Neuropathic pain involves long-lasting modifications of pain pathways that result in abnormal cortical activity. How cortical circuits are altered and contribute to the intense sensation associated with allodynia is unclear. Here we report a persistent elevation of layer V pyramidal neuron activity in the somatosensory cortex of a mouse model of neuropathic pain. This enhanced pyramidal neuron activity was caused in part by increases of synaptic activity and NMDA-receptor-dependent calcium spikes in apical tuft dendrites. Furthermore, local inhibitory interneuron networks shifted their activity in favor of pyramidal neuron hyperactivity: somatostatin-expressing and parvalbumin-expressing inhibitory neurons reduced their activity, whereas vasoactive intestinal polypeptide–expressing interneurons increased their activity. Pharmacogenetic activation of somatostatin-expressing cells reduced pyramidal neuron hyperactivity and reversed mechanical allodynia. These findings reveal cortical circuit changes that arise during the development of neuropathic pain and identify the activation of specific cortical interneurons as therapeutic targets for chronic pain treatment.
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We thank A. Sideris and B. Piskoun for surgical assistance and M. Santello, E. Recio-Pinto and J. Wang for discussions. We thank L. Looger (Janelia Farm Research Campus), the Genetically-Encoded Neuronal Indicator and Effector (GENIE) Project and the Janelia Farm Research Campus of the Howard Hughes Medical Institute for sharing GCaMP6 constructs. This work was supported by the Ralph S. French Charitable Foundation Trust (G.Y.), National Institutes of Health grants R01 GM107469 (G.Y.), R21 AG048410 (G.Y.), R01 NS047325 (W.-B.G.), R01 MH111486 (W.-B.G.) and U01 NS094341 (W.-B.G.).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Persistent elevation of L5 somatic Ca2+ activity in S1 after peripheral nerve injury.
(a) Representative two-photon images of active L5 pyramidal (PYR) neuron expressing GCaMP6s in SNI mice 1 month after surgery. Yellow arrowheads point to somata. Scale bar, 10 μm. (b) Average total integrated Ca2+ activity over 2.5 min in L5 PYR somata 1 month after surgery (SNI: n = 141 cells from 5 mice; Sham: n = 59 cells from 5 mice; t246 = 10.14, P < 0.001). Intraperitoneal (IP) injection of MK801 significantly reduced the activity of L5 PYR neurons in SNI (n = 29 cells from 2 mice; t246 = 9.681, P < 0.001) but not in sham mice (n = 21 cells from 2 mice; t246 = 0.9972, P = 0.63). (c) Representative fluorescence traces of L5 PYR somata expressing GCaMP6s in SNI or sham mice 1 week after surgery. (d) Average total integrated Ca2+ activity over 2.5 min in L5 PYR somata 1 week after surgery (SNI: 73.9 ± 5.5 ΔF, n = 51 cells from 4 mice; sham: 26.3 ± 1.6 ΔF, n = 30 cells from 2 mice; t79 = 6.535, P < 0.001). IP injection of MK801 reduced the activity of L5 PYR neurons (18.1 ± 1.0 ΔF, n = 12 cells; t61 = 4.895, P < 0.001). (e) Representative fluorescence traces of L5 PYR somata expressing GCaMP6s in SNI or sham mice 2 months after surgery. (f) Average total integrated Ca2+ activity over 2.5 min in L5 PYR somata 2 months after surgery (SNI: 54.8 ± 4.9 ΔF, n = 86 cells from 4 mice; sham: 20.4 ± 1.0 ΔF, n = 99 cells from 4 mice; t183 = 7.109, P < 0.001). Data are presented as means ± s.e.m. ***P < 0.001, two-way ANOVA followed by Bonferroni’s test (b), unpaired t test (d,f). (a,c,e) Representative images and traces from experiments carried out on at least 2 animals per group.
Supplementary Figure 2 Persistent elevation of L2/3 somatic Ca2+ activity in S1 after peripheral nerve injury.
(a–c) Representative fluorescence traces of L2/3 pyramidal (PYR) somata expressing GCaMP6s in SNI or sham at 1 week (a), 1 month (b), or 2 months (c) after surgery. (d) Average total integrated Ca2+ activity over 2.5 min in L2/3 PYR somata in SNI (1 week: 40.7 ± 1.9 ΔF, n = 138 cells from 4 mice; 1 month: 45.3 ± 3.1 ΔF, n = 36 cells from 5 mice; 2 months: 33.0 ± 2.7 ΔF, n = 89 cells from 4 mice) and sham mice (1 week: 27.7 ± 1.9 ΔF, n = 66 cells from 2 mice; 1 month: 28.8 ± 2.8 ΔF, n = 45 cells from 5 mice; 2 months: 17.1 ± 2.0 ΔF, n = 90 cells from 4 mice). SNI induced elevated Ca2+ activity at 1 week (t373 = 5.377, P < 0.001), 1 month (t79 = 2.991, P = 0.004), and 2 months (t177 = 4.973, P < 0.001) as compared to sham. Local application of MK801 to layer 1 significantly reduced L2/3 somatic Ca2+ activity in SNI (n = 134 cells from 4 mice; t373 = 10.48, P < 0.0001) and sham mice (n = 39 cells from 2 mice; t373 = 4.11, P < 0.001). L2/3 PYR neuron activity from ipsilateral S1 in SNI mice showed significant difference (n = 72 cells from 4 mice; t106 = 3.542, P < 0.001) from contralateral S1 at 1 month after surgery. Data are presented as means ± s.e.m. **P < 0.01, ***P < 0.001, two-way ANOVA followed by Bonferroni’s test for 1 week, unpaired t test for 1 month and 2 month. (a,b,c) Representative traces from experiments carried out on at least 2 animals per group.
Supplementary Figure 3 L5 apical tuft dendritic branch activity increases 1 week after peripheral nerve injury and persists for 2 months.
(a) Distribution of total integrated Ca2+ activity detected in apical tuft dendrites of SNI (n = 76 dendrites from 4 mice) and sham (n = 34 dendrites from 2 mice) mice 1 week after surgery (t108 = 5.527, P < 0.001). (b) Distribution of total integrated Ca2+ activity detected in apical tuft dendrites of SNI (n = 41 dendrites from 4 mice) and sham (n = 38 dendrites from 4 mice) mice 2 months after surgery (t77 = 3.729, P < 0.001). Data are presented as means ± s.e.m. ***P < 0.001, unpaired t test.
Supplementary Figure 4 SOM-expressing neuron activity 1 week and 1 month after peripheral nerve injury.
(a) Representative two-photon images of SOM-positive neurons expressing GCaMP6s in SNI mice 1 month after surgery. Yellow arrowheads point to active SOM axons in L1 (upper panel) and somata in L2/3 (lower panel). (b) Distribution of total integrated Ca2+ activity of SOM axonal boutons in L1 detected with GCaMP6s in SNI and sham mice 1 month after surgery (t139 = 9.008, P < 0.001). (c) Distribution of total integrated Ca2+ activity of L2/3 SOM somata detected with GCaMP6s in SNI (43.7 ± 3.8 F, n = 90 cells from 4 mice) and sham (95.3 ± 15.7 ΔF, n = 81 cells from 3 mice) mice 1 week after surgery (t169 = 3.267, P = 0.0013). (d) Distribution of total integrated Ca2+ activity of SOM somata detected with GCaMP6s in SNI and sham mice 1 month after surgery (t183 = 6.825, P < 0.001). **P < 0.01, ***P < 0.001, unpaired t test.
(a) Representative two-photon images of PV-positive interneurons expressing GCaMP6s 1 week after surgery. Arrowheads point to somata. Scale bar, 20 μm. Recording imaging fields yielded 3 to 10 PV cells per field. (b) Distribution of total integrated Ca2+ activity of PV somata in SNI and sham mice 1 week after surgery (SNI: 22.2 ± 1.3 ΔF, n = 149 cells from 4 mice; sham: 62.3 ± 3.6 ΔF, n = 130 cells from 3 mice; t277 = 11.14, P < 0.001). (c) Representative two-photon images of VIP-positive interneurons expressing GCaMP6s. Arrowheads point to somata. Scale bar, 20 μm. Typically, there were 3 to 6 VIP cells within an imaging field. (d) Distribution of total integrated Ca2+ activity of VIP somata 1 week after surgery (SNI: 85.1 ± 5.0 ΔF, n = 133 cells from 3 mice; sham: 63.5 ± 4.2 ΔF, n = 106 cells from 2 mice; t237 = 3.209, P = 0.001). **P < 0.01, ***P < 0.001, unpaired t test. (e) Cartoon depicting cortical interneuron circuit in S1 following SNI (magenta dashed box) and sham (green dashed box) operations. SNI induces shifts in local interneuron network activity (↑VIP, ↓SOM, ↓PV) that contribute to increased L2/3 and L5 PYR neuronal activity. (a,c) Representative two-photon images from experiments carried out on at least 2 animals per group.
(a) Representative confocal images of S1 following infection of Cre-dependent GCaMP6s in SOM-Cre mice. Scale bar, 100 μm. (b) The average number of GCaMP6-positive SOM cells in various cortical regions was quantified from 1 mm2 sections (slice thickness: 200 μm). There was no significant difference in the labeling of SOM cells with GCaMP6s between SNI and sham mice (t25 = 0.9813, P = 0.33, unpaired t test). (c) Representative confocal images of S1 following infection of Cre-dependent hM3Dq-mcherry in SOM-Cre mice. (d) Number of SOM cells expressing hM3Dq-mcherry in various cortical regions. The expression of hM3Dq was observed in S1, not in other cortical regions, such as frontal cortex, M1 and V1. There was no significant difference in the labeling of SOM cells with hM3Dq-mcherry between SNI and sham mice (t15 = 0.2372, P = 0.81, unpaired t test). Data are presented as means ± s.e.m.
Supplementary Figure 7 Acute activation of SOM neurons in S1 reduces L2/3 pyramidal neuron activity.
Percentage change in average total integrated Ca2+ activity of L2/3 pyramidal (PYR) neuron somata over 2.5 min following CNO application in SOM-Cre mice infected with hM3Dq-mcherry. PYR Ca2+ activity significantly decreased from baseline activity upon intraperitoneal CNO injection in SNI (-23.0 ± 5.4%, n = 42 cells from 2 mice, t41 = 4.226, P < 0.001, paired t test) and sham mice (-19.8 ± 4.9%, n = 46 cells from 3 mice, t45 = 4.032, P < 0.001). Data are presented as means ± s.e.m. ***P < 0.001.
Supplementary Figure 8 Expression of AAV-TurboRFP in cortex and effects of CNO on L5 pyramidal neuron activity and pain behavior.
(a) A control AAV vector (AAV-CMV-TurboRFP) was locally injected into S1 of SOM-Cre mice. The expression of TurboRFP in different cortical layers (L1 to L6) and regions (frontal, M1, S1, V1) was analyzed 2 weeks after injection. In both sham and SNI mice, the viral expression was mainly observed in L2/3 and L5 of S1. Scale bar, 100 μm. (b) Average signal intensity of TurboRFP signal across cortical sections (measured in AU). No significant difference was found between SNI and sham mice (P = 0.28, unpaired t test). (c) Dimensions of TurboRFP signal in cortical section, described in terms of depth (from pial surface) and width (horizontal spread) (measured in μm). No significant difference was found between SNI and sham infected mice (P = 0.99, two-way ANOVA followed by Bonferroni’s test). (d, e) In both sham and SNI, SOM-Cre mice infected with control vector AAV-CMV-TurboRFP, CNO injection had no effect on the activity of L5 PYR neurons (sham: P = 0.52, paired t test; SNI: P = 0.24, paired t test) (d) and the animals’ paw withdraw threshold in von Frey tests (sham: P = 0.51, paired t test; SNI: P = 0.96, paired t test) (e). Data are presented as means ± s.e.m.
(a) Representative confocal images of S1 following expression of hM3Dq-mCherry in PV-Cre mice. Scale bar, 100 μm. (b) Number of PV cells expressing hM3Dq-mCherry in various cortical regions. The expression of hM3Dq was mainly observed in S1, but not in other cortical regions such as frontal cortex, M1 and V1. There was no significant difference in the labeling of PV/hM3Dq-mcherry between SNI and sham mice (t11 = 1.459, P = 0.17, unpaired t test). Data are presented as means ± s.e.m.
Supplementary Figure 10 Peripheral nerve injury modulates the activity of interneuron networks to promote hyperactivity of pyramidal neurons in S1.
(a) A model showing the local circuitry of S1 in SNI (magenta dashed box) and sham (green dashed box) mice. VIP cells inhibit SOM and PV cells. PV cells inhibit the perisomatic region of L2/3 PYR neurons. SOM cells inhibit both PV and VIP cells as well as apical tuft dendrites of L2/3 and L5 PYR neurons. Following SNI, there is a reduction of SOM and PV cell activity and an increase in VIP cell activity (Fig. 4). This reduction of SOM-mediated inhibition on dendrites plus enhanced synaptic inputs associated with the pain state facilitate dendritic Ca2+ generation in apical tuft dendrites of L5 PYR neurons and promote a hyperactive state in S1 (Figs. 1, 2, 3). Additionally, increased VIP cell activity would further drive hyperactivity of PYR neurons through inhibition of SOM and PV cells. (b) Activation of SOM cells in L2/3 and L5 with DREADD variant hM3Dq (illustrated by orange syringe with CNO) restores SOM-mediated inhibition to the PYR dendrities and reduces PYR neuron activity in the neuropathic pain state (Figs. 5, 6).
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Cichon, J., Blanck, T., Gan, WB. et al. Activation of cortical somatostatin interneurons prevents the development of neuropathic pain. Nat Neurosci 20, 1122–1132 (2017). https://doi.org/10.1038/nn.4595
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