Swierczek, Nicholas et al. (2009) International Worm Meeting "High-throughput mostly-automated quantification of subtle behaviors."

Giles, Andrew, Swierczek, Nicholas, Kerr, Rex A., Rankin, Catharine

[International Worm Meeting, 2009]

We have designed and built a tool, the Multi-Worm Tracker (MWT), that enables analysis of subtle behavioral defects at speeds suitable for forward genetic screening. The Multi-Worm Tracker images a plate of worms with a 4 megapixel camera running at 10 Hz, delivers stimuli at user-specified intervals, and uses real-time image analysis to extract and save key parameters for each worm. We typically track dozens of worms on a single 5 cm plate; this allows rapid screening of baseline behaviors such as speed and turning frequency; of stimulus-driven behaviors such as reversals induced by tap to the plate; and of adaptations to repeated stimuli. Due to the low magnification, small-scale features such as shape of body bends cannot be reliably quantified; these require a high-magnification single-worm tracker. We are using the MWT to quantify in detail the tap habituation behavior of C. elegans. For instance, we have recorded from tens of thousands of wild-type worms to generate highly reliable statistics, and carefully sampled inter-stimulus intervals from seconds through minutes. We are also preparing to screen for tap habituation mutants, which would be highly impractical without an automated system. We demonstrate that we can detect and cluster existing mutants both for baseline behavior and tap habituation, and use analysis of wild-type variability to design an effective protocol for conducting a large-scale screen. (With luck, we will present preliminary results from a pilot screen.) Although the MWT was designed with tap habituation in mind, the system is generally useful for rapid quantification of behavior. We therefore have taken a number of steps to enable other labs to set up similar systems. The MWT software is open source (but requires commercial run-time libraries) and after the meeting will be available at http://sourceforge.net/projects/mwt; parts lists and installation instructions are also available. We also provide post-acquisition analysis software that plots or saves summary data with more sophistication than is possible online, and allows browsing of the data in 2D map format for easy manual validation of the results. We have conducted proof-of-principle experiments for foraging and chemotaxis; we hope that other labs will further develop these assays using the MWT or a similar system.

Nicholas Swierczek et al. (2007) International Worm Meeting "Automated behavioral analysis of populations of worms."

Catharine Rankin, Rex Kerr, Nicholas Swierczek

[International Worm Meeting, 2007]

We have built an automated C. elegans behavioral analysis system, the Multi-Worm Tracker (MWT), that follows a population of worms on a plate, analyzes their behavior, and reports summary statistics on that behavior, all in real time. The system uses a high speed, high resolution camera (Basler A403k, 2352x1736 at up to 50 Hz) to monitor a 5 cm plate. The basic tracking algorithm simply follows all dark moving objects of an appropriate size; further analysis can detect direction of motion and give a simple characterization of posture. Because the system is intended to run in real time and loss of individual data points is not critical with population-size data sets, we detect and discard difficult cases such as when worms collide with each other and omit those individuals from the analysis until they can be robustly distinguished from one another. Our first application of this system is to rapidly characterize tap habituation responses. For example, it takes the system about 6 minutes to characterize a plate of 30-40 worms with a protocol of 30 taps at 10 second inter-stimulus interval (one minute to set up initially, then 5 minutes to run the protocol). We will present a characterization of the consistency of the analysis and performance relative to hand-scored standards. Our eventual goal is to use this system to conduct screens for tap habituation mutants, and we will describe our plans and progress towards that goal. The MWT can be applied a variety of behaviors and organisms. We will show a variety of examples in worms (chemotaxis, response to noxious heat, etc.) and in Drosophila (thermotaxis in adults and vibration avoidance in larvae). We welcome suggestions for other uses of the MWT either in C. elegans or other organisms.

Nicholas Swierczek et al. (2008) C.elegans Neuronal Development Meeting "The Multi-Worm Tracker: A Tool for Rapid Screening of Subtle Behavior"

Nicholas Swierczek, Andrew Giles, Catharine Rankin, Rex Kerr

[C.elegans Neuronal Development Meeting, 2008]

Forward genetic screens for mutant behaviors require a rapid method for deciding whether an individual or population are significantly different from wild-type. For a wide variety of simple behaviors, clever assays coupled with visual inspection provide sufficiently rapid analysis. However, for more complex behaviors such as tap habituation, foraging, and chemotaxis, a simple visual inspection can only reveal the most egregious defects, especially if the behavior is best scored by accumulating data from many individuals. We have designed and built a tool, the Multi-Worm Tracker, that quantifies the behavior of each clearly visible worm on a plate. The MWT presents summary data for the population in real time and saves a set of simple parameters for each worm at each time point for later analysis if desired (the compact nature of the saved data makes it feasible to run protocols that last for hours or days). Optionally, the system can deliver up to three different types of stimuli to the plate at user-defined intervals; we currently are using tap and air puff as the stimuli. This allows extremely rapid screening of baseline behaviors such as movement and turning rate, as well as stimulus-driven responses and adaptation thereof. Offline tools let you browse the full data set as a 2D map, or plot and save summary data with more refined algorithms that are difficult to apply in real time without the benefit of hindsight. We will present details of the construction of the MWT and summarize the image processing and analysis methods. In addition, we will demonstrate that the MWT can be used to very rapidly collect data to cluster mutant phenotypes, score tap habituation, and assess chemotaxis parameters. None of these applications are new; the MWT simply allows these features to be investigated in a high-throughput way suitable for conducting RNAi or mutagenesis screens.

Giles, Andrew C. et al. (2011) International Worm Meeting "Secondary Allele Screen for Extreme Habituation Phenotypes after High Throughput Behavioral Characterization of a Nervous-system-biased Mutant Library."

Rankin, Catharine H., Swierczek, Nicholas A., Kerr, Rex A., Giles, Andrew C.

[International Worm Meeting, 2011]

Habituation is the most simple and fundamental form of learning and is measured as a decrease in response to a repeated stimulus. We assay habituation of the tap withdrawal response in C. elegans using the Multi-Worm Tracker (MWT), which allows rapid characterization of tap habituation and therefore enables the testing of large numbers of mutants. The assay consists of 10 minutes of recording prior to the stimulation protocol to assess locomotion and spontaneous reversal rates followed by thirty mechanical taps to the side of the plate at a 10 second inter-stimulus interval. In wild-type worms, the tap initially elicits a reversal response that gradually habituates with repeated presentations. We have collected and tested a nervous-system-biased mutant library (~700 strains) by cross-referencing a list of 2073 genes with predicted neural function based on domain structure (Sieburth et al. Nature:436, 2005) with the list of available strains at the Caenorhabditis Genetics Center. This identified a number of mutants with habituation phenotypes, but it was unclear whether these phenotypes were caused by the known mutation, or background mutations. We have now screened a set of secondary (and for some, tertiary) mutations to test if the phenotypes are consistent between alleles. The genes that cause the strongest effects on habituation and were consistent across all tested alleles encode the G-alpha-protein goa-1 and the regulator of G-protein signaling (RGS) known as eat-16. We are currently following up on these genes as well as others to develop new insights into the molecular mechanism of habituation.

Delia M O'Rourke et al. (2005) International Worm Meeting "Analysis of gene expression changes in C. elegans following infection with Microbacterium nematophilum."

Delia M O'Rourke, Richard Mott, Jonathan Hodgkin, Maria Demidova, Dilair Baban

[International Worm Meeting, 2005]

Microbacterium nematophilum is a bacterial pathogen of C. elegans (Hodgkin et al, 2000) that adheres to the rectum and post-anal region of the worm causing swelling of the underlying hypodermal tissue, mild constipation and slow growth rates. Affected worms are described as having a Dar (Deformed Anal Region) phenotype. C. elegans responds to this infection in part through activation of an ERK MAP kinase cascade which mediates tail swelling and prevents severe constipation (Nicholas and Hodgkin, 2004). To identify the downstream transcriptional changes that occur in C. elegans during this host/pathogen interaction we have conducted a genome wide analysis of gene expression using Affymetrix gene chips. We infected a synchronised population of larval C. elegans in liquid culture for 6hrs before harvesting the worms and extracting RNA. Our control sample was an identical experiment using an avirulent M. nematophilum generated in our laboratory by Tanya Akimkina and Steve Curnock. Analysis of data from triplicate microarray experiments has identified a number of statistically significant gene clusters whose expression changes upon infection. Clusters containing up-regulated genes are located on chromosomes IV and V and include C-type lectins, lysozyme-like proteins and proteins containing metridin-like ShK toxin and DUF141 domains. An RNAi feeding screen of these and other induced genes has identified a number that affect the Dar response. The microarray data suggest that although similar functional domains are found in proteins induced by both M. nematophilum and other pathogens, the response of C. elegans to M. nematophilum is specific. Hodgkin, J. Kuwabara, P. E. Corneliussen, B. Current Biology 200010 ;1615-1618 Nicholas, HR, Hodgkin, J. Current Biology 2004 14 ;1256-1261

Rothstein M et al. (1969) "Culture methods and nutrition of nematodes and Acanthocephala."

Nicholas WL, Rothstein M

[1969]

In order to study properly the nutrition and culture of nematodes, it is desirable to establish the organisms in axenic culture. Only in this way can the metabolic abilities of the nematodes be separated from those of coexisting and interacting organisms. One may settle for a mono-axenic culture, but the best way to attain this is to obtain axenic nematodes and then add the second organism or tissue, for example, alfalfa callus tissue for plant parasitic nematodes (Krusberg, 1961). This chapter will devote itself, in the main, to recent work on the culture and nutrition of nematodes, free-living and parasitic, and will refer only in passing to work already thoroughly reviewed (Dougherty et al., 1959; Nicholas, et al., 1959; Dougherty, 1960).

Aoki, Ichiro et al. (2021) International Worm Meeting "Specific (co-)transmission of two neuropeptide species from the AVK interneuron"

AOKI, Ichiro, Gottschalk, Alexander

[International Worm Meeting, 2021]

Communication among cells via neuropeptides is crucial for proper function of the nervous system, as is evident from human diseases caused by disruption of neuropeptide signaling. For example, Neuropeptide Y is involved in a variety of disorders such as hypertension, epilepsy, and obesity (Reichmann 2016; Yi 2018). Across diverse species, many neurons express multiple neuropeptides, raising the question whether these neurons simply co-release cocktails of neuropeptides, or whether some of them may release them separately and specifically. Examples for co-transmission are found in many neuron types, for example mammalian midbrain dopamine neurons, which also release glutamate from separate vesicles. We found that the AVK interneurons of C. elegans may specifically (co-)transmit at least two different neuropeptides, FLP-1 and NLP-49, both expressed at high levels in this neuron. However it remains largely unclear whether, in what context and by what mechanisms their release is differentially regulated. FLP-1 and NLP-49 are predominantly expressed in AVK and differentially affect behaviors such as locomotion and egg-laying (Oranth 2018; Chew 2018). We thus examined whether these peptides are differentially regulated. We found from locomotion analyses using the multi worm tracker (Swierczek 2013) that these peptides derived from AVK differentially affect the animal's response to sensory stimuli in a modality-specific manner. We also observed peptide precursors fused with fluorescent proteins and obtained evidence indicative of differential trafficking and regulation of those two neuropeptides in AVK. Reichmann F. and Holzer P. (2016) Neuropeptides Yi M. et al. (2018) Cell Physio. Biochem Chew, Y. L. et al. (2018) Philos Trans R Soc Lond B Biol Sci 373(1758). Oranth, A. et al. (2018) Neuron 100(6): 1414-1428 e1410. Swierczek, N. A. et al. (2011) Nat Methods 8(7): 592-598.

Partridge, Frederick A. et al. (2009) International Worm Meeting "Investigation of the mechanism of infection induced tail swelling."

Partridge, Frederick A., Hodgkin, Jonathan

[International Worm Meeting, 2009]

When C. elegans and certain other rhabditid nematodes are infected by various bacterial pathogens, notably the coryneform Microbacterium nematophilum, they respond by altering the development of the hindgut, producing a swollen tail. This is a protective innate immune response [1]. Our lab has previously shown that tail swelling involves activation of the ERK MAPK cascade in the rectal epithelial cells. The mechanism of tail swelling has been hard to address in genetic screens as mutants that abolish infectability are much more common than mutants affecting the response to infection. To avoid this problem we have stably expressed activated MAP kinase pathway genes in the hindgut, which phenocopies tail swelling in the absence of infection. This has allowed us to screen for modifiers of tail swelling without the confounding effects of susceptibility to infection. Results of RNAi and forward genetic screens will be presented. [1] Nicholas and Hodgkin (2004) Curr Biol 14 1256-61.

Ardiel, Evan L. et al. (2013) International Worm Meeting "Response to repeated activation of ASH requires glutamate, dopamine, and neuropeptide signaling."

Ardiel, Evan L., Lindsay, Theodore, Giles, Andrew C., Lockery, Shawn, Rabinowitch, Ithai, Schafer, William, Rankin, Catharine H.

[International Worm Meeting, 2013]

The objective of this research was a comparative analysis of habituation in two neural circuits that synapse onto the same interneurons. Previous work in the lab has focused on habituation to a non-localized mechanical stimulus - a plate tap. Here we studied the response to repeated activation of the ASH sensory neurons, which detect a variety of aversive stimuli. A strain expressing ChR2 exclusively in ASH (Ezcurra et al., 2011) was used for consistent and discrete delivery of simulated aversive stimuli to worm populations being tracked by real-time computer vision software (Swierczek et al., 2011). In addition to increasing throughput, this optogenetic approach allowed us to prevent sensory adaptation and specifically activate ASH. Whole-plate blue light illumination elicited backward crawling (reversal) in the majority of animals. As with the tap-withdrawal response, the magnitude of ASH-mediated reversals decremented with repeated stimulation in a manner dependent on the stimulus intensity and frequency and recovered to baseline in minutes. Electrophysiological recordings of repeatedly activated photocurrents in ASH demonstrated that the decrement was not caused by ChR2 desensitization. Furthermore, a tap could dishabituate decremented responding. Cross-modal habituation was evident in only one-direction, ie the tap-withdrawal response was decremented by repeated activation of ASH, thereby localizing the site of plasticity to the shared circuitry. Although their magnitude decremented over the trial, ASH reversals persisted far longer than tap-induced reversals. This response maintenance was dependent on glutamate and dopamine signaling. Repeated ASH activation also led to increased speed of forward locomotion and a greater suppression of spontaneous reversals in the periods between stimuli, as compared to tap. This change was dependent on neuropeptide signaling. These distinct behavioural strategies are hypothesized to facilitate escape from a hazardous area.

Sugi, Takuma (2013) International Worm Meeting "High-throughput behavioral imaging reveals the neurons responsible for mechanosensory memory in C. elegans."

Sugi, Takuma

[International Worm Meeting, 2013]

Behavioral genetic studies in C. elegans have provide a new paradigm by unveiling a role of genes and neurons in a neural circuit. The greatest advantage in C. elegans behavioral studies is that a variety of transgenic experiments, such as rescue experiments and RNAi-mediated gene disruption, can be done in a cell-specific manner. The throughput of these transgenic studies has been increased by developments of image processing software (Swierczek et al., Nat. Mathods., 2011), which accelerate analysis of acquired data. However, the throughput remains to be limited by behavioral assay itself, because discriminations of transgenic worms from nontransgenic worms are required for each strain before or during data acquisition, due to semi-stable inheritance of extrachromosomal transgenes. Here, I developed a multiplexed behavioral imaging system, which simultaneously quantifies behaviors of various different transgenic strains. In order to illuminate transgenic strains labeled by a fluorescent injection marker on the four assay plates placed side by side, I constructed the optical system by spatially multiplexing the beam from a high-power excitation laser and navigating them to all assay plates. Through the USB-based CCD cameras with the emission filters on each assay plate, I succeeded to record movements of fluorescence as behavior of transgenic strains in a wide-field of view (>30 worms), discriminating them from nontransgenic worms. I thus demonstrated high-throughput behavioral imaging by combining this system with the tapping system that delivers mechanical stimuli simultaneously to all assay plates in a temporally controlled manner and with machine-vision software. I tried to identify a site of action of CRH-1/CREB responsible for memory formation, on mechanosensory behavior. I created a variety of transgenic strains that each express the dominant negative form of CRH-1 under the various cell-specific promoters, and quantified their mechanosensory responses. The behavioral imaging indicated that CRH-1 acts in AVA and AVD neurons, suggesting that these neurons are responsible for mechanosensory memory. These results demonstrated that my system can be applied to high-throughput behavioral genetic studies.