daf-7 expression in ASI neurons is increased when nematodes are grown in the presence of endocannabinoid 2-arachidonoyl glycerol (2-AG). Left upper panel: representative image of reporter strain FK181 ksIs2 [Pdaf-7::GFP+rol-6(su1006)] captured under GFP epifluorescence microscopy. Right upper panel: same individual showing bright field microscopy together with GFP signal. Gray/black triangles mark ASI neurons expressing daf-7. Scale bars correspond to 0.03 mm. Lower panel: point plot of the adjusted GFP intensity/ASI neuron values gathered from worms treated either with the carrier solvent or with 2-AG. 2-AG treatment significantly increases daf-7 expression in ASI neurons. (*) indicates statistically significant difference with solvent control condition; the difference in the median values of the relative adjusted GFP fluorescence/ASI neuron between the two groups is greater than would be expected by chance; there is a statistically significant difference between solvent and 2-AG (Mann-Whitney Rank Sum Test, p=<0.001). The number of independent experiments carried out was three. The total number of ASI neurons analyzed was 70 for each condition.
Worms were raised on solid NGM plates supplemented with bacteria and the corresponding supplement for each condition tested. daf-7 expression was monitored following the protocol described by (Myers, 2012). Briefly, synchronized L1 larvae were fed with Escherichia coli HT115(DE3) supplemented with either 2-arachidonoyl glycerol (2-AG) 100 µM or with an equal volume of carrier solvent (acetonitrile) for 1,5 h at 20 ºC. GFP fluorescence viewed with confocal microscopy in ASI neurons of L1 FK181 animals. L1 images were captured with Zeiss LSM880 scan head on an axio observer Z1 inverted microscope with a 60x 1.4 AN oil immersion objective. A laser line 488 nm of an argon ion laser was used for the excitation, the detection was done in a GaAsP spectral detector with a
bandwidth between 508 and 588 nm. GFP intensity in ASI neurons was quantified using NIH Image J software. GFP intensity in each ASI cell body was subtracted from the intensity of a similarly sized background selection to get the adjusted GFP intensity /ASI neuron value.
Strain FK181 ksIs2 [Pdaf-7::GFP+rol-6(su1006)]
2-arachidonoyl glycerol (Cayman Chemical)
This work was supported by grants (PICTs 2155 and 3693) from the Agencia Nacional de Promoción Científica y Técnica (ANPCYT), C.G. and G.M.P., are fellows from Fundación Bunge y Born and CONICET respectively.
Reviewed ByEdith Myers
HistoryReceived: June 13, 2018
Accepted: November 19, 2018
Published: November 19, 2018