Callahorro, Fernando, Hu, Chunxiao, Dillon, James, O'Connor, Vincent, Holden-Dye, Lindy, Ferreiro, Teressa, Morgan, Hywel
[
International Worm Meeting,
2015]
The pharyngeal system of C.elegans provides a powerful in vivo route to investigate the biological function of a tractable neuroactive circuit. It consists of a muscular structure made up of the corpus isthmus and terminal bulb. This produces a rhythmic cycle of contraction and relaxation modulated by overriding neural activity. The pharynx expresses the major transmitter pathways and/or their cognate receptors that operate across the phyla. This makes its function sensitive to pharmacological regulation by many classes of neuroactive drugs. This includes those of general bio-medical and agrochemicals relevance and a more selective nematicidal potency. Thus, the pharynx lends it-self to use as a model for drug discovery, better mapping of mode of action and comparative approaches for selective and ecological toxicity. Measurement of pharyngeal function using extracellular measurement provides a powerful tool for the above approaches and supplements visual measurement of pumping. Microelectrode recording from intact C.elegans is a tractable route as it offers the potential to investigate chemically induced changes in the context of whole organism, to scope pharmacokinetics and comparative toxicity. The electropharyngeogram (EPG) is recorded by immobilizing the worm in glass electrode fabricated to make a tight seal around the nose of the worm. By analogy with a grease gap this allows recording of the potential difference between the two sides of the tight seal. We have adapted this principle and embedded it into microfluidic chip we have called neurochip. The central aspect of this device is the delivery of the worm into a PDMS trapping channels in which a double layer design allows the production of a compliant circular aperture that restrains the worm with a tight seal. The device has intrinsic platinum electrodes either side of the seal and when the pharynx contract a waveform with all the characteristic features of the EPG is recorded. We can record EPG in loading buffer or prime them by loading in 5-HT a potent stimulator of pumping. We have optimized a medium through put plate assays where worms are cultivated in drug overnight prior to investigation in the neurochip. This approach is particularly useful in instances where access across the cuticle is slow or mode of action of implies an important role of chronic exposure. Pre-exposure of the inhibitor with known and unknown candidates reveals a dose dependent inhibition of the EPG in which the frequency and pattern of activity are modified. A further iteration of the neurochip design incorporates supporting flow channels designed to direct efficient drug perfusion onto the pre immobilized worms. Restrictions to efficient flow in the immobilizing and recording micro channels is circumvented by incorporating perforated exit ports along the length of the trapped worm. Thus, the device and accompanied optimization of dosing regimes opens up a wide user base for previously technically challenging measurement of EPG. The format and ergonomics of loading are suited to investigation of both the chronic or acute effects of important classes of chemicals on wild type, mutant or genetically modified C.elegans sculpted to express targets of experimentally intractable nematode species. .