Arcuate Kisspeptin/Neurokinin B/Dynorphin (KNDy) Neurons Mediate the Estrogen Suppression of Gonadotropin Secretion and Body Weight

Abstract

Estrogen withdrawal increases gonadotropin secretion and body weight, but the critical cell populations mediating these effects are not well understood. Recent studies have focused on a subpopulation of hypothalamic arcuate neurons that coexpress estrogen receptor α, neurokinin 3 receptor (NK₃R), kisspeptin, neurokinin B, and dynorphin for the regulation of reproduction. To investigate the function of kisspeptin/neurokinin B/dynorphin (KNDy) neurons, a novel method was developed to ablate these cells using a selective NK₃R agonist conjugated to the ribosome-inactivating toxin, saporin (NK₃-SAP). Stereotaxic injections of NK₃-SAP in the arcuate nucleus ablated KNDy neurons, as demonstrated by the near-complete loss of NK₃R, NKB, and kisspeptin-immunoreactive (ir) neurons and depletion of the majority of arcuate dynorphin-ir neurons. Selectivity was demonstrated by the preservation of proopiomelanocortin, neuropeptide Y, and GnRH-ir elements in the arcuate nucleus and median eminence. In control rats, ovariectomy (OVX) markedly increased serum LH, FSH, and body weight, and these parameters were subsequently decreased by treatment with 17β-estradiol. KNDy neuron ablation prevented the rise in serum LH after OVX and attenuated the rise in serum FSH. KNDy neuron ablation did not completely block the suppressive effects of E₂ on gonadotropin secretion, a finding consistent with redundant pathways for estrogen negative feedback. However, regardless of estrogen status, KNDy-ablated rats had lower levels of serum gonadotropins compared with controls. Surprisingly, KNDy neuron ablation prevented the dramatic effects of OVX and 17β-estradiol (E₂) replacement on body weight and abdominal girth. These data provide evidence that arcuate KNDy neurons are essential for tonic gonadotropin secretion, the rise in LH after removal of E₂, and the E₂ modulation of body weight.

Estradiol suppression of gonadotropin secretion, known as estrogen negative feedback, is an essential component of the female reproductive cycle. Estrogens also act to suppress body weight by altering food intake and metabolism (1, 2). In the human, neurons in the infundibular (arcuate) nucleus that coexpress estrogen receptor (ER)α, neurokinin B (NKB), kisspeptin, and dynorphin have been proposed to mediate estrogen negative feedback on gonadotropin secretion (3–7). A homologous group of kisspeptin/NKB/dynorphin (KNDy) neurons resides in the arcuate nucleus of a variety of other species, including mice, rats, sheep, goats, and monkeys (8–15). Mutations in the genes encoding kisspeptin, NKB, or their receptors are associated with low serum gonadotropins, infertility, and absence of pubertal maturation in both men and women (16–20). Transgenic mouse models also illustrate the importance of kisspeptin and NKB signaling in reproductive regulation (16, 21). Because kisspeptin and NKB peptides are located in numerous brain regions, it is not known whether KNDy neurons represent the essential cell type for reproduction.

To investigate the role of KNDy neurons, we developed a method to selectively ablate these neurons based on their expression of the neurokinin 3 receptor (NK₃R) (10, 12, 22). Within the arcuate nucleus, the expression of NK₃R protein or mRNA is restricted to a small subpopulation of neurons (10, 23). Arcuate neuroendocrine neurons (24), proopiomelanocortin (POMC) neurons, or tyrosine hydroxylase-immunoreactive (ir) neurons do not express NK₃R (Krajewski-Hall, S. J., and N. E. Rance, unpublished observations). Moreover, NK₃R mRNA is exclusively expressed in kisspeptin neurons in the mouse arcuate nucleus (12). Based on these data, we reasoned that destruction of NK₃R-expressing cells in the arcuate nucleus should selectively ablate KNDy neurons.

Saporin (SAP) is a ribosome-inactivating toxin that can be conjugated to selective receptor ligands to produce targeted cell ablation (25). Because NK₃R internalizes after ligand binding, conjugation of SAP to a selective NK₃R agonist (NK₃-SAP) provides a selective toxin for NK₃R-expressing neurons. In the present study, we characterized the morphological and physiological effects of injecting NK₃-SAP into the rat arcuate nucleus to ablate KNDy neurons. Our primary goal was to determine the effects of KNDy neuron ablation on the changes in gonadotropin secretion in response to ovariectomy (OVX) and estradiol replacement. We were surprised to find that the 17β-estradiol (E₂) modulation of body weight was also strikingly altered in KNDy-ablated animals.

Materials and Methods

Young adult, female Sprague Dawley rats (∼12 wk old, 200–250 g; Harlan Laboratories, Indianapolis, IN) were individually housed in a quiet, temperature-controlled room (21.1–22.5 C) in the University of Arizona Animal Care Facility with a 12-h light, 12-h dark cycle (lights on at 0700 h). Rats had ad libitum access to water and a low-phytoestrogen diet (Harlan Teklad 2014; Harlan Laboratories). All protocols were approved by the University of Arizona Animal Care and Use Committee and conformed to National Institutes of Health guidelines.

Validation of NK₃-SAP for selective ablation of NK₃R-expressing neurons in rat brain

[MePhe⁷]NKB is a modified NKB peptide that has been established to be a potent and selective agonist for NK₃R (26, 27). A custom conjugate of SAP and [MePhe⁷]NKB (NK₃-SAP) was obtained from Advanced Targeting Systems (San Diego, CA). NK₃-SAP stock [40 ng/100 nl in 0.1 m PBS (pH 7.4)] was stored at −80 C until use. Blank-SAP, an 11-amino acid peptide conjugated to SAP (Advanced Targeting Systems), was injected as a control. The amino acid sequence of the peptide in Blank-SAP (scrambled αMSH) has no significant homology to known G protein-coupled receptor ligands.

To determine whether NK₃-SAP retains high affinity for NK₃R, competitive binding assays of [MePhe⁷]NKB (Calbiochem, Rokland, MA) or NK₃-SAP against the selective NK₃R radioligand, [³H]senktide (PerkinElmer, Waltham, MA), were conducted using freshly prepared crude membranes from rat brain neocortex. Competitive binding was carried out in a total assay volume of 0.5 ml using 10 nm [³H]senktide (50.6 Ci/mmol) and various concentrations, in duplicates, of [MePhe⁷]NKB or NK₃-SAP for 2 h at 22 C in a shaking water bath. Total binding of [³H]senktide was defined by the radioactivity bound in the absence of competing ligand, and nonspecific binding of [³H]senktide was defined as the radioactivity bound in the presence of 10 μm [MePhe⁷]NKB. Incubation was terminated by rapid filtration of the samples through polyethyleneimine-presoaked GF/B filters. Radioactivity on the filters was determined by liquid scintillation counting. Data were analyzed by nonlinear least squares analysis using GraphPad Prism software (Prism Software Corp., Irvine, CA). Data were based on two independent experiments.

Best-fit analysis of the data based on a two-site competition analysis (GraphPad Prism) showed that [MePhe⁷]NKB had a high-affinity IC₅₀ value of 11 nm (Log IC₅₀ = −7.96 ± 0.75, n = 2), and the NK₃-SAP had an IC₅₀ value of 6.4 nm (Log IC₅₀ = −8.20 ± 1.5, n = 2) in a parallel assay. Thus, both [MePhe⁷]NKB and NK₃-SAP exhibited comparable, high-affinity binding for the NK₃R receptor. These data show that conjugation of [MePhe⁷]NKB to SAP did not significantly alter its affinity for NK₃R. This high-affinity interaction of NK₃-SAP with NK₃R appeared well suited for selective ablation of NK₃R-expressing cells via ligand-mediated uptake.

Pilot experiments were conducted to determine the optimum dose of NK₃-SAP for selective ablation of NK₃R neurons. The medial septum was studied because NK₃R in this area is restricted to neurons that exhibit a distinctive magnocellular appearance (24, 28). Under general anesthesia, NK₃-SAP (2, 10, or 40 ng diluted in 100 nl of PBS) was injected unilaterally into the medial septum (0.0 mm posterior to Bregma, 0.2 mm lateral, and 7.5 mm below the skull surface; n = 2–4 rats/group). The rats were killed 1 wk later, and the brains were processed as described below. Inspection of Nissl-stained sections revealed that the 10-ng NK₃-SAP injections produced unilateral reduction of the magnocellular neurons in the medial septal nucleus, with preservation of small neurons, and no other discernible damage to brain parenchyma other than the small injection tract. Immunohistochemistry confirmed that unilateral injection of 10 ng produced unilateral loss of magnocellular NK₃R neurons in the medial septum. The 2-ng injections produced barely perceptible NK₃R cell loss, and there was unacceptable nonspecific damage with the highest dose of 40 ng. Therefore, 10 ng of NK₃-SAP (in 100 nl of PBS) was used for all further studies.

Experiment 1. Effects of arcuate NK₃-SAP injections on the E₂ modulation of gonadotropin secretion and body weight

Experimental day 0, OVX, and injection of NK₃-SAP or Blank-SAP in the arcuate nucleus

Twenty-four rats were anesthetized with a cocktail (1.0 ml/kg im) containing ketamine (33.3 mg/ml), xylazine (10.7 mg/ml), and acepromazine (1.3 mg/ml) and ovariectomized using a dorsal surgical approach. A DSI PhysioTel transmitter (TA10-F40; Data Sciences International, St. Paul, MN) was inserted into the peritoneal cavity for measurements of core temperature. Stereotaxic surgery was used to make four injections of 10 ng/100 nl PBS of either NK₃-SAP (n = 14 rats) or Blank-SAP (n = 10 rats). Injections were made bilaterally (two sites per side) to target the dorsolateral border of the arcuate nucleus (rostral coordinates: 2.4 mm posterior to bregma, ±0.5 mm lateral, and 9.8 mm ventral to skull surface; caudal coordinates: 3.5 mm posterior to bregma, ±0.5 mm lateral, and 9.7 mm ventral to skull surface). Microinjections were made using a NanoFil 10-μl syringe with a 33-gauge beveled tip needle (World Precision Instruments, Sarasota, FL) inserted into an UltraMicroPump with a Micro 4 controller (UMP3; World Precision Instruments). Each injection was made over a period of 5 min, and the needle was left in place for an additional 5 min before removal. The incision was closed, and animals were given postoperative analgesia (0.03–0.05 mg/kg sc of buprenorphine).

Estradiol replacement

Twenty to 23 d after the initial surgery, under isofluorane anesthesia, rats were implanted with two sc capsules (each 20 mm effective length, 1.57 mm inner diameter, and 3.18 mm outer diameter; Dow Corning, Midland, MI) containing 17β-estradiol dissolved in sesame oil (E₂, 360 μg/ml).

Blood collection and body weight measurements

Blood samples and body weight measurements were taken at three time points: immediately before OVX (d 0, intact), 20–23 d after OVX, and 11 d after E₂ treatment (OVX + E₂). Girth (circumference at the widest point of the torso) was measured in the OVX and OVX + E₂ conditions while animals were anesthetized and in the prone position. Blood samples were collected between 0700 and 1100 h. The first two blood samples (∼50 μl each) were collected from a saphenous vein puncture using a 5-mm lancet (Braintree Scientific, Braintree, MA) and a Microvette capillary tube (Sarstedt, Newton, NC) while the rats were anesthetized for the initial surgery or the capsule implantation. The final sample was taken at the time of killing via cardiac puncture. Blood from all samples was left to clot for 90 min and centrifuged. Serum was collected and stored at −20 C until assay.

Brain tissue collection for histology

After an overdose injection of sodium pentobarbital (100 mg/kg ip), animals were perfused through the ascending aorta with 200 ml of heparinized, 0.1 m PBS followed by 400 ml of 4% paraformaldehyde in phosphate buffer (pH 7.4). Brains were extracted, postfixed in the same fixative for 1 h, and cryoprotected in ascending solutions of sucrose (10, 20, and 30%) in PBS. Brains were blocked using a rat brain matrix (Braintree Scientific). Blocks containing the hypothalamus were frozen and sectioned (40-μm thickness) in the coronal plane with a sliding microtome. Every tenth section was Nissl stained, and the remaining tissue was stored in a cryoprotectant solution at −20 C until use (29).

Hormone assays

Serum samples were assayed by the Ligand Assay and Analysis Core Laboratory at the University of Virginia Center for Research in Reproduction. (Charlottesville, VA). Serum LH and FSH (10-μl sample, in singlet) were assayed using a multiplex panel assay (Milliplex MAP for Luminex xMAP Technology, RPT-86K-02). The LH and FSH assays had an intraassay variability of 6.9 and 6.7% and a sensitivity of 0.24 and 2.4 ng/ml, respectively. LH values that fell below the sensitivity of the assay (rats with arcuate NK₃-SAP injections, three OVX and five OVX + E₂ values) were assigned the minimal detectable value of 0.24 ng/ml.

Terminal serum samples (n = 12) were also assayed for estradiol (60 μl each, in duplicate, DSL Ultrasensitive E₂ rat-RIA). The estradiol RIA had an intraassay variability of 6.7%. The serum E₂ in these terminal samples (11 d after capsule implantation) was at physiological levels of 36.6 ± 14.9 pg/ml (30). There was no difference in serum E₂ between Blank-SAP and NK₃-SAP injected rats.

Immunohistochemical studies

Antibodies

For detailed information, see Table 1.

Table 1.

Antibodies used in immunohistochemical procedures

Primary antibody (host, dilution)	Immunogen	Source, lot	Secondary antibody
NK3R (polyclonal rabbit, 1:20,000)	Residues 443–452 of rat NK3R	Phillipe Ciofi IS-7/7, bleed 040595	Biotinylated goat antirabbit (Vector Laboratories, CABA-1000, lot W0117)
pro-NKB (polyclonal rabbit, 1:15,000)	Residues 50–79 of mouse pro-NKB	Novus Biologicals NB300-201, lot A2	Biotinylated goat antirabbit
Kisspeptin (polyclonal rabbit, 1:20,000)	Residues 43–52 of mouse kisspeptin	Millipore (Temecula, CA) AB9754, lot NMM1669485	Biotinylated goat antirabbit
Prodynorphin (polyclonal guinea pig, 1:1000)	Residues 235–248 of rat prodynorphin	Neuromics GP10110, lot Q10016	Goat antiguinea pig + Alexa Fluor 488 (Invitrogen A11073, lot 752761)
POMC (polyclonal rabbit, 1:50,000)	Residues 27–52 of porcine POMC precursor	Phoenix Pharmaceuticals (Burlingame, CA) H-029-30, lot 01181-3	Biotinylated goat antirabbit
NPY (polyclonal rabbit,1:100,000)	36 amino acid porcine NPY peptide	Peninsula Laboratories (Torrance, CA) IHC-7172, lot 991025	Goat antirabbit + Alexa Fluor 568 (Invitrogen A11036, lot 84C1-1)
GnRH/LHRH (monocloncal mouse, 1:5000)	LHRH decapeptide	Henryk Urbanski clone HU4U	Highly cross-adsorbed goat antimouse + Alexa Fluor 488 (Invitrogen A11029, lot 727756)

Neurokinin 3 receptor

The NK₃R antiserum showed staining comparable with previous reports of the distribution of NK₃R mRNA (23) and protein (24, 31). Labeling was abolished by preadsorption with a synthetic peptide corresponding to amino acids 443–452 of the C-terminal region of rat NK₃R protein (Bio-Synthesis, Lewisville, TX).

Neurokinin B

The distribution of pro-NKB immunoreactivity was similar to previous reports with this (10, 24) and other pro-NKB antibodies (32–34). Preadsorption with the synthetic peptide used for immunization (Novus Biologicals, Littleton, CO) prevented immunolabeling.

Kisspeptin

The pattern of staining using this antibody was similar to previous reports of kisspeptin immunoreactivity (35, 36). The characterization of this antibody has been previously described (35).

Dynorphin

Labeling with the prodynorphin antibody matched the distribution of dynorphin immunoreactivity using this (10) and other (37) prodynorphin antibodies. Staining was not observed in dynorphin knockout mice (38) or after preadsorption with the immunizing antigen (Neuromics, Edina, MN).

Proopiomelanocortin

The POMC antiserum revealed the expected distribution of labeling based on previous immunocytochemical (39) and in situ hybridization studies (8). Using this antibody, colocalization of POMC and green fluorescent protein was observed in enhanced green fluorescent protein-POMC mice (40).

Neuropeptide Y (NPY)

Within the arcuate nucleus, the NPY antiserum labeled predominantly fibers. The labeling was in agreement with previous descriptions of NPY immunoreactivity in the arcuate nucleus and median eminence (41). This antibody has been previously characterized, including preadsorption experiments (42).

Gonadotropin-releasing hormone

This extensively characterized antibody (43) labeled cells with the classic appearance of GnRH neurons (24). Labeling was prevented by preadsorption with the GnRH peptide (43).

NK₃R immunohistochemistry was performed on every tenth section from the level of the paraventricular nucleus to the mammillary bodies. For all other antibodies, sections were matched to middle (plate 56) and posterior (plate 64) levels of the arcuate nucleus using a rat brain atlas (44). NK₃R immunohistochemistry used an antigen retrieval step consisting of incubation in 15 mm sodium citrate for 30 min at 80 C and then cooling to room temperature (45). Kisspeptin, pro-NKB, and POMC immunohistochemistry used the same protocol but without the antigen retrieval step. Sections were rinsed in PBS, incubated in 0.3% H₂O₂ for the removal of endogenous peroxidases, rinsed, and blocked in 3% normal goat serum (NGS) + 0.4% Triton X-100 in PBS. Sections were incubated with the antibody in blocking solution at 4 C for 48 h (see Table 1 for antibody dilutions). Sections were then rinsed and incubated in blocking solution containing a biotinylated goat antirabbit antibody (1:600), followed by rinsing and incubation in an Avidin-Biotin Complex solution (Vector Laboratories, Burlingame, CA). After rinsing with PBS, then 0.175 m sodium acetate, filtered nickel-intensified 3,3′ diaminobenzidine (DAB) solution was used for visualization (1.25 g of nickel ammonium sulfate, 10 mg of DAB in 50 ml of sodium acetate solution, and 41.5 μl of 30% H₂O₂). Sections were mounted on subbed slides, dehydrated, and coverslipped.

Prodynorphin, NPY, and GnRH were visualized using fluorescent immunohistochemistry. Sections were rinsed in PBS, incubated in blocking solution for 1 h, and then incubated with a primary antibody diluted in blocking solution for 48 h at 4 C (see Table 1 for antibody dilutions). After PBS rinses, sections were incubated in the appropriate secondary antibody (1:500) for 4 h (Table 1). After final rinses in PBS, sections were mounted, allowed to dry, and coverslipped using ProLong Gold Antifade (Invitrogen, Carlsbad, CA).

Cell counts and fiber density measurements

Morphologic analysis was performed by an investigator blind to the treatment condition. For the nickel-DAB-labeled sections (NK₃R, NKB, kisspeptin, and POMC), an image-combining computer microscope (Nikon, Tokyo, Japan) equipped with a Ludl motorized stage (Ludl Electronic Products, Hawthorne, NY), a Lucivid miniature CRT, and Neurolucida software (Microbrightfield, Williston, VT) was used to count cells. Outlines of the sections and regions of interest were drawn bilaterally using a ×4 Plan objective. Nissl stains were used to facilitate identification of landmarks. Cells were then mapped and systematically counted using a ×20 Plan apochromat objective (numerical aperture 0.50).

Immunofluorescence images (prodynorphin, NPY, and GnRH) were captured using a Nikon E1000 microscope equipped with a VFM epifluorescent attachment, a LUDL motorized stage, a Uniblitz model VMM-D1 shutter driver (Vincent Associates, Rochester, NY) and a Photometrics Coolsnap FX camera (Roper Scientific, Trenton, NJ). Digital images of the sections were taken in systematic step-wise fashion using a ×20 Nikon Plan Fluor objective (N.A. 0.50) and the appropriate filter set: Alexa Fluor 488 (excitation, 480/30 nm; diachromatic mirror; 505 nm; and emission filter 535/40 nm) or Alexa Fluor 568 (excitation, 560/40; DM, 595; and emission filter 630/60). Images were assembled into montages using MetaMorph imaging software (Molecular Devices, Sunnyvale, CA). Prodynorphin neurons were counted within the arcuate nucleus using a ×40 Nikon Plan Fluor objective (numerical aperture 0.75). Because NPY immunoreactivity in arcuate cell bodies is not visible without previous colchicine injection, we evaluated the status of NPY neurons by measuring the density of their local projections. NPY fiber densities were evaluated by drawing a 250-μm² square within the arcuate nucleus. GnRH fiber densities were evaluated within a boundary drawn to include the bilateral arcuate nucleus and adjacent median eminence. The total area analyzed did not differ between groups. Thresholds were established so that the labeled fibers in focus were above threshold. The fiber density value consisted of the area (in pixels) covered by labeled fibers divided by total area (in pixels) within the boundaries.

Experiment 2. Effects of NK₃-SAP injections in the lateral hypothalamus on gonadotropin secretion and body weight

Injections of NK₃-SAP into the arcuate nucleus resulted in variable loss of large NK₃R neurons in the lateral hypothalamus and zona incerta. Because loss of these extraarcuate NK₃R neurons complicated the interpretation of the first experiment, a second experiment was designed to replicate the pattern of NK₃R neuron loss in the lateral hypothalamus and zona incerta, while preserving KNDy neurons in the arcuate nucleus. NK₃-SAP (10 ng/100 nl PBS) was injected bilaterally in the lateral hypothalamus (n = 4 rats; coordinates: 3 mm posterior to bregma, ±1.8 mm lateral to midline, and 7.0 mm ventral to skull surface). Control rats (n = 4) received bilateral injections of Blank-SAP (10 ng/100 nl PBS). The protocol for this experiment and the immunohistochemical procedures were identical to that described above.

To evaluate the amount of cell degeneration after lateral hypothalamic injections, tissue was evaluated using Nissl histology and NK₃R immunohistochemistry. NK₃R neurons were counted and mapped throughout the lateral hypothalamus and zona incerta using the image combining computer microscope described above. Counts were made at three levels corresponding to plates 56, 60, and 64 of a rat brain atlas (44). NKB and NK₃R immunohistochemistry was used to verify preservation of KNDy neurons in the arcuate nucleus.

Data analysis

Three rats with preservation of KNDy neurons after arcuate NK₃-SAP injections were considered to have missed injections and excluded from the study. The numbers of NK₃R, pro-NKB, kisspeptin, prodynorphin, and POMC neurons per unilateral arcuate section were calculated for each animal to determine group means. Fiber staining density (NPY and GnRH) was also averaged for each animal, and these values were used to obtain group means. To determine the percent loss of NK₃R neurons in the lateral hypothalamus/zona incerta, the total number of neurons counted in the three sections was subtracted from the average number counted in Blank-SAP controls, then divided by the average number counted in Blank-SAP controls. Groups were compared using a t test. Serum gonadotropins, body weight, and girth measurements were compared using a two-way ANOVA followed by Tukey's post hoc analysis (α = 0.05).

Results

Arcuate NK₃-SAP injections ablate NK₃R-expressing KNDy neurons, with preservation of POMC, NPY, and GnRH neuronal elements

The morphological appearance of Nissl-stained sections at the level of the arcuate nucleus was similar between NK₃-SAP and Blank-SAP rats (Fig. 1A). Other than the small needle tracts, destruction of brain parenchyma was not observed. These data are consistent with the small percentage of arcuate neurons expressing either the NK₃R protein (10) or its mRNA (23). In contrast, immunohistochemistry revealed near-complete loss of NK₃R, NKB, and kisspeptin cell bodies in the arcuate nucleus of NK₃-SAP rats (Fig. 1, B–D). This change was accompanied by the disappearance of virtually all NKB and kisspeptin fibers in the arcuate nucleus and adjacent median eminence, as well as fibers extending along the third ventricle in the periventricular pathway (46). Quantitative analysis confirmed loss of KNDy neurons: the number of NK₃R, NKB, and kisspeptin-ir neurons in anterior and posterior levels of the arcuate nucleus was reduced by 93–98% in NK₃-SAP rats, compared with Blank-SAP controls (Table 2). In addition, the number of prodynorphin cell bodies in the arcuate nucleus was reduced by 77% in NK₃-SAP rats (Fig. 1E and Table 2). Preservation of a small number of arcuate dynorphin neurons is consistent with previous studies showing that there is a subgroup of dynorphin neurons in the rat arcuate nucleus that do not express NK₃R (10) and are not KNDy neurons (47). Dynorphin neurons were also preserved in the adjacent ventromedial nucleus.

Fig. 1.

Representative photomicrographs of sections from rats with injections of Blank-SAP (left) or NK₃-SAP (right) in the arcuate nucleus, stained with Cresyl-violet (A) or immunohistochemistry (B–H). A, Cresyl violet-stained sections show preservation of the Nissl architecture in NK₃-SAP rats. B–D, NK₃-SAP rats exhibited near total depletion of NK₃R, NKB, and kisspeptin-ir neurons and fibers in the arcuate nucleus and median eminence. E, The majority of dynorphin-ir neurons and fibers in the arcuate nucleus and median eminence were ablated in NK₃-SAP rats. F and G, There was no difference in the number of POMC neurons or NPY fiber density between Blank-SAP and NK₃-SAP rats. H, GnRH fibers in the median eminence were preserved in NK₃-SAP rats. 3V, Third ventricle; ARC, arcuate nucleus; ME, median eminence; pro-DYN, prodynorphin; VMN, ventromedial nucleus. Scale bar, 100 μm.

Table 2.

Quantitative analysis of immunoreactive cell bodies or fiber densities in rats injected with either Blank-SAP or NK₃-SAP in the arcuate nucleus

Immunohistochemical phenotype	Mid-level arcuate		Posterior arcuate		Total percent loss
	Blank-SAP	NK3-SAP	Blank-SAP	NK3-SAP
NK3R	7.9 ± 0.7	0.3 ± 0.2a	13.4 ± 1.9	0.2 ± 0.1a	98.1 ± 0.8
NKB	10.4 ± 1.1	0.6 ± 0.5a	50.8 ± 3.6	3.1 ± 1.1a	92.6 ± 2.2
Kisspeptin	6.4 ± 1.0	0.1 ± 0.1a	31.4 ± 3.6	1.4 ± 0.6a	94.8 ± 1.6
Dynorphin	11.0 ± 1.5	3.6 ± 0.5a	24.0 ± 5.7	4.1 ± 1.0a	76.5 ± 2.9
POMC	33.7 ± 3.8	37.2 ± 3.2	29.7 ± 2.1	36.0 ± 3.0	0
NPY	0.07 ± 0.01	0.06 ± 0.02	0.03 ± 0.003	0.03 ± 0.004	0
GnRH	0.04 ± 0.004	0.04 ± 0.008	NA	NA	0

Values represent mean ± sem NK₃R, NKB, kisspeptin, dynorphin, and POMC values represent the average number of labeled neurons/unilateral arcuate section. NPY and GnRH values reflect average fiber density values (see Materials and Methods). NA, Not analyzed.

^aP < 0.01, Significantly different, Blank-SAP vs. NK₃-SAP-treated animals, n = 5–11 rats/group.

The selectivity of NK₃-SAP for ablation of NK₃R-expressing KNDy neurons was demonstrated by preservation of POMC, NPY, and GnRH-ir elements. NK₃-SAP had no qualitative or quantitative effect on the number of POMC neurons or NPY fibers within the arcuate nucleus (Fig. 1, F and G, and Table 2). Despite a previous demonstration of punctate NK₃R-staining on GnRH fibers (24), there were no qualitative differences in the distribution of GnRH fibers in the median eminence and no change in GnRH fiber density between NK₃-SAP and Blank-SAP rats (Fig. 1H and Table 2). These data agree with reports that SAP-peptide conjugates do not produce retrograde neuronal degeneration (48).

In Blank-SAP rats, large NK₃R neurons with intense dendritic labeling were identified in lateral hypothalamus (including the perifornical region) and zona incerta (see Ref. 24 for photomicrographs). No NK₃R neurons were identified in the ventral premamillary nucleus or the ventromedial nucleus, in agreement with previous reports (23, 24, 31, 49). In NK₃-SAP rats, there was variable and incomplete loss of the large NK₃R neurons in the lateral hypothalamus and zona incerta. NK₃R neurons in the supraoptic and paraventricular nuclei were intact, indicative of the limited diffusion of the toxin.

Arcuate KNDy neuron ablation prevents the rise in LH secretion after OVX and lowers serum gonadotropins

Blank-SAP rats exhibited a 13-fold increase in serum LH after OVX that returned to intact levels 11 d after E₂ treatment (Fig. 2A). Serum LH was not significantly elevated above intact levels in OVX rats that received injections of NK₃-SAP in the arcuate nucleus (Fig. 2A). E₂ treatment of OVX NK₃-SAP rats resulted in a small but significant decrease of serum LH. In both the OVX and OVX + E₂ conditions, serum LH was significantly lower in NK₃-SAP rats compared with Blank-SAP controls.

Fig. 2.

Effects of KNDy neuron ablation on serum LH (A) and FSH (B). Rats were ovariectomized and injected with NK₃-SAP or Blank-SAP in the arcuate nucleus on d 0, given E₂ capsules 20–23 d later (shown as d 22), and killed after 11 d of E₂. Serum LH and FSH were markedly increased by OVX and reduced by E₂ treatment in Blank-SAP animals. In contrast, serum LH after OVX was not significantly different from intact values in NK₃-SAP rats, and the rise in FSH was attenuated (mean ± sem, n = 6–11 rats/group for both A and B). Serum LH and FSH were significantly lower in KNDy-ablated rats than Blank-SAP controls in both OVX and OVX + E₂-treated groups. #, Significantly different, NK₃-SAP vs. Blank-SAP, P ≤ 0.05; +, significantly different from intact values on d 0, P ≤ 0.01; *, significantly different after E₂ (vs. OVX), P ≤ 0.05.

In Blank-SAP control rats, serum FSH was increased after OVX and decreased by E₂ treatment (Fig. 2B). In arcuate NK₃-SAP rats, serum FSH was also increased by OVX and reduced by E₂ treatment, but FSH levels were significantly lower after NK₃-SAP injections, compared with the control animals (Fig. 2B).

Arcuate KNDy neuron ablation prevents the E₂ modulation of body weight and girth observed in Blank-SAP controls

Three weeks after OVX, Blank-SAP rats were 25% heavier relative to their initial body weight (Fig. 3A), and E₂ treatment of these rats induced significant weight loss. In contrast, the body weight of arcuate NK₃-SAP rats was only 5% higher after OVX, and these values were not significantly different compared with those obtained on experimental d 0. In addition, E₂ treatment did not reduce the body weight of OVX NK₃-SAP rats. Overall, arcuate NK₃-SAP rats gained weight throughout the experiment in amounts expected from the standard growth charts of intact female Sprague Dawley rats (Growth Curve, Harlan Sprague Dawley).

Fig. 3.

Effects of KNDy neuron ablation on body weight and abdominal girth. Rats were ovariectomized and injected with NK₃-SAP or Blank-SAP in the arcuate nucleus on d 0, given E₂ capsules 20–23 d later (shown as d 22), and killed after 11 d of E₂. A, After OVX, Blank-SAP animals were 25% heavier relative to their initial body weight and lost weight after E₂ treatment. Rats with arcuate NK₃-SAP injections gained small amounts of weight throughout the study, regardless of E₂ status (mean ± sem, n = 9–11 rats/group). B, After OVX, the girth of NK₃-SAP rats was significantly smaller than Blank-SAP rats. Girth was significantly decreased by E₂ in Blank-SAP but not NK₃-SAP rats (mean ± sem, n = 3–4 rats/group). #, Significantly different, Blank-SAP vs. NK₃-SAP, P < 0.01; +, significantly different from intact values on d 0, P < 0.05; *, significantly different after E₂ (vs. OVX), P < 0.01.

Three weeks after OVX, the girth of Blank-SAP rats was significantly larger than in NK₃-SAP rats. E₂ treatment significantly decreased girth in Blank-SAP animals but had no effect on girth in NK₃-SAP rats (Fig. 3B).

Loss of NK₃R neurons in the lateral hypothalamus and zona incerta does not alter the E₂ modulation of gonadotropin secretion or body weight

NK₃-SAP injections in the lateral hypothalamus produced incomplete NK₃R neuron degeneration in lateral hypothalamus (including the perifornical area) and zona incerta (Fig. 4). The amount of neuronal loss was similar to that seen in experiment 1, but KNDy neurons were spared. Cell counts revealed that lateral hypothalamic NK₃-SAP injections depleted 80.6 ± 2.5% of the large NK₃R neurons in the lateral hypothalamus and zona incerta, compared with 71.3 ± 2.9% in rats with arcuate NK₃-SAP injections.

Fig. 4.

Representative maps of NK₃R neurons from a Blank-SAP-injected (A), arcuate NK₃-SAP-injected (B) or lateral hypothalamic NK₃-SAP-injected (C) rat. A, Map from Blank-SAP rat showing the location of NK₃R neurons in the arcuate nucleus, lateral hypothalamus, supraoptic nucleus, and zona incerta. B, Map from a rat with an arcuate NK₃-SAP injection with KNDy neuron ablation and incomplete loss of NK₃R neurons in the lateral hypothalamus and zona incerta. C, Map from rat with a lateral hypothalamic NK₃-SAP injection showing NK₃R neurons in the arcuate nucleus and incomplete loss of NK₃R cell bodies in the lateral hypothalamus and zona incerta. Each dot represents one labeled neuron. 3V, Third ventricle; ARC, arcuate nucleus; LHA, lateral hypothalamic area; ot, optic tract; SON, supraoptic nucleus; VMN, ventromedial nucleus; ZI, zona incerta. Scale bar, 250 μm.

Despite the extensive loss of the large NK₃R cells, lateral hypothalamic NK₃-SAP injections did not alter the E₂ modulation of gonadotropin secretion or body weight (Fig. 5). In both groups, serum LH and FSH levels were increased after OVX and decreased after E₂ treatment (Fig. 5, A and B). Moreover, rats with NK₃-SAP injections in the lateral hypothalamus gained a large amount of weight after OVX, and E₂ treatment resulted in significant weight loss (Fig. 5C).

Fig. 5.

Effects of NK₃-SAP injections in the lateral hypothalamus on serum LH (A), FSH (B), and body weight (C). Rats were ovariectomized and injected with NK₃-SAP or Blank-SAP on d 0, given E₂ capsules 20–23 d later (shown as d 22), and killed after 11 d of E₂. A–C, There were no significant differences between rats with lateral hypothalamic NK₃-SAP and Blank-SAP injections (mean ± sem, n = 4 values/group, except n = 2 for Blank-SAP gonadotropin levels). +, Significantly different from intact values on d 0, P < 0.05; *, significantly different after E₂ (vs. OVX), P < 0.05.

Discussion

To evaluate the role of NK₃R-expressing KNDy neurons in the E₂ modulation of gonadotropin secretion and body weight, we ablated these neurons using NK₃-SAP, a novel conjugate of a highly selective NK₃R agonist and SAP. Immunohistochemical studies documented the selectivity of NK₃-SAP injections for KNDy neurons in arcuate nucleus. In contrast to the marked rise in serum LH in Blank-SAP controls, serum LH was not significantly increased after OVX in KNDy-ablated rats. Moreover, LH and FSH were significantly lower in KNDy-ablated rats than controls, regardless of E₂ status. Surprisingly, KNDy neuron ablation blocked the pronounced effects of OVX and E₂ replacement on body weight and abdominal girth. Our data indicate an essential role for KNDy neurons in the regulation of gonadotropin secretion and the E₂ modulation of body weight.

The classic reciprocal relationship between the ovaries and anterior pituitary gland hormone secretion was demonstrated in Blank-SAP controls (50). Three weeks after OVX, serum LH and FSH were markedly increased in control rats. Replacement of physiological levels of E₂ suppressed LH to intact levels, consistent with numerous studies showing that that the rise in LH after OVX is largely due to removal of E₂ (51). Serum FSH was also decreased by E₂ replacement in control rats, although not to intact levels. These data reflect the dual ovarian control of FSH secretion, which includes protein hormones such as inhibin, as well as E₂ feedback (51).

Remarkably, in rats with KNDy neuron ablation, serum LH was not significantly increased 3 wk after OVX. These data indicate that the suppressive effects of E₂ on serum LH in OVX rats are predominantly mediated through KNDy neurons. E₂ treatment of KNDy-ablated rats still produced a small decrease in LH. Although these findings are consistent with redundant sites of E₂ negative feedback (52, 53), we cannot exclude E₂ effects on the few remaining KNDy neurons. Serum FSH was significantly modulated by OVX and E₂ replacement in KNDy-ablated rats but lower than Blank-SAP controls, regardless of E₂ status. Similarly, serum LH was lower in KNDy-ablated rats than in Blank-SAP rats in both the OVX and OVX + E₂ conditions. Thus, KNDy neurons play a role in stimulating the high levels of serum gonadotropins after withdrawal of E₂ as well as tonic stimulation of gonadotropin secretion in OVX E₂-treated rats.

Previous studies show that OVX markedly increases NKB and kisspeptin gene expression in the rodent arcuate nucleus, and these changes are reversed by E₂ replacement (8, 54). KNDy neurons express ERα (10, 54), the isoform required for E₂ suppression of GnRH mRNA, serum LH (55), and NKB and kisspeptin gene expression (56, 57). KNDy neurons do not directly modulate the anterior pituitary gland, because they do not project to fenestrated capillaries in the median eminence (24, 58), but they could influence LH secretion by projections to GnRH terminals in the median eminence (24, 46, 59–62). Pharmacological studies show that kisspeptin stimulates GnRH secretion (62, 63) and activates GnRH neurons in tissue slice preparations (64). Moreover, similar to the effects of KNDy neuron ablation, kisspeptin receptor knockout mice do not exhibit a rise in serum LH after OVX (65). Pharmacological studies have also shown NKB and dynorphin receptor activation to alter levels of serum gonadotropins, in addition to changing the frequency of LH pulses (13, 15, 66–68). Although the relative contribution of different hypothalamic cell groups in the regulation of GnRH secretion has been controversial, our data clearly underscore the importance of KNDy neurons in this circuitry.

In many mammalian species, ovarian hormone withdrawal causes obesity that can be reversed by E₂ treatment (1, 69, 70). E₂ decreases food intake and increases energy expenditure in OVX animals via an ERα-dependent mechanism (2, 71, 72). In our study, control rats gained a large amount of weight after OVX and lost weight after E₂ replacement. Based on abdominal girth measurements, the suppressive effect of E₂ on body weight was due (at least in part) to loss of visceral adiposity.

In contrast to the controls, KNDy-ablated rats did not gain a significant amount of weight 3 wk after OVX, and E₂ treatment did not cause weight loss or changes in abdominal girth. The KNDy-lesioned rats were not compromised in their ability to maintain body weight. Rather, they gained weight throughout the experiment at rates nearly identical to OVX + E₂-replaced rats under similar experimental conditions (73) and intact female rats (1). In addition, we have recently observed no effect of NK₃-SAP (vs. Blank-SAP) injections on the body weight of ovary-intact female rats within 4 wk after injection (Mittelman-Smith, M. A., and N. E. Rance, unpublished observations). Overall, the body weight regulation of KNDy-ablated rats was characterized by insensitivity to the effects of OVX and E₂ replacement.

Our data provide evidence that increased KNDy neuron signaling promotes visceral fat storage in parallel with the increase in serum LH after the removal of E₂. Further studies are necessary to determine whether the effects of KNDy neuron ablation are secondary to changes in food intake and/or energy expenditure. KNDy neurons have been proposed to relay metabolic signals to influence reproductive function (74–77), but there are no previous reports of a role for KNDy neurons in the E₂ modulation of body weight. Based on several studies, it is possible that KNDy neurons influence body weight via local projections to NPY and POMC neurons within the arcuate nucleus (78–80). However, because KNDy neurons project to multiple hypothalamic centers (46, 58), there are many potential pathways for these neurons to influence energy homeostasis.

The present study used targeted cell ablation to study the function of a subpopulation of neurons, rather than knockout of a specific gene. Kisspeptin cell ablation has also been accomplished using a transgenic mouse strategy, in which the cells are made selectively vulnerable to diphtheria toxin (81). Consistent with our findings, ablation of kisspeptin cells in mice on postnatal d 20 (and in adult mice) produced disrupted estrous cycles and infertility (81). However, ip administration of diphtheria toxin would have systemic effects on kisspeptin cells, producing ablation in the ovary, pituitary, arcuate nucleus, amygdala, and the anteroventral periventricular nucleus (AVPV), which has numerous kisspeptin mRNA-expressing neurons by postnatal d 18 (82). In contrast, our study used stereotaxic injections of NK₃-SAP in very small volumes (100 nl) to target NK₃R-expressing KNDy neurons. Limited diffusion of the toxin from the injection site was documented by sparing of the NK₃R-ir neurons in the supraoptic and parventicular nuclei. Thus, the NK₃-SAP could not have diffused in a high enough concentration (through tissue or the ventricle) to directly affect rostral areas, such as the AVPV, the medial preoptic area, or median preoptic nucleus. In addition, the AVPV kisspeptin neurons would not be vulnerable to NK₃-SAP because AVPV neurons do not express NK₃R (23, 49). We did observe variable and incomplete loss of large NK₃R cells in the lateral hypothalamus and zona incerta adjacent to the injection site. This was a potential confounding factor, because some of these cells contain melanin-concentrating hormone (31), a peptide involved in the regulation of body weight (83). However, a second experiment designed to replicate the extraarcuate NK₃R cell loss did not produce the same effects as the arcuate injections. Thus, the alterations in the E₂ modulation of gonadotropin secretion and body weight observed after injections of NK₃-SAP in the arcuate nucleus were not secondary to degeneration of NK₃R-expressing neurons in the lateral hypothalamus and zona incerta.

In postmenopausal women, KNDy neurons exhibit somatic hypertrophy and increased NKB and kisspeptin gene expression (4, 5). Experiments in cynomolgus monkeys indicate that the changes in postmenopausal women are due to loss of ovarian estrogen and not aging per se (5, 7, 9, 84). Based on these studies, we have hypothesized that increased activity of KNDy neurons stimulates the elevation in gonadotropin secretion that occurs in response to the ovarian failure of menopause (4, 5, 7). The present studies strongly support this hypothesis, because the rise in LH secretion after OVX was prevented in rats with KNDy neuron ablation. Dysfunction of KNDy neurons may also explain the low serum gonadotropins in patients with inactivating mutations in the genes encoding NKB, kisspeptin, or their receptors (16–20). A role of KNDy neurons in the E₂ modulation of body weight and abdominal girth is also relevant to women, because polymorphisms in the gene encoding ERα are associated with increased visceral adiposity and body mass index (85, 86). Moreover, postmenopausal women exhibit a change in body weight distribution characterized by an increased waist to hip ratio indicative of visceral adiposity (87). This is important, because increased visceral adiposity, independent of body mass index, is associated with hypertension, glucose intolerance, and increased risk for cardiovascular disease in women (88, 89).

In summary, we have demonstrated that arcuate KNDy neurons are essential for the rise in LH secretion after removal of ovarian E₂, supporting a role for these neurons in E₂ negative feedback. KNDy neurons are also required for the maintenance of tonic gonadotropin secretion and the E₂ modulation of body weight. Recent studies have implicated NK₃R signaling in the regulation of body temperature (90), and we are currently examining whether KNDy neuron ablation alters hypothalamic thermoregulation. Through integration of sex-steroid signals with various physiological systems, KNDy neurons could play a role in optimizing the internal environment for reproduction. Based on their location in the arcuate nucleus, expression of ERα and diverse projections to multiple homeostatic and neuroendocrine control centers (46, 58), KNDy neurons are ideally positioned for this task.