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

Original data for the graphs in Figs 2 and S3.

Each tab includes data for individual panels of Figs 2 and S3. (XLSX)</p

Summary of GABA_B-induced hyperpolarization of arcuate NPY neurons.

Summary of GABAB-induced hyperpolarization of arcuate NPY neurons.</p

Original data for the graphs in Figs 7 and S10.

Each tab includes data for individual panels of Figs 7 and S10. (XLSX)</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 Figs 4 and S7 and S8.

Each tab includes data for individual panels of Figs 4 and S7 and S8. (XLSX)</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