Fig 1.
Effects of ghrelin (5 nM) on membrane potential (MP in mV) and input resistance (IR in MΩ) of NPY GFP neurons in VMH slices.
The ΔMP (= MP+ghrelin - MPw/o ghrelin) and ΔIR (= IR+ghrelin - IRw/o ghrelin) values are plotted for individual neurons without (A) or with (B) added TTX (500 nM). (C) Unpaired, two-tailed t-test results for ghrelin-induced % Changes in MP and IR ± TTX in NPY-GFP neurons with decreased IR. (D) Paired, two-tailed t-test results indicate ghrelin can directly reduce the IR of NPY-GFP neurons when presynaptic inputs are blocked by TTX.
Fig 2.
Ghrelin directly activates NPY-GFP neurons.
(A) Representative field of isolated neurons from NPY-GFP mice. (B) Same field after addition of ghrelin. (C) Merged image of fields (A) and (B). Arrows point to non-NPY cells. (D) Average values (n = 4) of Relative [Ca2+]c after sequential addition of indicated concentrations of acylated ghrelin. (E) Ghrelin dose-response curve. (F) Ghrelin induced [Ca2+]c increases are blocked by the GHS-R antagonist [D-lys3]-GHRP-6 (50 μM). (G) CNQX (2 μM), an AMPA receptor antagonist, did not affect ghrelin action. Ghrelin (200 pM; red bars) was applied in F and G. Traces are mean values from 6–12 neurons.
Fig 3.
Ghrelin stimulation of GHS-R in isolated NPY-GFP neurons activates both adenylate cyclase (AC)-PKA and PLC-IP3 signaling pathways.
(A) The adenylate cyclase agonist, Forskolin (10 μM), mimics the effect of ghrelin. (B) The PKA inhibitor, H89 (10 μM) inhibits the effect of ghrelin. (C) U73122 (1 μM), an irreversible PLC inhibitor, reduced ghrelin action. (D) Thapsigargin (0.5 μM), an inhibitor of SERCA, the ER Ca2+ pump, did not inhibit ghrelin induced [Ca2+]c increases. Ghrelin was applied at 100 pM (red bars). Traces are mean values from 5–20 cells.
Fig 4.
AMPK and Ca2+/calmodulin (CaM)-dependent protein kinase kinases (CaMKK2) contribute to the ghrelin activation of NPY neurons.
(A) AICAR (400 μM), an AMPK activator, mimics the effect of ghrelin. (B) Compound-C (30 μM), an inhibitor of AMPK, completely blocks the effect of oligomycin. (C) STO-609 (1 μM), a CAMKK2 inhibitor, partially reverses the effect of ghrelin. Ghrelin was applied at 100 pM (red bars). Traces are mean values from 5–20 cells.
Fig 5.
GABA inhibits the stimulatory effects of acylated ghrelin (100 pM) in a dose-dependent manner.
The trace is the mean from 12 neurons.
Fig 6.
Extracellular Na+ influx is required to sustain ghrelin activation.
(A) Tetrodotoxin (TTX; 1 μM), an inhibitor of voltage-dependent Na+ channels, did not affect ghrelin induced [Ca2+]c increases. (B) The substitution of choline for Na+ rapidly and reversibly blocked the action of ghrelin. Ghrelin was applied at 200 pM (red bars). The traces are the mean values from 5–20 neurons.
Fig 7.
KATP channel modulators have SUR1 dependent effects on isolated NPY-GFP neurons.
(A) Diazoxide (200 μM) did not affect ghrelin action. (B) When applied alone, diazoxide (200 μM) mimics the effect of ghrelin, while glibenclamide (1μM) reduced the stimulatory effect of ghrelin. (C-D) Neither diazoxide (200 μM) nor glibenclamide (1μM) affected ghrelin action in SUR1-/- mice lacking SUR1. Ghrelin was applied at 200 pM as shown (red bars). (E) Comparison of the percentages of all ghrelin-activated cells in preparations of neurons isolated from SUR1-/- versus NPY-GFP mice shows the mean values are not significantly different, p > 0.7, using an unpaired, two-tailed t-test. About two-thirds of the activated neurons in NPY-GFP mouse preparations were GFP-positive. Values are the means ± S.D. from four NPY-GFP and six SUR1-/- preparations totaling more than 1000 cells.
Fig 8.
Antagonists of Trpm4 channels inhibit ghrelin action on isolated NPY-GFP neurons.
(A) 9-phenanthrol (50 μM) and (B) flufenamic acid (100 μM) potently block the effects of ghrelin (200 pM; red bars).
Fig 9.
NYP-expressing neurons of the arcuate nucleus express subunits of both SUR1-Kir6.2 (KATP) and SUR1-Trpm4 channels.
Coronal sections of the hypothalamus from an NPY-GFP mouse, showing NPY-GFP neurons (green) immunolabeled for SUR1(A), or Kir6.2 (B) or Trpm4 (C), as indicated (red); merged images show co-expression of GFP and all three channel subunits by NPY neurons (yellow) DAPI nuclear labeling shown in blue. The images (low power view, upper panels, and high power view, lower panels) shown are representative of findings in 2 NPY-GFP mice.
Fig 10.
Blockade of T-type and R-type voltage dependent channels reduces the effect of ghrelin.
(A) Ni2+ (100 μM), an inhibitor of T and R-type channels, and 5 μM mibefradil, an inhibitor of T and L type channels, inhibit the effect of ghrelin. (B) TTA-P2 (1 μM), a selective inhibitor of T-type channels, partially inhibited ghrelin action. (C) SNX482 (50 nM), an inhibitor of R-type channels, partially inhibits ghrelin induced [Ca2+]c increases. Ghrelin was applied at 100 pM (red bars).