Supplementary Figure 4: Pharmacology of acetylcholine-evoked currents.
From: Synaptic and circuit mechanisms promoting broadband transmission of olfactory stimulus dynamics
a) Mean acetylcholine-evoked currents recorded in voltage-clamp recordings from PNs (glomerulus VM2, DM6, or VM7, n=5 PNs, average of 5-6 trials per PN). Acetylcholine was iontophoresed into the neuropil for 5 ms at the time indicated (arrows). Bath application of curare (10 μM) slows the acetylcholine-evoked current. Adding IMI (100 nM) together with curare blocks a substantial fraction of the evoked current. Note that IMI (100 nM) increases the basal holding current, consistent with reports that it can act as a partial agonist (ref. 30). b) Same as panel a, but with baselines aligned to better illustrate changes in the timecourse of the evoked current.
We note that the two drugs did not completely block the response to iontophoresis of acetylcholine, likely because some of the current evoked by iontophoresis is due to metabotropic receptors. The two drugs together blocked 94.1+/-2.7% of the response to iontophoresis of nicotine (10 ms stimulus duration, n=4). In the presence of atropine (10 μM), the two drugs blocked 81% of the response to iontophoresis of acetylcholine (n=1). Metabotropic receptor antagonists (atropine at 10 μM and scopolamine at 10 μM) had no effect on responses to nerve stimulation. Together, these results imply that curare and IMI act on nicotinic acetylcholine receptors, and that most of the current evoked by nerve stimulation is nicotinic. Metabotropic receptors are evidently present on PNs and are activated by iontophoresis of acetylcholine but not by our nerve stimulation protocol.
Acetylcholine chloride was prepared as a 1M solution in water, nicotine hydrogen tartrate as a 0.25-1 M solution in water, atropine as a 5 mM stock in water, and scopolamine as a 1 mM stock in water. All drugs were obtained from Sigma and stored at −4°C as aqueous stocks. Iontophoretic ejection currents were +350-800 nA, and the constant holding current was -10 nA; these stimuli were delivered with a constant current generator (World Precision Instruments, Model 260).
