Demonstrating Connections Between Neuron Signaling and Behavior using C. elegans Learning Assays and Optogenetics in a Laboratory Class

Abstract

Due to its well-described neural circuitry and identified connectome, the Caenorhabditis elegans model is well-suited for demonstrating connections between neuron signaling and behavioral outcome. In the 2017 FUN workshop at Dominican University, three behavior-based techniques were introduced for their ease of introduction to students, the flexible data collection options they offer and the inexpensive cost to implement in an education setting. These behavioral assays were adapted to address some of the challenges of performing C. elegans behavior experiments in lab classes and included: an associative chemosensory avoidance task to examine behavior of groups of worms, a mechanosensory task to observe individual worm behavior and an optogenetics assay to directly manipulate neuron signaling and simultaneously observe resultant behavior. Methods for these assays as well as example data collected by undergraduate students in a lab class are provided. FUN Workshop feedback and assessment indicate these assays were well-received and overall seen as valuable for introducing neuroscience and behavior to undergraduates in a lab class.

Keywords: associative chemotaxis; biogenic amines; channelrhodopsin (ChR2); gustatory signaling; habituation; head touch; learning; mechanosensory.

Figure 1

C. elegans viewed under EZ4 Leica dissecting microscope captured at ~100× magnification (total). The head of the worm appears lighter in color than the narrower tail. Scale bar = 0.5 mm.

Figure 2

Identified circuits of sensory, interneuron and motor neurons involved in behavioral responses to specific types of stimuli (from Giles et al., 2006). Expression locations of genes mec-4 and unc-47 included for reference.

Figure 3

Associative Chemotaxis Protocol. Worm colonies washed from cultivation plates to ~1-hour incubation conditions, are then washed and collected to be placed in the test plate center origin where a 25 μL dry drop of 20 mM NaCl is located.

Figure 4

Sample data from an undergraduate lab class using the adapted chemoavoidance assay. Attraction index was calculated as: # of viable worms inside the Drop Zone/total # of worms for n = 8 test plates /condition. Data show a small decline in NaCl attraction for WT worms following NaCl incubation without food. Despite an overall greater NaCl attraction index, a similar decline in NaCl attraction is seen for cam-1 knockdown worms. * = p < 0.05 determined by planned comparisons.

Figure 5

Blue Flashlight setup. Sufficient light intensity to activate ChR2 (~2mW/mm2) was measured from these devices at ~21 cm from the plate surface, a distance that covered the plate area.

Figure 6

Sample data from an undergraduate lab class. Behavioral effects of repeated ChR2 activation of GABAergic motor neurons (_punc-47::ChR2). Behavior was measured as approximate speed of reversal responses resulting from head touch (n = 8/condition). Worms that underwent cam-1 RNAi knockdown show increased responsiveness to head touch after repeated _punc-47::ChR2 activation (5 s blue light exposure every 60 s for 5 min).

Figure 7

Histogram data of faculty assessment of two-hour FUN workshop. One assessment was omitted as individual rated ‘strongly disagree’ on all six items along with the following comment: Awesome! Can’t wait to work with worms. Total number of participants across four workshop presentations = 29.