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An amygdalar neural ensemble that encodes the unpleasantness of pain

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Science  18 Jan 2019:
Vol. 363, Issue 6424, pp. 276-281
DOI: 10.1126/science.aap8586
  • Fig. 1 A distinct nociceptive neural ensemble in the BLA represents diverse painful stimuli.

    (A) BLA neural activity was imaged in freely behaving mice with a microendoscope and the virally expressed fluorescent Ca2+ indicator GCaMP6m. Noxious mechanical (pin prick) and thermal (55°C H2O and 5°C H2O or acetone) stimuli were delivered to the left hindpaw, while reflexive and affective-motivational behavior were monitored via a scope-mounted accelerometer. (B) Microendoscope placement and GCaMP6m expression in the right, contralateral BLA. The red line marks the focal plane and is also a 1.0-mm scale bar. (C and D) Map of active BLA neurons (n = 131 neurons) with numbers in (C) matching independent component analysis–derived neuron activity traces in (D). Scale bar, 100 μm. (E) Spearman’s correlation between reflexive withdrawal and affective-motivational escape acceleration. (F) Mean Ca2+ response (Z-scored ∆F/F per trial) across all trials for all BLA neurons imaged during a single session (n = 215 neurons) from the same animal. Neurons are aligned from high to low Ca2+ responses in the noxious heat trials. Individual neuron identifications between different stimuli are consistent across the trial rows. (G) Stimulus-locked mean Ca2+ activity within the nociceptive ensemble (cyan) and mean affective-motivational escape acceleration (red). Shaded region, ±SEM. Pie charts indicate the percentages of significantly responding neurons. (H) Venn diagram of neural populations encoding nociceptive information in response to noxious heat, cold, and pin stimuli. Numbers show means ± SEM of percentages of significantly responding neurons across imaging sessions (see fig. S5E). (I) Neural populations within the nociceptive ensemble that encode innocuous light touch (0.07-g filament) and mild touch (a 1.4- or 2.0-g filament). (J) Divergent neural populations (versus the nociceptive ensemble) encoding appetitive stimuli (10% sucrose consumption). (K) Overlapping BLA populations between the nociceptive ensemble, electric footshock, and aversive stimuli (isopentylamine odor, facial air puff, 85-dB noise, and quinine consumption). A subset of nociceptive ensemble neurons were pain specific (~6% of the BLA neurons). (L) Accuracies of a nine-way Naïve Bayes decoder that distinguishes the ensemble activities for noxious, innocuous, aversive, anticipatory, and appetitive stimuli. The percentage of decoder accuracy to output for the actual stimuli (diagonal) was compared to that for the incorrect stimuli (off the diagonal) and normalized so that each actual stimuli column added up to 100%. Stars on the diagonal indicate the correct prediction of said stimulus was significantly greater than all off-diagonal stimuli within the same column (Wilcoxon sign-rank, Benjamini-Hochberg corrected). (M) Spearman’s correlation (ρ) between per trial pain behavioral responses and nociceptive ensemble activation. Error bars, ±SEM per session animal responses; n = 9 mice, 3 to 4 sessions each.

  • Fig. 2 The BLA nociceptive ensemble is necessary for generating protective and avoidance behavioral responses to painful stimuli.

    (A) Experimental strategy for inhibiting BLA nociceptive ensemble activity. Nociception-mediated targeted recombination in activity neural populations (noci-TRAP) of the inhibitory DREADD(hM4) receptor. CNO, clozapine N-oxide; 4-OHT, 4-hydroxytamoxifen. (B) noci-TRAPhM4 expression in the BLA nociceptive ensemble. CeA, central amygdala; ITC, intercalated neurons; Pir, piriform cortex. Scale bar, 50 μm. (C) Quantification of BLA noci-TRAPeYFP neurons following either no stimulus, innocuous touch (0.07-g filament), or noxious pin prick stimulation; n = 6 mice/group. (D and E) Effect of inhibiting the BLA nociceptive ensemble against reflexive behaviors, demonstrated by a von Frey mechanical threshold assay (D) and reflexive withdrawal frequency to increasing noxious mechanical stimuli (E). n = 14 mice per group. (F and G) Effect of inhibiting the BLA nociceptive ensemble against pain affective-motivational behaviors in response to increasingly noxious mechanical (F) and thermal stimuli (G). n = 14 mice per group. (H) Effect of inhibiting the BLA nociceptive ensemble on adaptive avoidance behavior to noxious thermal environments. The kymograph displays mouse location on a thermal gradient track over a 60-min trial following administration of saline (n = 6 mice) or CNO (n = 7 mice). Noxious temperature zones were areas at <17°C and >42°C. (I) Total number of visit entries (gray and light blue lines) and the occupancy time (black and dark blue lines) in the track’s 25 thermal zones. (J) Temporal cumulative visits and the mean occupancy time per visit (inset) to the noxious hot and cold zones. (K) Occupancy time within the open arms of an elevated plus maze (EPM). (L) The 10% sucrose spout lick rates and preference over a water choice. Overlaid dots and lines represent individual animals. Error bars, ± SEM. For (C) and (E) to (G) (CNO group baseline time points only), one-way analysis of variance (ANOVA; Friedman’s) plus Dunn’s correction. For (D) to (G) and (I), two-way repeated measures ANOVA with Bonferroni correction. For (J) and (K), data on left analyzed with Kolmogorov-Smirnov test; data on right analyzed with Student’s t test. Star, P < 0.05.

  • Fig. 3 Convergence of BLA neural ensemble representations of innocuous and noxious information during chronic pain.

    (A) Long-term tracking of BLA neural activity with microendoscopes throughout the development of chronic neuropathic pain. Peripheral nerve injury results in an increased sensitivity and perceived aversion to innocuous (allodynia) and noxious (hyperalgesia) stimuli. (B) Affective-motivational escape acceleration for neuropathic (top row; n = 5 mice) and uninjured (bottom row; n = 4 mice) animals in response to noxious pin or light touch stimuli before and after nerve injury. Dark lines, means; shaded regions, ±SEM. (C) Hyperalgesic and allodynic behavioral responses in neuropathic (n = 13 mice for paw withdrawal, n = 5 mice for escape acceleration) or uninjured (n = 4 mice for both measures) animals after application of light touch (0.07-g filament), noxious pin, or noxious cold (acetone or 5°C H2O drop) stimuli, respectively. Data were quantified by reflexive hypersensitivity (left axis) and affective-motivational escape acceleration (right axis). (D) Mean Ca2+ activity (Z-scored ∆F/F per trial) of all neurons from the same animal for that imaging session, before and after nerve injury, in response to noxious pin prick, noxious cold, and light touch stimuli. Neuron identifications were consistent between stimuli within a day, but not across days (n = 157 and 156 neurons, for days −7 and 42, respectively). (E) Mean Ca2+ response within the nociceptive ensemble for neuropathic (top row; n = 13 mice, 12,026 total neurons imaged) and uninjured (bottom row; n = 4 mice, 5370 total neurons imaged) animals in response to noxious pin or light touch stimuli. (F) Venn diagrams of percentages of significantly responding neurons to noxious pin, noxious cold, and light touch before and after nerve injury. (G) Overlapping neural populations responsive to light touch within the nociceptive ensemble (pin prick and 5°C water or acetone responsive neurons) after nerve injury (n = 13 mice) or in uninjured animals (n = 4 mice). Numbers indicate means ± SEM. (H) Percentages of nociceptive ensemble activated and escape acceleration per imaging session (light-colored points) and across animal groups and conditions (dark, larger points) show significant correlations [Spearman’s ρ = 0.54 (normal), 0.33 (Neuropathic), and 0.58 (Uninjured) groups]. All tests results in the figure were analyzed via Wilcoxon rank-sum with Benjamini-Hochberg correction unless otherwise noted. Stars, P < 0.01.

  • Fig. 4 Inhibition of neuropathic BLA ensemble activity reduces the aversive quality of chronic pain.

    (A) Utilization of light touch to gain genetic access to, and manipulate, the neuropathic nociceptive ensemble. (B) Quantification of light touch TRAP neurons in the BLA of neuropathic mice compared to uninjured mice; n = 7 per group. (C) Behavioral raster plots from neuropathic mice showing the effects of inhibiting the BLA nociceptive ensemble on reflexive and affective-motivational pain behaviors associated with cold allodynia. (D and E) Summary of the effects of ensemble inhibition against reflexive (D) and affective-motivational (E) pain behaviors in response to noxious pin prick, noxious cold (acetone drop), or formerly innocuous touch stimuli (0.07-g filament). Behavior was assessed before and 42 days after nerve injury and again at 60 min after CNO or saline administration on day 42; n = 14 per group. (F and G) Effects of neuropathic ensemble inhibition on adaptive avoidance during a cold place aversion assay. (F) Group mean exploration paths, color coded for the relative occupancy time, following CNO or saline treatments; (G) summary of the effects in response to decreasing floor plate temperatures; n = 6 per group. Stars, P < 0.05 for all panels. In (G), the black star indicates P < 0.05 versus the uninjured + saline group; open star, P < 0.05 versus the neuropathic + saline group. Overlaid dots and lines represent individual subjects. Error bars, ±SEM. For (B), Student’s t test; (D and E), two-way ANOVA with Bonferroni correction; (G) three-way ANOVA with Bonferroni correction.

Supplementary Materials

  • An amygdalar neural ensemble that encodes the unpleasantness of pain

    Gregory Corder, Biafra Ahanonu, Benjamin F. Grewe, Dong Wang, Mark J. Schnitzer, Grégory Scherrer

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Supplementary Text
    • Materials and Methods 
    • Figs. S1 to S17
    • Table S1
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