title: Caenorhabditis-in-drop array for monitoring C. elegans quiescent behavior.
abstract: OBJECTIVE
  To develop a method, called Caenorhabditis-in-Drop (CiD), encapsulating single worms in aqueous drops, for parallel analysis of behavioral quiescence in C. elegans nematodes.
  
  
  METHODS
  We designed, constructed, and tested a device that houses an array of aqueous droplets laden with individual worms. The droplets are separated and covered by immiscible, biocompatible oil. We modeled gas exchange across the aqueous/oil interface and tested the viability of the encapsulated animals. We studied the behavior of wild-type animals; of animals with a loss of function mutation in the cGMP-dependent protein kinase gene egl-4; of animals with a loss of function mutation in the gene kin-2, which encodes a cAMP-dependent protein kinase A regulatory subunit; of animals with a gain-of-function mutation in the gene acy-1, which encodes an adenylate cyclase; and of animals that express high levels of the EGF protein encoded by lin-3.
  
  
  RESULTS
  We used CiD to simultaneously monitor the behavior of 24 worms, a nearly 5-fold improvement over the prior best methodology. In support of our gas exchange models, we found that worms remain viable on the chip for 4 days, past the 12-h period needed for observation, but show reduced longevity to that measured on an agar surface. Measurements of duration of lethargus quiescence and total leth-argus quiescence showed reduced amounts as well as reduced variability relative to prior methods. There was reduced lethargus quiescence in animals that were mutant for kin-2 and for acy-1, supporting a wake-promoting effect of PKA in C. elegans, but no change in lethargus quiescence in egl-4 mutants. There was increased quiescence in animals that expressed kin-2 in the nervous system or over-expressed EGF.
  
  
  CONCLUSIONS
  CiD is useful for the analysis of behavioral quiescence during lethargus as well as during the adult stage C. elegans. The method is expandable to parallel simultaneous monitoring of hundreds of animals and for other studies of long-term behavior. Using this method, we were successful in measuring, for the first time, quiescence in kin-2(ce179) and in acy-2(ce2) mutants, which are hyperactive. Our observations also highlight the impact of environmental conditions on quiescent behavior and show that longevity is reduced in CiD in comparison to agar surfaces.
authors: Department of Neurology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.
authors: Samuel J
authors: SJ
authors: Belfer
authors: Han-Sheng
authors: HS
authors: Chuang
authors: Benjamin L
authors: BL
authors: Freedman
authors: Jinzhou
authors: J
authors: Yuan
authors: Michael
authors: M
authors: Norton
authors: Haim H
authors: HH
authors: Bau
authors: David M
authors: DM
authors: Raizen
mesh_terms: D000818:Animals; D001522:Behavior, Animal / Q000502:physiology*; D017173:Caenorhabditis elegans / Q000502:physiology*; D046508:Culture Techniques / Q000295:instrumentation; D004129:Dimethylpolysiloxanes; D004867:Equipment Design; D053609:Lethargy / Q000503:physiopathology*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008899:Mineral Oil; D015203:Reproducibility of Results; D012890:Sleep / Q000502:physiology*
chemical_list: D004129:Dimethylpolysiloxanes; C013830:baysilon; D008899:Mineral Oil
keywords: General; drop; elegans; lethargus; microfluidics; quiescence
references: Genetics. 2005 Feb;169(2):631-49
references: 15489510
references: Chem Biol. 2008 May;15(5):427-37
references: 18482695
references: Nature. 2006 Jun 8;441(7094):757-60
references: 16760980
references: Lab Chip. 2004 Aug;4(4):310-5
references: 15269796
references: Angew Chem Int Ed Engl. 2008;47(21):3900-4
references: 18412211
references: J Neurosci. 2010 Jun 9;30(23):7889-99
references: 20534837
references: Genetics. 1974 May;77(1):71-94
references: 4366476
references: Proc Natl Acad Sci U S A. 2009 Aug 25;106(34):14195-200
references: 19617544
references: PLoS Biol. 2012;10(4):e1001307
references: 22505849
references: Nature. 2008 Jan 31;451(7178):569-72
references: 18185515
references: J Neurosci. 2005 Aug 3;25(31):7159-68
references: 16079398
references: J Neurophysiol. 2003 Aug;90(2):1152-9
references: 12711709
references: Prog Neurobiol. 2000 Jul;61(4):339-51
references: 10727779
references: Lab Chip. 2010 Mar 7;10(5):589-97
references: 20162234
references: Curr Biol. 2011 Dec 20;21(24):2033-45
references: 22137474
references: Exp Gerontol. 1978;13(5):369-74
references: 153845
references: J Neurosci Methods. 2011 Sep 30;201(1):78-88
references: 21801751
references: Mol Reprod Dev. 2009 Dec;76(12):1178-87
references: 19672876
references: J Mol Biol. 1969 May 14;41(3):459-72
references: 4896022
references: Curr Biol. 2011 May 24;21(10):825-34
references: 21549604
references: Arch Environ Contam Toxicol. 1997 Jan;32(1):110-4
references: 9002442
references: J Exp Biol. 2007 Jul;210(Pt 13):2383-9
references: 17575043
references: J Neurophysiol. 2008 Jun;99(6):3136-43
references: 18337372
references: Nat Neurosci. 2001 Nov;4(11):1108-15
references: 11687816
references: Neuron. 2000 Jan;25(1):129-38
references: 10707978
references: Proc Natl Acad Sci U S A. 2008 Dec 30;105(52):20982-7
references: 19074276
references: Neuron. 2002 Dec 19;36(6):1079-89
references: 12495623
references: Sleep. 2008 Nov;31(11):1587-98
references: 19014079
references: J Biol Chem. 1990 Feb 25;265(6):3293-303
references: 2303451
references: Appl Microbiol. 1969 Feb;17(2):219-21
references: 5775905
references: Proc Natl Acad Sci U S A. 2009 Mar 10;106(10):4024-9
references: 19237558
references: EMBO J. 2002 Mar 1;21(5):986-94
references: 11867526
references: Nature. 2009 Oct 22;461(7267):1122-5
references: 19847264
references: Cell Metab. 2008 Mar;7(3):249-57
references: 18316030
references: Biochem Biophys Res Commun. 2005 May 13;330(3):839-43
references: 15809072
references: J Exp Biol. 1964 Mar;41:135-54
references: 14161604
references: Lab Chip. 2010 Nov 7;10(21):2855-63
references: 20882233
references: PLoS Biol. 2008 Aug 5;6(8):e198
references: 18687026
references: Nat Neurosci. 2007 Oct;10(10):1300-7
references: 17891142
references: J Exp Biol. 2008 Dec;211(Pt 23):3703-11
references: 19011210
references: J Exp Biol. 2006 Jan;209(Pt 1):89-102
references: 16354781
references: Genetics. 1983 Aug;104(4):619-47
references: 11813735
references: Development. 2003 Mar;130(6):1089-99
references: 12571101
references: Mol Reprod Dev. 1991 Apr;28(4):351-5
references: 2064778