Coburn C K et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Death Made Visible: A Burst Of Blue Fluorescence Accompanies Mortality In C. elegans"

Edwards S, Coburn C K, Cabreiro F, Taylor A, Gems D

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Under ultraviolet light (lambda~340nm) worms show punctate blue fluorescence (lambda ~430nm), particularly in the gut. In worm cohorts, mean intensity of blue fluorescence increases with age, and is commonly used as a biomarker of worm aging1,2. It has been suggested that this fluorescent material is lipofuscin1, a heterogeneous aggregate of damaged macromolecules which in mammals accumulates with age within lysosomes, and which also shows blue fluorescence3. In worms, blue fluorescence occurs within lysosome-related organelles4 We have reassessed the parallels drawn between blue fluorescence and lipofuscin. Firstly, we monitored blue fluorescence throughout life in individual worms, rather than measuring average levels in population cohorts. We saw no increase in blue fluorescence during most of the worms' lifetimes, but instead a dramatic, rapid burst (mean ~5-fold increase, p=2.8e-13) of blue fluorescence just as the worms died. A similar fluorescent burst was also seen when young adults were killed, e.g. by heat, freeze-thaw or pH<3. Thus, a burst of blue fluorescence seems to be typical of organismal death in C. elegans, irrespective of cause. We also found that exposure to hyperoxia sufficient to increase protein oxidation did not increase blue fluorescence. To better characterize the dynamics of the blue fluorescent burst at the organismal level, we used stop motion photography. The striking, rapid spread of the blue fluorescence appears to mark the passage of death through the organism. The fluorescent burst starts with a focal increase in fluorescence, typically in the anterior gut. A wave of fluorescence then moves rapidly (3-4hrs) along the intestine. The blue fluorescence eventually penetrates all tissues except the gonad, and then fades from the corpse of the worm. The entire duration of the burst is around 20 hours. These results imply that blue fluorescence is a marker of organismal death rather than of aging, and suggest that the blue fluorescent material is something other than lipofuscin. The chemical identity of the fluorescent material remains a mystery, although one property it possesses is pH sensitivity: its blue fluorescence is enhanced by low pH. 1. Klass, Mech Ageing Dev 6, 413 (1977). 2. Gerstbrein et al., Aging Cell 4, 127. 3. Brunk & Terman, Free Radic Biol Med 33, 611 (2002). 4. Hermann et al., Mol Biol Cell 16, 3273 (2005).

Robinson, Kristin et al. (2019) International Worm Meeting "The egg-counter: A microfluidic platform for analysis of egg-laying behavior."

Phillips, Patrick, Banse, Stephen, Jarrett, Cody, Blue, Benjamin, Robinson, Kristin

[International Worm Meeting, 2019]

For animals that do not provide parental care, when and where eggs are laid in the environment can have profound effects on the reproductive success of an individual. Because of its importance, it is not surprising that egg-laying is highly regulated in response to environmental cues. This regulation, coupled with the well described neural circuitry involved in the act of egg-laying, make it a great system for studying how animals interpret their environment and make behavioral choices. To facilitate this line of research we have developed a microfluidic "egg-counter" that allows 32 nematodes to reside in individual growth chambers while their egg-laying behavior is recorded at a sub-minute temporal resolution. The platform utilizes a perfusion-based feeding system that allows experimental control of the chemical environment as well as a built-in temperature control unit that allows the temperature to be patterned with 0.1 deg C accuracy without the use of incubators or temperature control rooms. Preliminary analysis of egg-laying behavior from wildtype animals in our microfluidic environment grossly fits that of the three-state model used to describe egg-laying on agar plates, with the log-tail distribution of egg-laying intervals exhibiting a bi-phasic distribution consistent with two interval types, inter and intra-cluster intervals. We will present a genetic characterization of the role of insulin signaling in regulating egg-laying behavior.

Ghosh, D. Dipon et al. (2017) International Worm Meeting "C. elegans detect the color of pigmented food sources to guide foraging decisions."

Nitabach, Michael N., Ghosh, D. Dipon, Jin, Xin

[International Worm Meeting, 2017]

Here we establish that contrary to expectations, Caenorhabditis elegans nematode worms possess a color discrimination system despite lacking any opsin or other photoreceptor genes. We found that simulated daylight guides C. elegans foraging decisions with respect to harmful bacteria that secrete a blue pigment toxin. By absorbing yellow-orange light, this blue pigment toxin alters the color of light sensed by the worm, and thereby triggers an increase in avoidance of harmful bacteria. These studies thus establish the existence of a color detection system that is distinct from those of other animals. In addition, these studies reveal an unexpected contribution of microbial color display to visual ecology.

Jee, Changhoon et al. (2015) International Worm Meeting "Activation of SEB-3 in LUA interneurons potentiates male mating drive of C. elegans."

Garcia, L. Rene, LeBoeuf, Brigitte, Jee, Changhoon

[International Worm Meeting, 2015]

The male C. elegans' sexual drive intensity determines his reproductive success under stressful conditions. To measure the strength of the male's sexual motivation, we developed the Mating Interference assay (Mi), which quantifies copulation persistence in noxious 475 nm (blue) light. Between copulations, free moving male will escape from 370-450mW/mm2 blue light illumination. However we found that the activated corticotropin-releasing factor (CRF) receptor homolog, SEB-3, causes the gender-common LUA interneurons to potentiate downstream male-specific reproduction circuits. This allows the male's copulatory behaviors to override the light-elicited escape response. SEB-3 also potentiates copulation in standard stress conditions, such as starvation and increased temperatures; thus we suggest that animals use the CRF receptor to acclimate and execute innate behaviors under non-optimal conditions.

Coburn, Cassandra et al. (2011) International Worm Meeting "Death Made Visible: The Necrosis Pathway Generates A Burst Of Blue Fluorescence Marking Organismal Death In C. elegans."

Gems, David, Matthijssens, Filip, Coburn, Cassandra, Nehkre, Keith, Braeckman, Bart, Cabreiro, Filipe, Fischer, Grahame, ARAIZ, Caroline

[International Worm Meeting, 2011]

Under UV light, blue fluorescence is visible in the intestinal cells of C. elegans. Mean levels of this fluorescence increase in aging worm cohorts(1). It has been suggested that the fluorescent substance is lipofuscin, a complex aggregate of oxidised proteins and lipids. Lipofuscin occurs in aging mammalian cells and has similar spectral properties(2). Blue fluorescence is thus often used as a biomarker of aging in C. elegans. When we monitored individual worms (rather than cohorts) as they aged, we saw no increase in fluorescence. Instead, as the worms died, we saw a rapid and striking increase in fluorescence (~5 fold, p = 2.8e-13; time lapse photography). This fluorescence was propagated in a wave along each worm as it died. A similar increase was also seen in young adult worms when we killed them. Hyperoxia caused increased protein oxidation but did not affect levels of blue fluorescence. Altogether, our findings strongly imply that the blue fluorescent material is not lipofuscin or any kind of biomarker of aging, but rather an indicator of death. We then asked how such death fluorescence is generated. In the necrotic calpain-cathepsin protease pathway of mammals(3) and nematodes(4), lysosomal lysis causes cytosolic acidosis, which leads to cell death via peptidase activation. We employed necrosis pathway mutants to block the pathway, and tested effects on death fluorescence. Knockdown of intracellular calcium release (crt-1, unc-68), or cysteine (tra-3) or aspartyl (cad-1) proteases, or inhibition of lysosomal acidification (vha-12) all significantly decreased death fluorescence levels. Moreover, cytosolic acidosis immediately precedes death fluorescence. This suggests that death fluorescence is a terminal product of the necrotic cell death pathway. The fluorescent material, which is non-proteinaceous, can be purified using HPLC. Mass spectrometry identifies a single molecular species, which we are now analyzing. Our work implies that the blue fluorescent substance in the worm intestine is not lipofuscin but, instead, a terminal product of necrotic death. Moreover, it suggests that intestinal necrosis, and its propagation along the worm, plays a major role in C. elegans organismal death. (1) Gerstbrein, Aging Cell, 2005 (2) Klass, Mech Ageing Dev, 1977 (3) Yamashima, Cell Calcium, 2004 (4) Xu, Neuron, 2001.

Husson, Steven J. et al. (2011) International Worm Meeting "Microbial proton pumps as hyperpolarizers complement the optogenetics toolbox in Caenorhabditis elegans."

Stirman, Jeffrey N., Husson, Steven J., Gottschalk, Alexander, Liewald, Jana F., Lu, Hang

[International Worm Meeting, 2011]

Optogenetic technologies use light to gain endogenous control of defined cells or tissues in a non-invasive manner. Neuronal activity can be manipulated at the millisecond timescale by expressing the light-activated depolarizing cation channel channelrhodopsin-2 (ChR-2) and subsequent illumination with blue light (1). In contrast, yellow light-triggered inhibition of neuronal activity can be achieved by activation of the hyperpolarizing halorhodopsin (NpHR) (2). However, only few successful attempts were undertaken to inhibit C. elegans neurons using NpHR, probably due to the insufficient trafficking to the plasma membrane, and thus need for high expression levels, or the limited hyperpolarizing power of this Cl- channel. An extensive screen of type I microbial opsins from archaebacteria, bacteria, plants and fungi recently revealed powerful outward directed proton pumps as valuable alternative hyperpolarisers (3). As cells and extracellular fluid are strongly buffered, shuffling protons across the membrane is not expected to cause any appreciable pH changes. The yellow-green light-sensitive archaerhodopsin-3 (Arch) from Halorubrum sodomense appears to be significantly more powerful than NpHR. Another proton pump from the fungus Leptosphaeria maculans, Mac, enables neuronal silencing by green-blue light. This opens the possibility to inhibit different neuronal populations, depending on the illumination wavelengths used. Here we present the use of these outward-directed proton pumps as potent circuit breakers in C. elegans. Electrophysiological recordings on dissected muscle cells allowed us to quantify the outward current evoked by either NpHR, Arch and Mac. As Mac can be stimulated using blue light, we can activate a subset of neurons using ChR2 while Mac could be used to simultaneously inhibit downstream neurons, using the same colour of light. Alternatively, illumination of predefined body segments with different colours of light using an integrated LCD projector as light source (4) allows using the more potent Arch (maximal hyperpolarizing power with green light) for neuronal inhibition and simultaneous ChR2-induced activation of other cells with blue light. A few examples for circuit dissection with either bacteriorhodopsin will be presented at the meeting. (1) G. Nagel et al., Curr. Biol. 15, 2279 (2005); (2) F. Zhang et al., Nature 446, 633 (2007); (3) B. Y. Chow et al., Nature 463, 98 (2010); (4) J. N. Stirman et al., Nat. Meth. 8, 153 (2011).

D Miller et al. (1999) International C. elegans Meeting "Two-color GFP expression in C. elegans"

D Piston, D Hardin, A Fire, J Fleenor, G Patterson, D Miller, N Desai

[International C. elegans Meeting, 1999]

The Green Fluorescent Protein (GFP) is now widely used for imaging cells and organelles in living animals. GFP variants with distinct excitation and emission spectra have been developed and should prove highly useful for simultaneous imaging of separate proteins labeled with different colored GFPs. Here we describe expression vectors and fluorescence filter sets that can be used to obtain bright, distinct images of "Cyan" GFP (CFP) and "Yellow" GFP (YFP) in C. elegans (1). GFP vectors specifically designed for expression in C. elegans (2) were modified to include amino acid substitutions appropriate for CFP (Y66W, N146I, M153T, V163A) or YFP (S65G, V68A, S72A, T203Y) (see www.ciwemb.edu ). Previously described promoter regions and localization signals were employed to create trangenic animals expressing CFP and YFP either in different cells or in separate intracellular compartments. In these experiments, CFP appears "Blue" and the YFP signal is "Green." For example, an unc-4 promoter element drives expression in VA motor neurons whereas a del-1 upstream region is specific for the adjacent VB motor neurons. Transgenic animals expressing both unc-4::CFP and del-1::YFP display side-by-side VA (Blue) and VB (Green) motor neurons in the ventral nerve cord. In another experiment, cytoplasmically localized YFP and nuclear-localized CFP were expressed under the control of the muscle-specific unc-54 promoter to produce Green bodywall muscle cells with Blue nuclei. It should now be possible to utilize these vectors for a wide variety of two-color labeling experiments. The fluorescence filter sets used in the work were developed in collaboration with Chroma Technology Corp. For CFP: excitation = 436/10 nm; dichroic = 450 nm; emission = 485/50 nm For YFP: excitation = 500/20 nm; dichroic = 515 nm; emission = 520 nm long pass. (1) Miller, et al. (1999) BioTechniques, in press. (2) Fire, et al. (1998). p. 153-168. In M. Chalfie and S. Kain (Eds.), GFP Strategies and Applications. John Wiley and Sons, NY

YU, B. et al. (2019) International Worm Meeting "An Upconversion Nanoparticle Enables Near Infrared-Optogenetic Manipulation of the C. elegans Motor Circuit."

Zeng, K., Tan, T., Xue, Y., Yu, Z., Zhang, Y., Zhen, M., Yu , M., YU, B., Hung, W., Xu, T., Yang, X., Ao, Y., Gao, S.

[International Worm Meeting, 2019]

Near-infrared (NIR) light penetrates tissue deeply, but its application to motor behavior stimulation has been limited by the lack of known genetic NIR light-responsive sensors. We designed and synthesized a Yb3+/Er3+/Ca2+-based lanthanide-doped upconversion nanoparticle (UCNP) that effectively converts 808 nm NIR light to green light emission. This UCNP is compatible with Chrimson, a cation channel activated by green light; as such, it can be used in the optogenetic manipulation of the motor behaviors of C. elegans. We show that this UCNP effectively activates Chrimson-expressing, inhibitory GABAergic motor neurons, leading to reduced action potential firing in the body wall muscle and resulting in locomotion inhibition. The UCNP also activates the excitatory glutamatergic DVC interneuron, leading to potentiated muscle action potential bursts and active reversal locomotion. Moreover, this UCNP exhibits negligible toxicity in neural development, growth and reproduction, and the NIR energy required to elicit these behavioral and physiological responses does not activate the animal's temperature response. This study shows that UCNP provides a useful integrated optogenetic toolset, which may have wide applications in other experimental system. In future, we continue to do related research in this area. Currently, we are developing a two-color upconversion nanoparticle in which one of the upconverted blue light can replace the blue light of visible light, and the neuron is damaged or even killed by activating the miniSOG protein.

Zeng, K. et al. (2019) International Worm Meeting "An Upconversion Nanoparticle Enables Near Infrared-Optogenetic Manipulation of the C. elegans Motor Circuit."

Gao, S, Tan, T, YU, B., Zhang, Y, Zeng, K., Zhen, M, Miao, Y, Yu, Z, Hung, W, Ao, Y, Xu, T, Xue, Y, Yang, X

[International Worm Meeting, 2019]

ABSTRACT: Near-infrared (NIR) light penetrates tissue deeply, but its application to motor behavior stimulation has been limited by the lack of known genetic NIR light-responsive sensors. We designed and synthesized a Yb3+/Er3+/Ca2+-based lanthanide-doped upconversion nanoparticle (UCNP) that effectively converts 808 nm NIR light to green light emission. This UCNP is compatible with Chrimson, a cation channel activated by green light; as such, it can be used in the optogenetic manipulation of the motor behaviors of C. elegans. We show that this UCNP effectively activates Chrimson-expressing, inhibitory GABAergic motor neurons, leading to reduced action potential firing in the body wall muscle and resulting in locomotion inhibition. The UCNP also activates the excitatory glutamatergic DVC interneuron, leading to potentiated muscle action potential bursts and active reversal locomotion. Moreover, this UCNP exhibits negligible toxicity in neural development, growth and reproduction, and the NIR energy required to elicit these behavioral and physiological responses does not activate the animal's temperature response. This study shows that UCNP provides a useful integrated optogenetic toolset, which may have wide applications in other experimental system. In future, we continue to do related research in this area. Currently, we are developing a two-color upconversion nanoparticle in which one of the upconverted blue light can replace the blue light of visible light, and the neuron is damaged or even killed by activating the miniSOG protein.

Vettkotter, Dennis et al. (2021) International Worm Meeting "Introducing optoSynC - a novel optogenetic tool for synaptic silencing"

Liewald, Jana F., Gottschalk, Alexander, Schneider, Martin W., Vettkotter, Dennis

[International Worm Meeting, 2021]

The investigation of neuronal circuits as well as molecular and cellular functions of neurons requires the control of activity in a spatio-temporal precise manner. The field of optogenetics opens the pathway to stimulate or inhibit specific types of neurons with light. In the last years, several tools have been developed that allow inhibition of specific neurons on time scales from milliseconds to minutes or long-term silencing. However, these optogenetic tools come at the cost of fast induction or reversibility of the altered neuronal function. Thus, a tool which allows for fast, long-term and spatially restricted neuronal silencing, while still allowing for fast reversibility, is of substantial need. Here, we developed an approach to achieve these goals. Using the ability of the Arabidopsis thaliana cryptochrome 2 (CRY2) to form homo-oligomers, we designed an optogenetic tool to cluster synaptic vesicles (SVs) and thus inhibit their function acutely. The tool, called OptoSynC (optogenetic synaptic vesicle clustering), comprises CRY2, fused to the synaptic vesicle intrinsic membrane protein synaptogyrin (SNG-1). The efficiency of OptoSynC was evaluated at the behavioral level. Blue light illumination of pan-neuronally expressed OptoSynC significantly reduced swimming cycles by 80% within seconds. Termination of blue light for more than 15 minutes allowed worms to recover their initial swimming behavior. In addition to behavioral assays, inhibition of synaptic transmission could be demonstrated by electrophysiology experiments. Using a combination of optogenetic activation of neurons with the red-light activated channel Chrimson and blue-light activated inhibition using OptoSynC, we could show the effect even in a single neuron, PVD, required for nociception. While behaviorally, OptoSynC evokes drastic effects, its mode of action has yet to be revealed, as it either inhibits synaptic transmission by SV clustering in the reserve pool, or it might clog-up release sites at the presynaptic terminal due to clustering of SNG-1 protein already present in the plasma membrane. Therefore, we currently employ electron microscopy to shed light on this mechanistic detail. With further optimization of this tool and the knowledge of its underlying mechanism OptoSynC can be a potent tool for synaptic silencing, that might also be used for the research of how SVs are guided to the plasma membrane or in which precise sequence of events SV recycling proceeds.