Melatonin attenuated chronic visceral pain by reducing Na v 1.8 expression and nociceptive neuronal sensitization

Animals

Male Sprague Dawley (SD) rats, 150–200 g body weight, were housed in plastic cages and under controlled conditions (a 12 h light–dark cycle from 8:00 to 20:00, room temperature: 24 ± 2°C) with a standard rodent diet and fresh water. Care and handling of these animals were approved by the Institutional Animal Care and Use Committee of the Soochow University and were strictly in accordance with the guidelines of the International Association for the Study of Pain. All efforts were made to minimize the suffering and the number of animals. Visceral hypersensitivity was induced by neonatal colonic injection of diluted acetic acid, as described previously. ⁷ In short, 10-days old pups received an infusion of 0.5% acetic acid solution in 0.2 mL into the colon 2 cm from the anus. Control rats received an equal volume of normal saline (NS). Experiments were performed on these rats at 6–8 weeks of age.

Measurement of visceral hyperalgesia response

Visceral hyperalgesia was measured at the age of 6 wk by recording the threshold of CRD as described previously.^30,31 Briefly, anesthetized with isoflurane (RWD, Shenzhen, China), a flexible latex balloon (6 cm) attached to a tygon tubing was inserted 8 cm into the descending colon and rectum via the anus and held in place by taping the tubing to the tail. Rats were placed in small lucite cubicles and allowed to adapt for 30 min. To minimize the possible insult from the repetitive distention stimuli of the colon, distention threshold (DT) was measured. DT was the minimal distention pressure to evoke abdominal visceromotor response. It was recorded in millimeters mercury by giving a steady increase in distention pressure by a sphygmomanometer. Blind method was used in all behavioral tests.

Drug administration

Melatonin (MT) first dissolved in dimethyl sulfoxide (1 mg/mL), and then diluted to the target concentration with 0.9% normal saline (1 nM, 10 nM, 100 nM, intrathecally, i.t.) was directly injected into CON or NCI rats once times or once daily for consecutive 7 days for behavioral experiments, molecular expression and electrophysiological experiments. 8-M-PDOT (8MP, a selective melatonin MT2 receptor agonist: 10 nM, 100 nM, 1000 nM, intrathecally, i.t.) was directly injected into the NCI rats once times or once daily for consecutive 7 days for behavioral experiments, molecular expression and electrophysiological experiments.

Western blotting

DRGs (T13–L2) from NCI-treated rats (6–8 wk) or age-matched control rats were dissected out and lysed in 100 μL MT-cellytics with 1% PIC (Bocai, Shanghai, China) and then ground on ice until there was no precipitation. Stand on the ice for 2 h to fully decompose the tissue. The cell lysates were then microfuged at 15,000 rpm for 30 min at 4°C. The concentration of protein in homogenate was determined using a BCA reagent (Beyotime, Shanghai, China). Calculate the amount of protein sample according to the measured protein concentration and then conduct SDS-PAGE gel electrophoresis (Bio-Rad, Hercules, CA). After electrophoresis, the proteins were electrotransferred onto polyvinyldifluoride membranes (Millipore, Massachusetts, USA) at 200 mA for 2 h at 4°C. Membranes were incubated in 25 mL of blocking buffer (1xTBS with 5% wt/vol fat-free dry milk) for 2 h at room temperature and then incubated with the primary antibodies for 2 h at room temperature. The primary antibodies used were anti-GAPDH (1:1000; Hangzhou Goodhere Biotechnology, Hangzhou, Zhejiang, China), anti-MT2 (1:500; Abcam, Cambridge, England) and anti-Na_v1.8 (1:200, Alomone Labs, Jerusalem, Israel). After incubation, membranes were washed with TBST (1xTBS and 5% Tween 20) three times for 15 min each and incubated with anti-rabbit peroxidase-conjugated secondary antibody (1:50,000; Jackson, USA) or anti-mouse horseradish peroxidase-conjugated secondary antibody (1:50,000; Jackson, USA) for 2 h at room temperature. The membranes were then washed with TBST three times for 15 min each. Put the strip on the gel imager, configure ECL chemiluminescence reagent solution (ECL kit; Tanon, Shanghai, China) to drop evenly on the strip, and use Image Lab software to develop. All samples were normalized to GAPDH as a control.

Cell labeling

Colon-specific DRG neurons were labeled by injection of 1, 1′-dioleyl-3, 3, 3′, 3-tetramethylindocarbocyanine methanesulfonate (DiI; Invitrogen) into the colon wall. ³¹ In brief, when the rats were 5 wk old, they were anesthetized with isoflurane. The abdomen was opened by midline laparotomy, and the colon was exposed. DiI, 25 mg in 0.5 mL methanol, was injected in an ∼1 μL volume at 10 sites on the exposed colon extending from the level of the bladder to about 6 cm in an oral direction. To prevent leakage and possible contamination of adjacent organs with the dye, the needle was left in place for 1 min, and each injection site was washed with NS following each injection. The colon was gently swabbed before closing of the abdomen. Animals were returned to their housing and given free access to drinking water and standard food pellets.

Immunofluorescence studies

Ten days after DiI injection, control and NCI rats were perfused transcardially with 150 mL phosphate-buffered saline followed by 150 mL ice-cold 4% paraformaldehyde in PBS. DRGs (T13-L2) were removed and post fixed for 4 h in PFA and then gradiently dehydrate with 20% and 30% sucrose (24 h each, sink to the bottom as the sign of dehydration completion). 14 μm frozen sections contained DRGs (T13-L2) area were used in immunofluorescence study as described previously. ³² The primary antibodies were anti-MT2 (1:200; Abcam, Cambridge, England), anti-Na_v1.8 (1:200, Alomone Labs, Israel). The secondary antibodies were Alexa Fluor 488 (1:100, Life Technologies Inc.) and Alexa Fluor 555 (1:500, Life Technologies Inc.). Images were captured with AXIO SCOPE A1 (20X, ZEISS, Oberkochen, Germany).

Acute dissociation of DRG neurons and whole-cell patch clamp recording

Ten days after DiI injection, NCI (6–8 wk) or age-matched control rats were killed by cervical dislocation, as described previously. ²⁹ DRGs (T13–L2) were bilaterally dissected out and transferred to an ice-cold, oxygenated fresh dissecting solution containing (in mM): 130 NaCl, 5 KCl, 2 KH₂PO₄, 1.5 CaCl₂, 6 MgSO₄, 10 glucose, and 10 HEPES, pH 7.2 (osmolarity: 305 mOsm). After removal of the connective tissue, the ganglia were transferred to a 5 mL dissecting solution containing collagenase D (1.8–2.0 mg/mL; Roche, Indianapolis, Indiana, USA) and trypsin (1.2–1.5 mg/mL; Amresco, USA) and incubated for 1.5 h at 34.5°C. DRGs were taken from the enzyme solution, washed, and transferred to 0.5 mL of the dissecting solution containing DNase (0.5 mg/mL; Sigma). A single cell suspension was subsequently obtained by repeated trituration through flame-polished glass pipettes. Cells were plated onto acid-cleaned glass cover slips. Cover slips containing adherent DRG cells were put in a small recording chamber (1 mL volume) and attached to the stage of an inverted microscope (Olympus IX71) fitted for both fluorescence and bright-field microscopy. DiI-labeled neurons were identified by their fluorescence under the fluorescent microscope.

For the patch-clamp recording experiments, cells were continuously cultured at room temperature with normal external solution containing (in mM): 130 NaCl, 5 KCl, 2 KH₂PO₄, 2.5 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose, with pH adjusted to 7.2 with NaOH, osmolarity: 295–300 mOsm. Recording pipettes were pulled from borosilicate glass tubing using a horizontal puller (P-97; Sutter Instruments, USA). Unless indicated, patch-clamp pipettes had a resistance of 5–10 MΩ when filled with the pipette solution containing (in mM): 140 K-gluconate, 10 NaCl, 10 HEPES, 10 glucose, 5 EGTA, and 1 CaCl₂, pH 7.25 adjusted with KOH; osmolarity: 290–295 mOsm. Resting potential and APs were recorded. The voltage was clamped at −60 mV by a HEKA EPC10 patch-clamp amplifier (HEKA Electronik GmBH; Germany). The leak currents at −60 mV were always <20 pA and were not corrected. The currents were filtered at 2–5 kHz and sampled at 50 or 100 μs/point. Whole cell current and voltage were recorded with a HEKA EPC10 patchclamp amplifier; and data were acquired and stored on a computer for later analysis using FitMaster (HEKA Electronik GmBH; Germany).

Isolation of Na_v currents

To record Na_v currents, amplifier was switched to the voltage clamp configuration. Neurons were superfused (2 mL/min) at room temperature with an external solution, containing (in mM): 60 NaCl, 80 choline chloride, 0.1 CaCl₂, 10 HEPES, 10 tetraethylammonium-Cl, 10 glucose, 0.1 CdCl₂ (pH = 7.4, adjustedwith tetraethylammonium-OH, osmolarity: 300 mOsm). The patch electrode had a resistance of 3–5 MΩ when filled with the pipette solution containing (inmM): 140 CsF, 1 MgCl₂, 5 EGTA, 3 Na-GTP, 10 glucose, 10 HEPES, pH = 7.2, adjusted with CsOH, osmolarity: 285–295 mOsm. TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) were separated by superfusing of external solution containing TTX (0.5 μM). The total sodium currents were recorded in response to depolarization steps to different testing potentials from −70 mV to +50 mV in 10 mV increments with a duration of 80 ms. TTX-R current was recorded using the above testing potentials from a holding potential of −60 mV in the presence of 0.5 μM TTX. To control for changes in cell size, the current density was measured by dividing the peak current amplitude by whole cell membrane capacitance (pA/pF), which was obtained by reading the value for whole cell input capacitance cancelation directly from the patch-clamp amplifier. To examine TTX-R channel conductance, activation curves were generated by voltage pulses in 10 mV steps from −70 mV to +30 mV and inactivation curves were generated by voltage pulses in 10 mV steps from −90 mV to +30 mV. The reversal potentials of sodium channels were measured directly or by extrapolation if necessary.

Data analysis

Origin 8 software (Origin Lab, Massachusetts, USA) and Prism 8 (GraphPad, California, USA) were used for statistical analysis. All data were represented as mean ± SEM, and the error line in the chart represented SEM. All data have been tested for normal distribution before analysis. Kruskal-Wallis ANOVA followed by Tukey post-hoc test, two-way repeated measures ANOVA followed by Tukey post-hoc test, two-sample t-test and Mann-Whitney test have been used to determine significance. A one-way repeated measures ANOVA followed by Bonferroni post-hoc test was also used for comparison. p < 0.05 was considered statistically significant.

Melatonin intensifies CRD threshold in NCI rats

In the present study, we firstly confirmed that NCI induces chronic visceral hypersensitivity (CVH) in adult rats, which was consistent with our previous study. ⁷ CVH was determined by measuring CRD threshold at 6 wk of age from control and NCI rats (Figure 1(a)). The CRD threshold of NCI rats was significantly decreased (Figure 1(b), n = 10 rats for each group, *p < 0.05 compared to CON, one-way repeated-measures ANOVA followed by Bonferroni post-hoc test). To determine whether melatonin (MT) activity was involved in NCI induced visceral hypersensitivity, melatonin was administrated intrathecally (i.t.). Injection of melatonin significantly increased CRD threshold in NCI rats, in a dose-dependent fashion (Figure 1(c), n = 8 for each group, *p < 0.05, **p < 0.01, ***p < 0.001 vs. NS, Tukey post-hoc test following Kruskal-Wallis ANOVA). The optimized dose for melatonin to produce the maximal effect was 10 nM in this study. We then determined the time course of melatonin effects. NCI rats received an intrathecal injection of melatonin (10 nM) or NS at the same volume once a day for 7 consecutive days. The effect of melatonin at doses of 10 nM lasted for 2 d (Figure 1(d), n = 8 for each group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. NS, Tukey post-hoc test following two-way repeated-measures ANOVA). To further determine the pharmacological effect of melatonin, we injected melatonin (10 nM, i.t.) to CON group for one time. The results showed that MT has no pharmacological effect on CON group (Figure 1(e), n = 5 for each group; p > 0.05, Tukey post-hoc test following two-way repeated-measures ANOVA). These data suggest that melatonin significantly increase colorectal distension threshold in NCI rats with a dose-dependent fashion.OPEN IN VIEWER

Melatonin treatment reverses hyperexcitability of colonic DRG neurons induced by NCI

Since melatonin attenuates NCI-induced CRD responses, we investigated whether melatonin affects the excitability of colonic DRG T13-L2 neurons derived from NCI rats. Under current-clamp conditions, colon-specific DRG neurons of NCI rats treated with NS (n = 5 rats) or MT (n = 5 rats) were recorded (Figure 2(a)). Resting Potentials (RPs) were −49.4 ± 0.9 and −53.8 ± 1.0 mV for the NS- and MT-treated groups, respectively. Therefore, MT treatment significantly hyperpolarized RPs (Figure 2(b), n = 24 and 20 cells from NS- and MT-treated groups, respectively, ***p < 0.001 compared to NS, two-sample t-test). The rheobase measurements were 0.018 ± 0.002 and 0.045 ± 0.005 nA for the NS- and MT-treated groups, respectively. The administration of MT markedly increased rheobase (Figure 2(c), n = 24 and 20 cells from NS- and MT-treated groups, respectively, ***p < 0.001 compared to NS, two-sample t-test). The action potential (AP) thresholds were −32.5 ± 0.5 and −26.4 ± 0.8 mV for NS- and MT-treated groups, respectively. The MT treatment also increased the AP thresholds (Figure 2(d), n = 24 and 20 cells from NS- and MT-treated groups, respectively, ***p < 0.001 compared to NS, two-sample t-test). MT treatment also greatly decreased the number of APs evoked by 2X and 3X rheobase current stimulation (Figures 2(e) and (g), n = 24 and 20 cells from NS- and MT-treated groups, respectively, **p < 0.01 compared to NS, two-sample t-test). The numbers of AP evoked by 2X rheobase current stimulation were 2.9 ± 0.2 and 1.7 ± 0.2 for NS- and MT-treated groups, respectively. The numbers of AP evoked by 3X rheobase current stimulation were 4.0 ± 0.4 and 2.7 ± 0.2 for NS- and MT-treated groups, respectively. In addition, MT treatment greatly reduced the number of APs evoked by 100, 300 and 500 pA ramp current stimulation. The numbers of APs evoked by 100 pA ramp current stimulation were 11.7 ± 1.5 and 3.7 ± 1.5 for the NS- and MT-treated groups, respectively. The numbers of APs evoked by 300 pA ramp current stimulation were 20.8 ± 2.1 and 8.6 ± 2.0 for the NS- and MT-treated groups, respectively. The numbers of APs evoked by 500 pA ramp current stimulation were 25.8 ± 2.8 and 13.8 ± 2.2 for the NS- and MT-treated groups, respectively. MT treatment remarkably reduced the numbers of APs in response to ramp current stimulation (Figures 2(f) and (h), n = 24 and 20 cells from NS- and MT-treated groups, respectively, ***p < 0.001 compared to NS,two-sample t-test).OPEN IN VIEWER

Melatonin reduces TTX-R sodium current density and modulates dynamics of TTX-R sodium current activation

Since melatonin reversed the hyperexcitability of colon-specific DRG neurons of NCI rats, we next investigated whether melatonin treatment suppressed current density of VGSCs in colon-specific DRG neurons. NCI rats were divided into two groups: NCI+MT group (n = 4 rats) treated with melatonin (10 nM, i.t.) and NCI+NS group (n = 4 rats) treated with the same volume of NS (i.t.). The total sodium current density was −225.3 ± 15.4 pA/pF (n = 10 cells) and −121.8 ± 10.0 pA/pF (n = 8 cells) for NS- and MT-treated group, respectively. MT treatment significantly reduced total sodium current density when compared with that of NS group (Figure 3(a), (b) and (c), *p < 0.05, two-sample t-test). To further determine the roles for MT on TTX-R current densities, TTX (0.5 μM) were used to identify TTX-R and TTX-S sodium currents in the present experiment. At the same voltage protocol as described above, TTX-R sodium currents were recorded in the presence of 0.5 μM TTX (Figure 3(d)). The peak current voltage (I–V) curves for TTX-R current were shown in Figure 3(e). TTX-R sodium current density was remarkably decreased in MT-treated rats (Figure 3(f), NS: −148.9 ± 17.8 pA/pF, n = 10 cells; MT: −95.2 ± 15.5 pA/pF, n = 8 cells, *p < 0.05, two-sample t-test). Because inhibition of peak current density of TTX-R sodium current was evident, we next characterized the effect of MT on voltage dependence of TTX-R currents of colon-specific DRG neurons. Activation dependence was constructed from I-V curves of neurons from NS and MT-treated NCI rats (Figure 3(g)). Currents at various test pulses were divided by the driving force for Na⁺ and the resulting conductance (G) was expressed as a percentage of that achieved at +30 mV for each condition. The G-V relationship curves were fitted with a modified Boltzmann equation. MT treatment led to a ∼10 mV positive shift in the steady state activation curve. The shift of the activation curve in a more depolarizing direction prevents the channel to open in response to weaker depolarization. We next determined the effect of MT on voltage dependence of steady-state inactivation of TTX-R sodium channels of colon-specific DRG neurons. The membrane potential was held at −60 mV and voltage steps were from −90 mV to +30 mV with 10 mV increments and 80 ms duration. Inactivation curves were obtained by plotting TTX-R sodium current during the test pulse (+20 mV) against the membrane potentials during the conditioning pulse. TTX-R sodium current was normalized to Imax during the test pulse to +20 mV after a conditioning pulse to −90 mV. Data were fitted with the negative Boltzmann function (Figure 3(h)). MT treatment did not significantly alter the inactivation curve, indicating the same channel availability in the potential domain between −50 mV and −10 mV of colon-specific DRG neurons from these two groups of rats.OPEN IN VIEWER

Melatonin suppresses Na_v1.8 expression

We next determined whether expression of Na_v1.8 was increased in colon DRGs after NCI treatment. Proteins isolated from both sides of T13-L2 DRGs of NCI and age-matched control rats were probed with anti-Na_v1.8 antibody. Anti-Na_v1.8 antibody labeled a 220 kDa molecular weight protein. Six weeks after NCI treatment, the relative densitometry of Na_v1.8 was 0.44 ± 0.05 (n = 4) and 0.69 ± 0.1 (n = 4) for control (CON) and NCI, respectively. The levels of expression of Na_v1.8 were increased significantly (Figure 4(a), *p < 0.05, two sample t-test). This is consistent with our previous report. ⁷ We then determined the role for melatonin in the upregulation of Na_v1.8 expression. Melatonin was administrated intrathecally (i.t. 10 nM) at age of 6 weeks once daily for consecutive 7 days. The same volume of normal saline (NS) was used as control. The relative densitometry of Na_v1.8 was 1.21 ± 0.03 (n = 3) and 0.78 ± 0.03 (n = 3) for NS and MT, respectively. Melatonin treatment significantly reduced expression of Na_v1.8 (Figure 4(b), *p < 0.05, two sample t-test). Thus, melatonin treatment reverses the upregulation of Na_v1.8 expression in colon DRGs isolated from NCI rats.OPEN IN VIEWER

NCI downregulates the expression of MT2 Receptors in DRGs

Then we assessed the protein expression of melatonin receptor MT2 after NCI (Figure 5(a)). The relative values of MT2 receptor proteins were 1.21 ± 0.2 (n = 4 rats) and 0.78 ± 0.15 (n = 3 rats) in the CON and NCI rats, respectively (Figure 5(a), *p < 0.05, two-sample t-test). There was a significant decrease in MT2 receptor protein levels between CON and NCI rats. Furthermore, to determine the possible interaction between MT2 and sodium channels, we first examined whether MT2 was co-expressed in Na_v1.8-positive colon specific DRG neurons. Triple-labeling techniques showed that colon specific DRG neurons that were immune reactive for MT2 also were positive for Na_v1.8 and that colon specific DRG neurons that were immune reactive for Na_v1.8 were also positive for MT2 (Figure 5(b), arrows). 8-M-PDOT (8MP) a selective melatonin MT2 receptor agonist, to further confirm the 8MP effect on NCI rats, 8MP at dose of 100 nM was intrathecally injected once daily for consecutive 7 days in NCI rats and assessed Na_v1.8 protein level. The same volume of normal saline (NS) was used as control. The relative densitometry of Na_v1.8 was 0.8 ± 0.01 (n = 4) and 0.49 ± 0.02 (n = 4) for NS and 8MP, respectively. 8MP treatment reverses the upregulation of Na_v1.8 expression in colon DRGs isolated from NCI rats (Figure 5(c), *p < 0.05, two-sample t-test).OPEN IN VIEWER

Agonist of 8MP treatment intensifies distention threshold in NCI rats

If melatonin generated endogenously contributes to the maintenance of visceral hyperalgesia in NCI animals, then application of agonist of 8MP to NCI rats should mimic the effects of melatonin. Therefore, we applied 8MP to NCI rats and assessed behavioral responses. Intrathecal administration of 8MP, led to a significant increase in distention threshold in NCI rats, in a dose-dependent manner (Figure 6(a), *p < 0.05, **p < 0.01, ***p < 0.001 vs. NS, n = 6 for each group, Tukey post hoc test following Kruskal-Wallis ANOVA). The optimized dose for 8MP to produce the maximal effect was 100 nM in this study. To further confirm the 8MP effect on NCI rats, 8MP at dose of 100 nM was intrathecally injected once daily for consecutive 7 days in NCI rats and assessed behavioral responses. Intrathecal administration of 8MP, led to a significant increase in distention threshold in NCI rats. (Figure 6(b), *p < 0.05, **p < 0.01, ***p < 0.001 vs. NS, n = 6 for each group, Tukey post hoc test following two-way repeated-measures ANOVA). The effect of 8MP at doses of 100 nM lasted for 7 hours. These data suggest that 8MP treatment significant intensifies distention threshold in NCI rats.OPEN IN VIEWER

8MP treatment reverses hyperexcitability of colonic DRG neurons induced by NCI

Since Melatonin attenuates hyperexcitability of colonic DRG neurons induced by NCI, we investigated whether 8MP affects the excitability of colonic DRG T13-L2 neurons derived from NCI rats. 8MP at dose of 100 nM was intrathecally injected once daily for consecutive 7 days in NCI rats, NCI+NS group treated with the same volume of NS (i.t.). Under current-clamp conditions, colon-specific DRG neurons of NCI rats treated with NS (n = 5 rats) or 8MP (n = 6 rats) were recorded. Resting Potentials (RPs) were −48.8 ± 0.8 and −55.1 ± 1.2 mV for the NS- and 8MP-treated groups, respectively. Therefore, 8MP treatment significantly hyperpolarized RPs (Figure 7(a), n = 18 and 22 cells from NS- and MT-treated groups, respectively, *p < 0.05 compared to NS, two-sample t-test). The rheobase measurements were 0.017 ± 0.003 and 0.035 ± 0.006 nA for the NS- and 8MP-treated groups, respectively. The administration of 8MP markedly increased rheobase (Figure 7(b), n = 18 and 22 cells from NS- and 8MP-treated groups, respectively, **p < 0.01 compared to NS, two-sample t-test). The action potential (AP) thresholds were −34.2 ± 0.8 and −29.7 ± 0.6 mV for NS- and 8MP-treated groups, respectively. The 8MP treatment also significantly increased the AP thresholds (Figure 7(c), n = 18 and 22 cells from NS- and 8MP-treated groups, respectively, *p < 0.05 compared to NS, two-sample t-test). 8MP treatment also greatly decreased the number of APs evoked by 2X and 3X rheobase current stimulation (Figures 7(d) and (f), n = 18 and 22 cells from NS and 8MP-treated groups, respectively, *p < 0.05 compared to NS, Mann-Whitney test). The numbers of AP evoked by 2X rheobase current stimulation were 2.3 ± 0.2 and 1.75 ± 0.25 for NS- and 8MP-treated groups, respectively. The numbers of AP evoked by 3X rheobase current stimulation were 3.3 ± 0.5 and 2.3 ± 0.2 for NS- and 8MP-treated groups, respectively. In addition, 8MP treatment greatly reduced the number of APs evoked by 100, 300 and 500 pA ramp current stimulation. The numbers of APs evoked by 100 pA ramp current stimulation were 11.4 ± 1.0 and 3.9 ± 0.5 for the NS- and 8MP-treated groups, respectively. The numbers of APs evoked by 300 pA ramp current stimulation were 20.4 ± 1.8 and 11.8 ± 1.6 for the NS- and 8MP-treated groups, respectively. The numbers of APs evoked by 500 pA ramp current stimulation were 26.6 ± 2.3 and 18.7 ± 2.8 for the NS- and 8MP-treated groups, respectively. 8MP treatment remarkably reduced the numbers of APs in response to ramp current stimulation (Figures 7(e) and (g), n = 18 and 22 cells from NS- and 8MP-treated groups, respectively, **p < 0.01 compared to NS, Mann-Whitney test).OPEN IN VIEWER

8MP reduces TTX-R sodium current density and modulates dynamics of TTX-R sodium current activation

Since melatonin reduces TTX-R sodium current density and modulates sodium current activation of colon-specific DRG neurons of NCI rats, we next investigated whether 8MP treatment suppressed current density of VGSCs in colon-specific DRG neurons. NCI+NS group (n = 4 rats), NCI+8MP group (n = 4 rats). The total sodium current density was −232 ± 20.6 pA/pF (n = 12 cells) and −156 ± 15.5 pA/pF (n = 10 cells) for NS- and 8MP-treated group, respectively. 8MP treatment significantly reduced total sodium current density when compared with that of NS group (Figure 8(a), (b) and (c), *p < 0.05, Mann-Whitney test). To further determine the roles for 8MP on TTX-R current densities, TTX-R sodium currents were recorded in the presence of 0.5 μM TTX. The TTX-R sodium current density was −139 ± 16.5 pA/pF (n = 12 cells) and −92 ± 21.3 pA/pF (n = 10 cells) for NS- and 8MP-treated group, respectively 8MP treatment significantly reduced TTX-R sodium current density when compared with that of NS group (Figure 8(d), (e) and (f), *p < 0.05, Mann-Whitney test). Because inhibition of peak current density of TTX-R sodium current was evident, we next characterized the effect of 8MP on voltage dependence of TTX-R currents of colon-specific DRG neurons. Activation dependence was constructed from I-V curves of neurons from NS and 8MP-treated NCI rats (Figure 8(g)). 8MP treatment led to a ∼10 mV positive shift in the steady state activation curve. The shift of the activation curve in a more depolarizing direction prevents the channel to open in response to weaker depolarization. We next determined the effect of 8MP on voltage dependence of steady-state inactivation of TTX-R sodium channels of colon-specific DRG neurons. 8MP treatment did not significantly alter the inactivation curve (Figure 8(h)), indicating the same channel availability in the potential domain between −50 mV and −10 mV of colon-specific DRG neurons from these two groups of rats.OPEN IN VIEWER