TRPA1 Agonist-Responsive Afferents Contribute to Central Sensitization by Suppressing Spinal GABAergic Interneurons Through Somatostatin 2A Receptors

Abstract

Altered nociception, a key feature of nociplastic pain, often involves central sensitization. We previously found that central sensitization underlying a nociplastic pain state in female mice depends on the ongoing activity of TRPA1 agonist-responsive afferents. Here, we investigated how the activity of these afferents induces and maintains central sensitization at the spinal level. We hypothesized that, in the superficial dorsal horn where somatostatin (SST) is expressed in excitatory interneurons and the SST2A receptor (SST2A-R) in GABAergic inhibitory interneurons (GABAn), TRPA1 agonist-responsive afferents stimulate SST-expressing excitatory interneurons (SSTn), leading to GABAn suppression through SST2A-R and resulting in altered nociception. We tested this hypothesis using ex vivo Ca²⁺ imaging of dorsal root-attached spinal cord slices expressing GCaMP6f in either SSTn or GABAn and in vivo assessment of mechanical hypersensitivity. The dorsal root was chemically (with allyl isothiocyanate [AITC]) and electrically stimulated to activate TRPA1-expressing nociceptors and all afferents, respectively. The stimulation of dorsal root with AITC excited SSTn. During activation of AITC-responsive afferents, a subset of SSTn showed potentiated responses to both low- and high-threshold afferent inputs, whereas a subset of GABAn showed suppressed responses to those afferents in an SST2A-R-dependent manner. Intrathecally administered SST2A-R antagonist inhibited the development of mechanical hypersensitivity by intraplantar AITC injection and alleviated persistent mechanical hypersensitivity in the murine model of nociplastic pain. These results suggest that the activity of TRPA1 agonist-responsive afferents induces and maintains central sensitization by activating dorsal horn SSTn and suppressing GABAn via SST2A-R, resulting in altered nociception that manifests as mechanical hypersensitivity. PERSPECTIVE: This article presents experimental evidence that TRPA1 agonist-responsive afferents induce and maintain central sensitization at the spinal level by activating SST-expressing excitatory interneurons and suppressing GABAergic inhibitory interneurons via SST2A-R. Spinal SST2A-R may represent a promising target for treating mechanical pain hypersensitivity due to central sensitization by TRPA1 agonist-responsive afferents.

Keywords: Somatostatin, GABAergic neuron, SST2A receptor, nociplastic pain, central sensitization.

Fig. 1.. Schematic illustration of our hypothesis and ex vivo spinal cord slice Ca²⁺ imaging.

(A) We hypothesized that TRPA1-expressing afferents excite somatostatin-expressing neurons (SSTn) in the dorsal horn, which in turn suppresses GABAergic inhibitory neurons (GABAn) through SST2A receptors (SST2A-R). This feedback disinhibition results in increased mechanical pain (altered mechanical nociception). (B) Schematic diagram of ex vivo Ca²⁺ imaging configuration. The sagittal spinal slice with a dorsal root was placed in an imaging chamber superfused with oxygenated artificial cerebrospinal fluid (ASCF). The dorsal root was electrically stimulated with a suction electrode and chemically stimulated by a drug locally applied via a PE tube. (C) Dye application using this local application approach stained only the dorsal root (broken line box), not the spinal cord. (D) A representative series of images showing a Ca²⁺ transient evoked by dorsal root stimulation (delivered immediately before ‘b’, marked as an electric bolt). The graph shows the corresponding fluorescent calcium signal.

Fig. 2.. TRPA1 agonist-responsive afferents excite SSTn in the dorsal horn and alter their responses to dorsal root electrical stimulation in female mice.

(A) A schematic showing Ca²⁺ imaging of SSTn (under an objective) and afferent manipulations for this experiment (AITC application to the dorsal root and dorsal root electrical stimulation [DR e-stim], LT, low-threshold, and HT, high-threshold). (B) The average (top) and individual (bottom) traces of fluorescent Ca²⁺ signals (ΔF/F₀) from SSTn in the SDH before, during, and after local application of the TRPA1 agonist AITC (50 μM for 3 minutes) to the dorsal root. The black bar indicates the AITC treatment. (C) Ca²⁺ transients were evoked by DR e-stim (0.5 ms pulse, 10–500 μA); three classes of SSTn were observed: their maximum Ca²⁺ transients increased (32.5%), decreased (10.0%), or within the normal variability range (57.5%) during AITC application. The top panels are representative images of SDH SST neurons activated by DR e-stim (500 μA) before, during, and after the AITC application. The magenta, white, and black arrows indicate neurons showing their Ca²⁺ transients increased, decreased, and within normal variability range activity, respectively (scale bar=20 μm). See Methods 2.5 for classification details. Data are expressed as mean (line) ± SD (shaded area). *p<0.05 and **p<0.01 vs. corresponding Ca²⁺ transients in the ‘before’ phase by sequential Sidak multiple comparison tests following linear mixed model analysis.

Fig. 3.. TRPA1 agonist-responsive afferents excite SSTn in the dorsal horn and alter their responses to dorsal root electrical stimulation in male mice.

(A) A schematic showing Ca²⁺ imaging of SSTn (under an objective) and afferent manipulations for this experiment (AITC application and dorsal root electrical stimulation [DR e-stim], LT, low-threshold, and HT, high-threshold). (B) The average (top) and individual (bottom) traces of fluorescent Ca²⁺ signals (ΔF/F₀) from SSTn in the SDH before, during, and after local AITC application to the dorsal root (50 μM, 3 min). The black bar indicates the AITC treatment. (C) Ca²⁺ transients were evoked by DR e-stim (0.5 ms pulse, 10–500 μA); two classes of SSTn were observed: maximum Ca²⁺ transients increased (19.0%) or within the normal variability range (81.0%) during AITC application. The top panels are representative images of SDH SST neurons activated by DR e-stim (500 μA) before, during, and after the AITC application. The magenta and black arrows indicate neurons showing their Ca²⁺ transients increased and within normal variability range activity, respectively (scale bar=20 μm). Data are expressed as mean (line) ± SD (shaded area). **p<0.01 vs. corresponding Ca²⁺ transients in the ‘before’ phase by sequential Sidak multiple comparison tests following linear mixed model analysis.

Fig. 4.. TRPA1 agonist-responsive afferents suppress GABAn activation by other afferent inputs.

(A) A schematic showing Ca²⁺ imaging of GABAn (under an objective) and afferent manipulations for this experiment (AITC application and dorsal root electrical stimulation [DR e-stim], LT, low-threshold, and HT, high-threshold). (B) In the SDH of GAD2-Cre;Ai95D mice, GCaMP6f was expressed in PAX2-immunoreactive neurons (scale bar=50 μm). Ca²⁺ transients were evoked in GABAn by DR e-stim (0.5 ms pulse, 10–500 μA) before, during, and after AITC application in (C) females and (D) males. Two classes of GABAn were observed: maximum Ca²⁺ transients decreased (39.7% in females and 10.7% in males) or within the normal variability range (60.3% in females and 89.3% in males) during AITC application. The top panels are representative images of SDH GABAn activated by DR e-stim (500 μA) before, during, and after the AITC application. The white and black arrows indicate neurons showing their Ca²⁺ transients decreased and within normal variability range activity, respectively (scale bar=20 μm). Data are expressed as mean (line) ± SD (shaded area). *p<0.05 and **p<0.01 vs. corresponding Ca²⁺ transients in the ‘before’ phase by sequential Sidak multiple comparison tests following linear mixed model analysis.

Fig. 5.. TRPA1 agonist-responsive afferents suppress GABAn via SST2A-R signaling.

(A) A schematic showing Ca²⁺ imaging of GABAn (under an objective) and experimental manipulations for this experiment (AITC application, dorsal root electrical stimulation [DR e-stim], and SST2A-R antagonist application, LT, low-threshold, and HT, high-threshold). Ca²⁺ transients were evoked in GABAn by DR e-stim (0.5 ms pulse, 10–500 μA) before, during, and after AITC application in the presence of the SST2A-R antagonist CYN 154806 (CYN) in (B) females and (C) males. Three classes of SSTn were observed: maximum Ca²⁺ transients increased (1.7% in females and 15.2% in males), decreased (0.9% in females only), or within the normal variability range (97.4% in females and 84.8% in males) during AITC application. The top panels are representative images of SDH GABAn activated by DR e-stim (500 μA) before, during, and after the CYN application with or without AITC. The magenta, white, and black arrows indicate neurons showing their Ca²⁺ transients increased, decreased, and within normal variability range activity, respectively (scale bar=20 μm). Data are expressed as mean (line) ± SD (shaded area). *p<0.05 and **p<0.01 vs. corresponding Ca²⁺ transients in the ‘before’ phase; #p<0.05 and ##p<0.01 vs. corresponding Ca²⁺ transients in the ‘CYN’ phase by sequential Sidak multiple comparison tests following linear mixed model analysis.

Fig. 6.. SST2A-R agonist suppresses GABAn activation by afferent inputs.

(A) A schematic showing Ca²⁺ imaging of GABAn (under an objective) and experimental manipulations for this experiment (dorsal root electrical stimulation [DR e-stim] and SST2A-R agonist application, LT, low-threshold, and HT, high-threshold). Ca²⁺ transients were evoked in GABAn by DR e-stim (0.5 ms pulse, 10–500 μA) before, during, and after the SST2A-R agonist L-054264 in (B) females and (C) males. Three classes of GABAn were observed: maximum Ca²⁺ transients increased (11.1% in males only), decreased (15.9% in females and 18.5% in males), or within the normal variability range (84.1% in females and 70.4% in males) during the L-054246 application. The top panels are representative images of SDH GABAn activated by DR e-stim (500 μA) before, during, and after the L-054264 application. The white and black arrows indicate neurons showing their Ca²⁺ transients decreased and within normal variability range activity, respectively (scale bar=20 μm). Data are expressed as mean (line) ± SD (shaded area). *p<0.05 and **p<0.01 vs. corresponding Ca²⁺ transients in the ‘before’ phase by sequential Sidak multiple comparison tests following linear mixed model analysis.

Fig. 7.. SST2A-R antagonism at the spinal level prevents TRPA1 agonist-induced mechanical hypersensitivity.

(A) Female and (B) male mice received an intrathecal injection of SST2A-R antagonist CYN 154806 (CYN 1 μg, injection marked by a blue dotted line) or its vehicle (saline). Thirty minutes later, AITC (0.1%, 3 μL, injection marked by a red broken line) or its vehicle was injected into the plantar side of a hind paw. Mechanical sensitivity was assessed using a von Frey filament (0.1 g force) before and at predetermined times after AITC injection (N=4–6 per group). In both sexes, pretreatment with intrathecal CYN 154806 (CYN+AITC) significantly inhibited the development of AITC-induced mechanical hypersensitivity compared to vehicle pretreatment (saline+AITC). *p<0.05 and **p<0.01 for saline+vehicle vs. saline+AITC groups; ^#p< 0.05 and ^##p<0.01 for saline+AITC vs. CYN+AITC groups by sequential Sidak multiple comparison tests following generalized linear mixed model analysis. (C) AITC at a low concentration (0.01%, 3 μL, injection marked by a red broken line) induced mechanical hypersensitivity only in females. **p<0.01 vs. male groups by sequential Sidak multiple comparison tests following generalized linear mixed model analysis.

Fig. 8.. SST2A-R antagonism at the spinal level alleviates nociplastic mechanical hypersensitivity.

(A) Female and (B) male mice in a nociplastic pain state received an intrathecal injection of SST2A-R antagonist CYN 154806 (1 μg, injection marked by a blue dotted line) or saline. The SST2A-R antagonist significantly attenuated mechanical hypersensitivity in both sexes. ^##p<0.01 vs. saline group by sequential Sidak multiple comparison tests following generalized linear mixed model analysis.