. We established a wholecell patch clamp preparation (25, 26) for the CEMs and
. We established a wholecell patch clamp preparation (25, 26) for the CEMs and performed electrophysiological recordings. We confirmed that the CEMs responded to each ascr3 and ascr8 but to not water (Fig. H).CEM Neurons Show Three Modes of Responses to Ascarosides. To measure the evoked electrical currents in CEMs in response to distinct concentrations of ascr8, we performed Valine angiotensin II voltage clamp recordings. CEM responses fell on a continuum that crosses zero: even though individually recorded neurons had stereotyped responses, the responses across the population varied in magnitude and sign (Fig. 2A and SI Appendix, Fig. S A and B). We classified the responses as depolarizing, hyperpolarizing, or no response (population averaged trials shown in Fig. 2C; instance traces in Fig. 2B and SI Appendix, Fig. S2). The depolarizing and hyperpolarizing responses don’t covary across concentration: The depolarizing current peaks at intermediate concentration of ascr8, that is the behaviorally most attractive, whereas the hyperpolarizing current is strongest in the highest tested concentration, which can be behaviorally much less attractive (Figs. D and 2D). The mode of response was depolarizing for approximately half the cells, regardless of the neuron’s anatomical identity (Fig. 2E; see also SI Appendix, Fig. S3). Similarly, CEM responses to ascr3 fall on a continuum crossing zero, and also can be classified into 3 modes (Fig. three A and C and SI Appendix, Fig. S C and D; instance traces in Fig. 3B and SI Appendix, Fig. S4) uncorrelated using the anatomical identity of your recorded CEM (Fig. 3D and SI Appendix, Fig. S5). The depolarizing existing also peaks at intermediate concentrations corresponding for the behavioral tuning curve (Figs. E and 3D). A few neurons had complex responses with each depolarizing and hyperpolarizing responses, at times inside the identical trial and occasionally on successive trials (ascr8, 44 neurons, 3.five of dataset; ascr3, 90 neurons, 2 of dataset, example neurons PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25819444 SI Appendix, Figs. S6 and S7). To observe membrane voltage fluctuations evoked by ascaroside application, we performed existing clamp recordings of CEMs. We observed big depolarizations and hyperpolarizations (200 mV modifications) as well as fast transient events (Fig. and SI Appendix, Fig. S8). Intact Worms Have Access to Each Depolarizing and Hyperpolarizing CEM Signals. To test no matter whether a given worm could potentially haveneous CEM responses, we recorded CEM responses towards the higher concentrations of ascarosides in worms deficient in UNC3, a syntaxinbinding protein that is important for fast synaptic transmission. We applied the unc3(s69) mutant that lacks each isoforms of UNC3 and has virtually no fast synaptic transmission (27). We found that the depolarizing responses to ascr8 have been enhanced in the absence of quick synaptic transmission, confirming our hypothesis that synaptic feedback plays a part in ascaroside representation (Fig. 5A). Further, we note that the depolarizing unc3 responses to ascr8 had been orders of magnitude bigger than wildtype ascr8 responses, responses to ascr3, and nondepolarizing unc3 responses (Fig. 5A and SI Appendix, Figs. S2, S4, and S5). This variety suggests that there may very well be largescale synaptic feedback inside the processing of ascr8 responses. The hyperpolarizing responses to ascr8 were also enhanced by the removal of synaptic transmission, despite the fact that to not the exact same extent as the depolarizing responses (Fig. 5A and SI Appendix, Figs. S2 and S5A). This enhancement sug.
