Methods

Animals collected from the same plate were divided into experimental or control conditions based on expression or lack thereof of RFP. Animals were then paralyzed with 10mM levamisole for 10 minutes, corralled together into rows, and then imaged at 50x under bright field and to measure GFP fluorescence. Images contained ~5 animals per image. Light level and exposure time (108ms) were selected so as to provide the brightest pictures without any saturation and were kept constant both within and across experiments. To analyze the images in as unbiased a manner as possible, the entire body of each animal was outlined under a brightfield image in imageJ and then the average intensity of that outline was measured in the corresponding fluorescent image. A large section of the background fluorescence was measured and this number was subtracted from all measurements. Significance was measured by comparing the control and experimental group utilizing a two-tailed t-test. While the tetanus toxin construct itself was bound to GFP, the intensity of that construct was exceedingly dim and we required the use of an RFP co-injection marker to separate Tetx-expressing animals from controls at magnifications of 50x and lower. In addition, the increase in expression was not restricted to the ASI neuron pair alone but also appeared throughout the head as well as the nerve cord, parts of the vulva, and the tail, all locations the psod-3::GFP construct is expressed under control conditions (Figure Panel A; Libina et al. 2003). Due to both these pieces of evidence, we conclude that the increased GFP measured is due to the effects of tetanus toxin in ASI.