5-HT2A Receptor-Induced Morphological Reorganization of PKCγ-Expressing Interneurons Gates Inflammatory Mechanical Allodynia in Rat

Abstract

Mechanical allodynia, a widespread pain symptom that still lacks effective therapy, is associated with the activation of a dorsally directed polysynaptic circuit within the spinal dorsal horn (SDH) or medullary dorsal horn (MDH), whereby tactile inputs into deep SDH/MDH can gain access to superficial SDH/MDH, eliciting pain. Inner lamina II (II_i) interneurons expressing the γ isoform of protein kinase C (PKCγ⁺) are key elements for allodynia circuits, but how they operate is still unclear. Combining behavioral, ex vivo electrophysiological, and morphological approaches in an adult rat model of facial inflammatory pain (complete Freund's adjuvant, CFA), we show that the mechanical allodynia observed 1 h after CFA injection is associated with the following (1) sensitization (using ERK1/2 phosphorylation as a marker) and (2) reduced dendritic arborizations and enhanced spine density in exclusively PKCγ⁺ interneurons, but (3) depolarized resting membrane potential (RMP) in all lamina II_i PKCγ⁺/PKCγ^- interneurons. Blocking MDH 5HT_2A receptors (5-HT_2AR) prevents facial mechanical allodynia and associated changes in the morphology of PKCγ⁺ interneurons, but not depolarized RMP in lamina II_i interneurons. Finally, activation of MDH 5-HT_2AR in naive animals is enough to reproduce the behavioral allodynia and morphological changes in PKCγ⁺ interneurons, but not the electrophysiological changes in lamina II_i interneurons, induced by facial inflammation. This suggests that inflammation-induced mechanical allodynia involves strong morphological reorganization of PKCγ⁺ interneurons via 5-HT_2AR activation that contributes to open the gate for transmission of innocuous mechanical inputs to superficial SDH/MDH pain circuitry. Preventing 5-HT_2AR-induced structural plasticity in PKCγ⁺ interneurons might represent new avenues for the specific treatment of inflammation-induced mechanical hypersensitivity.SIGNIFICANCE STATEMENT Inflammatory or neuropathic pain syndromes are characterized by pain hypersensitivity such as mechanical allodynia (pain induced by innocuous mechanical stimuli). It is generally assumed that mechanisms underlying mechanical allodynia, because they are rapid, must operate at only the level of functional reorganization of spinal or medullary dorsal horn (MDH) circuits. We discovered that facial inflammation-induced mechanical allodynia is associated with rapid and strong structural remodeling of specifically interneurons expressing the γ isoform of protein kinase C (PKCγ) within MDH inner lamina II. Moreover, we elucidated a 5-HT_2A receptor to PKCγ/ERK1/2 pathway leading to the behavioral allodynia and correlated morphological changes in PKCγ interneurons. Therefore, descending 5-HT sensitize PKCγ interneurons, a putative "gate" in allodynia circuits, via 5-HT_2A receptor-induced structural reorganization.

Keywords: 5-HT2A; PKC-gamma; inflammation; medullary dorsal horn; pain; serotonin.

Figure 1.

Methodology for morphological analysis. A, Field area of neuronal arborization calculated as the product of rostrocaudal and dorsoventral extents of neuritic arbor. B, Fractal dimension (Df) was measured from the reconstructed interneurons (black on white) transformed in skeletonized drawings. Fractal Box count was used to determine the Df score of each interneurons, estimated as the negative slope of the logarithm of the number of non-empty box [log (count)] versus the logarithm of the box size of grids [log (box size)]. C, Sholl analysis of reconstructed interneurons was performed by counting the number of branches crossing concentric circles traced around the soma (radii increasing at 3.0 μm steps). D, Neuritic branching order corresponding to primary (red), secondary (yellow), tertiary (blue), and quaternary (green) branches.

Figure 2.

PKCγ and 5-HT_2AR are involved in CFA-induced facial mechanical allodynia. A1–A3, Time courses of facial mechanical sensitivity thresholds measured by von Frey filaments before (Bas, baseline) and after subcutaneous injection into the vibrissa pad of CFA (2.5 mg/kg) or saline. GammaV5–3 (20 μm, PKCγ inhibitor) or aCSF (vehicle of γV5-3) (A2) and 4F4PP (100 nm, 5-HT_2AR antagonist) or DMSO (0.05%, vehicle of 4F4PP) (A3) were intracisternally applied 30 min before subcutaneous CFA or saline. Note that CFA-induced mechanical allodynia was prevented by prior administration of γV5-3 or 4F4PP. Symbols represent mean ± SEM of n animals per group. ⁺⁺p ≤ 0.01, ⁺⁺⁺p ≤ 0.001 versus corresponding baseline by Dunnett's post test following two-way repeated-measures ANOVA; ^ap ≤ 0.05, ^bp < 0.01, and ^cp < 0.001 versus saline, saline+aCSF or saline+DMSO groups, respectively, by Tukey's HSD post test following two-way repeated-measures ANOVA; ^$$p ≤ 0.01 versus CFA+γV5-3 group and ^&&p ≤ 0.01 versus CFA+4F4PP group, respectively, by Tukey's HSD post test following two-way repeated-measures ANOVA. B1, Representative confocal fluorescence images of PKCγ (blue, left) and pERK1/2-IR (red, middle) cells in MDH lamina II_i after subcutaneous injection of saline, CFA, and CFA+4F4PP (100 nm i.c. 30 min before CFA). White arrowheads show PKCγ/pERK1/2 double-labeled interneurons (overlay images at right). Immunolabeling was performed in parasagittal slices (350 μm thick). Dashed lines represent lamina limits: II_o, II_i, and III. Scale bar, 10 μm. B2, Bar histograms showing the density of double PKCγ/pERK1/2-IR cells (PKCγ⁺/pERK⁺) (left bars) and PKCγ⁻/pERK⁺ (right bars) interneurons within MDH lamina II_i after subcutaneous injections of saline, CFA, and CFA+4F4PP. Note that innocuous mechanical stimulation of the face by 0.07 g von Frey filament in CFA animals elevated pERK1/2-IR within PKCγ⁺ interneurons, but not PKCγ⁻ interneurons, and such elevation was prevented by 4F4PP. Each symbol is the mean value of three to four slices for a single animal. *p < 0.05 by Tukey's HSD post test following one-way ANOVA.

Figure 3.

PKCγ is involved in TCB-2-induced facial mechanical allodynia. A1, A2, Time courses of facial mechanical sensitivity thresholds measured by von Frey filaments before (Bas, baseline) and after intracisternal administrations of aCSF or increasing doses of TCB-2 (100, 200, and 300 μm; 5-HT_2AR agonist) (A1), or either TCB-2 (300 μm) or TCB-2 (300 μm) + 4F4PP (100 nm, 15 min before TCB-2) or TCB-2 (300 μm) + γV5-3 (20 μm, 15 min before TCB-2) (A2). Note that TCB-2-induced facial mechanical allodynia is dose dependent and is prevented by prior administration of γV5-3 or 4F4FP. Symbols represent mean ± SEM of n animals per group. ⁺p ≤ 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 versus corresponding baseline by Dunnett's post test following two-way repeated-measures ANOVA; ^ap ≤ 0.05 and ^bp < 0.01 versus aCSF group by Tukey's HSD post test following two-way repeated-measures ANOVA; ^&p ≤ 0.05, ^&&p ≤ 0.01 TCB-2 versus TCB-2+4F4PP group by Tukey's HSD post test following two-way repeated-measures ANOVA. B, Bar histograms showing the density of double-labeled PKCγ/pERK1/2-IR interneurons (PKCγ⁺/pERK⁺) and PKCγ⁻/pERK⁺ ones within MDH lamina II_i after intracisternal administration of aCSF, TCB-2 (300 μm), or TCB-2+γV5-3 (20 μm, 15 min before TCB-2). Note that innocuous mechanical stimulation of the face by 0.07 g von Frey filament after intracisternal TCB-2 injection elevates pERK1/2-IR in PKCγ⁺ interneurons, but not in PKCγ⁻ interneurons, and such elevation is prevented by intracisternal γV5-3. Bars represent mean ± SEM n animals per group. Each symbol is the mean value of three to four slices for a single animal. **p < 0.01 TCB-2 versus aCSF and TCB-2 versus TCB-2+γV5-3 by Tukey's HSD post test following one-way ANOVA.

Figure 4.

No cumulative effects of 5-HT_2AR activation and CFA injection were seen on facial mechanical allodynia and neuronal activation. A, Time courses of facial mechanical sensitivity thresholds measured by von Frey filaments before (Bas, baseline) and after subcutaneous injection into the vibrissa pad of saline, CFA (2.5 mg/kg), or CFA+TCB-2 (200 μm, 5-HT_2AR agonist, injected i.c. 30 min before CFA). Note that direct activation of 5-HT_2AR by TCB-2 has no impact on the time course of CFA-induced mechanical allodynia, suggesting that 5-HT_2AR are already fully recruited in the CFA-induced inflammation. Symbols represent mean ± SEM of n animals per group. ⁺p ≤ 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 versus corresponding baseline by Dunnett's post test following two-way repeated-measures ANOVA; ^ap ≤ 0.05, ^bp < 0.01 and ^cp < 0.001 versus saline+aCSF group by Tukey's HSD post test following two-way repeated-measures ANOVA. B, Bar histograms showing the density of double PKCγ/pERK1/2-IR interneurons (PKCγ⁺/pERK⁺) and PKCγ⁻/pERK⁺ ones within the MDH lamina II_i after subcutaneous injection of saline, CFA, or CFA+TCB-2 (200 μm, injected i.c. 30 min before CFA). Bars represent mean ± SEM n animals per group. Each symbol is the mean value of three to four slices for a single animal. *p ≤ 0.05 by Tukey's HSD post test following one-way ANOVA.

Figure 5.

CFA-induced facial inflammation modifies intrinsic properties of MDH lamina II_i interneurons. A: Representative confocal fluorescence images showing PKCγ (blue,), 5-HT_2AR (red), and NeuN (turquoise) immunolabeling in MDH lamina II_i in naive animals. White arrowheads show PKCγ/5-HT_2AR/NeuN triple-labeled interneurons (see overlay in fourth image). Immunolabeling was performed in parasagittal slices (350 μm thick). Scale bar, 20 μm. The fifth confocal image (on the right) is a high magnification of the dashed rectangle of overlayed image exhibiting 5-HT_2AR-immunoreactivity into PKCγ⁺ interneuron membrane (yellow arrowhead). Scale bar, 2 μm. B, C, RMP (B) and current-voltage curves (C) obtained from whole-cell patch-clamp recordings in MDH lamina II_i interneurons after subcutaneous injection into the vibrissa pad of saline, CFA (2.5 mg/kg), or CFA+4F4PP (100 nm, injected intracisternally 30 min before CFA). Bars and symbols in B represent mean ± SEM of n neurons per group. Symbols are RMP of single recorded neurons. Note that CFA produces depolarization in both PKCγ⁺ and PKCγ⁻ interneurons; 5-HT_2AR blockade does not prevent such CFA-induced depolarization in either PKCγ⁺ or PKCγ⁻ interneurons. *p ≤ 0.05 and ***p < 0.001 by Tukey's HSD post test following one-way ANOVA. ^ap ≤ 0.05, ^bp < 0.01, and ^cp < 0.001 versus saline group; ^$p ≤ 0.05 CFA+4F4PP versus CFA group by Tukey's HSD post test following two-way repeated-measures ANOVA. D, E, RMP (D) and current-voltage curves (E) obtained from whole-cell patch-clamp-recorded MDH lamina II_i interneurons before (aCSF) and after bath-applied TCB-2 (10 μm, 5-HT_2AR agonist). Note that TCB-2 has no effect on RMP or current-voltage curve in either PKCγ⁺ nor PKCγ⁻ interneurons. For D, symbols at the left and right of each graph represent mean ± SEM of n neurons per group. Symbols in the middle of graphs show RMP values before and after TCB-2 for each individually recorded neuron. For C and E, symbols represent mean ± SEM of n PKCγ⁺ (C1, E1) and n PKCγ⁻ (C2, E2) interneurons per group.

Figure 6.

CFA-induced facial inflammation produces structural modifications of MDH lamina II_i interneurons. A–H, Morphological features of 3D-reconstructed neuritic arbors of neurobiotin-labeled PKCγ⁺ and PKCγ⁻ interneurons after subcutaneous injection into the vibrissa pad of saline (vehicle) or CFA (2.5 mg/kg) or CFA+4F4PP (100 nm, injected i.c. 30 min before CFA). For bar histograms, bars represent mean ± SEM of n neurons per group and each symbol is the value for a single neuron. A, B, Total field area (A) and fractal dimension (B) of PKCγ⁺ and PKCγ⁻ interneurons in the different experimental groups. *p ≤ 0.05 and **p < 0.01 by Tukey's HSD post test following one-way ANOVA. C1, C2, Sholl analysis curves of PKCγ⁺ (left) and PKCγ⁻ (right) interneurons showing the number of branching intersections as a function of path length from soma. Symbols represent mean ± SEM of n neurons per group. D, Number of neuritic branches of PKCγ⁺ and PKCγ⁻ interneurons in the different experimental groups. *p ≤ 0.05 and ^$p ≤ 0.05 by Tukey's HSD post test following one-way ANOVA. E, Representative neuronal reconstructions showing the neuritic aborizations of PKCγ⁺ interneurons. F, Number of primary, secondary, tertiary, and quaternary branches of PKCγ⁺ and PKCγ⁻ interneurons in the different experimental groups. *p ≤ 0.05 and ^$p ≤ 0.05 by Tukey's HSD post test following one-way ANOVA. G, Curves showing the primary, secondary, tertiary, and quaternary branch distribution of PKCγ⁺ interneurons according to their length. Symbols represent the number of branches per 5 μm bin. ^ap ≤ 0.05 and ^bp < 0.01 versus vehicle group by Tukey's HSD post test following two-way repeated-measures ANOVA. H1, Representative confocal fluorescence images showing dendritic spines (red arrowheads) in a PKCγ⁺ interneurons after CFA injection into the vibrissa pad. H2, Number of spines per 10 μm in PKCγ⁺ interneurons. Symbols represent mean ± SEM of spine numbers on the corresponding primary, secondary, and tertiary branches. *p ≤ 0.05 and p = 0.07 by Tukey's HSD post test following one-way ANOVA.

Figure 7.

5-HT_2AR activation induces structural modifications of MDH lamina II_i interneurons. A–I, Morphological features of 3D-reconstructed neuritic arbors of neurobiotin-labeled PKCγ⁺ and PKCγ⁻ interneurons after bath-applied (6–10 min) TCB-2 (10 μm; 5-HT_2AR agonist) or 5-HT (10 μm). For bar histograms, bars represent mean ± SEM of n neurons per group and each symbol is the value for a single neuron. A, B, Total field area (A) and fractal dimension (B) of PKCγ⁺ and PKCγ⁻ interneurons in the different experimental conditions. p = 0.08 and *p ≤ 0.05 by Tukey's HSD post test following one-way ANOVA. C1, C2, Sholl analysis curves of reconstructed PKCγ⁺ (left) and PKCγ⁻ (right) interneurons showing the number of branching intersections as a function of path length from soma. Symbols represent mean ± SEM of n neurons per group. D, Number of neuritic branches of PKCγ⁺ and PKCγ⁻ interneurons. *p ≤ 0.05 by Tukey's HSD post test following one-way ANOVA. E, Representative neuronal reconstructions showing the neuritic aborization of PKCγ⁺ interneurons in the different experimental conditions. F, Number of primary, secondary, tertiary, and quaternary branches of PKCγ⁺ and PKCγ⁻ interneurons in vehicle, TCB-2, or 5-HT groups. **p < 0.01 by Tukey's HSD post test following one-way ANOVA. G, Curves showing the primary, secondary, tertiary, and quaternary branch distribution of PKCγ⁺ interneurons in vehicle, TCB-2, or 5-HT groups according to their length. Symbols represent the number of branches per 5 μm bin. ^ap ≤ 0.05 and ^bp < 0.01 versus vehicle group by Tukey's HSD post test following one-way ANOVA.

Figure 8.

Schematic diagram illustrating the contribution of segmental and suprasegmental mechanisms mediating mechanical allodynia. Activation of primary afferents following peripheral CFA injection activates nociceptive fibers (1). Dorsal horn neuronal circuits activate supraspinal structures (2) and engage a network of descending pathways, including serotoninergic (5-HT) fibers (3). Activation of 5-HT_2AR (4) to the PKCγ and ERK1/2 phosphorylation pathway triggers morphological reorganization (5) of specifically MDH lamina II_i PKCγ⁺ interneurons and transforms PKCγ⁺ interneurons from a physiological to a pathological state. These morphological changes in PKCγ⁺ interneurons could explain the mechanisms of pain induced by non-nociceptive stimulations. RMg, Nucleus raphe magnus.