GIRK channels stabilize RMP of NPY neurons.

(A) Brightfield illumination (Brightfield), fluorescent (FITC) illumination (Npy-hrGFP), fluorescent (TRITC) illumination (Alexa Fluor 594), and merged (Merge) images of targeted NPY neuron. Arrows indicate the cell targeted for whole-cell patch clamp recording. (B) Image demonstrates a depolarizing effect of tertiapin-Q. Dotted line indicates RMP. (C) Voltage deflections in response to small hyperpolarizing current steps (from −25 pA to 0 pA by 5 pA increments) before (control, black) and after (tertiapin-Q, red) the perfusion with tertiapin-Q as indicated by arrows in (B). (D) The voltage–current (V-I) relationship demonstrates increased input resistance by tertiapin-Q. Erev = reversal potential. (E) Lines and dots summarize effects of tertiapin-Q on RMP (from −47.7 ± 3.0 mV to −44.9 ± 2.1 mV, n = 11, df = 10, t = 2.787, p = 0.019). Red and black lines indicate changes of membrane potential in depolarized and nonresponsive neurons, respectively. (F) Lines and dots summarize effect of tertiapin-Q on input resistance (from 2.75 ± 0.27 GΩ to 3.03 ± 0.30 GΩ, n = 11, df = 10, t = 4.370, p = 0.001). Red and black lines indicate changes of input resistance in depolarized and nonresponsive neurons, respectively. (G, H) Lines and dots summarize effects of 100 nM tertiapin-Q (G) (from −41.2 ± 0.8 mV to −40.0 ± 1.1 mV, n = 11, df = 10, t = 2.040, p = 0.069) and 500 nM tertiapin-Q (H) (from −42.9 ± 1.2 mV to −40.5 ± 1.1 mV, n = 13, df = 12, t = 3.292, p = 0.006) on RMP. Red and black lines indicate changes of membrane potential in depolarized and nonresponsive neurons, respectively. (I) Histogram summarizes responses (no effects or depolarization) of NPY neurons to different concentrations of tertiapin-Q. (J) Bar graphs and dots summarize effects of K+ channel blockers. Each neuron was tested with only 1 K+ channel blocker. Data are presented as mean ± SEM. Paired t test was used for statistical analyses. *p p S1 Data. GIRK, G protein-gated inwardly rectifying K+; NPY, neuropeptide Y; RMP, resting membrane potential.</p

Dominant expression of <i>Girk2</i> over <i>Girk1</i> by the arcuate AgRP neurons.

(A) Image demonstrates DAPI (blue) and mRNA of Agrp (white), Girk1 (green), and Girk2 (magenta) detected by FISH experiments within the arcuate nucleus. 3V = third ventricle. Scale bar = 50 μm. (B) Magnified images of red rectangular area in (A). Dotted circles indicate Agrp (+) neurons (white) with Girk1 (green), Girk2 (magenta), or both Girk1 and Girk2 (yellow) mRNA. Scale bar = 10 μm. (C) Bar graph demonstrates numbers of Agrp (+) neurons in the arcuate nuclei of 3 wild-type mice. (D) Venn diagram demonstrates the numbers of Girk1- and/or Girk2-expressing Agrp (+) neurons. Data were pooled from neurons of 3 mice shown in (C), and 12 hypothalamic slices from each mouse (from bregma −1.58 mm to −2.02 mm) were included for analyses. The numerical data for Fig 2C can be found in S2 Data. AgRP, agouti-related peptide; FISH, fluorescence in situ hybridization; GIRK, G protein-gated inwardly rectifying K+.</p

Expression of <i>Girk</i> mRNA by arcuate AgRP neurons.

Related to Fig 2. (A) Graph demonstrates percentage of Agrp (+) neurons that express mRNA of Girk1 and/or Girk2. Girk1 (green): Girk1-containing Agrp (+) neurons; Girk2 (magenta): Girk2-containing Agrp (+) neurons; Girk1 and Girk2 (gray): Agrp (+) neurons containing both Girk1 and Girk2. n = 3. (B) Graph demonstrates percentage of Agrp (+) neurons that express mRNA of Girk1 and/or Girk3. Girk1 (green): Girk1-containing Agrp (+) neurons; Girk3 (cyan): Girk3-containing Agrp (+) neurons; Girk1 and Girk3 (gray): Agrp (+) neurons containing both Girk1 and Girk3. n = 3. (C) Graph demonstrates percentage of Agrp (+) neurons that express mRNA of Girk1 and/or Girk4. Girk1 (green): Girk1-containing Agrp (+) neurons; Girk4 (orange): Girk4-containing Agrp (+) neurons; Girk1 and Girk4 (gray): Agrp (+) neurons containing both Girk1 and Girk4. n = 3. (D) Graph demonstrates percentage of Agrp (+) neurons that express mRNA of Girk2 and/or Girk3. Girk2 (magenta): Girk2-containing Agrp (+) neurons; Girk3 (cyan): Girk3-containing Agrp (+) neurons; and Girk2 and Girk3 (gray): Agrp (+) neurons containing both Girk2 and Girk3. n = 3. Data are presented as mean ± SEM. Twelve hypothalamic slices from each mouse (from bregma −1.58 mm to −2.02 mm) were included for analyses. See text for specific values. The numerical data for S3A–S3D Fig can be found in S2 Data. (TIF)</p

Locomotion and anxiety-like behavior of GIRK2^WT and GIRK2^AgRP-KO mice.

Related to Fig 6. (A) Bar graphs and dots summarize ambulatory movement of GIRK2WT (n = 14) and GIRK2AgRP-KO (n = 16) mice (260.8 ± 48.5 counts, n = 14, for GIRK2WT and 256.9 ± 48.8 counts, n = 16, for GIRK2AgRP-KO, df = 28, t = 0.057, p = 0.955 in dark cycle; 58.0 ± 10.3 counts, n = 14, for GIRK2WT and 51.6 ± 8.1 counts, n = 16, for GIRK2AgRP-KO, df = 28, t = 0.4921, p = 0.627 in light cycle). (B) Bar graphs and dots summarize rearing activity of GIRK2WT (n = 14) and GIRK2AgRP-KO (n = 16) mice (148.3 ± 27.1 counts, n = 14, for GIRK2WT and 146.3 ± 32.0 counts, n = 16, for GIRK2AgRP-KO, df = 28, t = 0.049, p = 0.962 in dark cycle; 26.7 ± 9.6 counts, n = 14, for GIRK2WT and 18.7 ± 3.9 counts, n = 16, for GIRK2AgRP-KO, df = 28, t = 0.804, p = 0.428 in light cycle). (C) Trajectory of freely moving GIRK2WT (n = 8) and GIRK2AgRP-KO (n = 9) mice in the OFT chamber in dark and light cycles. (D) Bar graphs and dots summarize total moving distance of GIRK2WT (n = 8) and GIRK2AgRP-KO (n = 9) mice (95.1 ± 9.0 m, n = 8, for GIRK2WT and 108.1 ± 4.1 m, n = 9, for GIRK2AgRP-KO, df = 15, t = 1.370, p = 0.191 in dark cycle; 113.8 ± 6.6 m, n = 8, for GIRK2WT and 123.9 ± 7.8 m, n = 9, for GIRK2AgRP-KO, df = 15, t = 0.980, p = 0.343 in light cycle). (E) Image demonstrates a view of chamber by a camera that is installed on the ceiling of sound-proof booths. (F) Heat-maps demonstrate zone preference of GIRK2WT and GIRK2AgRP-KO mice in the chamber. (G) Bar graphs and dots summarize proportions of duration in center and outer zones of GIRK2WT (n = 8) and GIRK2AgRP-KO (n = 9) mice (10.6 ± 1.8%, n = 8, for GIRK2WT and 8.4 ± 0.6%, n = 9, for GIRK2AgRP-KO, df = 15, t = 1.224, p = 0.240 in dark cycle and center; 13.4 ± 1.4%, n = 8, for GIRK2WT and 12.3 ± 1.6%, n = 9, for GIRK2AgRP-KO, df = 15, t = 0.523, p = 0.609 in light cycle and center; 89.4 ± 1.8%, n = 8, for GIRK2WT and 91.6 ± 0.6%, n = 9, for GIRK2AgRP-KO, df = 15, t = 1.224, p = 0.240 in dark cycle and outer; 86.6 ± 1.4%, n = 8, for GIRK2WT and 87.8 ± 1.6%, n = 9, for GIRK2AgRP-KO, df = 15, t = 0.523, p = 0.609 in light cycle and outer). Data are presented as mean ± SEM. Unpaired t test was used for statistical analyses. ns = not significant. The numerical data for S9A, S9B, S9D, and S9G Fig can be found in S6 Data. (TIF)</p

Effects of K⁺ channel blockers on RMP of NPY neurons.

Related to Fig 1. (A) Trace demonstrates depolarizing effects of linopirdine and XE991, M channels blockers. (B) Trace demonstrates no effects of PK-THPP, a TASK-3 channel blocker. (C) Trace demonstrates no effects of spadin, a TREK-1 channel blocker. (D) Trace demonstrates no effects of tolbutamide, a KATP channel blocker. (E–H) Bar graphs and dots summarize effects on RMP change of linopirdine and XE991 (from −40.4 ± 0.7 mV to −39.5 ± 0.7 mV, n = 12, df = 11, t = 1.650, p = 0.127) (E), PK-THPP (from −42.5 ± 1.0 mV to −42.1 ± 0.8 mV, n = 12, df = 11, t = 0.890, p = 0.393) (F), spadin (from −41.9 ± 1.1 mV to −42.3 ± 1.0 mV, n = 13, df = 12, t = 1.866, p = 0.087) (G), and tolbutamide (from −42.2 ± 0.7 mV to −41.7 ± 0.8 mV, n = 13,df = 12, t = 1.879, and p = 0.085) (H). Red and black lines indicate changes of membrane potential in depolarized and nonresponsive neurons, respectively. Data are presented as mean ± SEM. Paired t test was used for statistical analyses. ns = not significant. The numerical data for S2E–S2H Fig can be found in S1 Data. (TIF)</p

Effects of CGP54626 on NPY^G2WT neurons.

Related to Fig 3. (A) Image demonstrates no effects of CGP54626 on NPYG2WT neurons. Dotted line indicates RMP. (B) Lines and dots summarize effects of CGP54626 on RMP (from −42.9 ± 0.8 mV to −43.2 ± 0.8 mV, n = 12, df = 11, t = 2.191, p = 0.051). (C) Lines and dots summarize effect of CGP54626 on input resistance (from 2.68 ± 0.20 GΩ to 2.71 ± 0.21 GΩ, n = 12, df = 11, t = 0.519, p = 0.614). Paired t test was used for statistical analyses. ns = not significant. The numerical data for S6B and S6C Fig can be found in S3 Data. (TIF)</p

Contribution of GIRK2-containing channels to RMP and GABA_B-induced inhibition of NPY neurons.

(A) Traces demonstrate spontaneous firing and RMP of NPYG2WT (black) and NPYG2KO (red) neurons. Dotted line indicates RMP. (B, C) Bar graphs and dots summarize RMP (−47.9 ± 0.9 mV, n = 64, for NPYG2WT and −44.5 ± 0.7 mV, n = 41, for NPYG2KO, df = 103, t = 2.556, p = 0.012) (B) and input resistance (2.35 ± 0.11 GΩ, n = 64, for NPYG2WT and 2.78 ± 0.11 GΩ, n = 41, for NPYG2KO, df = 103, t = 2.590, p = 0.011) (C) of NPYG2WT (n = 64, black) and NPYG2KO (n = 41, red) neurons. (D) Image demonstrates a hyperpolarization of NPYG2WT neuron membrane potential by baclofen (10 μm). Arrows indicate interruptions to apply current step pulses. (E) Small hyperpolarizing current steps (from −50 pA to 0 pA by 10 pA increments) were applied before (control) and after (baclofen) applications of baclofen. (F) Voltage–current relationship demonstrates decreased input resistance and Erev close to EK. (G) Image demonstrates a hyperpolarization of NPYG2KO neuron membrane potential by baclofen (10 μm). (H) Summary of GABAB-induced hyperpolarization of NPYG2WT (black) and NPYG2KO (red) neurons. Changes of membrane potential by 10 μm baclofen was −11.9 ± 2.2 mV for NPYG2WT (n = 14) and −20.9 ± 2.4 mV for NPYG2KO (n = 8) (df = 20, t = 2.655, p = 0.015). Solid lines indicate fitting of dose-response curve (Hill slope = 1.0, Y = Bottom + (Top-Bottom)/(1+10^(logEC50-X)). Both hyperpolarizing and no responses were included for analyses. See Table 1 for hyperpolarizing responses only. Data are presented as mean ± SEM. Unpaired t test was used for statistical analyses. *p S3 Data. GIRK, G protein-gated inwardly rectifying K+; NPY, neuropeptide Y; RMP, resting membrane potential.</p

Role of GIRK2-containing GIRK channels in GABA_B-activated K⁺ current recorded from NPY neurons.

Related to Fig 3. (A) Image demonstrates outward currents by local application of 100 μm baclofen. Voltage ramp pulses (from −120 mV to −10 mV, 100 mV/s) were applied as indicated by arrows, a and b, to obtain current responses, Ia and Ib. (B) Image demonstrates current–voltage (I-V) relationship of baclofen-activated currents (IBac); IBac was calculated by subtracting current responses (Ib- Ia) obtained in (A). (C) Rectification index was calculated by obtaining the ratio of amplitudes at −120 mV (I-120 mV) and −60 mV (I-60 mV) in 12 NPY neurons. (D, E) Images demonstrate IBac recorded from NPYG2WT (black) and NPYG2KO (red) neurons using 10 μm (D) or 100 μm (E) baclofen. (F, G) Image summarizes normalized amplitudes of IBac recorded from NPYG2WT (black) and NPYG2KO (red) neurons using 10 μm baclofen (1.4 ± 0.1 pA/pF, n = 32, for NPYG2WT and 1.4 ± 0.1 pA/pF, n = 23, for NPYG2KO, df = 53, t = 0.276, p = 0.783) (F) and 100 μm baclofen (1.8 ± 0.1 pA/pF, n = 53, for NPYG2WT and 1.8 ± 0.2 pA/pF, n = 26, for NPYG2KO, df = 77, t = 0.021, and p = 0.984) (G). Data are presented as mean ± SEM. Unpaired t test was used for statistical analyses. ns = not significant. The numerical data for S4C, S4F, and S4G Fig can be found in S3 Data. (TIF)</p

Deletion of GIRK2, but not GIRK1, leads to increased Fos expression by the arcuate AgRP neurons.

(A) Images demonstrate Fos IHC results from AgrptdTomato, AgrptdTomato/Girk1KO, and AgrptdTomato/Girk2KO mice, as indicated. 3V = third ventricle. Scale bar = 50 μm. (B) Bar graphs and dots summarize proportion of Fos-expressing AgRP neurons in AgrptdTomato (56.0 ± 3.2%, n = 6, black), AgrptdTomato/Girk1KO (64.6 ± 2.6%, n = 4, gray), and AgrptdTomato/Girk2KO (71.7 ± 4.8%, n = 4, red). Twelve hypothalamic slices from each mouse (from bregma −1.46 mm to −2.06 mm) were included for analyses. Data are presented as mean ± SEM. Ordinary one-way ANOVA with Bonferroni correction was used for statistical analyses (df = 2, F2, 11 = 4.961, p = 0.029). *p S4 Data. AgRP, agouti-related peptide; GIRK, G protein-gated inwardly rectifying K+; IHC, immunohistochemistry.</p

Original data for the graphs in Fig 5.

Each tab includes data for individual panels of Fig 5. (XLSX)</p