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New lab announcement: Kevin Collins

Kevin Collins
1Department of Biology University of Miami, Coral Gables FL
Correspondence to: Kevin Collins ([email protected])

I started my independent research group from August 2014 in the Department of Biology at the University of Miami in Coral Gables, Florida. Our fundamental goal is to understand how neurons communicate in circuits to establish an appropriate level of activity that produces a robust, stable behavior. Our approach is to analyze in detail a model neural circuit that controls egg-laying behavior in C. elegans. We are taking advantage of the optical clarity and powerful genetics in this experimental system to literally watch the activity of every cell in the circuit in behaving animals using fluorescent Ca2+ reporters, and also to manipulate their activity using optogenetic tools. Using mutations and transgenes to discover and alter molecular signaling events between cells, we are determining how the complex pattern of activity in a circuit creates a coherent, regulated behavior. We expect these studies will reveal general principles of neurotransmitter signaling and neural circuit function with applications to understanding the human nervous system and its dysfunction in disease.

Website:  http://www.as.miami.edu/biology/people/faculty/kevin-collins/

Ph.D. student and Postdoc positions are currently available.  To apply, please send a CV and description of research interests to [email protected]

A modified leaving assay to study pathogen avoidance

Alexandra Anderson1 and Rachel McMullan1
1Department of Life Science, Imperial College London, London, UK

Correspondence to: Rachel McMullan ([email protected])

C. elegans constantly encounter pathogenic threats and the ability to recognize and avoid these threats is crucial to survival. C. elegans have evolved a variety of immediate and learned behavioral responses to minimize pathogen exposure (Yook and Hodgkin, 2007; McMullan et al., 2012; and others). Behavioral responses to pathogens are commonly investigated using a food choice assay where animals are given a choice between their standard laboratory food, OP50, and the pathogen of interest (Yook and Hodgkin, 2007; McMullan et al. 2012). The results of this type of assay integrate both the known attractive effect of OP50 and the attractive/repellent effect of the pathogen under investigation. In order to examine only potential repellent cues, we have adapted the male leaving assay (Lipton et al., 2004) to analyze the response of naïve animals upon exposure to Microbacterium nematophilum, a gram positive bacteria which binds to and colonizes the rectal opening of C. elegans.

Method: 90 mm NGM assay plates were prepared using 13 ml NGM in order to make animals tracks easier to observe. Bacterial cultures were grown to OD600 = 1.0 before plating 20 µl in the center of the assay plate.

Single synchronized young adult hermaphrodites were transferred onto individual plates. 10 plates were prepared for each bacterial lawn to be tested.

Plates were kept at 20 oC and examined at 1 hr intervals to determine if the animal was a leaver, meaning that the animal or its tracks can been seen 1 cm or less from the edge of the plate. Animals determined to have left were not scored again at later time points

Results: Animals placed on plates seeded with OP50 show little leaving behaviour over 24 hrs. An avirulent M. nematophilum strain (Akimkina et al., 2006) induces a limited leaving response, whilst virulent M. nematophilum induces a rapid response over 24 hrs (Figure 1).

Using this assay we tested the role of the cyclic nucleotide gated ion channel TAX-2/TAX-4 in this behaviour. This pathway has been shown to be required for wild type responses to M. nematophilum using a food choice assay (Yook and Hodgkin 2007). Avoidance of M. nematophilum depends on TAX-2/TAX-4, since mutations in either TAX-2 or TAX-4 abrogate the leaving response (Figure 2). In wild-type animals TAX-2/TAX-4 is expressed in AWC, AFD, ASE, ASG, ASJ, AQR, BAG, ASK, ASI, AWB, and PQR (Coburn and Bargmann, 1996). The allele tax-2(p694) has a deletion in the promoter region and first exon of tax-2 abolishing its expression in ASE, AQR, AFD, and BAG (Coburn and Bargmann, 1996). In contrast to the response to M. nematophilum in a food choice assay (Yook and Hodgkin, 2007), tax-2(p694) animals show a defective leaving response particularly at longer time points (Figure 2), suggesting these four cells are important for the normal leaving response to this pathogen.

This simple behavioural assay could be adapted to investigate the response to numerous pathogens and removes the influence of the attractive cue from OP50 in food choice assays.

Acknowledgements: We thank Arantza Barrios (University College London) and Richard Poole (University College London) for suggestions and assistance in setting up this assay. We thank Jonathan Hodgkin for M. nematophilum strains.

Figures
Figure 1: Leaving assay response to OP50, avirulent M. nematophilum and virulent M. nematophilum over 24 hrs. Results are average of the mean of three independent experiments, n=30.

Leaving assay Figure 2
Figure 2: Leaving assay response of tax-2/tax-4 mutants to virulent M. nematophilum over 24 hrs. Results are average of the mean of five independent experiments, n=50

References

Akimkina T, Yook K, Curnock S, and Hodgkin J. (2006). Genome characterization, analysis of virulence and transformation of Microbacterium nematophilum, a coryneform pathogen of the nematode Caenorhabditis elegans. FEMS Microbiol. Lett. 264, 145–151. PubMed

Coburn CM, and Bargmann CI. (1996). A putative cyclic nucleotide-gated channel is required for sensory development and function in C. elegans. Neuron 17, 695–706. PubMed

Lipton J, Kleemann G, Ghosh R, Lints R, and Emmons SW. (2004). Mate searching in Caenorhabditis elegans: a genetic model for sex drive in a simple invertebrate. J. Neurosci. 24, 7427-7434. PubMed

McMullan R, Anderson A, and Nurrish S. (2012). Behavioral and immune responses to infection require Gαq-RhoA signaling in C. elegans. PLoS Pathog 8, e1002530. PubMed

Yook, K. and Hodgkin J. (2007). Mos1 mutagenesis reveals a diversity of mechanisms affecting response of Caenorhabditis elegans to the bacterial pathogen Microbacterium nematophilum. Genetics 175, 681-697. PubMed

Freed from the image plane: A low-cost optical technique for studying nematode behavior

Jenny Magnes1, Alicia Jago1, Tewa Kpulun1 and Kathleen Raley-Susman2
1Department of Physics, Vassar College, Poughkeepsie NY, 2Department of Biology, Vassar College, Poughkeepsie NY

Correspondence to: Kathleen Raley-Susman ([email protected])

The soil environment of Caenorhabditis elegans in its native habitat is much more complex than either an agar or a liquid medium. Nematodes move within the three-dimensional soil space, responding not only to changes in medium (soil particles, water, colloidal mixtures), but also likely altering locomotory patterns in response to gravity, temperature, humidity, and other environmental cues. Notably, C. elegans accelerate away from blue and even shorter wavelengths of light, which are the wavelengths most likely to penetrate into soil (Edwards et al., 2008). The neural circuitry of the photophobic response is only just beginning to be studied. How other coincident information, like gravitational cues, might modulate locomotion is not known.

Most behavioral studies with C. elegans are performed using microscope-based imaging analysis, which are typically limited to a narrow vertical plane of focus. When observing worms in small droplets of liquid or in specially constructed channeled chambers, more locomotory behaviors arise than seen on flat surfaces (Lockery et al., 2008; Pierce-Shimomura et al., 2008). The worms navigate this more realistic environment with novel locomotory patterns, so-called “enhanced swimming” that relies on mechanosensory neuron activity (Park et al., 2008). However, the contribution or influence of the gravitational field and the movement of the worm within three-dimensional space cannot be studied easily with these microscope-based approaches (Kim et al., 2007).

How do worms behave when in a larger column of liquid, considerably larger than the dimensions of the worm? Do nematodes detect and respond to gravity? To study these kinds of questions, we have developed an inexpensive non-microscope-based imaging system that requires a laser, a cuvette, a lens and some mirrors, as well as a digital camera to record real-time images focused on a screen (Fig. 1). The full measurement technique is described in our two recent publications (Magnes et al., 2012b; Magnes et al., 2012a).

The diffraction-based measurements recorded the shape of the worms in space and time. We used shadow imaging, a modification of the diffraction-based method, to study the velocity of nematodes in a gravitational field. A small number of nematodes were placed in a quartz cuvette positioned in front of a laser of coherent light. The nematodes slowly fall down the column of water because of gravity, passing through the expanded laser beam. A digital camera records the moving patterns, which result from the locomotion of the worm. The velocities (speed and direction) are analyzed using Newtonian mechanics.

We assessed vertical velocity in wildtype worms using four different wavelengths of light and compared to velocities of worms killed with chloroform to control for passive effects of gravity on similar mass. We found that living nematodes locomote at twice the rate of passive fall in the column due to gravity alone. We also noted that velocities were greater when shorter wavelength lasers were used (Table 1).

Our preliminary findings indicate that worms are oriented “head down” towards the direction of the gravitational field. In contrast, dead worms, passively falling, fall in random orientations. These results suggest that nematodes actively move in the direction of gravity.

Figures
Figure 1: Figure 1: Diagram of light diffraction/scattering experimental set up
A) Equipment and position of cuvette with nematodes B) Representative diffraction pattern of a single nematode
Figure 2: Table 1

References

Edwards SL, Charlie NK, Milfort SC, Brown BS, Gravlin CN, Knecht, JE, Miller KG. (2008) A novel molecular solution for ultraviolet light detection in Caenorhabditis elegans. PLOS Biol 6, 1715-1729. PubMed

Kim N, Dempsey DM, Kuan CJ, Zoval JV, O’Rourke E, Ruvkin G, Madou MJ, Sze JY. (2007) Gravity force transduced by the MEC-4/MEC-10 DEG/ENaC channel modulates DAF-16/FoxO activity in Caenorhabditis elegans. Genetics 177, 835-845. PubMed

Lockery SR, Lawton KJ, Doll JC, Faumont S, Coulthard SM, Thiele TR, Chronis N, McCormick KE, Goodman MB, Pruitt BL. (2008) Artificial dirt: microfluidic substrates for nematode neurobiology and behavior. J Neurophysiol 99, 3136-3143. PubMed

Magnes J, Susman K, Eells R. (2012) Quantitative locomotion study of freely swimming micro-organisms using laser diffraction. J Vis Exp 68, e4412. PubMed

Park S, Hwang H, Nam SW, Martinez F, Austin RH, Ryu WS. (2008) Enhanced Caenorhabditis elegans locomotion in a structured microfluidic environment. PLOS One 3, e2550. PubMed

Pierce-Shimomura JT, Chen BL, Mun JJ, Ho R, Sarkis R, McIntire SL. (2008) Genetic analysis of crawling and swimming locomotory patterns in C. elegans. Proc Natl Acad Sci U S A 105, 20982-20987. PubMed

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