Scaling Up Cortical Control Inhibits Pain

Abstract

Acute pain evokes protective neural and behavioral responses. Chronic pain, however, disrupts normal nociceptive processing. The prefrontal cortex (PFC) is known to exert top-down regulation of sensory inputs; unfortunately, how individual PFC neurons respond to an acute pain signal is not well characterized. We found that neurons in the prelimbic region of the PFC increased firing rates of the neurons after noxious stimulations in free-moving rats. Chronic pain, however, suppressed both basal spontaneous and pain-evoked firing rates. Furthermore, we identified a linear correlation between basal and evoked firing rates of PFC neurons, whereby a decrease in basal firing leads to a nearly 2-fold reduction in pain-evoked response in chronic pain states. In contrast, enhancing basal PFC activity with low-frequency optogenetic stimulation scaled up prefrontal outputs to inhibit pain. These results demonstrate a cortical gain control system for nociceptive regulation and establish scaling up prefrontal outputs as an effective neuromodulation strategy to inhibit pain.

Keywords: PFC; chronic pain; cortical gain control; neuromodulation; prefrontal cortex.

Figure 1. Neurons in the PFC Increase Firing Rates in Response to an Acute Pain Stimulus

(A) Experimental paradigm for electrophysiological recordings in free-moving rats. Neural activities in the PFC were recorded before and after peripheral noxious stimulation (with PP using a 28G needle) to the hind paw through a mesh table. Each trial lasted until paw withdrawal. (B) Histology showing the location of recording tetrodes in the prelimbic PFC contralateral to peripheral stimulations. Scale bar, 1,000 μm. (C) PP caused nocifensive paw withdrawals, in contrast to stimulation with a non-noxious stimulus using 2-g VF filament. n = 7, p < 0.0001, paired Student’s t test. (D) Raster plots and PSTHs of a representative prefrontal neuron. Time 0 indicates the onset of noxious (PP) stimulation. Inset shows representative single-cell recordings. FRs, firing rates. (E) A total of 31.38% of recorded neurons in the PFC (n = 290 from three rats) demonstrated increased FRs in response to acute pain (pain-responsive neurons). See Experimental Procedures. (F) Raster plots and PSTHs before and after non-noxious (VF filament) stimulation. (G) A total of 15.17% of recorded neurons (n = 290) demonstrated increased FRs in response to VF filaments. See Experimental Procedures. (H) The difference in the proportion of neurons that increased their FRs in response to VF filaments and PP is statistically significant. p = 0.0002, Fisher’s exact test. See Experimental Procedures. (I) Most of the neurons that respond to the mechanical pain stimulus (PP) did not respond to non-painful (VF filament) stimulation. ↑: neurons that increase their FRs; ↓: neurons that decrease their FRs; ↔: neurons that did not alter their FRs in response to a peripheral stimulus. p < 0.0001, Fisher’s exact test. (J) In neurons that responded to both stimuli, PP induced higher FRs than VF filaments. n = 25, p = 0.0011, Wilcoxon paired signed-rank test. See Experimental Procedures for calculations of stimulus-evoked FRs. (K) A representative session of unbiased SVM-based population-decoding analysis to distinguish between painful and non-painful stimulations. Time 0 denotes the stimulus (PP or VF filaments) onset. The blue curve denotes the decoding accuracy (n1 = 28 trials for PP, n2 = 27 trials for VF filaments; C = 7 PFC neurons) derived from the data with true labels; the error bar denotes the SEMs from 50 Monte Carlo simulations based on 5-fold cross-validation; the maximum decoding accuracy was 0.79. See Experimental Procedures for details. (L) SVM-based population-decoding analysis demonstrated the ability to distinguish between painful and non-painful stimulation. n = 20 sessions from three rats. Data in (C), (K), and (L) are represented as mean ± SEM. Insets in (D) and (F) are represented as mean ± SD. Data in (J) are represented as medians with interquartile ranges. See also Figure S1.

Figure 2. Chronic Pain Suppresses the Prefrontal Response to Acute Pain

(A) Schematic for recording nociceptive responses in the PFC in the chronic pain state. CFA was injected into the hind paw of a rat to induce persistent pain, and recordings were performed before and after peripheral stimulation of the contralateral, uninjected paw. Thus, CFA injection was ipsilateral to implanted recording electrodes. (B) CFA induced chronic inflammatory pain, as indicated by mechanical allodynia in the affected limb. n = 6, p < 0.0001, two-way ANOVA with repeated measures and Bonferroni post-tests. (C) Raster plot and PSTH of a representative prefrontal neuron in the chronic pain state. (D) A total of 19.73% of recorded prefrontal neurons (n = 365 from three rats) responded to acute pain in the chronic pain state. (E) Chronic pain reduced the number of pain-responsive neurons in the PFC. p = 0.0007, Fisher’s exact test. (F) Chronic pain reduced pain-evoked FRs of prefrontal neurons. Left: histogram showing the distribution of neurons. Right: median ± interquartile range for evoked FRs in rats with (red) or without (blue) chronic pain. p = 0.0002, Mann-Whitney U test. (G) Chronic pain decreased the basal FRs of PFC neurons. Left: histogram showing the distribution of neurons. Right: median ± interquartile range for basal FRs of PFC neurons. p = 0.0071, Mann-Whitney U test. See Experimental Procedures for calculations of basal FRs. (H) Linear regression analysis shows a strong positive correlation between basal firing and acute pain-evoked FRs. See Experimental Procedures. Slope = 1.621, R² = 0.8471. (I) CFA-treated rats maintained a strong positive correlation between basal firing and pain-evoked FRs in PFC neurons. Slope = 1.463, R² = 0.7616. (J) The difference in the slopes of the two linear regressions suggests a further decline in the ability to respond to acute pain signals in the chronic pain state. p = 0.0077, analysis of covariance (ANCOVA). Data in (B) and (J) are represented as mean ± SEM. Data in (C) are represented as mean ± SD. See also Figure S2.

Figure 3. Increasing Basal Activities Can Scale Up the Prefrontal Response to Acute Pain

(A) Schematic for in vivo optrode recordings. (B) Representative recording trace shows that 2-Hz optogenetic stimulation increased the basal FRs of a pyramidal neuron in the PFC. (C) Low-frequency (2 Hz) optogenetic stimulation increased the basal FRs of a number of prefrontal neurons. n = 36 (out of a total of 69 from two rats), p ≤ 0.0001, Wilcoxon paired signed-rank test. (D) Low-frequency (2 Hz) activation increased the FRs of prefrontal neurons in response to acute pain. n = 36, p = 0.0421, Wilcoxon paired signed-rank test. (E and F) The strong correlation between basal and pain-evoked FRs was preserved without (E) and with (F) optogenetic stimulation. Slope = 2.42, R² = 0.733 (E) and slope = 2.02, R² = 0.8725 (F). Data are represented as medians with interquartile ranges. See also Figure S3.

Figure 4. Increasing Basal Activities in the PFC Relieves Pain

(A) Histologic expression of ChR2-eYFP in the prelimbic PFC. Scale bar, 1000 μm. (B) Low-frequency (2 Hz) optogenetic activation of the PFC increased the latency to paw withdrawal on the Hargreaves test. n = 11–12, p = 0.0427, unpaired Student’s t test. (C) Low-frequency activation of the PFC relieved mechanical allodynia. n = 8–11, p = 0.0014, unpaired Student’s t test. (D) Schematic for a two-chamber CPA test to assess the aversive response to acute pain. (E) Rats displayed aversive responses to acute mechanical pain. One of the chambers was paired with PP stimulation; the other chamber was not paired with a painful stimulus (NS). n = 17, p < 0.0001, paired Student’s t test. (F) Rats were injected with CFA in one hind paw, and the CPA test was conducted by conditioning with noxious stimulus in the opposite uninjected paw. n = 17, p < 0.0001, paired Student’s t test. (G) Chronic pain induced enhanced aversive response to acute pain, demonstrated by an increase in the CPA score. n = 17, p = 0.0377, paired Student’s t test. (H and I) Low-frequency PFC activation decreased the aversive response to noxious stimulation. One of the chambers was paired with 2-Hz stimulation of the PFC and noxious stimulus; the other chamber was paired with the noxious stimulus without PFC activation. YFP-treated rats demonstrated no preference for either chamber. n = 8, p = 0.7745, paired Student’s t test (H). In contrast, ChR2-treated rats spent more time during the test phase than at baseline in the chamber-paired associated with PFC activation. n = 10, p < 0.0001, paired Student’s t test (I). (J) Low-frequency PFC activation decreased the CPA score. n = 8–10, p = 0.0118, unpaired Student’s t test. (K and L) Low-frequency PFC activation decreased the aversive response to noxious stimulation. One of the chambers was paired with 2 Hz stimulation of the PFC and PP; the other chamber was not paired with an acute pain stimulus (NS). YFP-treated rats displayed preference for the no-pain chamber. n = 9, p = 0.0021, paired Student’s t test (K). In contrast, ChR2-treated rats displayed no preference for either chamber. n = 10, p = 0.9473, paired Student’s t test (L). (M) Low-frequency PFC activation decreased the aversive response to noxious stimulation, which was demonstrated by diminished CPA score. n = 9–10, p = 0.0195, unpaired Student’s t test. (N and O) Low-frequency PFC stimulation decreased the aversive response to PP in CFA-treated rats. CFA-treated rats underwent the same CPA test as in (H)–(J), with noxious stimulation in the uninjured paw. YFP-treated rats demonstrated no preference. n = 8, p = 0.8402, paired Student’s t test (N). In contrast, ChR2-treated rats preferred the chamber paired with PFC activation. n = 10, p = 0.0027, paired Student’s t test (O). (P) Low-frequency PFC activation decreased the aversive response to noxious stimulation in the chronic pain state. n = 8–10, p = 0.0175, unpaired Student’s t test. (Q and R) Low-frequency PFC activation decreased the aversive response to noxious stimulation in CFA-treated rats. CFA-treated rats underwent the same CPA test as in (K)–(M), with stimulation in the uninjured paws. YFP-treated rats displayed preference for the no-pain chamber. n = 8, p = 0.0006, paired Student’s t test (Q). In contrast, ChR2-treated rats displayed no preference. n = 10, p = 0.0889, paired Student’s t test (R). (S) Low-frequency PFC activation decreased the aversive response to noxious stimulation in the chronic pain state. n = 8–10, p = 0.0147, unpaired Student’s t test. All of the data are presented as means ± SEMs. See also Figures S3–S5.

Figure 5. Inhibition of the PL-PFC Enhances Both Sensory and Affective Components of Pain

(A) Expression of halorhodopsin from Natronomonas (NpHR)-eYFP in the PL-PFC. Scale bar, 1,000 μm. (B) Optogenetic inhibition of the PFC decreased the latency to paw withdrawal on the Hargreaves test. n = 9–11, p = 0.0081, unpaired Student’s t test. (C) Inhibition of the PFC increased the aversive response to acute pain. During the conditioning phase of the CPA, one of the chambers was paired with optogenetic inhibition of the PFC and peripheral noxious stimulation (PP); the other chamber was paired with PP without PFC inhibition. NpHR-treated rats demonstrated avoidance of the chamber associated with optogenetic inhibition. n = 7, p = 0.0263, paired Student’s t test. (D) YFP-treated rats did not show any preference for or avoidance of either chamber. n = 8, p = 0.7745, paired Student’s t test. (E) Inhibition of PL-PFC increased the aversive response to acute pain, as demonstrated by an increased CPA score in NpHR-treated rats compared with YFP-treated rats. n = 7–8, p = 0.0239, unpaired Student’s t test. All of the data are presented as means ± SEMs.