Y1R expressed in GnRH-1 neurons. Panel A, Schematic of nasal explant obtained from E11.5 mouse and maintained for 7 div, with nasal pit epithelium (NPE) and nasal midline cartilage (NMC) surrounded by mesenchyme. GnRH-1 neurons (dots) migrate out of the NPE along olfactory axons to the NMP and into the periphery. Panel B, 7-div nasal explant immunostained for GnRH-1 cells (brown, inset). Panel C, Representative gel of PCR products from single-cell GnRH-1 cDNAs using GnRH-1-, Y1R-, and Y5R-specific primers. All tested cells were positive for GnRH-1, whereas 65% of the cells were positive for Y1R transcript (n = 20). All cells were negative for Y5R transcript. B, Adult brain cDNA; W, water, negative control. Panel D, Staining for Y1R in the ARC: D₁, Y1R-positive cells (brown) at higher magnification in inset; D₂, negative control, omission of Y1R primary. Panel E, Double-label immunocytochemistry of GnRH-1 neurons (green) and Y1R (red) indicating colocalization: E₁, single wavelength for 488-conjugated secondary used to label GnRH-1; E₂, single wavelength for Cy3-conjugated secondary used to label Y1R; E₃, 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei of GnRH-1 neurons; E₄, merged image of GnRH-1 cells and Y1R. Panel F, Negative control for double-label immunocytochemistry of GnRH-1 neurons (green) and omission of Y1R primary (red): F₁, single wavelength for 488-conjugated secondary used to label GnRH-1 neurons; F₂, single wavelength for Cy3-conjugated secondary after omission of Y1R primary; F₃, DAPI-stained nuclei of GnRH-1 neurons; F₄, merged image of GnRH-1 cells (green) and Y1R negative control (red). Scale bars, 100 μm (panel B), 10 μm (inset in panel B), 50 μm (panel D₁ and inset in panel D₂), and 5 μm (panels E and F).

Inhibitory effect of NPY is mimicked by Y1R agonist Pro³⁴NPY and prevented by Y1R antagonist BIBP3226. A, Representative calcium imaging recordings showing a decrease in the frequency of calcium oscillations in two GnRH-1 neurons during a 5-min perfusion of Pro³⁴NPY (1 nm). B, Nasal explants exposed to increasing concentrations of Y1R agonist Pro³⁴NPY, Y2R agonist NPY_13-36, and Y5R agonist BWX 46. Similar to NPY, the Y1R agonist decreased the frequency of calcium oscillations in a dose-dependent manner, whereas neither Y2R nor Y5R agonists altered GnRH-1 neuronal activity. C, Representative calcium imaging recording showing that BIBP3226 (BIBP, Y1R antagonist, 200 nm, 5 min) had no effect on the number of calcium peaks per minute in a GnRH-1 neuron and that coapplication of BIBP3226 (200 nm) with NPY (1 nm) prevented the inhibitory effect of NPY. D, Summary of BIBP3226 experiments. The Y1R antagonist prevented the inhibitory effect of NPY. [Results are normalized to control (values in peaks per minute in Tables 2 and 3; paired t test, P < 0.05). Dotted line indicates the response of GnRH-1 neurons to application of 1 nm NPY alone. *, Significant difference vs. control.]

Inhibitory effect of NPY on GnRH-1 neuronal activity is mediated by activation of GIRK channels. Panel A, Schematic of Y1R pathways that could elicit the inhibitory effect of NPY. Panel B, Pretreatment of explants with 250 ng/ml PTX, a blocker of Gα_i/o coupling, for 2 h and subsequent application of NPY (1 nm) reduced the inhibitory effects exerted by NPY. A 6-h preincubation with PTX (250 ng/ml) prevented the inhibitory effect of NPY on GnRH-1 neuronal activity. Panel C, Coapplication of DDA (80 μm, a pharmacological blocker of AC) together with NPY (1 nm) did not prevent the inhibitory effect of NPY on the frequency of calcium oscillations in GnRH-1 neurons. Panel D, Coapplication of TPN-Q (100 nm, a specific GIRK channel blocker) together with NPY (1 nm) decreased the response of GnRH-1 neurons to NPY. Panel E, Representative gel showing PCR products from single-cell GnRH-1 cDNAs using Kcnj9-specific primers. Sixty percent of the tested cells were positive for Kcnj9 transcript (7-div cells, n = 10). B, Adult brain cDNA; W, water, negative control. [Results in B–D are normalized to control (values in peaks per minute in Table 3; paired t test, P < 0.05; a, no significant difference vs. control; b, significantly different from control and other letters). Dotted line indicates the response of GnRH-1 neurons to application of 1 nm NPY alone.]

GnRH-1 neuronal activity is inhibited by NPY. Panel A, Calcium Green-1 imaging of GnRH-1 neurons; cells were identified by their bipolar morphology (A₁), loaded with fluorescent calcium-sensitive dye (A₂), and their identity verified after imaging by immunocytochemistry (A₃). Arrows indicate identical cells in all fields. B, Representative recordings showing spontaneous baseline oscillations in intracellular calcium levels in three GnRH-1 neurons during 5 min in SFM and subsequent decrease in activity during a 5-min superfusion of NPY (1 nm) (y-scale = OD units). C, Exposure to increasing concentrations of NPY (1 pm to 100 nm) caused a significant decrease in the frequency of spontaneous calcium oscillations in GnRH-1 neurons in a dose-dependent manner. Dotted line indicates the inhibitory effect of 1 nm NPY application. [Results are normalized to control (values in peaks per minute in Table 2; paired t test, P < 0.05; a, no significant difference vs. control; b–d, significantly different from control and other letters).]

GABAergic inputs do not mediate the response of GnRH-1 neurons to NPY. A, Representative calcium imaging recording showing spontaneous baseline oscillations in intracellular calcium levels of a GnRH-1 neuron during 5 min of SFM, 5 min of Bic (20 μm, a GABA_A receptor antagonist), and 5 min coapplication of Bic and NPY (1 nm). B, Summary of Bic/NPY experiments. Bic (20 μm) decreased the frequency of calcium oscillations in GnRH-1 neurons by more than 50%. However, coapplication with NPY (1 nm) further decreased the frequency (∼50%). [Results are normalized to control (values in peaks per minute in Table 3; paired t test, P < 0.05; a, no significant difference vs. control; b and c, significantly different from control and other letters). Dotted line indicates the response of GnRH-1 neurons to application of 1 nm NPY alone.]