Activation of cortical somatostatin interneurons prevents the development of neuropathic pain - PubMed (original) (raw)

Figure 1

Figure 1. Elevated L5 pyramidal somatic Ca2+ activity in the S1 after peripheral nerve injury

(a) Schematic of spared nerve injury (SNI) and two-photon Ca2+ imaging timeline in the primary somatosensory cortex. Imaging was performed at different cortical depths (dashed boxes) from pial surface in awake, head-restrained mice expressing GCaMP6s in L5 pyramidal (PYR) neurons. (b) Measures of hind limb paw withdrawal threshold before and after SNI (n = 17 mice) and Sham operations (n = 12 mice) (2 d: P = 0.025; 7 d: P < 0.001; 14 d: P < 0.001; 31 d: P < 0.001, two-way ANOVA followed by Tukey’s test). (c) Fluorescence traces of representative L5 PYR soma expressing GCaMP6s in SNI/Sham mice at 1 month post-surgery. (d) Average total integrated Ca2+ activity over 2.5 min in L5 PYR 1 month after surgery (SNI: 116.2 ± 8.5 Δ_F, n_ = 141 cells from 5 mice; sham: 41.6 ± 2.9 Δ_F, n_ = 59 cells from 5 mice; t_198 = 5.581, P < 0.001, unpaired t test; SNI ipsilateral: 20.2 ± 1.0 Δ_F, n = 106 cells, t_245 = 9.694, P < 0.001, unpaired t test). (e) L5 PYR soma Ca2+ activity at 1 month correlates with the hind limb paw withdrawal threshold in SNI mice (Pearson r = -0.33, P < 0.001), but not in Sham mice (Pearson r = 0.046, P = 0.609). (f) L5 PYR soma total integrated Ca2+ activity before (SNI: 40.3 ± 3.6 Δ_F, n = 50 cells from 2 mice; Sham: 17.1 ± 1.2 Δ_F, n_ = 45 cells) and after local application of TTX to sciatic nerve (SNI: 41.9 ± 3.7 Δ_F, t49_ = 1.928, P = 0.06, paired t test_;_ Sham: 11.6 ± 0.9 Δ_F, t44_ = 12.08, P < 0.001, paired t test) at 2 weeks post-Sham/SNI. Data are presented as means ± s.e.m. *P < 0.05, ***P < 0.001. (c) Representative traces from experiments carried out on at least 5 animals per group.

Figure 2

Figure 2. Enhanced dendritic spine and branch activities in the S1 of neuropathic pain mice

(a) Representative two-photon images of active, apical tuft dendrites of L5 PYR neurons expressing GCaMP6s in SNI/Sham mice at 1 month post-surgery. Arrowheads point to spine heads measured. (b) Fluorescence traces of dendritic spines and adjacent shaft show increased Ca2+ transients in spines and shaft of SNI as compared to Sham mice. Light magenta bars indicate Ca2+ elevations in both spine heads and shaft, indicating Ca2+ spike generation. (c) Distribution of peak Ca2+ amplitude of dendritic spines over 1 minute (SNI: 282 ± 21 peak Δ_F/F_0, n = 54 spines from 3 mice; Sham: 183 ± 13 peak Δ_F/F_0, n = 47 spines from 3 mice; t_231 = 4.116, P < 0.001). (d) Distribution of average total integrated Ca2+ activity of dendritic spines over 1 minute (SNI 125 ± 8.2 Δ_F, Sham 66.5 ± 4.1 Δ_F_, _t_99 = 6.078, P < 0.001). (e) Average spine co-activity index in SNI/Sham mice (SNI 0.8 ± 0.02, n = 43 spines; Sham 0.63 ± 0.04, n = 40 spines; t_81 = 3.368, P < 0.01). (f) In both SNI and Sham mice, L5 cell spine co-activity correlates with the generation of apical tuft dendritic Ca2+ spike (SNI: Pearson r = 0.54, P < 0.001; Sham: Pearson r = 0.33, P < 0.05). (g) Fast-scanning of individual tuft Ca2+ events in SNI (rise time, 0.7 ± 0.2 s, decay time, 4.8 ± 1.5 s, n = 11 dendrites) and Sham mice (rise time, 0.6 ± 0.1 s, decay time, 4.5 ± 0.5 s, n = 9 dendrites). Average trace shown as magenta for SNI and green for Sham. (h) Average number of dendritic Ca2+ spikes per minute recording on dendrites (SNI, 4.1 ± 0.5 transients/min; Sham, 1.7 ± 0.2 transients/min; t_32 = 4.509, P < 0.001). (i) Distribution of peak Ca2+ amplitudes of dendritic Ca2+ spikes (SNI, 534 ± 38.5 Δ_F/F_0, n = 147 spikes from 5 mice; Sham, 192 ± 19.4 Δ_F/F0, n = 117 spikes from 5 mice, t_262 = 7.393, P < 0.001). (j) Average total integrated Ca2+ activity detected with GCaMP6s over 2.5 min in active tuft apical dendrites (SNI, 80.2 ± 6.6 Δ_F, n = 147 dendrites; Sham, 37.0 ± 4.0 Δ_F, n = 117 dendrites, t_408 = 6.613, P < 0.001). Local application of either MK801 or TTX to L1 in SNI/Sham mice at 1 month post-surgery (SNI+MK801: 4.0 ± 0.6 Δ_F, n = 30 dendrites from 2 mice, t_408 = 7.22, P < 0.001; Sham+MK801: 8.4 ± 1.2 Δ_F, n = 38 dendrites from 2 mice, t_408 = 2.918, P = 0.011; SNI+TTX: 6.6 ± 0.7 Δ_F, n = 37 dendrites from 4 mice, t_408 = 7.593, P < 0.0001; Sham+TTX: 5.6 ± 0.5 Δ_F, n = 45 from 2 mice, _t_408 = 3.402, P = 0.0022). Data are presented as means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test in (c-e, h, i), two-way ANOVA followed by Bonferroni's test in (j). (a-b) Representative images and traces from experiments carried out on at least 3 animals per group.

Figure 3

Figure 3. Elevated dendritic Ca2+ spike generation in apical tuft branches and trunks of L5 pyramidal neurons in the S1 of neuropathic pain mice

(a) Two-dimensional projection of multiple sibling branches from an individual L5 PYR neuron in SNI mice. Five regions-of-interests (ROIs) (magenta box) corresponding to different dendritic branches were analyzed. All ROIs generated multiple Ca2+ transients during recording. (b) Percentages of local or global Ca2+ spikes observed on sibling branches from individual PYR neurons (SNI, 9.8 ± 1.2 transients/min, n = 24 cells from 3 mice; Sham, 4.8 ± 1.0 transients/min, n = 16 cells from 3 mice). Ca2+ events were analyzed over 2.5 min. (c) Fluorescence traces of apical dendritic trunks imaged at ∼300 μm below the pial in SNI/Sham mice. (d) Average total integrated Ca2+ activity over 2.5 min in apical trunk (SNI, 77.2 ± 4.8 Δ_F, n_ = 102 trunks from 5 mice; Sham, 32.9 ± 2.3 Δ_F_, n = 103 trunks from 5 mice; t_255 = 9.34, P < 0.001). Local application of MK801 in SNI (16.7 ± 0.8 Δ_F, n = 26 trunks from 2 mice, t_255 = 8.111, P < 0.001, two-way ANOVA followed by Bonferroni's test) and Sham mice (8.3 ± 0.9 Δ_F, n = 28 trunks from 2 mice, _t_255 = 3.4, P = 0.0016). (e) TTX locally applied to L1 (blue shaded circle) significantly reduced Ca2+ activity in apical trunk and L5 soma (noted by grey dashed lines) in SNI mice (before TTX: 1; trunk after TTX: 0.51 ± 0.02, n = 50 trunks, _t_49 = 23.77, P < 0.001, paired t test; soma after TTX: 0.53 ± 0.03, n = 54 cells, _t_53 = 17.19, P < 0.001; trunks and cells not related to same cells). Data are presented as means ± s.e.m. **P < 0.01, ***P < 0.001. (a,c) Representative traces from experiments carried out on at least 3 animals per group.

Figure 4

Figure 4. Alterations in inhibitory neuronal Ca2+ activity in the S1 of neuropathic pain mice

(a–d) Cartoon (left of traces) depicting cortical interneuron subtype imaged with GCaMP6s (highlighted by color). Representative fluorescence traces (right) and average total integrated Ca2+ activity over 2.5 min recordings of L2/3 SOM-positive somata and axon fibers expressing GCaMP6s in SNI/Sham mice at 1 month (soma: SNI 36.0 ± 4.7 Δ_F_, n = 117 cells from 4 mice, Sham 75.2 ± 7.7 Δ_F_, n = 68 cells from 3 mice; t_183 = 6.825, P < 0.001; axon: SNI 19.1 ± 1.1 Δ_F, n = 80 fibers from 4 mice, Sham 42.5 ± 3.1 Δ_F, n_ = 61 fibers from 3 mice; t_139 = 9.008, P < 0.001). (e–h) Representative fluorescence traces and quantification of average total integrated Ca2+ activity of L2/3 PV-positive (e–f; SNI, 40.0 ± 3.0 Δ_F, n = 81 cells from 3 mice; Sham, 55.5 ± 5.1 Δ_F, n_ = 66 cells from 3 mice; t_145 = 2.707, P < 0.01) and L2/3 VIP-positive (g–h; SNI, 90.0 ± 5.0 Δ_F, n = 133 cells from 4 mice; Sham, 47.9 ± 3.6 Δ_F_, n = 144 cells from 4 mice; _t_275 = 6.899, P < 0.001) neurons at 1 month. Data are presented as means ± s.e.m. **P < 0.01, ***P < 0.001, unpaired t test. (a, c, e, g) Representative traces from experiments carried out on at least 3 animals per group.

Figure 5

Figure 5. Acute activation of SOM neurons in the S1 reduces L5 pyramidal neuron activity in neuropathic pain mice

(a) Representative fluorescence traces of SOM neurons expressing GCaMP6s/hM3Dq-mcherry before and after CNO injection in SNI mice. (b) Percent change in average Ca2+ activity of SOM somata over 2.5 min following CNO injection in SNI (382 ± 78%, n = 93 cells from 4 mice) and Sham mice (361 ± 114%, n = 61 cells from 5 mice). SOM Ca2+ activity significantly increased from baseline activity upon CNO injection (SNI: _t_92 = 4.912, P < 0.001; Sham: _t_60 = 3.168, P < 0.01, paired t test). No significant difference was found between SNI and Sham mice after CNO injection (_t_152 = 0.163, P = 0.87, unpaired t test). (c) Representative fluorescence traces of apical dendrites of L5 PYR neurons expressing GCaMP6 before and after CNO injection to activate SOM/hM3Dq-positive neurons in SNI mice. (d) Dendritic Ca2+ activity of L5 PYR neurons following CNO injection in SNI (-14.5 ± 5.5%, n = 29 dendrites from 3 mice; _t_28 = 2.522, P = 0.0176, paired t test) and Sham mice (-11.5 ± 5.3%, n = 43 dendrites from 4 mice; _t_42 = 2.149, P = 0.0374). (e) Representative fluorescence traces of L5 PYR neuron somata expressing GCaMP6s before and after CNO injection to activate SOM/hM3Dq-positive neurons in SNI mice. (f) L5 PYR somatic Ca2+ activity following CNO injection in SNI (-17.9 ± 4.0%, n = 94 cells; _t_93 = 4.449, P < 0.001, paired t test) and Sham mice (-13.7 ± 5.9 %, n = 87 cells; _t_86 = 2.310, P < 0.05). (g) Percent change in average total integrated Ca2+ activity of SOM somata following CNO injection in naïve mice infected with hM4Di. SOM Ca2+ activity significantly decreased from baseline activity upon CNO injection (-25.4 ± 10.6%, n = 20 cells from 2 mice, _t_19 = 2.381, P = 0.03). (h, i) Inactivating SOM cells with hM4Di exacerbated Ca2+ activity of L5 PYR dendrites (h, % change in L5 dendritic Ca2+ activity after CNO: Sham: 32.9 ± 14.1 %, n = 60 dendrites, _t_59 = 2.35, P = 0.02, paired t test; SNI: 21.6 ± 6.6 %, n = 40 dendrite, t39 = 3.195, P = 0.003, paired t test) and somata (i, % change in L5 PYR somata Ca2+ activity after CNO: Sham: 31.7 ± 5.0%, n = 60 soma, _t_59 = 6.289, P < 0.001, paired t test; SNI: 13.9 ± 6.5%, n = 52 soma, _t_51 = 2.126, P < 0.05, paired t test) in naïve mice. (j) Cartoon depicting design for determining mechanical allodynia before and after CNO-induced SOM cell activation or inactivation. Thresholds measured before and 20 min after CNO injection in SNI/Sham mice. (k) Withdrawal threshold after SOM activation by CNO in SNI (before CNO: 1.7 ± 0.1 g, after 20-min CNO: 4.0 ± 0.2 g, n = 14 mice; _t_13 = 12.97, P < 0.001, paired t test) and Sham mice (before CNO: 4.8 ± 0.4 g, after 20-min CNO: 5.2 ± 0.2 g, n = 14 mice; _t_13 = 1.685, P = 0.12, paired t test). (l) Withdrawal threshold before and after CNO injection in naïve mice infected with hM4Di. Inactivating SOM cells had no significant effects on the animals' withdraw threshold (contralateral paw: P = 0.12, paired t test; ipsilateral paw: P = 0.13, paired t test). (m) Percent change in average total integrated Ca2+ activity of PV somata following CNO injection in SNI (185 ± 29.7%, n = 81 cells from 3 mice) and Sham mice (131 ± 24.5%, n = 48 cells from 3 mice). PV Ca2+ activity significantly increased from baseline activity upon CNO injection (SNI: _t_80 = 6.243, P < 0.001; Sham: _t_47 = 5.339, P < 0.001, paired t test). No significant difference was found between SNI and Sham mice after CNO injection (_t_127 = 1.262, P = 0.21, unpaired t test). (n) Withdrawal threshold after PV activation by CNO in Sham (before CNO: 5.4 ± 0.3 g, after 20-min CNO: 4.5 ± 0.4 g, n = 8 mice; _t_7 = 2.305, P = 0.06, paired t test) and SNI mice (before CNO: 1.9 ± 0.3 g, after 20-min CNO: 2.1 ± 0.1 g, n = 8 mice; _t_7 = 0.7693, P = 0.47, paired t test). Data are presented as means ± s.e.m. *P < 0.05. **P < 0.01, ***P < 0.001. (a, c, e) Representative traces from experiments carried out on at least 3 animals per group.

Figure 6

Figure 6. Daily activation of SOM neurons following perpherial nerve injury prevents the development of neuropathic pain

(a) Top panel, experimental design for daily activation of SOM neurons following SNI or sham surgeries (CNO, orange syringe; vF, von Frey). For control, SNI and sham mice received saline injections. Bottom panel, cartoon of two-photon imaging planes corresponding to SOM somata (b), pyramidal somata (c,d) and pyramidal tuft dendrites (e,f). (b) Percent change in average total integrated Ca2+ activity of SOM somata over 2.5 min following single CNO injection in SNI (n = 23 cells from 2 mice) and Sham mice (n = 26 cells from 2 mice). CNO injection significantly increased from baseline activity of SOM cells for at least 12 h (P < 0.001) but not 24 h (Sham, P = 0.19; SNI, P = 0.29). (c) Fluorescence traces of L5 PYR neuron somata expressing GCaMP6s after 1-week of daily CNO activation of SOM cells in SNI/Sham mice. (d) Average total integrated Ca2+ activity over 2.5 min recording of L5 PYR somata following 1-week daily SOM activation in SNI (14.4 ± 0.7 Δ_F, n_ = 170 soma from 5 mice) and Sham mice (14.4 ± 1.2 Δ_F , n_ = 54 soma from 5 mice). No significant difference between SNI and Sham mice treated with CNO (t_377 = 7.171, P = 0.99). (e) Fluorescence traces of L5 apical tuft dendrites expressing GCaMP6s after 1-week of daily SOM activation in SNI and Sham mice. (f) Average total integrated Ca2+ activity over 2.5 min recording of L5 dendrites following 1-week daily SOM activation in SNI (14.1 ± 0.7 Δ_F, n = 159 dendrites from 5 mice) and Sham mice (12.9 ± 0.8 Δ_F, n_ = 37 dendrites from 5 mice). No significant difference between SNI and Sham mice treated with CNO (_t_279 = 0.7357, P = 0.9251). (g) Withdrawal threshold under various conditions. Daily SOM activation significantly increased withdrawal threshold in SNI mice as compared to SNI mice with IP saline (P < 0.05 at day 5 and P < 0.001 at day 7, 14, 21, and 28). Data are presented as means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, paired t test in (b), two-way ANOVA followed by Bonferroni's test in (d, f), and Tukey’s test in (g). (c, e) Representative traces from experiments carried out on at least 3 animals per group.