Opposite regulation of medullary pain-related projection neuron excitability in acute and chronic pain

Abstract

Pain hypersensitivity is associated with increased activity of peripheral and central neurons along the pain neuroaxis. We show that at the peak of acute inflammatory pain, superficial medullary dorsal horn projection neurons (PNs) that relay nociceptive information to the parabrachial nucleus reduce their intrinsic excitability and, consequently, action potential firing. When pain resolves, the excitability of these neurons returns to baseline. Using electrophysiological and computational approaches, we found that an increase in potassium A-current (I_A) underlies the decrease in the excitability of medullary dorsal horn PNs in acute pain conditions. In chronic pain conditions, no changes of I_A were observed, and medullary dorsal horn PNs exhibit increased intrinsic excitability and firing. Our results reveal a differential modulation of the excitability of medullary dorsal horn projection neurons in acute and chronic pain conditions, suggesting a regulatory mechanism that, in acute pain conditions, tunes the output of the dorsal horn and, if lacking, could facilitate pain chronification.

Fig. 1.. Targeting medullary dorsal horn PNs.

(A) Schematic illustration and representative images of experimental procedure for retrograde labeling of medullary dorsal horn PNs. (B) Representative image of tdTomato expressing PN with the recording pipette in acute medullary dorsal horn slice and representative traces of typical delayed firing pattern of the medullary dorsal horn PN (representative of 70 of 88). (C) Distribution of medullary dorsal horn PNs based on their firing patterns, n cells/N mice: 88/29. Scale bars, 20 mV, 200 ms.

Fig. 2.. UV photokeratitis leads to acute ongoing pain and hyperalgesia.

(A) Scheme depicting the induction of acute pain model of UV photokeratitis. (B) Left, representative images of mice eyes in sham and acute pain conditions two days after UV irradiation. Middle, eye-closing ratio of mice in acute pain or sham conditions (N = 6 mice per group, P_group = 0.010879; P_{group × days} = 0.037225). Right, relative change (day 2 relative to day 0) in eye-closing ratio in acute pain (UV_day2) and sham conditions (N = 6 mice per group, P = 0.0203). (C) Left, scheme of capsaicin installation to mice eye. Middle, number of eye wipes following capsaicin application to the eyes of sham and acute pain mice. N_Sham = 7, N_{UV-photokeratitis} = 9, P = 0.050538. Right, the change in the number of eye wipes between day 0 and day 2 (N_Sham = 7; N_UV,day2 = 9, P = 0.0484). Significance was assessed by two-way analysis of variance (ANOVA) for repeated measures with Tukey’s multiple comparisons test in [(B) middle]; two-tailed unpaired Student’s t test in [(B) right] and [(C) right]; linear mixed effects model in [(C) middle]. Data are presented as means ± SEM. *P < 0.05, n.s. not significant. See table S1 for full statistical information.

Fig. 3.. Medullary dorsal horn PNs decrease their excitability during acute pain.

(A) Representative traces of AP firing in response to a depolarizing current step from identified medullary dorsal horn PNs in slices from mice in acute pain (UV_day2) and sham conditions. Note decreased AP firing in medullary dorsal horn PNs in acute pain conditions. (B) Frequency-intensity (f-I) curves of medullary dorsal horn PNs in acute pain (UV_day2) and sham conditions. n cells/N mice: sham: 60/26, UV_day2: 55/20, P = 0.0085998. Inset, calculated f-I slopes of medullary dorsal horn PNs from both groups. Sham: 61/26, UV_day2: 52/20, P = 0.0157. (C) Latencies to the first AP in mice in acute pain (UV_day2) and sham conditions. n cells/N mice: sham: 60/26, UV_day2: 47/19, P = 0.00023576. (D) Left, representative traces of voltage responses following hyperpolarizing current step in medullary dorsal horn PNs in sham and acute pain conditions. Right, calculated R_in of medullary dorsal horn PNs from both groups. n cells/N mice: sham: 59/26, UV_day2: 54/20, P = 0.0024. (E) Excitable properties of medullary dorsal horn PNs (specified above each panel) from sham and acute pain conditions. n cells/N mice: AP half-width, sham: 62/26, UV_day2: 55/20, P = 0.012; AP threshold, sham: 62/26, UV_day2: 55/20, P = 0.1377; RMP, sham: 56/26, UV_day2: 49/20, P = 0.1374. Significance was assessed by fitting a linear mixed effects model in (B) and (C); two-tailed unpaired Student’s t test in [(B) inset], [(D) right], and (E). Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001. n.s. not significant. See table S2 for full statistical information.

Fig. 4.. The change in the excitability of medullary dorsal horn PNs follows the timeline of acute pain.

(A) Representative traces of AP firing of identified medullary dorsal horn PNs in slices from sham mice and mice recovering from acute pain (UV_day7). (B) Frequency-intensity (f-I) curves of medullary dorsal horn PNs from sham, acute pain conditions (UV_day2; data from Fig. 3B), and recovery groups (UV_day7). n cells/N mice: sham: 60/26, UV_day2: 55/20, UV_day7: 19/4, P = 0.024343. Inset, calculated f-I slope of medullary dorsal horn PNs from all groups. n cells/N mice: sham: 61/26, UV_day2: 51/20, UV_day7: 19/4, P = 0.0043783. (C) Latencies to the first AP in mice in sham, acute pain conditions (UV_day2), and recovery (UV_day7). n cells/N mice: sham: 60/26, UV_day2: 47/19, UV_day7: 19/4, P = 0.0056189. (D) Left, representative traces of voltage responses to hyperpolarizing current steps in medullary dorsal horn PNs from sham and mice recovering from acute pain (UV_day7). Right, calculated R_in of medullary dorsal horn PNs from all groups. n cells/N mice: sham: 59/26, UV_day2: 54/20, UV_day7: 19/4, P = 0.0033831. (E) Excitable properties of medullary dorsal horn PNs (specified above each panel) from sham mice, mice 2 days (acute pain), and 7 days (recovery) after UV irradiation. n cells/N mice: AP half-width, sham: 62/26, UV_day2: 55/20, UV_day7: 19/4, P = 0.049391; AP threshold, sham: 62/26, UV_day2: 55/20, UV_day7: 19/4, P = 0.35523; RMP, sham: 56/26, UV_day2: 49/20, UV_day7: 18/4, P = 2.4391 × 10⁻⁰⁵. In all panels, significance was assessed by fitting a linear mixed effects model. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, n.s. not significant. See table S3 for full statistical information.

Fig. 5.. Medullary dorsal horn PNs increase their excitability in chronic neuropathic pain conditions.

(A) A scheme depicting the induction of chronic pain conditions (CCI-dION). (B) Eye-closing ratio of sham or CCI-dION mice 4 weeks after surgery (N = 6 mice per group, P = 7.382 × 10⁻⁰⁴). (C) Mechanical withdrawal thresholds of sham or CCI-dION mice (N = 7 mice per group, P_group = 2.2214 × 10⁻⁰⁶; P_{group × time} = 1.6972 × 10⁻⁰⁷). (D) Representative traces of AP firing of identified medullary dorsal horn PNs in slices from mice 2 to 4 weeks after sham or CCI-dION procedure. (E) Frequency-intensity (f-I) curves of PNs in chronic pain and sham conditions. n cells/N mice, sham: 22/4, CCI-dION: 18/4, P = 0.016999. Inset, calculated f-I slopes from both groups. n cells/N mice, sham: 22/4, CCI-dION 18/4, P = 0.0131. (F) Latencies to the first AP in mice in sham and chronic pain conditions. n cells/N mice, sham: 22/4, CCI-dION: 18/4, P = 0.0068754. (G) Left, representative traces of voltage responses following hyperpolarizing current steps in medullary dorsal horn PNs in sham and chronic pain conditions. Right, calculated R_in in both groups. n cells/N mice, sham: 21/4, CCI-dION: 18/4, P = 0.0204. (H) Excitable properties of medullary dorsal horn PNs from sham and chronic pain conditions. n cells/N mice, AP half-width, sham: 22/4, CCI-dION: 18/4, P = 0.0013; AP threshold, sham: 22/4, CCI-dION: 18/4, P = 0.5358; RMP, sham 21/4, CCI-dION 18/4, P = 0.6035. Significance was assessed by two-tailed unpaired Student’s t test in (B), [(E) inset], [(G) right], and (H); two-way ANOVA for repeated measures with Tukey’s multiple comparisons test in (C); linear mixed effects model in (E) and (F). Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, n.s. not significant. See table S4 for full statistical information.

Fig. 6.. A-type potassium current in medullary dorsal horn PNs increases in acute pain conditions.

(A) Representative traces of isolated I_A in medullary dorsal horn PNs from mice in acute pain (UV_day2) and sham conditions. (B). I_A peak amplitudes from both groups. n cells/N mice, sham: 9/7, UV_day2: 10/4, P = 0.5808. (C) Voltage dependence of I_A’s activation and inactivation in acute pain and sham conditions. n cells/N mice, activation curves, sham: 9/7, UV_day2, 10/4; inactivation curves, sham: 9/7, UV_day2: 9/4. (D) V_1/2 (left), and slope factor k (right) of the I_A inactivation curve in mice from both groups. n cells/N mice, sham: 9/7, UV_day2: 9/4, V_1/2: P = 0.0405; slope factor k: P = 0.4636. (E) Left, activation and inactivation curve fits from (B) of both groups. Shaded area under the curves depicts the window currents. Right, I_A window current of medullary dorsal horn PNs in acute pain and sham conditions. n cells/N mice, sham: 7/7, UV_day2: 8/4. P = 0.041. (F) Representative traces of isolated I_A in response to a voltage step to +10 mV in both conditions. Superimposed gray traces are two-exponential fits to the decay of the current. (G) I_A kinetics in acute pain and sham conditions. n cells/N mice, rise time, sham: 9/7, UV_day2: 10/4. P = 0.0113; fast τ, sham: 7/7, UV_day2: 6/4. P = 0.0492; slow τ, sham: 7/7, UV_day2: 6/4. P = 0.1981. (H) I_A charge in both groups. n cells/N mice, sham: 9/7, UV_day2: 10/4. P = 0.0485. Significance was assessed by two-way ANOVA for repeated measures in (B); two-tailed unpaired Student’s t test for (D), [(E) right], and (H); two-tailed unpaired Student’s t test with unequal variance for (G). Data are presented as means ± SEM. *P < 0.05, n.s. not significant. See table S5 for full statistical information. a.u., arbitrary units.

Fig. 7.. A-type potassium current in medullary dorsal horn PNs does not change in chronic pain conditions.

(A) Left, representative traces of the isolated I_A in medullary dorsal horn PNs in slices from mice in chronic pain (2 to 4 weeks after CCI-dION) and sham conditions. Right, voltage dependence of I_A’s activation and inactivation in chronic pain and sham conditions. n cells/N mice, activation curves, sham: 14/5, CCI-dION: 7/5; inactivation curves, sham: 9/5, CCI-dION: 7/5. The shaded area under the curves depicts the window currents. (B) V_1/2 of the I_A inactivation curve (left) and I_A window current (right) in medullary dorsal horn PNs from mice in chronic pain and sham conditions. n cells/N mice, sham: 9/5, CCI-dION: 7/5, inactivation V_1/2: P = 0.7468; I_A window current: P = 0.5185. (C). I_A charge in chronic pain and sham conditions. n cells/N mice, sham: 14/5, CCI-dION: 7/5. P = 0.8171. Significance was assessed by two-tailed unpaired Student’s t test with unequal variance [(B) left]; two-tailed unpaired Student’s t test for [(B) right], and (C). Data are presented as means ± SEM. n.s. not significant. See table S6 for full statistical information.

Fig. 8.. A rightward shift in the inactivation curve of I_A is sufficient to reduce AP firing.

(A) Representative traces of firing responses from a modeled PN in simulated acute pain (red) and sham conditions (black), i.e., when the V_1/2 of the I_A inactivation was set to the values experimentally measured in acute pain (V_1/2 = −45.2 mV) or sham conditions (V_1/2 = −49.3 mV; see Fig. 6C). (B) Frequency-intensity (f-I) curves of modeled PN in simulated acute pain (red) and sham conditions (black). (C) The relation between voltage dependence of I_A inactivation and medullary dorsal horn PNs firing at different stimulations. Note that the depolarizing shift of V_1/2 of I_A inactivation leads to a decrease in medullary dorsal horn PNs’ firing. (D) Latency to the 1st AP for different stimulation intensities from modeled PN in simulated acute pain (red) and sham conditions (black). (E) The relation between voltage dependence of I_A inactivation and the latency to 1st AP at different current intensities. Note that the depolarizing shift of V_1/2 of I_A inactivation leads to an increase in the latency to 1st AP.