Kisspeptin, neurokinin B, and dynorphin act in the arcuate nucleus to control activity of the GnRH pulse generator in ewes

Abstract

Recent work has led to the hypothesis that kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the arcuate nucleus play a key role in GnRH pulse generation, with kisspeptin driving GnRH release and neurokinin B (NKB) and dynorphin acting as start and stop signals, respectively. In this study, we tested this hypothesis by determining the actions, if any, of four neurotransmitters found in KNDy neurons (kisspeptin, NKB, dynorphin, and glutamate) on episodic LH secretion using local administration of agonists and antagonists to receptors for these transmitters in ovariectomized ewes. We also obtained evidence that GnRH-containing afferents contact KNDy neurons, so we tested the role of two components of these afferents: GnRH and orphanin-FQ. Microimplants of a Kiss1r antagonist briefly inhibited LH pulses and microinjections of 2 nmol of this antagonist produced a modest transitory decrease in LH pulse frequency. An antagonist to the NKB receptor also decreased LH pulse frequency, whereas NKB and an antagonist to the receptor for dynorphin both increased pulse frequency. In contrast, antagonists to GnRH receptors, orphanin-FQ receptors, and the N-methyl-D-aspartate glutamate receptor had no effect on episodic LH secretion. We thus conclude that the KNDy neuropeptides act in the arcuate nucleus to control episodic GnRH secretion in the ewe, but afferent input from GnRH neurons to this area does not. These data support the proposed roles for NKB and dynorphin within the KNDy neural network and raise the possibility that kisspeptin contributes to the control of GnRH pulse frequency in addition to its established role as an output signal from KNDy neurons that drives GnRH pulses.

Figure 1.

Left panels, Top panels depict LH pulse patterns in two ewes receiving empty (open bar) and Kiss1r antagonist-containing (shaded bar) microimplants into the ARC. Bottom panels present LH pulse patterns in two ewes receiving microinjections (arrows) of saline (Cont) and 2 nmol Kiss1r antagonist into the ARC. Solid circles depict peaks of identified LH pulses. Right panels, Mean (± SEM) LH IPIs (left) and AMPLs (right) in response to control and Kiss1r antagonist treatments into the ARC; top two panels are from microimplants and bottom two from microinjections. Open bars, Pretreatment values; shaded bars, values for 0–2 hours after microimplantation or microinjection; black bars, values for 2–4 hours after microimplantation or microinjection; striped bar, not analyzed due to low animal numbers. *, P < .05 vs pretreatment values.

Figure 2.

Panels on left depict LH pulse patterns in two ewes receiving empty (open bars) and SB222200-containing (shaded bars) microimplants into the ARC. Ewes were selected to depict shortest (left panels) and longest (right panels) interruption of LH pulses after antagonist treatment. Solid circles depict peaks of identified LH pulses. Bars on right present mean (± SEM) LH interpulse IPIs (left) and AMPLs (right) in response to control (top panel) and SB222200 (bottom panel). Open bars, Pretreatment values; shaded bars, values for 0–2 hours after microimplantation; black bars, values for 2–4 hours after microimplantation; striped bar, not analyzed due to low animal numbers. *, P < .05 vs pretreatment values

Figure 3.

Close contacts between a GnRH fiber and a KNDy neuron. An example of serial optical sections in a confocal Z-stack (A–C) and orthogonal views (D) through a kisspeptin-positive neuron (blue) receiving close contacts from a GnRH fiber (green) containing varicosities that are colabeled with synaptophysin (red). In addition, numerous dual-labeled kisspeptin/synaptophysin (magenta) inputs also surround this cell, representing KNDy-KNDy reciprocal contacts.

Figure 4.

LH pulse patterns in one ewe from each replicate in experiment 5 with duration of treatment indicated by bars. In first replicate (left panels), ewes received empty (cont), NKB, and BNI-filled microimplants into the ARC. Treatments in second replicate (middle panels) were empty implants, an NMDA receptor antagonist (MK801), BNI, and acyline (pulse pattern shown in Figure 6). Ewes in the final replicate received control microimplants that were empty, or contained NKB, or the antagonist to the orphanin-FQ receptor (UFP-101). Solid circles depict peaks of identified LH pulses.

Figure 5.

LH pulse IPIs (left) and AMPLs (right) in response to NKB (top panel), and antagonists for the κ-opioid receptor (middle panel), the NKDA receptor (bottom panel). Bar codes are the same as in Figure 2. *, P < .05 vs pretreatment values.

Figure 6.

Left, Top panels depict LH pulse patterns in one ewe receiving empty (open bar) and acyline-containing (shaded bar) microimplants into the ARC; note these data are from same ewe shown in Figure 4 (middle panel). Data in bottom panels are from one ewe given control (open bars) and an antagonist for the orphanin-FQ receptor (UFP-101); results from similar treatments in another ewe are illustrated in Figure 4, left panels. Right, Mean (± SEM) LH IPIs (left) and AMPLs (right) in response to control and antagonist treatments into the ARC; bar codes are the same as in Figure 2. There were no statistical differences with either treatment.