lin, Mehat, Pavan, Gabel, Christopher, Chung, Samuel

[International Worm Meeting, 2013]

Employing high-resolution laser surgery techniques, we are studying how the C. elegans mechanosensory reflex circuitry recovers from specific neuronal lesion. We find that in vivo laser severing of the lateral synaptic branch of the posterior lateral microtubule neurons (PLM, the prominent posterior mechanosenory neurons) results in an initial hyperactivation of the downstream circuitry and elevated execution of posterior touch avoidance behaviors (i.e. increased forward movement as well as suppression of both spontaneous and anterior touch induced reversals). Over time the animal recovers and its behavior returns to its original baseline level within 24 h. Interestingly, this effect is seen only in surgeries that eliminate all sensory input from the PLM neurons, as single neuron surgery or severing the axon near the cell body have no effect. In addition, post-surgery hyperactivity is eliminated in glutamate receptor mutants suggesting that the effect maybe the results of neuronal modulation following sensory deprivation. Neuronal damage and sensory deprivation are known to elicit hyper-excitability and neuronal remodeling within the mammalian central nervous system. Prominent examples include phantom pain of amputated limbs, acute seizures and epilepsy resulting from traumatic brain damage and cortical rewiring after spinal cord injury or sensory deprivation. Here we are studying similar effects within a well defined, simple and tractable sensory transduction pathway in C. elegans.

Civale, Sabrina et al. (2021) International Worm Meeting "Removal of cell body haze with inverse square fit"

Chung, Samuel, Civale, Sabrina, Wang, Yao, Zhang, Chi

[International Worm Meeting, 2021]

Unwanted background fluorescence in microscopy can occur when light emitted by fluorescent structures is scattered by nearby tissues. In our in vivo imaging of green fluorescent protein (GFP)-tagged neurons in the roundworm C. elegans, scattered light produces a haze surrounding the cell body that can obscure the imaging of target structures, such as an axon or dendrite. The thin fibers appear dimmer than the much larger cell body and cannot be clearly visualized due to the low contrast between it and the bright background. Here, we describe a method to model and remove the cell body haze utilizing an inverse square intensity distribution. Such distributions are common in nature and can describe the intensity of light that emanates away from a point or sphere, such as a fluorescent cell body. Assuming that scattering is proportional to intensity, it follows that an inverse square distribution also describes the intensity of the scattered light. Utilizing this model, we have developed a post processing procedure to subtract background from an image. Removal of the haze surrounding the bright cell body enhances contrast of the dim axon, particularly in the region close to the cell body. Preliminary algorithms demonstrate a signal to background ratio improvement of >5x. This improvement in image quality allows us to more clearly visualize the axon. We intend to broadly disseminate this technique via an ImageJ extension and in MATLAB. We may further release our fitting technique on other imaging platforms. We are also investigating methods to accelerate the computations for real-time image improvement.

Jaklitsch, Erik et al. (2021) International Worm Meeting "Immobilization of C. elegans by thermoelectric cooling for high-throughput microscopy"

Jaklitsch, Erik, Grooms, Noa, Chung, Samuel, Wang, Yao

[International Worm Meeting, 2021]

Microscopy, surgical techniques, and imaging in vivo are broadly utilized for model organisms. However, despite widespread usage, these strategies for multicellular organisms remain low-throughput and require significant manual involvement. Here, we report the implementation of a novel cooling stage to immobilize Caenorhabditis elegans on typical agar cultivation plates for these purposes. This device can effectively cool C. elegans to between 1-2 degrees Celsius for immobilization and maintain the temperature with minimal fluctuations. We demonstrate the ability to perform imaging and surgical techniques without classic anesthetic agents like sodium azide. This technique decreases animal processing time while maintaining organism viability and fecundity, using an intuitive device built with attainable materials. Our thermoelectric cooling stage, which is highly effective and built to combine with standard microscopy setups, can enable high-throughput microscopy and surgical techniques with decreased manual and chemical interventions.

Grooms, Noa et al. (2021) International Worm Meeting "Expression of trx-1 correlates with intrinsic regenerative capacity"

Urena, Samuel, Chung, Samuel, Grooms, Noa, Schulting, Leilani, Fitzgerald, Michael

[International Worm Meeting, 2021]

A conditioning lesion of the peripheral sensory axon triggers robust central axon regeneration in mammals. Lesion conditioning could be utilized to drive powerful therapies for neuroinjuries. Despite being studied for >30 years, lesion conditioned regeneration remains poorly understood, and progress is severely limited by low throughput in vertebrate models. To expedite research in the field, we are developing a model for lesion-conditioned regeneration in C. elegans. Our model employs green fluorescent protein (GFP) to label the ASJ neuron. When we condition the neuron, we see increased fluorescence in the ASJ, indicating a correlation between GFP expression and regenerative capacity. Meanwhile, following prior work from our laboratory, disruptions to the sensory pathway can also chronically condition the neuron, increasing regenerative capacity without the need of a conditioning lesion. We saw increased fluorescence in chronically conditioned strains, further supporting the correlation between GFP expression and regenerative capacity. We used ethyl methanesulfonate to stochastically introduce mutations into a chronically conditioned strain and selected for offspring with decreased ASJ fluorescence, indicating a mutation in a gene potentially in the conditioning pathway. We isolated twelve strains, originating from six distinct F1s. Six of these strains show reduced frequency of ectopic axon outgrowths compared to the pre-mutagenized strain. A reduction in ectopic outgrowths suggests a disruption in the conditioning pathway, as previously characterized by our laboratory. To quantify the fluorescent correlation, we imaged the mutagenized strains with calibrated fluorescent beads and compared fluorescent intensities of the ASJ neurons to strains with increased outgrowths and regenerative potential. We found significantly decreased brightness in cell bodies and dendrites in strains with reduced regenerative potential compared to chronically conditioned strains. This correlation provides a powerful proxy for evaluating regenerative capacity and to identify genes potentially implicated in the lesion conditioned pathway.

Sun, Lin et al. (2011) International Worm Meeting "Characterization of intracellular calcium signaling within damage C. elegans neurons ."

Gao, Fay, Roodhouse, Kevin, Sun, Lin, Chung, Samuel, Gabel, Christopher V.

[International Worm Meeting, 2011]

Traumatic neuronal damage triggers large intracellular calcium transients that are associated both with subsequent neuronal degeneration and cell death, and alternatively with regenerative repair and outgrowth [1, 2]. Combining femtosecond laser ablation with the use of genetically encoded calcium sensitive fluorophores, we can optically measure intracellular calcium signaling within a specific target neuron of an intact adult C. elegans in response to precision laser damage [3-5]. Here we characterize the damage induced calcium signal across a variety of laser ablation experiments. This includes variations in the proximity of the damage point to the cell soma, targeting of distinct morphological structures and different neuronal types, modulation of laser power, different animal ages, and multiple surgeries to the same neuron. Results are revealing complex subcellular calcium dynamics that are precisely tuned to control cell fate. We find that in general large, extended calcium transients correlate with cell degeneration, while particularly small transients are associated with reduced regenerative outgrowth. This suggests an optimum window in which elevation of cytoplasmic calcium successfully facilitates neuronal repair and outgrowth without initiating cell death. Our results are helping to pinpoint the critical aspects of this signaling pathway that dictate neuronal survival and regeneration following traumatic damage. 1.Coleman, M., Axon degeneration mechanisms: commonality amid diversity. Nat Rev Neurosci, 2005. 6(11): p. 889-98. 2.Kamber, D., H. Erez, and M.E. Spira, Local calcium-dependent mechanisms determine whether a cut axonal end assembles a retarded endbulb or competent growth cone. Exp Neurol, 2009. 219(1): p. 112-25. 3.Yanik, M.F., et al., Neurosurgery: functional regeneration after laser axotomy. Nature, 2004. 432(7019): p. 822. 4.Gabel, C.V., et al., Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C. elegans. Development, 2008. 135(6): p. 1129-36. 5.Ghosh-Roy, A., et al., Calcium and Cyclic AMP Promote Axonal Regeneration in Caenorhabditis elegans and Require DLK-1 Kinase. Journal of Neuroscience, 2010. 30(9): p. 3175-3183.

Sun, Lin et al. (2013) International Worm Meeting "UNC-68/RyR channels modulate critical sub-cellular calcium signals during normal and optogenetically enhanced neuronal regeneration."

Gabel, Christopher V., Chung, Samuel, Alkema, Mark, Shay, James, Sun, Lin, Clark, Christopher

[International Worm Meeting, 2013]

Sub-cellular calcium signaling plays an important role in a neuron's ability to recover from traumatic damage. Employing femtosecond laser surgery, time-lapse microscopy, fluorescent calcium imaging and optogenetic photo-stimulation, we can damage a single neuron, quantify its regeneration, measure sub-cellular calcium signals and dynamically manipulate cell physiology in vivo. Extending previous C. elegans studies that measured an initial (~5 min) damage induced calcium transient within an axotomized neuron, we observe a prolonged, localized calcium signal within the vicinity of the damage point for 5 h following laser axotomy of the ALM neuron. This signal is eliminated by mutation of unc-68 encoding the C. elegans homologue of the ryanodine receptor (RyR) channel, a calcium release channel in the endoplasmic reticulum membrane. In addition, the same unc-68(e540) mutants exhibit a >50% reduction in regeneration outgrowth at 5h and 24h post-lesion as well as severely disrupted regeneration guidance. Employing optogenetic techniques we periodically stimulate axotomized ALM neurons by photo-activation of the cation channel channelrhodopsin-2 (ChR2). This results in a robust, >30%, increase in regeneration outgrowth over the 24 h following laser surgery. The effect is eliminated in unc-68(e540) mutants, or by pharmacologically blocking UNC-68 with dantrolene. Calcium induced calcium release via RyR channels plays an important role in amplifying sub-cellular calcium signals within a neuronal growth cone to modulate axon guidance. In C. elegans, UNC-68 is necessary for ChR2 photo-activation of muscle. In our experiments UNC-68 calcium release may amplify the ChR2 initiated signal to stimulate additional outgrowth. Taken as a whole, our results demonstrate an important role for UNC-68/RyR calcium release in stimulating early regeneration outgrowth and point to possible therapeutic control of cellular calcium physiology to enhance neuronal regeneration in vivo.