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

Original data for the graphs in Figs 2 and S3.

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

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

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

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