Christopher Gabel et al. (2008) C.elegans Neuronal Development Meeting "Calcium Dynamics During Laser Axotomy and Neural Regeneration in C. elegans"

Monica Driscoll, Christopher Gabel, Berangere Pinan-Lucarre, Aravinthan Samuel

[C.elegans Neuronal Development Meeting, 2008]

We are investigating the role of intracellular calcium transients in the acute response to nerve damage as well as subsequent nerve regeneration. Using tightly-focused pulses from an ultrafast laser, we can snip individual nerve fibers with minimal collateral damage. By targeting neurons expressing the FRET based calcium indicator cameleon YC3.60, we are characterizing calcium transients within the neuron immediately following laser axotomy and during axon regrowth. We discovered that the temporal dynamics of calcium transients strongly depend on the precise site of axotomy, and also correlate with eventual cell fate, i.e., whether the cell dies or repairs itself. Calcium has long been known to play a role in axon guidance and necrosis, and we have also shown that regenerative capacity depends on calcium regulatory molecules such as crt-1. Our observations establish a novel framework and technological tools for investigating the role of intracellular calcium in the neuron''s ability to detect, respond to, and recover from damage.

Christopher Gabel et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Electrotaxis: C. elegans Response to Fixed and Time Varying Electric Fields"

Aravinthan Samuel, Christopher Gabel

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

In the nematode C. elegans, the best studied navigational behaviors are chemotaxis and thermotaxis. While it has long been known that the worm responds to external electric fields, the investigation of "electrotaxis" has thus far remained limited. Here, we investigate the motile response of individual worms to fixed and time-varying electric fields. We find that C. elegans crawls toward the negative pole of an electric field with directed forward movement along a constant trajectory. We also find that C. elegans orients itself towards its preferred crawling direction using the standard maneuvers of abrupt turns and reversals. Using systematic laser surgical analysis, quantitative behavioral assays, and imaging of neuronal activity, we have uncovered and analyzed several neuronal correlates of electrotaxis, from amphid neurons involved in sensation to motor neurons which dictate specified reorientation maneuvers. The robust, deterministic, and highly stereotyped patterns of electrotactic behavior in C. elegans suggest its underlying sensorimotor pathways may be eventually mapped with relative ease, precision, and completeness.

Christopher Gabel et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Rewiring of Sensory and Motor Neural Circuits Following Femtosecond Laser Axotomy"

Christopher Gabel, Phuong Anh Nguyen, Aravinthan Samuel, Chieh Chang

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

To date, the most widely used animal models for nervous system regeneration are the mouse and rat, in which lesions are produced by scalpel or contusion. We study C. elegans, which is a far more tractable animal model in terms of genetics and molecular biology, using lesions delivered by femtosecond laser ablation, a new surgical technique which delivers exquisite precision and reliability. We have shown that neurons in the peripheral nervous system of C. elegans - e.g., motor neurons and sensory neurons - regrow after being snipped by femtosecond laser ablation. However, neurons in the animal's head do not regenerate. Therefore, the dichotomy between the regenerating neurons of the peripheral nervous system and the non-regenerating neurons of the central nervous system, a well known property of higher organisms, might also be conserved in this simple worm. This project aims to identify genes that confer the capacity to regenerate in certain classes of neurons in C. elegans. Such molecular determinants might permit switching of adult neurons from a non-regenerative state to a regenerative state. Long-term imaging methods have been developed to monitor and quantify the spatiotemporal dynamics of severed axon reorganization in nematodes following femtosecond laser axotomy. The time-lapse description of dynamics of axon morphogenesis has revealed several distinct cell biological changes that take place during axon degeneration and regeneration. Not much happens for ~7hrs following injury although the severed axon seems to explore the environment by alternating local retraction and regrowth. After that the cell body of the injured neuron sends out the big fan of growth cones potentially to survey overall guidance gradient in the surrounding areas. Competition of axon branches followed by pruning of unwanted axon branches can be often observed. In more than 50% of cases, within 24 hours after laser axotomy, the distal end of the severed axons had degenerated while the proximal end had regenerated to the normal synaptic targets. The exuberant axon sprouting that accompanies several forms of posttraumatic epilepsy in mammalian models can also be observed in our system. These results suggest that C. elegans and mammalians might share similar mechanisms for axon regeneration as well. We will present data describing differential contribution of central guidance molecules in axon regeneration versus axon development.

Christopher V. Gabel et al. (2007) International Worm Meeting "Rewiring of Adult Neuronal Circuits by Distinct Wiring Mechanisms Following Femtosecond Laser Axotomy."

Ju-Ling Liu, Aravinthan D.T. Samuel, Christopher V. Gabel, Chieh Chang

[International Worm Meeting, 2007]

We utilize femtosecond laser ablation, a new optical scalpel with exquisite precision and reproducibility to axotomize specific neurons of interest. The submicrometer precision of femtosecond laser ablation, as well as the million-fold shorter exposure interval, allows us to snip individual nerve fibers without damaging collateral fibers. We apply femtosecond laser ablation to the study of nervous system regeneration in C. elegans, which is more tractable than vertebrate injury models for genetic and optical analysis. The transparency and small size of C. elegans allows us to target any axon with femtosecond laser ablation, as well as visualize axonal degeneration and regeneration in live animals using time-lapse fluorescence microscopy. As our surgical approach is relatively high-throughput, we can examine axon regeneration in a large number of mutant animals at almost all the developmental stages. In this study, we have shown that axons in the peripheral nervous system of C. elegans - e.g., motor neurons and sensory neurons - regrow after being snipped by femtosecond laser ablation. However, axons in the animal''s head do not regenerate. Therefore, the dichotomy between the regenerating neurons of the peripheral nervous system and the non-regenerating neurons of the central nervous system, a well known property of higher organisms, might be conserved in this simple worm. Here, we study regenerative axonal growth and guidance in the AVM mechanosensory axons. In contrast to early development, axon regeneration is strikingly random, requiring competition and pruning of unwanted axon branches. Nevertheless, at least one of the new axons regrows to its normal targets with &#62;50% reliability and regenerates synaptic specifications. Also in contrast to early development, axon guidance during regeneration becomes heavily dependent on cytoplasmic protein MIG-10/Lamellipodin but surprisingly independent of UNC-129/TGF-<font face=symbol>b</font> repellent and UNC-40/DCC receptor, and axon growth during regeneration becomes largely reliant on UNC-34/Ena and CED-10/Rac actin regulators. Therefore, axon regeneration in adult neurons requires novel regulation of axon guidance molecules.

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.

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.

Yadav, Dilip Kumar et al. (2021) International Worm Meeting "Identification of Metabolic Pathways Involved in Neuronal Regeneration"

Gabel, Christopher, Taub, Daniel, Yadav, Dilip Kumar

[International Worm Meeting, 2021]

The inability of some neurons to sufficiently regenerate is in part due inefficient activation of intrinsic regeneration programs. A regenerating neuron must molecularly reprogram to allow the production of cellular components and meet the increased energy demands as well. Recent evidence highlights the critical role of metabolism in neuronal regeneration. We recently discovered that modifications in O-linked beta- N-acetylglucosamine (O-GlcNAc) signaling can strongly enhance axon regeneration. The findings indicate that O-GlcNAc transferase (OGT-1) mutation enhances axon regeneration due to shifts in cellular glycolysis. To identify the important genetic and metabolic factors involved in this effect, we adapted unbiased approach. We performed whole worm RNAseq analysis for wild-type and ogt-1(-/-) or O-GlcNAcase, oga-1(-/-), mutants. A relatively smaller number of genes were differentially expressed in oga-1(-/-) compared to wildtype, but notably they were enriched in carbohydrate, energy, and amino acid metabolism. On the other hand, we identified a large number of differentially expressed genes in the ogt-1(-/-) compared to wild-type. These genes are involved in numerous cellular processes, metabolic and signaling pathways such as development, cell growth and death, and signal transduction, as well as lipid, carbohydrate, amino acid and energy metabolism. Importantly we find differential expression of several genes with known roles in regeneration (including ced-3, daf-18, lin-12, cex-1, fax-1 etc.) confirming the validity of this approach. In addition, we find genes involved in One Carbon Metabolism (OCM), transmethylation pathways (folr-1, sams-5, set-1 etc.) and associated lipid metabolism (fat-7, ckb-2, dod-20 etc.). Performing single neuron regeneration experiments in the major S-Adenosyl Methionine Synthase (sams-1) deletion mutant, we find a significant reduction in regeneration following laser axotomy of the ALM neuron. Importantly sams-1 mutation appears to also block the enhanced regeneration effects of ogt-1 in a sams-1;ogt-1 double mutant. Our findings begin to identify genes and transcriptional regulatory pathways underlying enhanced regeneration via O-GlcNAc signaling. Our results predict that knockdown of OCM or glycolysis genes will either block or reduce, the enhanced regeneration of ogt-1(-/-), highlighting and defining the importance of cellular metabolism in neuronal regeneration.

Sun, Lin et al. (2015) International Worm Meeting "Activity dependent modulation of neuronal regeneration."

Shay, James, Sun, Lin, Chung, Sam, Gabel, Christopher

[International Worm Meeting, 2015]

Neuronal activity plays a critical role during development modulating neurite outgrowth and the formation of synaptic connections. Following traumatic cellular damage, neuronal activity can also have a profound effect on the ability of a neuron to regenerate. Employing laser surgery, fluorescent calcium imaging and optogenetic techniques in C. elegans, we can optically damage individual nerve processes, measure and manipulate their activity levels and quantify the regenerative response. We find highly varied results dependent on neuron type, damage location and pattern of excitation. Periodic excitation of laser damaged ALM and PLM neurons with Channelrhodospin-2 results in an increase in regeneration that is dependent on the frequency of stimulation. In addition this enhance regeneration is link to cellular calcium signaling as it is block by disruption of either unc-68/RyR (encoding an ER calcium release channel) or L-type voltage gated calcium channels (VGCC). In contrast we find that innate sensory signaling of the ASJ amphid neuron restricts a DLK-1 independent regenerative pathway. Disruption of this sensory activity through laser severing of the sensory dendrite or genetic disruption of egl-19/VGCC results in robust neurite sprouting following axotomy. Interestingly, the mechanisms modulating this outgrowth appear similar to that of activity dependent ectopic outgrowth observed in the same neurons during development. Taken as a whole our work is elucidating how activity mediated mechanisms controlling regeneration are highly dependent on temporal characteristics and cellular context. .

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.

Chang, Andrew et al. (2021) International Worm Meeting "Imaging neuronal dynamics during recovery from anesthesia in C. elegans"

Wirak, Gregory, Gabel, Christopher, Awal, Mehraj, Chang, Andrew, Connor, Christopher

[International Worm Meeting, 2021]

The innovation of anesthesia with volatile anesthetic agents has made modern surgical practice possible. The anesthetized state is defined by behavioral criteria and is today largely monitored through physiological measurements such as hemodynamics and gas exchange. These measurements are sometimes supplemented by EEG derived metrics, but the relationship between these metrics and the neurophysiological mechanisms of anesthesia is entirely unknown. We have used cellular resolution multi-neuronal functional imaging in C. elegans as a powerful model for bridging the gap between single-neuron activity and whole nervous system dynamics under anesthesia. We previously found that the anesthetic state in C. elegans corresponds to a loss of synchrony in global neural dynamics as opposed to generalized depression of individual neuron activity. Here we extend this work by examining how the nervous system of C. elegans recovers from the anesthetic state of dissynchrony to normal function. Employing a light-sheet diSPIM microscope we can measure the activity of the majority of neurons in the C. elegans head as the animal emerges from anesthesia. We observe that correlation in activity between neuron pairs, which is significantly suppressed in the anesthetized state, recovers to near pre-exposure levels in a non-linear, cyclical manner over several hours. Notably, the time required for this recovery is significantly longer then the time needed to induce the neurological anesthetic state. This finding recapitulates the concept of "neural inertia", a proposed hysteresis in the processes of anesthesia induction and emergence well-characterized in humans by the observation that emergence from anesthesia tends to occur at lower concentrations of drug than are required for induction. Because C. elegans perform gas exchange through diffusion, the differences we observe in time needed for induction and emergence are consistent with the phenomenon characteristic of neural inertia in humans. This finding further demonstrates the utility of C. elegans as a model system for investigating the effects of anesthesia on neuronal function and the physiological basis of those effects.