Albrecht, Dirk R et al. (2009) International Worm Meeting "C. elegans behavior in dynamic microfluidic environments."

Bargmann, Cori, Albrecht, Dirk R

[International Worm Meeting, 2009]

Neuronal circuits that govern goal-directed behaviors such as chemotaxis are highly interconnected, suggesting that their emergent functions may not be evident from the properties of individual neurons or connections. To study the underlying neural computation and dynamics, an engineering approach would systematically quantify output responses to many precise inputs under many circuit perturbations. While C. elegans locomotory behaviors are easily measured, and genetic tools enable precise circuit modification, the presentation of input stimuli in typical agar-plate assays is often poorly controlled. To address this limitation, we developed microfluidic liquid-filled arenas that enable the study of freely-moving animals in highly precise and dynamic microenvironments. We first optimized arena geometry to mimic C. elegans crawling motion, speed, and behavioral responses on agar surfaces. Microfluidic features create controlled liquid gradients that span several cm for population behavior or change sharply across the animal (<50 micron), and remain stable for hours or change rapidly within seconds. The transparent arenas are compatible with light-based neural control (via genetically-encoded rhodopsins) and fluorescent readouts of neural activity. We are characterizing wildtype C. elegans responses to complex spatial and temporal odorant patterns (steps and ramps) to understand how modulation of specific behaviors (e.g., speed, types of turns) influences chemotaxis strategy. Similar studies of genetic mutants with disrupted neurons or neuronal connections are revealing the role of perturbed information flow in directing these behaviors. For example, we found new behaviors (gradient-directed turning), new circuit pathways (glutamate-independent speed regulation), and strong phenotypes in subtle neuromodulatory mutants. Overall, the vast improvement in stimulus control in these microfluidic arenas enables new studies to understand the flow of information in neural circuits governing behavior.

Dirk Albrecht et al. (2008) C.elegans Neuronal Development Meeting "C. elegans Behavior in Dynamic Microfluidic Environments"

Dirk Albrecht, Cori Bargmann

[C.elegans Neuronal Development Meeting, 2008]

We seek to understand goal-directed behaviors such as chemotaxis at the organism, neuronal, and genetic levels, using the soil-dwelling nematode C. elegans as a model system. Typical behavioral assays on agar dishes are limited by difficulties in the control and measurement of input stimuli and by sensitivity to physical conditions such as humidity. To address these limitations, we developed a series of microfluidic liquid-filled arenas that enable the study of subtle locomotory behaviors in precise and dynamic microenvironments. The dimensions of microchannels enclosing C. elegans were found to define the animal''s speed, mode of locomotion (swimming or crawling), and turning behavior. We identified conditions in which animals mimic crawling motion and speed on agar and display stereotyped locomotory behaviors (e.g., forward runs, short and long reversals, and omega turns). In these arenas, controlled liquid gradients can span several cm for population behavior or change sharply across an individual animal (50 microm). Further, chemical conditions can remain stable for hours or change rapidly within seconds. The transparent arenas are compatible with light-based neuronal control (via genetically-encoded rhodopsins) and readouts of neural activity (at high magnification via fluorescent calcium sensors). We initially characterized the unrestrained locomotory behavior of wildtype N2 animals in the microfluidic arenas when challenged with complex spatial and temporal odorant patterns (e.g., steps and ramps). These experiments yield far more detail than standard endpoint chemotaxis assays, including measurements of speed and behavioral state over space and time for each precise stimulus condition. Our ongoing analysis of specific mutants with disrupted sensation, integration, or motor activity should yield further insights about the flow of information during chemotaxis and other behaviors. With spatiotemporal control over input environments, improved imaging of behavioral output, and both measurement and manipulation of neuronal activity, microfluidic assays expand the study of the neural basis of behavior in small organisms.

Lagoy, Ross et al. (2017) International Worm Meeting "An automated high-throughput functional screen of neuromodulators for neuropsychiatric disease models."

Lagoy, Ross, Kim, Heesun, Albrecht, Dirk

[International Worm Meeting, 2017]

Mental health disorders, like autism, depression, and epilepsy, affect millions of people worldwide. Some of these illnesses are highly associated with channelopathies, or dysfunction in ion channels, that affect neural activity in the brain. To accelerate the discovery of compounds that modulate neuronal activity in a living whole-organism, we are developing functional screening methods and establishing novel C. elegans models of human channelopathies. To rapidly and cheaply screen many compounds in C. elegans, we developed two automated approaches based on calcium imaging in a single neuron with precise odor or optical stimulation. Our first method adapts a high-throughput microfluidic device that allows for automated serial drug exposure (seconds-minutes) to animals while recording neural responses when stimulated by odor or light. Our second method is an automated 384-well plate-based screen that allows for increased drug exposure (hours-days) and records neural responses by optogenetic stimulation. To address drug bioavailability, we are using these methods to observe dynamics of drug effects in various mutants. For example, we found nemadipine-A (an L-type calcium channel blocker) to inhibit calcium responses time-dependently in wild-type animals over 14 hours of exposure. Currently, we are scaling-up this process to screen hundreds of FDA approved small-molecules to detect other compounds that modulate neuronal activity in C. elegans for secondary screens. In parallel, we are establishing various C. elegans models of channelopathies in the orthologus voltage-gated calcium channel (VGCC) EGL-19/CACNA1C. We used CRISPR-mediated homologus recombination to generate the first C. elegans model of Timothy syndrome (TS), a human disorder caused by a missense VGCC mutation that results in autism, arrhythmia, and developmental disorders. Shown at the cellular level in various model systems, this mutation leads to decreased channel inactivation. In our whole-organism TS model, we observe a severe developmental phenotype; animals are arrested immediately post-hatching stage, while heterozygous animals develop as wild-type. With our high-throughput microfluidic pulse device, we can quantify residual calcium, channel activation and inactivation rate, and detect altered channel kinetics in various EGL-19 channelopathies. Altogether, we aim to rescue these phenotypes with novel compounds derived from our screens and suggest their use for mammalian models.

Larsch, Johannes et al. (2013) International Worm Meeting "Maintaining sensitivity: dissecting sensory adaptation using high-throughput in vivo calcium imaging."

Bargmann, Cori I., Larsch, Johannes, Albrecht, Dirk R.

[International Worm Meeting, 2013]

Sensory neurons face a dilemma: Biologically relevant changes in stimulus strength are often small compared to the possible range of stimulus intensities. One solution to this problem is sensory adaptation, in which the sensory neuron continuously adjusts its dynamic range based on stimulus history to maximize sensitivity while avoiding saturation. During chemotaxis, C. elegans relies on known chemosensory neurons to detect odors over at least five orders of magnitude in concentration, suggesting a sophisticated molecular machinery to keep ongoing sensory activity within dynamic range. To probe adaptation in C. elegans, we wanted to deliver a broad range of odor stimulus concentrations and patterns while monitoring neural responses. To this end, we developed a high throughput system for in vivo calcium imaging that allows us to record up to 20 animals simultaneously for hours, enabling quantitative mapping of sensory receptive fields and their adjustment to constant or changing stimulus levels. We observed odor-evoked calcium dynamics in AWA sensory neurons across a million-fold range of concentrations. Neurons adapted upon repeated stimulation with odor pulses on two different time scales: fast inactivation of the calcium transient during each odor pulse and slow adjustment of response magnitude and dynamics across repeated pulses. We have screened mutants and pharmacological interventions for effects on odor-evoked calcium transients and their adaptation, seeking to define mechanisms for odor sensing and fast vs. slow adaptation. Paradoxically, several chemotaxis mutants with structurally defective cilia (che-2, che-3, osm-6) had stronger and more slowly adapting calcium responses to odor than wild-type animals suggesting that IFT cilia genes are more important for rapid adjustment of sensory sensitivity than for primary transduction in AWA.

Chalasani, Sreekanth H. et al. (2009) International Worm Meeting "AWC sensory neurons release two neurotransmitters to regulate exploratory behavior upon removal from food."

Albrecht, Dirk, Bargmann, Cornelia I., Chalasani, Sreekanth H., Kato, Saul

[International Worm Meeting, 2009]

Animals increase their pirouette frequency in response to removal from food stimulus for a period of 15 min. The AWC and ASK sensory neurons and the AIB interneurons stimulate pirouettes immediately after removal from food, while the AIY and AIA interneurons inhibit pirouettes (Wakabayashi et al 2004, Gray et al 2005). We have found that AWC sensory neurons become active in response to removal of stimulus, releasing two neurotransmitters (glutamate and a neuropeptide NLP-1). The released glutamate acts to activate AIB and inhibit AIY interneurons, promoting reversals (Chalasani et al 2007). In contrast to glutamate, AWC-released NLP-1 acts on AIA interneurons to suppress reversals, suggesting that reversal frequencies are regulated by at least two opposing signaling systems. AWC calcium responses are modulated in these neurotransmitter mutants, suggesting that feedback pathways affect AWC neuronal activity. References: Chalasani, S. H., Chronis, N., Tsunozaki, M., Gray, J. M., Ramot, D., Goodman, M. B., and Bargmann, C. I. (2007). Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans. Nature 450, 63-70. Gray, J.M., Hill, J.J., and Bargmann, C.I. (2005). A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 102, 3184-3191. Wakabayashi, T., Kitagawa, I., and Shingai, R. (2004). Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111.

Lagoy, Ross C. et al. (2018) MicroPubl Biol

Lagoy, Ross C., Kim, Heesun, Albrecht, Dirk R., Mello, Craig C.

[MicroPubl Biol, 2018]

Timothy syndrome (TS) type 1 is a rare genetic human disease caused by a gain-of-function (GOF) missense mutation G406R in the L-type voltage-gated calcium channel (VGCC) 1 subunit gene CACNA1C, which results in severe cardiac arrhythmia and autism (Splawski et al. 2004). The C. elegans ortholog is EGL-19 (Fig. 1a), which is expressed in muscle and neurons (Lee et al. 1997). GOF mutations in egl-19 cause myotonic phenotypes, while reduction-of-function (ROF) mutants are flaccid, variably elongated, and egg-laying defective, and lethal mutations cause paralysis with arrested elongation at the two-fold stage (Pat) (Lee et al. 1997). Additionally, treatment of embryos with nemadipine-A, an antagonist of L-type VGCCs, causes a severe variable abnormal (Vab) phenotype like some ROF hatchlings (Kwok et al. 2006). Neural and muscle cells derived from patients with TS type 1 also yield impaired channel inactivation (Yazawa et al. 2011; Paca et al. 2011). Therefore, we hypothesized that insertion of the human TS type 1 GOF mutation in the C. elegans genome (G369R) by CRISPR-Cas9 homologous recombination (HR) would cause observable changes in calcium dynamics and serve as a new animal disease model of TS to broadly investigate molecular mechanisms in vivo. Instead, introduction of the TS type 1 human GOF mutation in C. elegans resulted in homozygous animals that closely resemble the Vab phenotype (Fig. 1b-d), similar to ROF mutant hatchlings and wild type embryos treated with the L-type VGCC antagonist nemadipine-A. This phenotype was observed in three independently edited lines, suggesting it resulted from the human TS mutation rather than a cis loss-of-function mutation in egl-19 generated by the CRISPR-Cas9 editing. Further, heterozygous animals appeared wild type, which is consistent with recessive inheritance of ROF and lethal mutations in egl-19. Taken together, this human mutation appears to dysregulate egl-19 function in C. elegans indirectly, such as through expression, trafficking, or channel kinetics at the cell membrane (Fig. 1e). Evaluation of gene expression, protein localization, and functional imaging or electrophysiology in this new animal model of TS type 1 are needed to distinguish among these possibilities. This result demonstrates that CRISPR-Cas9 can be used to generate human VGCC disease mutations in C. elegans, although unexpected phenotypes may result from introducing human mutations in these animals. Nonetheless, this new whole organism model of TS type 1 may provide a foundation for investigating molecular mechanisms involved in this severe genetic disease, screening of additional genetic and therapeutic suppressors as potential treatments for translation, and studying other human channelopathies in C. elegans.

Flavell, Steven W et al. (2013) International Worm Meeting "Serotonin and PDF are opposing neuromodulators that control a bistable foraging behavior in C. elegans."

Pokala, Navin, Albrecht, Dirk A, Macosko, Evan Z, Bargmann, Cornelia I, Larsch, Johannes, Flavell, Steven W

[International Worm Meeting, 2013]

Foraging animals have distinct exploration and exploitation behaviors that are organized into discrete, long-lasting behavioral states. Here we characterize a neuromodulatory circuit that generates such long-lasting roaming and dwelling states in Caenorhabditis elegans. We find that two opposing neuromodulators, serotonin and the neuropeptide pigment dispersing factor (PDF), each initiate and extend one behavioral state. Serotonin promotes dwelling states through the MOD-1 serotonin-gated chloride channel. The spontaneous activity of serotonergic neurons correlates with dwelling behavior, and optogenetic modulation of the critical MOD-1-expressing targets induces long-lasting dwelling states. PDF promotes roaming states through the Gas-coupled PDFR-1 receptor; optogenetic activation of cAMP production in PDFR-1-expressing cells induces long-lasting roaming states. The neurons that produce and respond to each neuromodulator form a distributed circuit orthogonal to the classical wiring diagram, with several essential neurons that express each molecule. The slow temporal dynamics of the neuromodulatory circuit supplement fast motor circuits to give rise to long-lasting behavioral states.

Narayanaswamy N et al. (2019) Nat Methods "Author Correction: A pH-correctable, DNA-based fluorescent reporter for organellar calcium."

Leung K, Chakraborty K, Narayanaswamy N, Devany J, Krishnan Y, Zeichner E, Saminathan A

[Nat Methods, 2019]

The originally published paper has been updated to include the following new reference, added as ref. 18: Albrecht, T., Zhao, Y., Nguyen, T. H., Campbell, R. E. &amp; Johnson, J. D. Fluorescent biosensors illuminate calcium levels within defined beta-cell endosome subpopulations. Cell Calcium 57, 263-274 (2015). Subsequent references have been renumbered in the reference list and throughout the text. Minor text changes were made in the sentence in which this new reference is first cited: "Previous attempts used endocytic tracers bearing either pH- or Ca²⁺-sensitive dyes to serially measure population-averaged pH and apparent Ca²⁺ in different batches of cells, thus scrambling information from individual endosomes^13-17" in the original introduction was changed to "Previous attempts used endocytic tracers bearing either pH- or Ca²⁺-sensitive dyes^13-17 or fluorescent-protein-based sensors¹⁸ to serially measure population-averaged pH and apparent Ca²⁺ in different batches of cells, thus scrambling information from individual endosomes." These changes have been made in the HTML and PDF versions of the article.

Sato, Hirofumi et al. (2015) International Worm Meeting "A gustatory neural circuit for experience-dependent salt chemotaxis in C. elegans."

Hashimoto, Koichi, Iino, Yuichi, Fei, Xianfeng, Kunitomo, Hirofumi, Sato, Hirofumi

[International Worm Meeting, 2015]

Caenorhabditis elegans shows experience-dependent behaviors to many environmental cues. For sodium chloride, worms are known to memorize a particular salt concentration and approach the memorized concentration. In this study, we therefore searched for the neural circuit required for the memory of salt concentration. First, we conditioned worms in different salt concentrations, and monitored the activity of the salt-sensing chemosensory neuron ASER and three downstream interneurons; AIA, AIB, and AIY. We found that ASER, AIB, and AIY changed the responses depending on the previously exposed salt concentrations. We investigated the response of ASER in more detail, and found that the basal calcium level of ASER might change depending on cultivation concentration, and the plasticity of ASER response seemed to be independent of inputs from other neurons. Next, to assess the contribution of the three interneurons to the behavior, we ablated them individually, and compared behavioral responses of those worms with wild type. As a result, the reversal frequency of cell-ablated worms was different from that of the wild type. However, cell-ablated worms showed normal salt chemotaxis under the tested conditions, indicating that there are redundancies in the neural circuit that processes the salt perception signal. Furthermore, we investigated the relationship between the neural response and locomotion of worms. We used a tracking-imaging system with microfluidic arena that allowed worms to crawl in a controlled liquid environment (Albrecht et al., 2011), and recorded locomotion of worms and neural responses simultaneously. The result showed that the speed of worms decreased only when salt concentration was decreased below cultivation concentration. However, ASER always showed an off-response to salt, indicating that there is an experience-dependent plasticity in the process that links the ASER response to moving velocity.

Calahorro, F. et al. (2017) International Worm Meeting "TrakBox: A new modular option for behavioural monitoring of freely moving C. elegans."

James, C., Calahorro, F., O'Connor, V., Holden-Dye, L.

[International Worm Meeting, 2017]

Tracking the movement of C. elegans is a well-stablished route to identify and refine understanding of microcircuits that control behaviour. Over several years a number of different experimental platforms and associated software for video analysis have been developed to facilitate this (1). To contribute to this we have designed a relatively low cost modular system for real-time tracking and analysis of worm locomotory behaviour. TrakBox (EMbody Biosignals Ltd) (2) is assembled from 3-D printed components in which a robotic arm moves a USB camera to maintain the animal being tracked within a target site. Reverse kinematics is used to decode the coordinates of the worm as it moves around the plate with continuous tracking being possible for at least 24 h. Videos may also be captured but are not required for the behavioural analyses. Advanced signal processing filters and corrects the worm position over time and constantly updates a series of behaviour parameters calculated 'on the fly'. TrakBox software derives parameters over the entire course of tracking that describe worm position, instantaneous velocity, instantaneous direction of travel, dwelling and roaming times and number of reversals. The user may define the behavioural parameters of interest and they may be displayed as a graphical representation. As a proof of concept we compared the behaviour of N2 and a mod-1 mutant. mod-1 encodes a serotonin-gated chloride channel and a loss of function mutant exhibits increased exploration and extended roaming time (3). TrakBox efficiently extracted this phenotype by analysing roaming/dwelling fractions of time, velocity and reversal events and their duration. Thus, we show TrakBox permits discrete real-time analysis of C. elegans locomotory behaviour that circumvents the need for post-hoc analysis of videos. This experimental platform also has applications for investigation of the effects of drugs on behaviour, may be applied to analysis of other microscopic nematodes including pest species, have uses in field studies and provide an affordable option for class room demonstration of C. elegans biology. (1) Husson, S. J. et al. Keeping track of worm trackers (September 10, 2012), WormBook, ed. (2) http://embody-biosignals.com/ (3) Flavell, S. W., N. Pokala, E. Z. Macosko, D. R. Albrecht, J. Larsch and C. I. Bargmann (2013). "Serotonin and the neuropeptide PDF initiate and extend opposing behavioral states in C. elegans." Cell 154(5): 1023-1035