The mediation path of the effect of physical pain on moral judgment.

<p>Path model shows that the effect of physical pain on moral judgment is partially mediated by the state perspective taking subscale of the state empathy. Bias-corrected and accelerated 95% confidence intervals (CIS) from 5,000 bootstrap samples are reported for the indirect effect.</p

PAD-mediated inhibition of spike propagation in a multi-compartment axon model.

<p><b>(A)</b> Cartoon depicts our three-compartment axon model. One or more spikes were initiated by current injection applied to the left end of the axon. Voltage was measured at the midpoint of each compartment; color of traces in B and C correspond to compartment colors shown in A. GABA conductance was distributed uniformly throughout the middle (blue) compartment. <b>(B)</b> For <i>E</i>_GABA = -35 mV (left column), <i>g</i>_GABA blocked the propagation of the evoked spike under all three combinations of <i>ḡ</i>_GABA and <i>p</i> that were tested, where <i>p</i> represents the proportion of sodium channels susceptible to inactivation. The <i>g</i>_GABA step did not elicit its own spiking in any condition. On the other hand, for <i>E</i>_GABA = -20 mV (right column), PAD-induced transient spiking was observed for all three conditions yet propagation of the stimulus-evoked spike was blocked in two of the three conditions. Comparing the top and middle panels shows that modest <i>ḡ</i>_GABA relies on sodium channel inactivation to block spike propagation, whereas stronger <i>ḡ</i>_GABA could block propagation without any contribution from sodium channel inactivation. <b>(C)</b> During a spike train, sodium channel inactivation accumulates between spikes such that spikes early in the train can propagate whereas later spikes do not. Comparing with combinations of <i>ḡ</i>_GABA and <i>p</i> required to block propagation of a single spike (see B), these results show that partial blockade during a spike train can be mediated by even comparatively weak PAD.</p

Summary of descriptive statistics and correlation analysis results.

<p>Summary of descriptive statistics and correlation analysis results.</p

PAD-mediated inhibition in DRG neurons.

<p><b>(A)</b> Sample responses from a typical neuron made hyperexcitable by 4-AP and virtual sodium conductance (<i>ḡ</i>_Na = 0.3 nS/pF). The repetitive spiking elicited by the <i>I</i>_stim step was reduced by application of a small <i>g</i>_GABA step (middle row) and was altogether stopped by larger <i>g</i>_GABA steps (top row). For equivalent <i>ḡ</i>_GABA, stronger inhibition was evident with <i>E</i>_GABA = -35 mV than with <i>E</i>_GABA = -20 mV (compare left and right columns). <b>(B)</b> Sequence of <i>I</i>_stim and <i>g</i>_GABA steps was changed to verify that the latter could elicit transient spiking yet still inhibit the repetitive spiking driven by <i>I</i>_stim. Note that repetitive spiking starts after the <i>g</i>_GABA step ends and lasts until the <i>I</i>_stim step ends. <b>(C)</b> PAD-mediated inhibition of transient spiking was assessed using the same protocol as in B but we varied the amplitude of the <i>I</i>_stim step to determine rheobase (i.e. the minimum <i>I</i>_stim required to evoke spiking). Only responses to rheobasic stimulation are shown. Note that rheobase increases with increases in <i>ḡ</i>_GABA, whereas spike height decreases. <b>(D)</b> Change in rheobase (mean ± SEM) is plotted against <i>ḡ</i>_GABA for <i>E</i>_GABA = -35 mV (blue, <i>n</i> = 3 cells) and -20 mV (red, <i>n</i> = 4 cells). Rheobase was significantly increased by <i>ḡ</i>_GABA (<i>p</i> = 0.013, one-way repeated measures ANOVA; <i>p</i> = 0.013 (*), <i>p</i> = 0.002 (**), Holm-Sidak post-hoc tests vs no <i>g</i>_GABA) but <i>E</i>_GABA did not have a significant effect (<i>p</i> = 0.52).</p

PAD-mediated inhibition in a neuron model.

<p><b>(A)</b> 2-D bifurcation diagram showing the combinations of <i>E</i>_GABA and <i>p</i> associated with PAD-induced spiking (grey region) and PAD-mediated inhibition of other spiking (green region), where <i>p</i> represents the proportion of sodium channels susceptible to inactivation. Simulations here are based on a neuron model with sodium channel inactivation (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005215#pcbi.1005215.e007" target="_blank">Eq 7</a>) with <i>β</i>_w = 0 mV and <i>ḡ</i>_GABA = 2 nS/pF. Note that the green and grey regions overlap, indicating that PAD can initiate its own spikes yet still inhibit spikes initiated by other means (e.g. by stimulating current <i>I</i>_stim). Labels <i>a-e</i> indicate parameter combinations for which sample responses are shown in B. <b>(B)</b> Responses to <i>g</i>_GABA steps occurring alone or during <i>I</i>_stim steps are shown down the left and right columns, respectively. <b>(C)</b> Boundary between inhibitory and non-inhibitory region (as in A) re-plotted for different <i>ḡ</i>_GABA. Higher <i>ḡ</i>_GABA enables GABA to be inhibitory despite less inactivating sodium current (i.e. smaller <i>p</i>) and more depolarized <i>E</i>_GABA.</p

ANO-1 channels do not contribute to PAD.

<p>For all panels, responses in the presence of the ANO-1 antagonist T16Ainh-A01 (A01) are shown in red for comparison against responses in normal aCSF shown in black. <b>(A)</b> Traces show responses in a typical neuron to the minimum virtual <i>ḡ</i>_GABA required to evoke spiking based on a fast synaptic waveform and <i>E</i>_GABA = -20 mV before and after ANO-1 blockade. Summary data show that the minimum <i>ḡ</i>_GABA to evoke spiking was not significantly changed by A01 (<i>p</i> = 1.0; paired <i>t</i>-test) based on all TRPV1+ neurons (<i>n</i> = 5) that spiked in response to virtual GABA. <b>(B)</b> Traces show responses in a typical neuron to different <i>ḡ</i>_GABA based on slow synaptic waveform and <i>E</i>_GABA = -35 mV. Summary data show mean (± SEM) depolarization at different <i>ḡ</i>_GABA for all (<i>n</i> = 7) TRPV1+ neurons tested. Blockade of ANO-1 did not significantly affect depolarization (<i>p</i> = 0.77; two-way repeated measure ANOVA). <b>(C)</b> At the end of each experiment, the recorded cell was stimulated with capsaicin. Traces show typical data from a responsive (TRPV1+) and unresponsive (TRPV1-) neuron. Because ANO-1 is expressed predominantly in TRPV1+ neurons, only data from capsaicin-responsive neurons were included for analysis in panels A and B. <b>(D)</b> To confirm the efficacy A01, we verified that it reduced the response to capsaicin, consistent with Takayama et al. [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005215#pcbi.1005215.ref049" target="_blank">49</a>].</p

PAD-induced transient spiking in DRG neurons.

<p><b>(A)</b> Sample responses to virtual GABA conductance applied via dynamic clamp. Labels <i>a-d</i> on cartoon indicate testing conditions and are referred to in all subsequent panels. Most neurons, like the typical one illustrated here, spiked only for <i>E</i>_GABA = -20 mV and after being made hyperexcitable by exposure to 2.5 mM 4-AP (point <i>c</i>). <b>(B)</b> Summary of the proportion of neurons responding with or without spikes to virtual PAD. Numbers inside each bar indicate the number of cells. A total of 29 neurons from naïve rats were tested before and after 4-AP and for each <i>E</i>_GABA. A total of 10 neurons from nerve-injured rats were tested for each <i>E</i>_GABA. The proportion of spiking/non-spiking cells was compared between conditions using Fisher’s exact tests (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005215#sec002" target="_blank">Results</a>). <b>(C)</b> Left panel summarizes the minimum <i>ḡ</i>_GABA required to elicit spiking in cells that spiked both before and after 4-AP for <i>E</i>_GABA = -20 mV. Minimum <i>ḡ</i>_GABA was significantly reduced from 0.76 ± 0.19 to 0.20 ± 0.05 nS/pF (mean ± SEM) by 4-AP (<i>p</i> = 0.005, two-way repeated measures ANOVA and Tukey test). These values are lower than observed in simulations in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005215#pcbi.1005215.g001" target="_blank">Fig 1</a>; therefore, we adjusted the neuron model to reproduce this higher sensitivity to <i>ḡ</i>_GABA. As illustrated in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005215#pcbi.1005215.s001" target="_blank">S1 Fig</a>, this revised model shows the same relationship between <i>E</i>_GABA and excitability (<i>β</i>_w) as seen in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005215#pcbi.1005215.g001" target="_blank">Fig 1</a>. Right panel shows minimum <i>ḡ</i>_GABA plotted against soma diameter. Soma diameter, which correlates with fiber type, did not significantly affect minimum <i>ḡ</i>_GABA or the effect of 4-AP (<i>p</i> = 0.61 and 0.29, respectively; two-way repeated measures ANOVA). <b>(D)</b> Summary of the minimum <i>ḡ</i>_GABA required to elicit spiking in cells that spiked for each <i>E</i>_GABA value after 4-AP. The depolarizing shift in <i>E</i>_GABA from -35 mV to -20 mV caused a significant reduction (<i>p</i><0.022, paired <i>t</i>-test) from 0.30 ± 0.07 nS/pF to 0.11 ± 0.02 nS/pF. <b>(E)</b> Sample responses from a typical neuron tested with <i>g</i>_GABA steps and ramps. The minimum <i>ḡ</i>_GABA required to elicit transient spiking when applied as a step was 40 nS (or 0.96 nS/pF after normalization by membrane capacitance) but a ramp with 2.5x greater peak amplitude failed to elicit spiking. <i>E</i>_GABA = -20 mV.</p

Summary of descriptive statistics and correlation analysis results.

<p>Summary of descriptive statistics and correlation analysis results.</p

Means and Std. Deviation of the three subscales of C-IRI.

<p>Means and Std. Deviation of the three subscales of C-IRI.</p

The bar graph of moral judgment in study two.

<p>This bar graph provides comparative view of the moral judgment across experimental conditions.</p