Serotonin 5-HT2C receptor-mediated inhibition of the M-current in hypothalamic POMC neurons

Abstract

Hypothalamic proopiomelanocortin (POMC) neurons are controlled by many central signals, including serotonin. Serotonin increases POMC activity and reduces feeding behavior via serotonion [5-hydroxytryptamine (5-HT)] receptors by modulating K(+) currents. A potential K(+) current is the M-current, a noninactivating, subthreshold outward K(+) current. Previously, we found that M-current activity was highly reduced in fasted vs. fed states in neuropeptide Y neurons. Because POMC neurons also respond to energy states, we hypothesized that fasting may alter the M-current and/or its modulation by serotonergic input to POMC neurons. Using visualized-patch recording in neurons from fed male enhanced green fluorescent protein-POMC transgenic mice, we established that POMC neurons expressed a robust M-current (102.1 ± 6.7 pA) that was antagonized by the selective KCNQ channel blocker XE-991 (40 μM). However, the XE-991-sensitive current in POMC neurons did not differ between fed and fasted states. To determine if serotonin suppresses the M-current via the 5-HT(2C) receptor, we examined the effects of the 5-HT(2A)/5-HT(2C) receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) on the M-current. Indeed, DOI attenuated the M-current by 34.5 ± 6.9% and 42.0 ± 5.3% in POMC neurons from fed and fasted male mice, respectively. In addition, the 5-HT(1B)/5-HT(2C) receptor agonist m-chlorophenylpiperazine attenuated the M-current by 42.4 ± 5.4% in POMC neurons from fed male mice. Moreover, the selective 5-HT(2C) receptor antagonist RS-102221 abrogated the actions of DOI in suppressing the M-current. Collectively, these data suggest that although M-current expression does not differ between fed and fasted states in POMC neurons, serotonin inhibits the M-current via activation of 5-HT(2C) receptors to increase POMC neuronal excitability and, subsequently, reduce food intake.

Fig. 1.

The M-current does not run down in proopiomelanocortin (POMC) neurons. A: whole cell patch-clamp recording of deactivation of the M-current in POMC neurons. From a holding potential of −60 mV, a voltage jump to −20 mV (300 ms) was followed by steps from −30 to −75 mV in 5-mV increments (500 ms) (31). B: K⁺ currents evoked by the deactivation protocol do not run down over the 30-min recording period in 3 cells. T = 0, 3 min after whole cell access. In 1 cell, 20 μM 2,5-dimethoxy-4-iodoamphetamine (DOI) was perfused for 15 min; then 20 μM XE-991 was perfused for 15 min to demonstrate the efficacy of DOI and the activity of the M-current.

Fig. 2.

XE-991 (40 μM)-sensitive M-current in POMC neurons is not affected by fasting. A: currents were recorded under control conditions and after 10 min of perfusion of 40 μM XE-991 in the presence of 0.5 μM TTX in fed and 24-h-fasted male mice. B: XE-991 (XE) depolarized POMC neurons significantly in fed and fasted male mice. RMP, resting membrane potential. *P < 0.05; **P < 0.01 (by Student's t-test).

Fig. 3.

Lower concentrations of XE-991 illustrate a potential difference in KCNQ subunit composition in POMC neurons between fed and fasted male mice. A: representative traces of deactivation protocol in POMC neurons from fed and fasted male mice before and after perfusion of 20 μM XE-991. B: as in Fig. 2A, currents were recorded under control conditions and after 10 min of perfusion of 20 μM XE-991 in fed and 24-h-fasted male mice. Current-voltage plots were analyzed by 2-way ANOVA (P < 0.05, F = 4.5, df = 1) followed by Bonferroni-Dunn multiple comparison tests: *P < 0.05; **P < 0.01; ***P < 0.001 vs. fed. C: 20 μM XE-991 depolarized POMC neurons significantly in fed and fasted male mice. **P < 0.01; ***P < 0.001 (by Student's t-test). D: maximum XE-991-sensitive peak current at −35 mV in fed and fasted male mice perfused with 20 or 40 μM XE-991. Data were analyzed by 1-way ANOVA (P < 0.0001, F = 21.1, df = 3) followed by Bonferroni-Dunn multiple comparison tests: **P < 0.01; ****P < 0.0001.

Fig. 4.

Agonists of serotonin [5-hydroxytryptamine (5-HT)] type 2C (5-HT_2C) receptors suppress activity of the M-current in POMC neurons from fed male mice. A: DOI suppressed activity of the M-current in 19 of 22 POMC neurons from fed male mice. Currents were recorded in control conditions and after 10 min of perfusion with 20 μM DOI. A subsample of DOI-inhibited cells was perfused for 10 min with 20 μM XE-991. Current-voltage plots were analyzed by 2-way ANOVA (P < 0.001, F = 9.8, df = 2) followed by Bonferroni-Dunn multiple comparison tests: **P < 0.01; ***P < 0.001; ****P < 0.0001 vs. control. B: representative traces of deactivation protocol in POMC neurons from fed male mice before and after perfusion with 20 μM DOI. C: perfusion for 10 min with another 5-HT_2C receptor agonist, m-chlorophenylpiperazine (mCPP, 20 μM), suppressed the M-current in 7 of 8 POMC neurons from fed male mice. Plots were analyzed by 2-way ANOVA (P < 0.05, F = 6.9, df = 1) followed by Bonferroni-Dunn multiple comparison tests: *P < 0.05; ***P < 0.001. D: representative traces of deactivation protocol in POMC neurons from fed male mice before and after perfusion with 20 μM mCPP. E: to determine if the 5-HT_2C receptor is mediating the effects of DOI, slices were incubated for 30 min with a selective antagonist to the 5-HT_2C receptor, RS-102221 (RS, 20 μM), prior to 10 min of coperfusion with 20 μM DOI. In the presence of RS-102221, DOI did not suppress the M-current in POMC neurons (n = 8 cells). Data (control vs. DOI) were analyzed by 2-way ANOVA (P < 0.01, F = 31.7, df = 1) followed by Bonferroni-Dunn multiple comparison tests: **P < 0.01; ***P < 0.001; ****P < 0.0001 vs. control. F: change in maximum peak current (−35 mV) after perfusion of DOI (n = 19 cells), DOI + RS-102221 (RS102, n = 8 cells), or mCPP (n = 7 cells) in fed male mice. Data were analyzed by 1-way ANOVA (P < 0.01, F = 6.6, df = 2) followed by Bonferroni-Dunn multiple comparison tests: **P < 0.01 vs. DOI and mCPP.

Fig. 5.

DOI suppresses the M-current in fasted male mice similar to fed male mice, and expression of DOI-sensitive M-current is regionally dependent in arcuate POMC neurons. A: in fasted male mice, 20 μM DOI suppressed M-current activity. After DOI treatment, 20 μM XE-991 was perfused for 10 min. Current-voltage plots were analyzed by 2-way ANOVA (P < 0.0001, F = 17.5, df = 2) followed by Bonferroni-Dunn multiple comparison tests: **P < 0.01; ***P < 0.001; ****P < 0.0001 vs. control. B: percentage of control current suppressed by DOI at −35 mV in POMC neurons from fed and fasted male mice. C: DOI did not significantly suppress more M-current after XE-991 perfusion in POMC neurons from fed male mice. D: amount of M-current suppressed by DOI (DOI-sensitive current) is dependent on the region of the arcuate (Arc) nucleus. Suppression of the M-current was more robust in the caudal (n = 7 cells) and middle (n = 16 cells) regions than in the rostral region (n = 8 cells). Current-voltage plots were analyzed by 2-way ANOVA comparing arcuate regions (P < 0.01, F = 7.3, df = 2) followed by Bonferroni-Dunn multiple comparison tests: *P < 0.05; **P < 0.01; ***P < 0.001 vs. caudal.

Fig. 6.

Effects of XE-991 and the transient receptor potential channel (TRPC) blocker 2-aminoethoxydiphenyl borate (2-APB) on DOI-induced depolarization and firing in POMC neurons. A: in current clamp, perfusion with 20 μM DOI significantly depolarized POMC neurons from fed male mice after 10 min by 7.3 ± 0.6 mV (n = 3) and induced firing. B: coperfusion of DOI and 40 μM XE-991, which fully inhibited the M-current, caused a depolarization of 10.5 ± 0.4 mV (n = 3) and robustly increased firing. C: DOI also depolarized POMC neurons (n = 4) in the presence of 100 μM 2-APB by 5.8 ± 0.9 mV and increased firing. D: DOI + XE-991 perfusion significantly depolarized membrane potential compared with DOI alone and DOI + 2-APB. Data were analyzed by 1-way ANOVA (P < 0.01, F = 11.2, df = 2) followed by Bonferroni-Dunn multiple comparison tests: *P < 0.05; **P < 0.01.