Mapping Cortical Integration of Sensory and Affective Pain Pathways

Abstract

Pain is an integrated sensory and affective experience. Cortical mechanisms of sensory and affective integration, however, remain poorly defined. Here, we investigate the projection from the primary somatosensory cortex (S1), which encodes the sensory pain information, to the anterior cingulate cortex (ACC), a key area for processing pain affect, in freely behaving rats. By using a combination of optogenetics, in vivo electrophysiology, and machine learning analysis, we find that a subset of neurons in the ACC receives S1 inputs, and activation of the S1 axon terminals increases the response to noxious stimuli in ACC neurons. Chronic pain enhances this cortico-cortical connection, as manifested by an increased number of ACC neurons that respond to S1 inputs and the magnified contribution of these neurons to the nociceptive response in the ACC. Furthermore, modulation of this S1→ACC projection regulates aversive responses to pain. Our results thus define a cortical circuit that plays a potentially important role in integrating sensory and affective pain signals.

Keywords: anterior cingulate cortex; chronic pain; pain; somatosensory cortex.

Figure 1.. S1 inputs enhance the nociceptive response in the ACC.

(A) Schematic for in vivo optrode recording experiments. (B) Raster plots and peri-stimulus time histograms (PSTHs) of a representative ACC neuron without activation of S1 inputs. Time 0 indicates the onset of noxious pin prick (PP) stimulation. FR: firing rates. Inset shows representative single cell recordings. (C) Representative recording trace shows that optogenetic activation of the presynaptic S1 inputs increased the firing rates of a pyramidal neuron in the ACC, in response to PP. (D) Raster plots and PSTHs of a representative ACC neuron without activation of S1 inputs. Time 0 indicates the onset of non-noxious von Frey filament (vF) stimulation. (E) Representative recording trace shows that optogenetic activation of the S1 inputs did not change the firing rate response to vF in an ACC neuron. (F) Activation of the presynaptic S1 inputs increased the firing rates of ACC neurons, in response to PP. n = 623 from 5 rats; p < 0.0001, Wilcoxon matched-pairs signed rank test. In contrast, activation of the presynaptic S1 inputs had no impact on the firing rates of ACC neurons, in response to vF. n = 567 from 5 rats; p = 0.3299. (G) A representative session of SVM-based population-decoding analysis to distinguish between PP and vF in the presence of S1 activation, compared to a session without S1 activation. Time 0 denotes the onset of stimulus (PP or vF). The blue curve denotes the decoding accuracy in the presence of S1 activation, (n1 = 25 trials for PP, n2 = 25 trials for vF; C = 8 ACC neurons) derived from the data with true labels; the error bar denotes the SEM from 50 Monte Carlo simulations based on 2-fold cross-validation. (H) S1 activation increases the decoding accuracy to distinguish between noxious and non-noxious stimulation. n1 = 40, n2 = 48; p = 0.0406, Wilcoxon matched-pairs signed rank test. (I) Proportion of ACC neurons that received S1 inputs and their responsiveness to noxious stimulations. See Methods for criteria of responsiveness to S1 inputs. (J) A table illustrating the number of ACC neurons that respond to noxious inputs and number of neurons that respond to S1 activation. (K) ACC neurons that received S1 inputs (20 out of 54 total) were more likely to respond to noxious stimulations that neurons that did not receive S1 inputs (78 out of 569). p < 0.0001, Fisher’s exact test. (L) ACC response to noxious stimulations in the presence of S1 activation. (M) Activation of the S1 inputs increase the ACC response to PP. n = 98 vs 131 out of 623 neurons from 5 rats. p = 0.0191, Fisher’s exact test. (N) S1 inputs increased pain-evoked firing rates of ACC neurons. n = 54; p < 0.0001, Wilcoxon matched-pairs signed rank test. Data represented as mean ± SEM. See also Figures S1 and S2.

Figure 2.. S1→ACC projection regulates aversive pain behaviors.

(A) Schematic of the conditioned place preference (CPA) assay. (B) Rats display aversive response to acute mechanical pain. One of the chambers was paired with PP, the other chamber was not paired with a noxious stimulus (NS). n = 19; p < 0.0001, paired Student’s t test. (C) Schematic of injection of channelrhodopsin (ChR2) and halorhodopsin (NpHR) into the S1 hind limb (S1-HL), and insertion optic fibers into the ACC. (D) Schematic of CPA assay with optogenetic activation of the S1→ACC projection in the presence of PP. One of the chambers was paired with optogenetic activation of the S1→ACC projection and PP; the other chamber was paired with PP alone. (E) Rats avoided the chamber associated with S1→ACC activation, when presented with PP. n = 10; p = 0.0114, paired Student’s t test. (F) CPA score for S1→ACC activation in the presence of mechanical pain. n = 10–14; p = 0.0415, unpaired Student’s t test. (G) Schematic of CPA assay with inhibition of S1→ACC circuit. One of the chambers was paired with optogenetic inhibition of the S1→ACC projection and PP; the other chamber was paired with PP alone. (H) Rats preferred the chamber associated with S1→ACC inhibition, when presented with PP. n = 11; p = 0.0486, paired Student’s t test. (I) CPA score for S1→ACC inhibition in the presence of mechanical pain. n = 11–14; p = 0.0495, unpaired Student’s t test. Data represented as mean ± SEM. See also Figures S3 and S4.

Figure 3.. Persistent pain increases S1-ACC connectivity.

(A) Schematic of the CFA model. (B) CFA treatment induces mechanical allodynia, n = 6 (CFA), n= 6 (Saline). (C) Raster and PSTH of a representative ACC neuron in a CFA-treated rat, in response to PP. (D) Chronic pain increased the peak firing rates of ACC neurons in response to PP. n = 623 (−CFA), n = 294 (+CFA) from 3 rats; p < 0.0001, Mann-Whitney U test. (E) Representative recording trace shows that optogenetic activation of the presynaptic S1 inputs increased the firing rates of a pyramidal neuron in the ACC in response to PP, in a CFA-treated rat. (F) Activation of the S1 inputs increased the firing rates of ACC neurons, in response to PP, in CFA-treated rats. n = 294; p = 0.0083, Wilcoxon matched-pairs signed rank test. (G) Proportions of ACC neurons that receive S1 inputs in the chronic pain condition. (H) Chronic pain increases the proportion of ACC neurons that received S1 inputs. p = 0.0021, Fisher’s exact test. (I) Chronic pain increases the pain-responsiveness of ACC neurons that received S1 inputs. p = 0.0487, Fisher’s exact test. (J) Activation of S1 inputs further enhances the firing rates of pain-responsive ACC neurons. n = 98; p < 0.0001, Wilcoxon matched-pairs signed rank test. Data represented as mean ± SEM.

Figure 4.. Enhanced S1-ACC connectivity contributes to chronic inflammatory pain.

(A) Schematic of the CPA with CFA-treated rats. One of the chambers was paired with PP; the other chamber was paired with NS alone. (B) CFA-treated rats spent significantly less time in the chamber paired with PP. n = 9; p = 0.0007, paired Student’s t test. (C) CFA increases the aversive value of PP, as indicated by a higher CPA score. n = 9–19; p = 0.0460, unpaired Student’s t test. A CPA score was calculated by subtracting the amount of time spent during the test phase from baseline in the chamber paired with PP in CFA-treated and control rats. (D) Schematic of the CPA assay with one chamber paired with optogenetic activation of the S1→ACC projection and PP; the other chamber was paired with NS alone. (E) Rats spent significantly less time during the test phase than at baseline in the chamber paired with S1→ACC activation and PP. n = 15; p < 0.0001, paired Student’s t test. (F) S1→ACC activation caused a similar increase in the aversive response to PP as chronic pain. A CPA score was calculated by subtracting the amount of time spent during the test phase from baseline in the chamber paired with simultaneous S1→ACC activation and PP in (E), compared with the CPA score calculated in (C) n = 9–15; p = 0.4746, unpaired Student’s t test. (G) Schematic of the CPA for tonic-aversive response. One of the chambers was paired with activation of the S1→ACC projection; the other chamber was not. No peripheral stimulus was given. (H) CFA-treated rats avoided the chamber associated with S1→ACC activation. n = 7; p = 0.0017, paired Student’s t test. (I) CPA score for CFA-treated rats which received S1→ACC activation. n = 7; p= 0.0021, unpaired Student’s t test. (J) Schematic of the conditioned place preference (CPP) assay for tonic pain. One of the chambers was paired with inactivation of the S1→ACC projection; the other chamber was not. No peripheral stimulus was given. (K) CFA-treated rats preferred the chamber associated with S1→ACC inhibition. n = 9; p = 0.0128, paired Student’s t test. (L) CPP score for CFA-treated rats which received S1→ACC inhibition. n = 7–9; p = 0.0256, unpaired Student’s t test. Data represented as mean ± SEM. See also Figure S5.

Figure 5.. Enhanced S1-ACC connectivity contributes to chronic neuropathic pain.

(A) SNI treatment induces mechanical allodynia, n = 6 (SNI), n = 6 (Sham). (B) Schematic of the CPA assay with SNI-treated rats. One of the chambers was paired with PP; the other chamber was paired with NS. (C) SNI treatment gives rise to the aversive value of PP. n = 7; p < 0.0001, paired Student’s t test. (D) SNI increased the aversive value of PP, as indicated by a higher CPA score. n = 7–19; p = 0.0034, unpaired Student’s t test. (E) Schematic of the CPA assay with one chamber paired with optogenetic activation of the S1→ACC projection and PP; the other chamber was paired with NS alone. (F) Rats spent significantly less time during the test phase than at baseline in the chamber paired with S1→ACC activation and PP. n = 15; p < 0.0001, paired Student’s t test. (G) S1→ACC activation caused a similar increase in the aversive response to PP as chronic pain. n = 7–15; p = 0.3012, unpaired Student’s t test. (H) Schematic of the CPA assay for tonic pain in the SNI model. (I) SNI-treated rats avoided the chamber associated with S1→ACC activation. n = 7; p < 0.0001, paired Student’s t test. (J) CPA score for SNI-treated rats which received S1→ACC activation. n = 7 (YFP), n = 6 (ChR2); p = 0.0009, unpaired Student’s t test. (K) Schematic of the CPP assay for tonic pain in the SNI model. One of the chambers was paired with inactivation of the S1→ACC projection; the other chamber was not. No peripheral stimulus was given. (L) SNI-treated rats preferred the chamber associated with S1→ACC inhibition. n = 7; p = 0.0111, paired Student’s t test. (M) CPP score for SNI-treated rats which received S1→ACC inhibition. n = 7; p = 0.0260, unpaired Student’s t test. Data represented as mean ± SEM. See also Figure S5.