Regulation of Gonadotropin-Releasing Hormone Secretion by Kisspeptin/Dynorphin/Neurokinin B Neurons in the Arcuate Nucleus of the Mouse (original) (raw)

J Neurosci. 2009 Sep 23; 29(38): 11859–11866.

Victor M. Navarro

Departments of 1Physiology and Biophysics,

Michelle L. Gottsch

2Obstetrics and Gynecology, and

Charles Chavkin

3Pharmacology, University of Washington, Seattle, Washington 98195, and

Hiroaki Okamura

4Laboratory of Neurobiology, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-0901, Japan

Donald K. Clifton

2Obstetrics and Gynecology, and

Robert A. Steiner

Departments of 1Physiology and Biophysics,

2Obstetrics and Gynecology, and

Departments of 1Physiology and Biophysics,

2Obstetrics and Gynecology, and

3Pharmacology, University of Washington, Seattle, Washington 98195, and

4Laboratory of Neurobiology, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-0901, Japan

corresponding authorCorresponding author.

Correspondence should be addressed to Robert A. Steiner, University of Washington, Department of Physiology and Biophysics, Box 357290, Seattle, WA 98195-7290., ude.notgnihsaw.u@reniets

Received 2009 Apr 1; Revised 2009 Jun 6; Accepted 2009 Jul 30.

Copyright © 2009 Society for Neuroscience 0270-6474/09/2911859-08$15.00/0

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 κ 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.

Introduction

Kisspeptin signaling plays a critical role in the neuroendocrine regulation of reproduction (Popa et al., 2008). Kisspeptin activates gonadotropin-releasing hormone (GnRH) neurons (Gottsch et al., 2004; Han et al., 2005), which are the final common pathway by which the brain regulates the gonadotropin secretion, and pulsatile secretion of kisspeptin and GnRH are temporally linked (Keen et al., 2008). The Kiss1 gene encodes kisspeptin, which is expressed by neurons in the arcuate nucleus (Arc) of the hypothalamus (Smith et al., 2005a,b), the nodal point for controlling the negative feedback regulation of GnRH secretion by estradiol (E2) and for generating pulsatile GnRH secretion (Freeman, 2005). Kiss1 neurons in the Arc express estrogen receptor-α (ERα) (Smith et al., 2005b), which mediates negative feedback and the inhibition of Kiss1 expression in the Arc (Smith et al., 2005b; Glidewell-Kenney et al., 2007). Thus, Kiss1 neurons in the Arc may be conduits for receiving E2 signaling from the gonad and relaying that information to GnRH neurons (Smith et al., 2005b). However, Kiss1 neurons in the Arc also express several cotransmitters, which may have important, independent functions.

In the sheep, Kiss1 neurons in the Arc coexpress dynorphin A (Dyn) and neurokinin B (NKB) (Goodman et al., 2007), and both Dyn and NKB have been implicated in the regulation of luteinizing hormone (LH) secretion. Dyn, which acts via the κ opioid receptor (KOR) (Chavkin et al., 1982), inhibits LH release (Schulz et al., 1981; Kinoshita et al., 1982), as does NKB (Sandoval-Guzmán and Rance, 2004). Moreover, disabling mutations of either NKB (known as TAC3 in humans) or the NKB type 3 receptor gene (NK3) (known as TAC3R in humans) are associated with reproductive failure (Topaloglu et al., 2009). Thus, three neuropeptides are produced by a single population of cells in the Arc (in the sheep), and all three have been independently linked to GnRH secretion. However, the nature of the interaction among these cotransmitters and their physiological significance remains a mystery. We postulated that kisspeptin, Dyn, and NKB work together to generate episodic kisspeptin secretion, which drives the pulsatile secretion of GnRH, based on several lines of reasoning. First, in the rat, Dyn/NKB-containing fibers contact Dyn/NKB-containing soma in the Arc (Burke et al., 2006), suggesting that recurrent collaterals shape and synchronize periodic firing of Kiss1/Dyn/NKB neurons. Second, Dyn/NKB-containing fibers project from the Arc and terminate near GnRH fibers or terminals in the median eminence (ME) (Burke et al., 2006). Finally, GnRH neurons express both the kisspeptin receptor (Kiss1r) and NK3 (Irwig et al., 2004; Krajewski et al., 2005). The studies described here were designed to test this model. First, we evaluated whether Dyn and NKB are coexpressed in Kiss1 neurons in the Arc of the mouse. Second, we determined whether Kiss1 neurons express KOR and NK3. Third, we tested whether Dyn and NKB are targets for regulation by E2, and fourth, we studied the effects of KOR and NK3 agonists and the impact of genetically targeted deletions of Dyn and KOR on GnRH/LH secretion.

Materials and Methods

Animals

Animals were housed at the University of Washington, and all procedures and surgeries were approved by the Animal Care Committee of the University of Washington School of Medicine in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Adult female C57BL/6 mice (8 weeks old) were purchased from The Jackson Laboratory. C57BL/6 mice with a prodynorphin (Dyn) gene deletion were generated as described previously (McLaughlin et al., 2003), and homozygous (−/−) and paired wild-type (+/+) littermate controls were generated by heterozygote crosses. Homozygous KOR gene deletion (−/−) mice (C57BL/6 background) were prepared by homologous recombination as described previously (Clarke et al., 2002) and provided for this study. Female _ER_α null mice (C57BL/6 background) were produced by breeding heterozygote pairs, each carrying a single copy of the disrupted _ER_α gene, as described previously (Lubahn et al., 1993; Smith et al., 2005b). Animals were housed in groups of three to five, maintained on a 14/10 light cycle with lights on at 4:00 A.M. and had access to standard rodent chow and water ad libitum.

Ovariectomy and steroid replacement

Ovariectomy (OVX) was performed on adult female mice through bilateral lumbar incisions, while maintaining the animals under isoflurane inhalation anesthesia (Abbott Laboratory) delivered by a vaporizer (Veterinary Anesthesia Systems). Vasculature to the ovary was sutured, and wound clips were used to close the incision. Immediately after OVX, oil-filled capsules (sham) or E2 plus oil-filled capsules were implanted subcutaneously via a small midscapular incision at the base of the neck; wound clips were used to close the incision. For E2 implants, SILASTIC tubing (inner diameter, 1.47 mm; outer diameter, 1.95 mm; Dow Corning) was cut to 9 mm; one end was sealed with silicone cement and allowed to cure overnight. Crystalline E2 (Sigma) at a dose of 1 mg/ml was dissolved in safflower oil based on previous studies (Miller et al., 1995; Smith et al., 2005b; Dungan et al., 2007). After capsules were filled with E2 in oil, the end of the capsule was sealed with silicone cement and allowed to cure overnight. The day before surgery, implants were washed two times for 10 min in changes of 100% ethanol and then placed in sterile physiological saline overnight.

Tissue preparation

Blood was centrifuged for 5 min (3000 rpm), and the serum was stored at −20°C until hormone measurements. Brains were removed for in situ hybridization (ISH), frozen on dry ice, and then stored at −80°C until sectioned. Five sets of 20 μm sections in the coronal plane were cut on a cryostat (from the diagonal band of Broca to the mammillary bodies), thaw mounted onto SuperFrost Plus slides (VWR Scientific), and stored at −80°C. A single set was used for in situ hybridization (adjacent sections 100 μm apart).

Radioimmunoassays

Serum levels of LH were measured at Northwestern University (Evanston, IL). Reagents for the LH assay were obtained from NIH. For LH, the antiserum used was anti-rLH–S-11, and the standard was rLH–RP3. The assay sensitivity was 0.2 ng/ml, and the intra-assay coefficient of variation was 4%.

Detection of Kiss1 mRNA

The Kiss1 probe used for detection of Kiss1 mRNA was described previously (Gottsch et al., 2004). The _Kiss1_-specific sequence of the probe spans bases 76-486 of the mouse cDNA sequence (GenBank accession number AF472576). The procedure for ISH is outlined below.

Detection of Dyn mRNA

The Dyn probe used for detection of Dyn mRNA was described previously (Gottsch et al., 2009). The Dyn-specific sequence of the probe spans bases 132-540 of the mouse cDNA sequence (GenBank accession number NM_018863). The procedure for in situ hybridization is outlined below.

Detection of KOR, NKB, and NK3 mRNAs

Total RNA was extracted from mouse brain using an RNAqueous kit (Ambion). RNA was reverse transcribed into cDNA with a RetroScript kit (Ambion) primed with oligodeoxythymidine for subsequent PCR. Primers were designed based on the published sequence of the KOR mouse gene (GenBank accession number NM_011011) with forward primers starting at 127 bp and reverse primers starting at 964 bp, NKB mouse gene (GenBank accession number NM_009312) with forward primers starting at 177 bp and reverse primers starting at 440 bp, and NK3 mouse gene (GenBank accession number NM_021382) with forward primers starting at 286 bp and reverse primers starting at 691 bp. Primers were custom synthesized (OPERON). PCR reactions contained the following in a volume of 25 μl:2 μl of reverse transcriptase reaction product, 0.2 μm of each primer, 12.5 μl of RediTaq polymerase (Sigma-Aldrich), and 8.5 μl of water. Reactions were performed in a PTC-100 thermal cycler (MJ Research) using the following protocol: cDNA was denatured for 5 min at 94°C, and then 35 cycles were performed at 94°C for 1 min, 55°C for 1.5 min, and 72°C for 2 min, with a final 5 min extension at 72°C. After electrophoresis on a 2% agarose (w/v) gel, a single DNA fragment was obtained of approximately the expected size and gel purified with a QiaQuick gel extraction kit (Qiagen). The PCR product was confirmed to be the mouse KOR, NKB, or NK3 probe by sequencing. Clamp polymerase sequences for T7 or T3 polymerase were added for the final primer product sequence and transcribed for ISH.

Single-label in situ hybridization of Dyn mRNA

Dyn mRNA sense and antisense probes were transcribed with T7 or T3 polymerase (Fermentas) as described previously by Gottsch et al. (2009). Briefly, radiolabeled probes were synthesized in vitro by inclusion of the following ingredients in a volume of 20 μl:250 μCi [33P]UTP (PerkinElmer Life and Analytical Sciences), 1 μg of linearized DNA (or 1 μg of PCR product), 0.5 mm each ATP, CTP, and GTP, and 40 U of polymerase. Residual DNA was digested with 4 U of DNase (Ambion), and the DNase reaction was terminated by addition of 2 μl of 0.5 m EDTA, pH 8.0. The riboprobes were separated from unincorporated nucleotides with NucAway Spin Columns (Ambion).

Slides with mouse hypothalamic sections from the different experimental groups were processed as reported previously (Cunningham et al., 2002; Gottsch et al., 2004).

Double-label in situ hybridization

Antisense mouse Kiss1 probe was transcribed from linearized pAMP1 plasmid containing the mouse Kiss1 insert with T7 polymerase (Fermentas) (Gottsch et al., 2004). The cDNA template for the Dyn, KOR, NKB, and NK3 riboprobes were generated by PCR with primers that were designed to contain promoters for T7 RNA polymerase in the antisense direction and T3 RNA polymerase in the sense direction. Radiolabeled riboprobes for KOR, NKB, and NK3 were synthesized as described above for the Dyn riboprobe. Digoxigenin (DIG)-labeled Kiss1 antisense riboprobe was synthesized with T7 RNA polymerase and DIG labeling mix (Roche) according to the instructions of the manufacturer. Slides were processed for double-labeled in situ hybridization as described previously (Irwig et al., 2004). Slides were stored at 4°C and developed 8–12 d later.

Quantification and analysis of Kiss1, Dyn, KOR, NKB, and NK3 mRNAs

The brain sections were analyzed bilaterally unless otherwise specified. Slides from all of the animals were assigned a random three-letter code, alphabetized, and read under dark-field illumination with custom-designed software designed to count the total number of cells and the number of silver grains (corresponding to radiolabeled Dyn, KOR, NKB, or NK3 mRNA) over each cell (Chowen et al., 1990). Kiss1 mRNA-containing cells were visualized under fluorescent illumination, and custom-designed software was used to count the number of silver grains over each Kiss1 cell depending on which radiolabeled probe was used. The number of cells reported for each experiment represents the number of cells within the coronal sections containing the Arc nucleus for each set, not the total number of cells in the Arc. The starting and ending point of quantification was determined according to Paxinos and Franklin (2001). Signal-to-background ratios (SBRs) for individual cells were calculated; an individual cell was considered to be double labeled if it had an SBR of three or more. For each animal, the number of double-labeled cells was calculated as a percentage of the total number of Kiss1 mRNA-positive cells and then averaged across animals to produce a mean ± SEM.

Statistical analysis

All data are expressed as the mean ± SEM for each group. One-way ANOVA was used to assess variation among experimental groups in each experiment. Significance level was set at p < 0.05. All analyses were performed with Statview 5.0.1 for Macintosh (SAS Institute).

Experimental design

Experiment 1: coexpression of Kiss1/Dyn mRNAs in the forebrain.

The purpose of this experiment was to determine whether Kiss1 neurons in the mouse female forebrain [Arc and anteroventral periventricular nucleus (AVPV)] coexpress Dyn mRNA. Wild-type (WT) mice were divided into two groups (n = 5 per group): OVX + sham and OVX + E2 replacement. At approximately 9:00 A.M. on 7 d after castration, mice were anesthetized with isoflurane, blood was collected for LH radioimmunoassays by retro-orbital bleeding to confirm gonadectomy, and mice were killed by decapitation. Brains were collected as described above.

Experiment 2: regulation of Dyn mRNA by E2.

The purpose of this experiment was to analyze the regulation of Dyn mRNA by E2 and determine whether E2 can regulate this expression in _ER_α knock-out (KO) female mice. Seven female _ER_α KO mice and seven WT littermates were ovariectomized, and four from each group received E2 replacement. Tissue collection and preparation for Dyn mRNA in situ hybridization was performed as described in experiment 1.

Experiment 3: coexpression of Kiss1/KOR mRNAs in the Arc.

The purpose of this experiment was to determine whether Kiss1 and KOR are coexpressed in cells in the Arc and to assess the possible regulation of KOR mRNA by E2. We performed double-labeled in situ hybridization on a set of coronal sections from brains of OVX + sham and OVX + E2 adult female mice (n = 8 per group), generated as described above. Kiss1 mRNA was visualized via DIG-labeled riboprobes.

Experiment 4: coexpression of Kiss1/NKB/NK3 mRNAs in the AVPV and the Arc.

The purpose of this experiment was to determine whether Kiss1, NKB, and NK3 are coexpressed in cells in the Arc (as well as the AVPV for Kiss1/NKB) and to assess their possible regulation by E2. We performed double-labeled in situ hybridization on a set of coronal sections from brains used in the previous experiment (OVX + sham and OVX + E2 mice; n = 8 per group). Kiss1 mRNA was visualized by the use of DIG-labeled riboprobes.

Experiment 5: postcastration LH response in adult female Dyn KO and KOR KO mice.

The purpose of this experiment was to determine the effect of long-term deficiency of either Dyn or KOR in controlling the GnRH/LH response to reduced circulating levels of E2. Ten adult female Dyn KO mice, 8 adult female KOR KO mice, and 10 adult WT littermates (between 8 and 12 weeks of age) were ovariectomized, and blood was collected after 7 d as described in experiment 1 (for LH determinations). This experiment showed a reduced rise in serum levels of LH after OVX in Dyn KO and KOR KOs compared with their respective WT controls. To investigate whether this phenomenon might be attributable to a decrease in kisspeptin drive to GnRH neurons, WT and Dyn KO animals (five adult female mice for each group) were subjected to the same protocol described above. At 1 week after OVX, brain levels of Kiss1 mRNA were measured and compared between genotypes by quantitative ISH (after confirming that the postcastration rise in LH was compromised in the Dyn KOs).

Experiment 6: response of LH serum levels to U50488.

The purpose of this experiment was to assess the effect of the KOR agonist U50488 [(trans)-3,4-dichloro-_N_-methyl_-N_-[2-1-pyrrolidinyl)-cyclo-hexyl] benzeneacetamide] (Tocris Biosciences) on serum LH levels in adult (8 weeks old) female WT mice, in the presence and absence of E2. Animals were ovariectomized as described above (n = 10 per group) and allowed to recover for 7 d. Each group received an intraperitoneal injection of vehicle (0.9% NaCl) or U50488 (5 mg · kg−1 · ml−1). After 30 min, animals were bled and serum was collected as described in experiment 1. In addition, adult WT OVX mice were implanted with E2-containing capsules and treated intraperitoneally with vehicle or U50488 (n = 10 per group) following the same protocol.

Experiment 7: LH response to NK3 agonist.

The purpose of this experiment was to assess the effect of the NK3 agonist senktide (Sigma-Aldrich) on LH secretion in adult (8 weeks old) female WT mice, in the presence and absence of E2. Animals were ovariectomized and implanted with empty capsules (OVX + sham) or E2 capsules (OVX + E2) as described above (n = 7–10 per group). Each group was equally divided and received an intracerebroventricular injection (Gottsch et al., 2004) of vehicle (0.9% NaCl) or senktide (600 pmol/3 μl) as described previously (Sandoval-Guzmán and Rance, 2004). After 30 min, the animals were bled and serum was collected as described in experiment 1.

Results

Coexpression of Kiss1/Dyn genes and the regulation of Dyn gene by E2

The percentage of Kiss1 neurons expressing Dyn in OVX mice with and without E2 treatment was assessed by double-labeled ISH. In the Arc, nearly all (92%) of the Kiss1 neurons coexpressed Dyn (Fig. 1A). Dyn was also found in some _Kiss1_-containing cells in the AVPV, but the extent of coexpression was only 33%. Levels of Dyn mRNA in Kiss1 cells of the AVPV—as indicated by the number of silver grains per cell—was low, amounting to <10% of that found in Kiss1 cells of the Arc (OVX + sham AVPV, <2 gpc; OVX + E2 AVPV, <2 gpc; OVX + sham Arc, 15 ± 2 gpc; OVX + E2 Arc, 17 ± 2 gpc). E2 treatment did not affect the percentage of Dyn coexpressing Kiss1 cells in either the Arc or AVPV.

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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 _ER_α KO and WT controls. Data are presented as the mean ± SEM.

Single-labeled ISH was used to evaluate the ability of E2 to regulate Dyn expression in adult WT and _ER_α KO mice. As shown in Figure 1B, the number of Dyn mRNA-containing cells in the Arc was high in OVX WT mice (574 ± 27), and E2 treatment reduced the number of cells by ∼80% (112 ± 21; p < 0.01). E2 also reduced the per cell content of Dyn mRNA in the Arc (OVX + sham, 125 ± 4 gpc vs OVX + E2, 44 ± 4 gpc; p < 0.01). Dyn expression in the Arc of _ER_α KO mice was unaffected by E2 treatment (Fig. 1B), both in terms of cell numbers (_ER_α KO OVX + sham, 365 ± 62 cells vs _ER_α KO OVX + E2, 330 ± 122 cells; p = 0.17) and gpc (_ER_α KO OVX + sham, 181 ± 13 gpc vs _ER_α KO OVX + E2, 133 ± 22 gpc; p = 0.66).

Coexpression of Kiss1/KOR genes and regulation of KOR by E2

The coexpression of KOR mRNA in Kiss1 neurons of OVX mice with and without E2 was also determined by double-labeled ISH. KOR mRNA was found in ∼20% of the Kiss1 neurons in the Arc, regardless of E2 treatment. Although this would seem to be a relatively small fraction of the total number of Kiss1 cells, it is significant and may reflect the relatively weak limits of detectability of the KOR riboprobe. Using the single-labeled approach, we found that E2 inhibited the expression of KOR in cells inside the Arc, presumably including some that do not contain Kiss1 (OVX + sham, 57 ± 11 cells vs OVX + E2, 19 ± 4 cells; p < 0.02) (Fig. 2). There were several hypothalamic areas that showed a greater expression of KOR than the Arc, including the ventromedial nucleus, dorsomedial nucleus, and the caudal part of the periventricular nucleus (data not shown).

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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.

Coexpression of Kiss1, NKB, and NK3 genes and regulation by E2

In the Arc, double-labeled ISH revealed that ∼90% of the Kiss1 neurons of OVX mice also expressed NKB mRNA (regardless of E2 treatment) (Fig. 3A). Some cells in the Arc that contained NKB did not colabel with Kiss1 (data not shown). Only ∼10% of the Kiss1 neurons in the AVPV coexpressed NKB, and the level of NKB expression in those cells was much less than in the Arc (OVX + sham AVPV, <1 gpc; OVX + E2 AVPV, <1 gpc; OVX + sham Arc, 28 ± 5 gpc; OVX + E2 Arc, 30 ± 35 gpc). It has been reported previously that NKB in the Arc is regulated by E2 (Danzer et al., 1999; Goubillon et al., 2000; Pillon et al., 2003; Dellovade and Merchenthaler, 2004). In the present work, we studied the effect of E2 specifically in Kiss1/NKB colabeled neurons. We found that E2 replacement decreased the population of these neurons in the Arc of OVX animals by 53% when compared with the OVX + sham group (OVX + sham, 308 ± 57 vs 145 ± 18 cells; p = 0.027) (Fig. 3C).

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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).

We also found that virtually all (96%) of the Kiss1 neurons in the Arc of OVX mice coexpressed high levels of NK3 mRNA in the absence of E2 (Fig. 3B). Within the Arc, NK3 appeared to be expressed exclusively in Kiss1 neurons (data not shown). In the presence of E2, there were too few detectable _Kiss1_-expressing cells to determine the degree of NK3 mRNA coexpression. In any case, there were no visible clusters of silver grains in the Arc of E2-treated animals, suggesting that NK3 mRNA was profoundly suppressed by E2, as was the case for Kiss1 mRNA in this region.

Effect of a long-term deficit of Dyn/KOR signaling on the LH response to OVX

Serum LH levels in adult WT, Dyn KO, and KOR KO female mice were elevated 7 d after OVX compared with those in diestrus animals. However, the post-OVX LH levels observed in both Dyn KO and KOR KO mice were only 37 and 51% of the LH level in the WT group (WT, 1.95 ± 0.3 ng/ml; Dyn KO, 0.73 ± 0.09 ng/ml; p < 0.01; KOR KO, 1.0 ± 0.3 ng/ml; p < 0.05) (Fig. 4). In OVX mice treated with E2, LH levels were not significantly different between WT and Dyn KO (WT, 0.202 ± 0.001 ng/ml vs Dyn KO, 0.27 ± 0.04 ng/ml; p = 0.29).

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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.

To determine whether reduced expression of Kiss1 in Dyn KOs might account for their reduced LH response to OVX, we compared levels of Kiss1 mRNA between WT and Dyn KOs, following the same protocol used to assess serum LH levels. We confirmed that, 7 d after OVX, Dyn KOs had reduced LH levels compared with WTs (WT, 1.93 ± 0.34 ng/ml vs Dyn KO, 0.92 ± 0.26 ng/ml; p < 0.05). Furthermore, we found no significant difference in Kiss1 mRNA between genotypes (WT, 59.2 ± 18.17 cells vs Dyn KO, 65.0 ± 15.93 cells; p = 0.81).

Action of KOR and NK3 agonists on LH secretion

Although it has been shown in rats that KOR and NK3 agonists inhibit LH release, this has not been demonstrated in the mouse. In OVX (7 d) female mice, we found that administration of either the KOR agonist U50488 or the NK3 agonist senktide reduced serum LH levels after 30 min (vehicle + OVX, 1.92 ± 0.27 ng/ml vs U50488 + OVX, 0.88 ± 0.29 ng/ml; p < 0.01; vehicle + OVX, 1.60 ± 0.53 ng/ml vs senktide + OVX, 0.81 ± 0.17 ng/ml; p < 0.01) (Fig. 5). When this study was repeated in E2-treated OVX mice, serum levels of LH were exceedingly low (at or near the minimum detectable limits of the assay) and were indistinguishable among groups, regardless of treatment (vehicle + OVX + E2, 0.28 ± 0.09 ng/ml vs U50488 + OVX + E2, 0.24 ± 0.03 ng/ml; senktide + OVX + E2, 0.20 ± 0.0 ng/ml) (Fig. 5).

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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.

Discussion

Based on evidence from this and previous studies, we have proposed that Kiss1 neurons in the Arc of the mouse participate in the generation and regulation of pulsatile kisspeptin release that drives ultradian GnRH release (Fig. 6). Central to this model is the coexpression of Dyn and NKB in Kiss1 neurons of the Arc, which has been reported previously to occur in sheep (Goodman et al., 2007). Here, we show that this is also the case in the mouse, suggesting that the presence of Kiss1/Dyn/NKB neurons of the Arc may be common across diverse species. Furthermore, because we found that most of the cells containing Dyn and/or NKB in the Arc are Kiss1 neurons, we can reasonably infer that previous descriptions of the distribution of kisspeptin-, Dyn-, and NKB-containing fibers in the Arc also describe the distribution of fibers from Kiss1/Dyn/NKB neurons, which should contain all three neuropeptides. Using immunohistochemistry coupled with confocal microscopy, Burke et al. (2006) found that a plexus of axons and terminals containing Dyn and NKB surround and abut Dyn/NKB cell bodies in the Arc of the rat. These observations suggest that Kiss1/Dyn/NKB neurons are interconnected via recurrent collaterals, as shown in Figure 6. In addition, Dyn/NKB fibers project from the Arc to the ME in the rat, sheep, and horse (Burke et al., 2006; Foradori et al., 2006; Decourt et al., 2008), and in the monkey kisspeptin fibers have been shown to have extensive and intimate associations with GnRH axons in the ME (Ramaswamy et al., 2008). Thus, we deduce that axons from Kiss1/Dyn/NKB soma in the Arc of the mouse project to one another and to the ME in the vicinity of GnRH fibers and terminals.

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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.

Presumably, kisspeptin, Dyn, and NKB are all available to act as cotransmitters or neuromodulators at the targets of projections from Kiss1/Dyn/NKB soma in the Arc, and any specific action would occur as a function of the presence (or absence) of Kiss1r, KOR, and NK3 in those potential targets. Remarkably, the targets of the recurrent collaterals appear to be Kiss1/Dyn/NKB cells themselves. Dyn cells in the Arc express NK3 (Burke et al., 2006), and our results confirm that _Kiss1_-expressing cells coexpress NK3, implying that the recurrent collaterals could signal through an NKB/NK3 signaling pathway. In addition, KOR is expressed in Kiss1 neurons (albeit at low levels), so it is plausible that Dyn/KOR signaling is also involved in communication through these same recurrent collaterals (or conceivably through interneurons). Kiss1r mRNA does not appear to be expressed in the Arc (d'Anglemont de Tassigny et al., 2008); thus, Kiss1/Dyn/NKB cell bodies in this region are unlikely to be targets for the direct action of kisspeptin. However, the kisspeptin receptor itself (as opposed to its mRNA) is concentrated in the Arc/ME region—most likely in GnRH terminals—based on the observation that kisspeptin can elicit GnRH secretion from explants of the mediobasal hypothalamus, which contains few, if any, GnRH cells bodies (Irwig et al., 2004; d'Anglemont de Tassigny et al., 2008). Thus, it seems reasonable to infer that Kiss1r appears in the ME through axonal transport, originating from sites of production in GnRH cell bodies in the medial preoptic area (Irwig et al., 2004). GnRH fibers and terminals in the ME also contain NK3 (Burke et al., 2006), suggesting that GnRH fibers and terminals are sites of action for both kisspeptin and NKB, but not dynorphin, because GnRH neurons apparently do not express KOR (Mitchell et al., 1997; Sannella and Petersen, 1997).

Based on what is known about kisspeptin, Dyn, and NKB signaling, we can make some predictions about their roles in this model. Assuming that NKB stimulates and Dyn inhibits Kiss1/Dyn/NKB neurons, the recurrent collaterals constitute a potentially oscillatory feedback loop. Any spontaneous activity in the Kiss1/Dyn/NKB cells would be amplified through regenerative feedback, involving NKB/NK3 signaling. Through interconnections between Kiss1/Dyn/NKB cells, regenerative activity would propagate throughout the entire population of cells, and, as a result, a burst of kisspeptin, Dyn, and NKB would be delivered to the GnRH terminals in the ME. This would be followed by a delayed inhibition of Kiss1/Dyn/NKB cells, mediated by Dyn acting through KOR. As this subsides, the cells would become active, restarting the entire process. The kisspeptin released near GnRH terminals would bind to Kiss1r and initiate prolonged volleys of action potentials, and thus produce sustained GnRH secretion (Han et al., 2005; Pielecka-Fortuna et al., 2008; Zhang et al., 2008). To limit the release of GnRH to a discrete pulse, the activity induced by kisspeptin must somehow be extinguished. Although this could be accomplished by an unidentified inhibitory neurotransmitter, it could also occur through NKB binding to NK3 and activating a delayed inhibitory signaling pathway, as indicated in Figure 6.

If Kiss1/Dyn/NKB neurons participate in the negative feedback regulation of GnRH/LH secretion, it would seem (at first glance) that E2 should induce Dyn expression, because Dyn is an inhibitory factor. Nevertheless, we found that E2 suppressed levels of Dyn mRNA, consistent with previous results (Spampinato et al., 1995). This paradoxical observation can be readily explained by the model in Figure 6. Accordingly, the GnRH pulse generator is most active when E2 levels are low (to drive GnRH and LH secretion). Under these conditions, there is a demand for all three neurotransmitters (kisspeptin, Dyn, and NKB) and their receptors, as reflected by increases in the expression of their respective mRNAs. Not only was the expression of Dyn, KOR, and NK3 in the Arc high when E2 levels were low, but so too was the expression of NKB and Kiss1, confirming previous reports (Rance and Bruce, 1994; Dellovade and Merchenthaler, 2004; Smith et al., 2005b).

The model would also predict that exogenously administered Dyn or a Dyn agonist would suppress Kiss1/Dny/NKB neurons in the Arc, thus reducing drive to GnRH and LH secretion. In accord with this prediction, we found that the Dyn agonist U50488 inhibited LH levels, confirming previous observations in rats (Schulz et al., 1981; Kinoshita et al., 1982). We would also forecast that a permanent disruption of Dyn/KOR signaling would make the Kiss1/Dyn/NKB neurons continuously active, causing a desensitization of the kisspeptin → GnRH → LH cascade, resulting in low LH levels. Indeed, we observed reduced levels of LH in both Dyn KO and KOR KO mice, whereas Kiss1 expression remained unaltered. Furthermore, the model predicts that NKB, acting through NK3, activates Kiss1/Dyn/NKB cells in the Arc but inhibits GnRH fibers and terminals in the ME. Thus, the administration of the NKB agonist senktide would be expected to activate Kiss1/Dyn/NKB neurons but also block GnRH (and thus LH) secretion. Consistent with this expectation, senktide inhibited LH release in our mice, as it has been shown previously to do in rats (Kalra et al., 1992; Sandoval-Guzmán and Rance, 2004).

The results of this study accentuate the phenotypic differences between the two major _Kiss1_-expressing populations in brain located in the Arc and the AVPV. We have shown previously that, although both populations express ERα (Smith et al., 2005b), E2 acts via an estrogen response element (ERE)-independent mechanism to inhibit Kiss1 expression in the Arc and through an ERE-dependent mechanism to induce Kiss1 expression in the AVPV (Gottsch et al., 2009). Here, we have shown that Kiss1 neurons in the AVPV express virtually no NKB and much less Dyn compared with those in the Arc. These phenotypic differences likely reflect the different physiological roles these neurons play in the regulation of gonadotropin secretion. In the Arc, Kiss1 cells stimulate basal GnRH/LH release and are negatively regulated by E2, whereas in the AVPV, they are induced by E2 and participate in generating the preovulatory GnRH/LH surge.

In conclusion, we report that kisspeptin-expressing neurons in the Arc of the mouse coexpress Dyn and NKB, as well as KOR and NK3, and that these neuropeptides and their receptors are targets for regulation by E2. We also demonstrate that pharmacological activation of Dyn and NKB signaling disrupts LH secretion and that animals bearing genetically targeted deletions of Dyn and KOR have compromised abilities to regulate GnRH secretion. We present a model whereby kisspeptin, Dyn, and NKB act autosynaptically on kisspeptin neurons in the Arc to shape the pulsatile secretion of kisspeptin and hence GnRH release. This model is consistent with the present observations and with previous reports that bursts of multiple unit activity (MUA) in the Arc of monkeys, rats, and goats are coincident with LH pulses (for review, see Nishihara et al., 1991). Nevertheless, direct testing of this model with regard to GnRH/LH pulse generation remains to be performed in a species better suited for serial blood sampling and the measurement of MUA. Details of this model will evolve as we learn more about the specific actions of Dyn and NKB on Kiss1/Dyn/NKB neurons in the Arc and GnRH fibers and terminals in the ME.

Footnotes

This research was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development/National Institutes of Health (NICHD/NIH) through Cooperative Agreement U54 HD12629 (to the University of Washington Center for Research in Reproduction and Contraception), NICHD/NIH Grant R01 HD27142, National Institute on Drug Abuse/NIH Grants RO1 DA016898 and KO5 DA020570, the Fulbright/MEC Program of the United States Department of State and Ministerio Español de Ciencia e Innovación, the Marie Curie Outgoing International Fellowship supported by the 7th Frame Programme of the European Union, and the Program for Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN) of Japan. We are grateful for the technical assistance provided by Sarah McConkey, Sarah Ahmad, and Dan Messinger at the University of Washington (Seattle, WA) and Brigitte Mann at Northwestern University (Evanston, IL). We thank Drs. Martin Kelly and Oline Ronnekleiv at the Oregon Health and Science University (Portland, OR) for their constructive comments on a previous draft of this manuscript.

References

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