Regulation of gonadotropin-releasing hormone secretion by kisspeptin/dynorphin/neurokinin B neurons in the arcuate nucleus of the mouse

Abstract

Kisspeptin is encoded by the Kiss1 gene, and kisspeptin signaling plays a critical role in reproduction. In rodents, kisspeptin neurons in the arcuate nucleus (Arc) provide tonic drive to gonadotropin-releasing hormone (GnRH) neurons, which in turn supports basal luteinizing hormone (LH) secretion. Our objectives were to determine whether preprodynorphin (Dyn) and neurokinin B (NKB) are coexpressed in Kiss1 neurons in the mouse and to evaluate its physiological significance. Using in situ hybridization, we found that Kiss1 neurons in the Arc of female mice not only express the Dyn and NKB genes but also the NKB receptor gene (NK3) and the Dyn receptor [the kappa opioid receptor (KOR)] gene. We also found that expression of the Dyn, NKB, KOR, and NK3 in the Arc are inhibited by estradiol, as has been established for Kiss1, and confirmed that Dyn and NKB inhibit LH secretion. Moreover, using Dyn and KOR knock-out mice, we found that long-term disruption of Dyn/KOR signaling compromises the rise of LH after ovariectomy. We propose a model whereby NKB and dynorphin act autosynaptically on kisspeptin neurons in the Arc to synchronize and shape the pulsatile secretion of kisspeptin and drive the release of GnRH from fibers in the median eminence.

Figure 1.

A, Representative photomicrographs showing coexpression of Kiss1 mRNA with Dyn in the Arc and AVPV of the female mouse. Kiss1 mRNA-expressing cells are fluorescent with Vector Red substrate, and clusters of silver grains reflect the presence of Dyn mRNA. Scale bars, 50 μm. B, Effect of E₂ replacement on the number of Dyn-expressing cells in coronal sections of the Arc in OVX ERα KO and WT controls. Data are presented as the mean ± SEM.

Figure 2.

Effect of E₂ replacement on the number of KOR-expressing cells in coronal sections of the Arc in OVX WT mice. Data are presented as the mean ± SEM.

Figure 3.

Representative photomicrographs showing coexpression of Kiss1 mRNA with NKB (A) and NK3 (B) in the Arc of the female mouse. Kiss1 mRNA-expressing cells are fluorescent with Vector Red substrate, and clusters of silver grains reflect the presence of NKB mRNA (A) or NK3 mRNA (B). Scale bars, 50 μm. Effect of E₂ replacement on the number of Kiss1/NKB cells in coronal sections of the Arc in OVX WT mice. Data are presented as the mean ± SEM (C).

Figure 4.

Serum LH levels in adult female WT, Dyn KO, and KOR KO mice 7 d after OVX compared with diestrus LH levels. Data are presented as the mean ± SEM.

Figure 5.

Serum LH levels in female WT mice (OVX + sham and OVX + E₂) 30 min after treatment with vehicle or senktide (top), and vehicle or U50488 (bottom). Data are presented as the mean ± SEM.

Figure 6.

Schematic representation of the role of Kiss1/Dyn/NKB neurons in the generation of the GnRH pulses. According to this model, Kiss1/Dyn/NKB neurons receive autosynaptic input from NKB and Dyn and target GnRH fibers in the ME, which are responsive to kisspeptin and NKB (through Kiss1r and NK3, respectively). When E₂ levels decline, Kiss1/Dyn/NKB neurons become spontaneously active. This activity would be amplified by positive autosynaptic feedback through NKB/NK3 signaling, which would also propagate by collaterals to trigger synchronized firing in the ensemble of Kiss1/DYN/NKB neurons in the Arc (as represented by the faded neuron in the diagram). DYN, which would be released along with NKB, would act on Kiss1/DYN/NKB neurons (or interneurons that express KOR) with a small phase lag to clamp further discharge from Kiss1/Dyn/NKB neurons and thus extinguish their activity. In the absence of additional activity, Dyn release would cease, and eventually the inhibitory effect of Dyn would wane, causing the Kiss1/Dyn/NKB neurons to reactivate and initiate another cycle of regenerative activity, followed by inhibition. Each time Kiss1/Dyn/NKB neurons would undergo a burst of activity, a “pulse” of kisspeptin, Dyn, and NKB would be released in the ME, where kisspeptin and NKB would act directly on GnRH fibers or terminals. It is unlikely that Dyn acts directly on GnRH neurons, because GnRH neurons apparently do not express KOR. Kisspeptin would evoke prolonged activation of GnRH fibers or terminals, which, if unchecked, would last for hours. Thus, some mechanism must subsequently inactivate GnRH neurons so that a discrete pulse of GnRH can be delivered into the portal circulation. We postulate that NKB acts via NK3 on GnRH fibers or terminals to accomplish this task. Although this model is consistent with the results presented here, as well as the published observations of others, its validity requires additional critical investigation.