Constructing a rapid solution exchange system

Abstract

The detailed study of ligand-gated channels often requires the reproducible, rapid, and temporally precise application of defined concentrations of ligands. There are numerous different systems for ligand application offering trade-offs between cost, ease of use, volume of compound needed, etc. When studying the extremely rapid kinetics of activation and desensitization in ionotropic ligand-gated receptors (iGluR), a prime consideration is how fast solutions can be exchanged. Of the available systems, rapid piezo-driven translation of parallel solutions streams over an outside-out patch offers the fastest and most versatile option, with open tip current rise-times in the 100-300 μs range, and occasionally as fast 20 μs (Bowie and Lange, J Neurosci 22:3392-3403, 2002; Carbone and Plested, Neuron 74:845-857, 2012; Maclean and Bowie, J Physiol 589:5383-5390, 2011; Robert and Howe, J Neurosci 23:847-858, 2003), and pulse durations ≤1 ms (Bowie and Lange, J Neurosci 22:3392-3403, 2002; Carbone and Plested, Neuron 74:845-857, 2012; Robert and Howe, J Neurosci 23:847-858, 2003). Indeed, under ideal conditions agonist applications as short as 100 μs may be possible (Auzmendi et al., PLoS One 7:e42275, 2012). However, from the experimenter's perspective a "fast" system may also mean a system which rapidly changes test solutions, i.e., how fast can one switch between the various agonists or concentrations being tested. And while the former sense of "fast" is certainly more important in that it is necessary for certain experiments, time spent optimizing the latter yields substantial rewards. With fast switching amongst test solutions, one can complete experiments quicker, easier, and with fewer stressful moments hoping your best patch of the week hangs on for that last data point. This protocol outlines the construction of a double or multi-barrel solution exchange system which can rapidly jump between solutions (10-90 % rise-time 50-300 μs) but can also quickly switch amongst several test solutions (<0.5 s). This protocol can also be modified for constructing application pipettes for whole-cell recording.