Glutamate-induced Ca²⁺ influx does not interact with DHPG-induced Ca²⁺ mobilization.

<p>(A) Ca²⁺ influx by glutamate was determined by comparing the amplitudes of two glutamate-induced Ca²⁺ transients in the presence or absence MPEP, which blocks DHPG-induced Ca²⁺ mobilization (blue box), and was compared to Ca²⁺ mobilization (red box). (B) Ca²⁺ influx by glutamate was determined by comparing the amplitudes of two glutamate-induced Ca²⁺ transients in the presence or absence of ryanodine, which blocks DHPG-induced Ca²⁺ mobilization (blue box), and was compared to Ca²⁺ mobilization (red box). (C) Bar graphs summarizing the relative contribution of Ca²⁺ mobilization (red), Ca²⁺ influx (blue) and estimated supralinear Ca²⁺ mobilization (black) in both conditions. (D) Ca_Glu was not inhibited by U73122. Scale bars indicate 10 sec (horizontal) and 50 nM (vertical).</p

Glutamate-induced Ca²⁺ transients are significantly larger than DHPG-induced Ca²⁺ transients.

<p>(A) Cells were treated with both DHPG (50 µM) and glutamate (30 µM). (B) Bar graphs summarizing the average amplitudes of DHPG- and glutamate-induced Ca²⁺ transients from 74 cells. Scale bars indicate 10 sec (horizontal) and 50 nM (vertical). Glu = glutamate. ** indicates <i>p</i><0.01.</p

L-type Ca²⁺ channels are responsible for glutamate-induced Ca²⁺ influx.

<p>(A) Ca_Glu was greatly inhibited in the presence of nimodipine. (B) Bar graphs represent the ratio between first and second Ca_Glu. Scale bars indicate 10 sec (horizontal) and 50 nM (vertical). Nimo = nimodipine, Cono = ω-conotoxin GVIA, Aga = ω-agatoxin IVA. ** indicates <i>p</i><0.01.</p

DHPG-induced intracellular Ca²⁺ mobilization occurs via the cADPR/RyR signaling pathways.

<p>DHPG-induced Ca²⁺ transients were completely inhibited when cells were pretreated with 5 mM nicotinamide (A), 100 µM 8-NH₂-cADPR (B), or 20 µM ryanodine (C) prior to the second application of DHPG. (D) Bar graphs represent the relative peak amplitude of second Ca²⁺ transients to those of first (Ca_DHPG,2/Ca_DHPG,1). Scale bars indicate 10 sec (horizontal) and 50 nM (vertical). EP = electroporation, NA = nicotinamide, cADPR = 8-NH₂-cADPR, RYA = ryanodine. ** indicates <i>p</i><0.01.</p

mGluR5 activation by DHPG leads to intracellular Ca²⁺ mobilization.

<p>Acutely dissociated rat hippocampal CA1 neurons were loaded with 2 µM Fura 2-AM for Ca²⁺ measurements. Cells were pretreated with 25 µM MPEP (A), 100 µM LY367385 (B), 0 Ca²⁺ external solutions (C) or 2 µM thapsigargin (D) prior to the second application of DHPG (50 µM). (E) Bar graphs represent the relative peak amplitude of second Ca²⁺ transients to those of first (Ca_DHPG,2/Ca_DHPG,1). Scale bars indicate 10 sec (horizontal) and 50 nM (vertical). CTRL = control, LY = LY367385, SKF = SKF96365, TG = thapsigargin. ** indicates <i>p</i><0.01.</p

AMPA receptors, but not NMDA receptors, are responsible for glutamate-induced Ca²⁺ influx.

<p>(A) The amplitudes of glutamate-induced Ca²⁺ transients (Ca_Glu) were significantly attenuated in Ca²⁺-free solutions. Ca_Glu was not affected by AP-5 (B), but was significantly decreased by the pretreatment with CNQX (C). (D) Ca_Glu was not inhibited by NASPM. (E) Bar graphs represent the ratio between first and second Ca_Glu. Scale bars indicate 10 sec (horizontal) and 50 nM (vertical). ** indicates <i>p</i><0.01.</p

PLC/IP₃R signaling pathways are not involved in DHPG-induced Ca²⁺ mobilization.

<p>U73122 (1 µM), a PLC inhibitor, did not inhibit induction of Ca²⁺ transients by DHPG (A), but blocked those induced by the muscarinic receptor agonist CCh (10 µM, B). (C) The Ca₂/Ca₁ ratio was 90.0±0.9% (n = 4) for CCh. It was 100.4±5.3% (n = 6) for DHPG and 4.8±1.0% (n = 6) for CCh when pretreated with U73122. (D) Cells were loaded with heparin (20 mg/ml) by single cell electroporation. Alexa Fluor-488 was added to the pipette solutions to confirm successful loadings. (E) CCh-induced Ca²⁺ transients were almost completely inhibited in cells loaded with heparin, a competitive antagonist of the IP₃Rs, whereas DHPG still caused Ca²⁺ transients in cells loaded with heparin. (F) The Ca_DHPG,2/Ca_DHPG,1 ratio was 97.1±24.6% (n = 4) and 103.2±14.7% (n = 4) when cells were patched with pipette solutions with or without heparin and were voltage-clamped at −60 mV at the second DHPG application. (G) DHPG still induced Ca²⁺ transients in cells isolated from PLCβ1 (upper) or PLCβ4 (lower) knockout mice. Scale bars indicate 10 sec (horizontal) and 50 nM (vertical). ** indicates <i>p</i><0.01.</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

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

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