Comparison of the kinetics of olfactory responses during inhibitor application.

<p>Representative electroolfactogram (EOG) traces from rat in response to the odorant mixture of 10⁻⁴ M before and during MDR inhibitor application. EOG traces were normalized to their maximum amplitude and averaged for the comparison of their time course. Inhibitors applied were verapamil at 200 µM, CsA  =  cyclosporin A at 5 µM, probenecid at 2.5 mM, and MK571 at 25 µM. Each averaged waveform represents the independent assessment from four different animals.</p

Effect of various MDR inhibitors on calcein accumulation in olfactory slices of rat and mouse.

<p>Olfactory tissue slices were incubated with 1 µM calcein-AM in the absence or presence of the specific MDR inhibitors. Bars represent average accumulation rates as determined from slopes over all time points of the experiment, in (<b>A</b>) from mice, and in (<b>B</b>) from rats. The following inhibitors were tested: vera  =  verapamil at 100 and 200 µM, CsA  =  cyclosporin A at 5 and 10 µM, prob  =  probenecid at 2.5 and 5 mM, and MK  =  MK571 at 25 and 50 µM. Values from at least three independent assays for each inhibitor and concentration. * p<0.05.</p

Primer pairs used for the amplification of cDNAs for rat and mouse Mdrs and Mrps.

<p>Primer pairs used for the amplification of cDNAs for rat and mouse Mdrs and Mrps.</p

Modulatory effect of MDR inhibitors on the olfactory response to odorants in rat and mouse.

<p><b>A:</b> Representative superimposed electroolfactogram traces recorded in the septal olfactory mucosa of a rat (left) and a mouse (right) under control conditions. Responses are recorded to a sequence of stimuli (inset, left) as KCl, IBMX, a mixture of odorants, isoamyl acetate and 2,5-dimethyl pyrazine, at concentrations of 10⁻⁴ M. <b>B:</b> Representative selection of EOG recordings from rats in response to the mixture at a concentration of 10⁻⁴ M before (control, solid line), during (inhibitor, dashed line), and after (recovery, dot-dash line) application of four MDR inhibitors: verapamil at 200 µM, cyclosporin A (CsA) at 5 µM, probenecid at 2.5 mM and MK571 at 25 µM. Recordings are from four different specimen.</p

Calcein fluorescence in olfactory slices is elevated in the presence of an MDR inhibitor. A:

<p>Time series images in pseudocolor of two individual mouse olfactory slices incubated with 1 µM calcein–AM (left panel), or concomitantly with 25 µM MK571 (right panel). The numbered images correspond to individual frames at indicated time points. Some pixel saturation occurred at the 60 min time point in the center portion of the OE in MK-treated slice. The two slices are from the same specimen, recorded with identical exposure times. <b>B:</b> Time-dependent accumulation of fluorescence intensity (arbitrary units, a.u) for calcein in the absence or presence of 25 µM MK571 as evaluated from regions of interest (ROIs), saturated pixels were not considered for evaluation. Data derived from above experiment. Values of the pseudocolor code bar scales from 0 (low, blue) to 4095 (high, red), scale bar in A = 200 µm.</p

Distribution of mdr and mrp mRNAs in rat and mouse olfactory and liver tissue.

<p>The presence of <i>mdr1a</i>, <i>mdr1b</i> and <i>mrp1</i> mRNA analyzed by means of RT-PCR from samples of olfactory mucosa and liver tissue. Electrophoresis of PCR products <b>A:</b> from newborn and adult rat, <b>B:</b> from newborn and adult mouse. The cDNAs were amplified with rat- and mouse-specific primers for the Mdr1a, Mdr1b and Mrp1 genes. All tissues were positive for the tested cDNAs (note that staining in A, lane 12 for <i>mdr1b</i> is weak, but visible on the gel). c  =  cyclophilin A used as positive control, w  =  negative control containing water in place of cDNA, m = DNA size markers. All gels were run together except for the newborn rat, which was run on a separate gel. Results from a single experiment.</p

Properties of the voltage-gated sodium current from honeybee ALNs.

<p>A, Sodium current traces obtained from an ALN for voltage steps to the indicated potential from a holding potential of −80 mV. B, Mean relative I/V curve (n = 24). C, Mean steady-state inactivation curve (n = 12). D, Recovery from fast inactivation in ALN (n = 12). E, Use-dependent decrease in the amplitude of the sodium current observed in an ALNs submitted to a 10-pulses train at 13 Hz. I, Average decrease in the amplitude of the sodium current in ALNs submitted to a 10-pulse train at 13 Hz (filled triangles, n = 36) or 35 Hz (filled circles n = 7).</p

Use-dependent effects of type II (cypermethrin) and type I (permethrin and tetramethrin) pyrethroids in ALNs.

<p>Sodium current recordings in response to a 10-pulse train (3 ms, from −80 mV to −10 mV, 13 Hz) in three different ALNs in the presence of 10 µM cypermethrin (A), 10 µM permethrin (B) or 10 µM tetramethrin (C).</p

Effect of pyrethroids on the use-dependent decrease in the sodium current peak.

<p>Mean evolution of the relative amplitude of the peak current along with protocol shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112194#pone-0112194-g002" target="_blank">Figure 2</a> (current normalized to the amplitude obtained at the first pulse of the train) from ALNs in a control solution (empty triangles) and after exposure to pyrethroids (filled circles). All pyrethroids tested amplify the decrease in current amplitude and tetramethrin (n = 10) has a stronger effect than cypermethrin (n = 6) or permethrin (n = 10).</p

Pyrethroids decrease deactivation rate constants.

<p>A. State model used to fit the experimental traces (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112194#s2" target="_blank">Methods</a> and text for details). Pyrethroids can only bind to open channel. Once bound they modify channel kinetics to and from open and inactivated states by given factors, i.e. KFwr, Kbck, KiF, KIB, Kin and Kout by p, q, r, s, t, and u respectively to give KFwrb, Kbckb, KiFb, KIBb, Kinb and Koutb. Voltage dependence is not changed. B-C-D. Fitting of experimental traces obtained under differents pyrethroids (cypermethrin, permethrin, tetramethrin) using the above model allows to determine the changes in the different kinetic parameters. Scale bar 200 pA (B and C) or 500 pA (D) and 150 ms. E. Radargraph of the changes (logarythmic scale) in the different kinetic parameters produced by pyrethroids. Fitting for KpyrF KpyrB, the binding and unbinding rate constants at a single concentration (10 µM), are used to calculate Kd.</p