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Molecular Endocrinology, doi:10.1210/me.2007-0207
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Molecular Endocrinology 21 (12): 3062-3070
Copyright © 2007 by The Endocrine Society

Expression of a Functional G Protein-Coupled Receptor 54-Kisspeptin Autoregulatory System in Hypothalamic Gonadotropin-Releasing Hormone Neurons

Samuel Quaynor1, Lian Hu1, Po Ki Leung1, Hao Feng, Nadia Mores, Lazar Z. Krsmanovic and Kevin J. Catt

Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510

Address all correspondence and requests for reprints to: Lazar Z. Krsmanovic, Ph.D., Endocrinology and Reproduction Research Branch, Building 49, Room 6A-36, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892. E-mail: lazar{at}mail.nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The G protein-coupled receptor 54 (GPR54) and its endogenous ligand, kisspeptin, are essential for activation and regulation of the hypothalamic-pituitary-gonadal axis. Analysis of RNA extracts from individually identified hypothalamic GnRH neurons with primers for GnRH, kisspeptin-1, and GPR54 revealed expression of all three gene products. Also, constitutive and GnRH agonist-induced bioluminescence resonance energy transfer between Renilla luciferase-tagged GnRH receptor and GPR54 tagged with green fluorescent protein, expressed in human embryonic kidney 293 cells, revealed heterooligomerization of the two receptors. Whole cell patch-clamp recordings from identified GnRH neurons showed initial depolarizing effects of kisspeptin on membrane potential, followed by increased action potential firing. In perifusion studies, treatment of GT1–7 neuronal cells with kisspeptin-10 increased GnRH peak amplitude and duration. The production and secretion of kisspeptin in cultured hypothalamic neurons and GT1–7 cells were detected by a specific RIA and was significantly reduced by treatment with GnRH. The expression of kisspeptin and GPR54 mRNAs in identified hypothalamic GnRH neurons, as well as kisspeptin secretion, indicate that kisspeptins may act as paracrine and/or autocrine regulators of the GnRH neuron. Stimulation of GnRH secretion by kisspeptin and the opposing effects of GnRH on kisspeptin secretion indicate that GnRH receptor/GnRH and GPR54/kisspeptin autoregulatory systems are integrated by negative feedback to regulate GnRH and kisspeptin secretion from GnRH neurons.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
KISSPEPTINS, A FAMILY OF peptides encoded by the KiSS-1 gene, promote GnRH secretion and are endogenous ligands for the G protein-coupled receptor, GPR54 (1, 2, 3). Both KiSS-1 and GPR54 are expressed in the mammalian hypothalamus, consistent with observations that kisspeptins and GPR54 are involved in activation and regulation of the hypothalamic-pituitary-gonadal axis (4, 5).

Disrupted GPR54 signaling causes hypogonadotrophic hypogonadism in rodents and humans (6, 7). Central or peripheral administration of kisspeptin potently stimulates the hypothalamic-pituitary-gonadal axis, causing increased circulating gonadotropin concentrations in a number of animal models (8). Although these effects appear likely to be mediated via the hypothalamic gonadotropin-releasing hormone system, kisspeptins may also have direct effects on the anterior pituitary gland (9). GPR54 is widely expressed in many tissues related to reproductive function, and there is increasing evidence that kisspeptin acts predominantly at the level of the central nervous system to regulate GnRH secretion (10, 11, 12).

In the present studies, the expression of mRNAs for GnRH, kisspeptin, and GPR54 was analyzed by single-cell RT-PCR in visually identified hypothalamic GnRH neurons. Bioluminescence resonance energy transfer between Renilla luciferase (Rluc) tagged GnRH-R and GPR54 tagged with green fluorescent protein [bioluminescence resonance energy transfer (BRET2)] was used to estimate constitutive and GnRH agonist (des-GLy10-[D-Ala6]GnRH N-ethylamide ([D-Ala6]Ag-) induced heterooligomerization of GnRH-R and GPR54 expressed in human embryonic kidney (HEK)-293 cells. Modulation of spontaneous electrical activity by kisspeptins was monitored by whole-cell patch-clamp recordings from identified GnRH neurons. The cell content and secretion of GnRH and kisspeptin from cultured hypothalamic and GT1–7 cells were measured in static and perifused cell cultures. The data from these studies indicate that the GPR54 receptors expressed in hypothalamic GnRH neurons form heterooligomers with GnRH-R and. in accord with secreted kisspeptin and GnRH, regulate GnRH neuronal activity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of GnRH, GPR54, and KiSS-1 mRNAs in Cultured Hypothalamic Cells, GT1–7 Neurons, and Identified Hypothalamic GnRH Neurons
RT-PCR analysis of total RNA derived from cultured hypothalamic cells using gene-specific primers based on the sequences of GnRH, GPR54, and KiSS gave the expected fragment size of 383 bp for GnRH, 235 bp for GPR54, and 136 bp for KiSS-1 (Fig. 1AGo; lanes 2 and 3). Probing of RNA extracts from GT1–7 cells with primers for GPR54 and KiSS-10 revealed expression of both gene products (Fig. 1BGo; lanes 1 and 2). No such products were obtained in the absence of reverse-transcribed mRNA, indicating that the RNA preparation was free of genomic DNA contamination (Fig. 1AGo, lane 1; and Fig. 1BGo, lane 3).


Figure 1
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  		Fig. 1. RT-PCR Analysis of GnRH, GPR54, and KiSS-1 Expression in Hypothalamic GnRH Neurons

A, mRNAs for GnRH, GPR54, and KiSS-1 were detected in total RNA extracted from cultured hypothalamic cells (lane 1, negative control; lanes 2 and 3, positive bands for GnRH, GPR54, and KiSS-1). B, Expression of GPR54 mRNA (lane 1) and KiSS-1 mRNA (lane 2) C, Expression of GnRH mRNA in seven visually identified single hypothalamic GnRH neurons. D, KiSS-1 mRNA was also detected in seven of eight hypothalamic GnRH neurons. D, mRNA for GPR54 was less abundant and was found in three of eight individual hypothalamic GnRH neurons.

 
Individual hypothalamic GnRH neurons were selected by differential interference contrast microscopy, which permits their morphological identification in hypothalamic cell cultures with an accuracy of more than 95%. After being harvested, the cytoplasmic content of each neuron was subjected to multiplex single-cell PCR to confirm the presence of GnRH, kisspeptin, and GPR54 transcripts. Seven individual GnRH neurons were collected from cultured hypothalamic cells by their morphology and single-cell RT-PCR revealed that all seven GnRH neurons expressed GnRH mRNA (Fig. 1CGo, lanes 1–7). Multiplex single-cell RT-PCR also confirmed that mRNA for kisspeptin was expressed in all seven GnRH neurons. (Fig. 1DGo, lanes 1–7). Moreover, about 50% of individual GnRH neurons express mRNA for GPR54 (Fig. 1EGo, lanes 1, 4, and 7).

BRET2 Analysis of the Interaction between GnRH Receptors (GnRH-Rs) and GPR54
A constitutive kisspeptin- and GnRH-activated BRET2 signal was observed in HEK-293 cells stably expressing GnRH-R-Rluc, after transient transfection with GPR54-GFP2. Single-point BRET2 measurements showed a significant net BRET2 ratio increase during treatment with 10 nM and 100 nM kiSS-10 (Fig. 2AGo). Similarly, treatment with 10 nM and 100 nM GnRH agonist analog, [D-Ala6]-Ag, caused significant increases in net BRET2 ratio (Fig. 2BGo). In contrast, treatment with a GnRH-R antagonist had no effect on BRET2 signaling (Fig. 2CGo). To evaluate the specificity of constitutive and agonist-induced interactions between GnRH-R-Rluc and GPR54-GFP2, saturation assays was used in which BRET2 signal was monitored in HEK-293 cells stably about 300 fmol/mg of GnRH-R-Rluc and increasing amounts of GPR54-GFP2 varying between 300 and 1500 fmol/mg. As shown in Fig. 3AGo, increasing concentrations of GFP2-fused GPR54 gave a hyperbolic BRET2 signal with BRET250 at 2.1 ± 0.2 GPR54-GFP2/GnRH-R-Rluc ratios. The maximum BRET2 signal was reached at 1:4 ratios, when all GnRH-R-Rluc engaged in oligomerization is in complex with GPR54-GFP2 (Fig. 3AGo, open circles). The maximal BRET2 signal increased significantly from 0.09 ± 0.008 (constitutive) to 0.11 ± 0.007 during treatment with 100 nM [D-Ala6]Ag (P < 0.05, n = 3), with unchanged half-maximal response (Fig. 3AGo, solid circles). In dynamic studies, the constitutive BRET2 signal increased over 40 sec with a half-time value of 15.3 ± 2.4 sec, reached maximal net BRET2 ratio at 26 sec and saturated thereafter (Fig. 3BGo, open circles). The mean net BRET2 ratio increased significantly over the constitutive BRET2 level during agonist treatment with 0.1 nM [D-Ala6]Ag (0.16 ± 0.01 vs. 0.23 ± 0.01 0.1; P < 0.001, n = 37). The half-maximal response was observed at 16.9 ± 1.8 sec, maximal net BRET2 ratio was at 30 sec, and the net BRET2 ratio saturated thereafter (Fig. 3BGo, solid circles). Treatment with increasing [D-Ala6]Ag concentrations caused further increases in net BRET2 ratio (0.28 ± 0.01, 10 nM [D-Ala6]Ag vs. 0.16 ± 0.01, constitutive; P < 0.01, n = 3, Fig. 3BGo, open squares) and (0.32 ± 0.01, 1 mM [D-Ala6]Ag vs. 0.16 ± 0.01, constitutive; P < 0.01, n =3, Fig. 3BGo, solid squares). At increased concentrations of [D-Ala6]Ag, the net BRET2 ratio did not saturate during the 40-sec treatment period and estimated EC50 values were 125.8 ± 14.2 and 128.1 ± 14.8 sec for 10 nM and 1 mM [D-Ala6]Ag, respectively (Fig. 3BGo, open and solid squares).


Figure 2
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  		Fig. 2. BRET2 Analysis of the Interaction between Rluc-Tagged GnRH-R and GFP2-Tagged GPR54 Receptor

The BRET2 signal from HEK-293 cells transfected with Rluc-GnRH-R and GFP-GPR54 was measured in the presence of DeepBlueC coelenterazine. A, Activation of GPR54 by KiSS-10 caused significant increase in BRET signal. Data are means ± SE of three experiments. Asterisks indicate significant differences compared with the control. B, Significant increases in BRET2 signal were also observed during treatment with [D-Ala6]-Ag. Asterisks indicate significant differences compared with the control. Data are means ± SEs of four experiments. C, There was no significant changes in BRET2 signal during treatment with CDB, a GnRH-R antagonist. Data are means ± SE of three experiments.

 

Figure 3
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  		Fig. 3. Characterizations of GnRH-R-Rluc and GPR54-GFP2 Interactions in Saturation and Dynamic Studies

A, An increasing amount of GPR54-GFP2 was transiently expressed in HEK-293 cells stably expressing GnRH-R-Rluc (300 fmol/mg), and the BRET2 signal was measured 48 h after transfection. Expression levels of GPR54-GFP2 varied between 300 and 1500 fmol/mg. The BRET signal is plotted as a function of the expression ratio of GPR54-GFP2 over GnRH-R-Rluc. Data are means ± SEs of three experiments. B, In dynamic studies, BRET2 measurements were collected at 1-sec intervals under basal conditions and during stimulation with [D-Ala6]-Ag for up to 40 sec. The constitutive BRET2 signal increased monotonically, reached a maximum at 26 sec, and saturated thereafter (open circles). Agonist treatment caused a dose-dependent increase in BRET2 signal that increased significantly during treatment with 0.1 nM [D-Ala6]-Ag (solid circles), followed by 10 nM [D-Ala6]-Ag (open squares), and was maximal during treatment with 1 µM [D-Ala6]-Ag (solid squares). Data are means ± SE of three experiments.

 
Patch-Clamp Recording from Identified Hypothalamic GnRH Neurons
Whole-cell recordings from identified hypothalamic GnRH neurons consistently revealed spontaneous action potential (AP) firing, with most of the cells (75%) showing irregular spiking activity (Fig. 3Go, A and E). Treatment of spontaneously active GnRH neurons with 100 nM kisspeptin-10 increased AP firing from the basal 0.8 ± 0.09 Hz to 1.8 ± 0.2 Hz (P < 0.01; n = 10; Fig. 3BGo). These kisspeptin-10-induced changes in AP firing were reversible, and basal AP firing resumed in both cell types during the washout period (Fig. 3CGo). In quiescent hypothalamic GnRH neurons, treatment with kisspeptin-10 caused membrane depolarization followed by a prominent increase in AP firing that continued during washout of kisspeptin (Fig. 3DGo). Treatment of identified GnRH neurons with 100 nM human metastin-54 also increased spontaneous AP firing from 1.4 ± 0.16, n =8 (Fig. 3EGo) to 1.9 ± 0.22, n =8 (Fig. 3FGo). In quiescent hypothalamic GnRH neurons, treatment with metastin-54 caused membrane depolarization followed by an increase in AP firing (Fig. 2GGo). In contrast to kisspeptin-10, metastin-54 did not have a long-lasting effect on AP firing, which diminished during metastin-54 washout (Fig. 3GGo).

Secretion of GnRH and Kisspeptin from Cultured Hypothalamic Cells and GT1–7 Neurons
The 145-amino acid kiSS-1 peptide is a primary translation product of the KiSS-1 gene. It is processed into various C-terminal fragments, such as kisspeptin-54, -14, -13, and -10, that bind to and activate GPR54 (1). In cell lines expressing transfected human or rat GPR54, kisspeptin-54 (bp 68–121), i.e. metastin, induced an increase in [Ca2+]i. N-terminally truncated peptides; kisspeptin-14 (bp 40–54) and kisspeptin-10 (bp 45–54) are 3 to 10 times more active than metastin (13). In human first-trimester trophoblast, which expresses endogenous GPR54, only kisspeptin-10 caused an increase in intracellular Ca2+ (14). In our studies metastin and kisspeptin-10 were used as low- and high-potency ligands, respectively, to activate the GPR54 receptor endogenously expressed in native and immortalized GnRH neurons.

Perifused GT1–7 neurons consistently exhibited spontaneous pulsatile GnRH release in the absence of exogenous stimuli (Fig. 4Go, A–C, open circles). Analysis of the dynamic profile of GnRH release in perifused GT1–7 cells revealed that exposure to 100 nM kisspeptin-10 increased the average peak height from 7.1 ± 2.1 pg/ml to 25.5 ± 2.3 pg/ml (P < 0.01, Fig. 4AGo, solid circles) and from 12.5 ± 2.3 pg/ml to 39.6 ± 3.8 pg/ml, with the occasional appearance of a single prominent peak (Fig. 4BGo, solid circles). In contrast, treatment with 100 nM human metastin-54 had no significant effect on pulsatile GnRH release from perifused GT1–7 neurons (Fig. 4CGo, solid circles). Static cultures of both GT1–7 cells and hypothalamic cells released measurable quantities of kisspeptin in incubation medium (Fig. 4DGo). In both cases, treatment with GnRH caused a significant decrease in kisspeptin secretion from 2.3 ± 0.3 pg/ml to 0.95 ± 0.2 pg/ml (P < 0.05, n = 6) for GT1–7 cells and from 1.4 ± 0.2 pg/ml to 0.8 ± 0.08 pg/ml (P < 0.05, n = 6) for cultured hypothalamic cells (Fig. 4DGo).


Figure 4
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  		Fig. 4. Whole-Cell Patch Clamp Recordings from Identified GnRH Neurons

A, Spontaneous AP firing in hypothalamic GnRH neurons. B, Stimulation of AP firing by KiSS-10 in a spontaneously active GnRH neuron. C, Recovery of basal AP firing rate during KiSS-10 washout. D, Induction of AP firing by KiSS-10 in an identified quiescent GnRH neuron. E, Spontaneous AP firing in a hypothalamic GnRH neurons. F, Stimulation of AP firing by metastin-54 in a spontaneously active GnRH neuron. G, Induction of AP firing by metastin-54 in an identified quiescent GnRH neuron.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The generation of pulsatile GnRH release at the median eminence depends on the coordinated activity of the hypothalamic GnRH neurons (15, 16, 17). GnRH/GnRH-R autocrine regulation, agonist activation of receptors and channels expressed in the plasma membrane of GnRH neurons, and synaptic inputs from other hypothalamic and extra hypothalamic neurons, are major pathways that regulate GnRH neuronal activity.

Visually identified cultured GnRH neurons and their immortalized counterparts, GT1–7 cells, consistently expressed mRNAs for GnRH as well as for kisspeptin and GPR54. Both cell types secreted kisspeptin into the incubation medium, suggesting that an autocrine regulatory system for GPR54-kisspeptin is operative in hypothalamic GnRH neurons. Expression of GPR54 and KiSS-1 has also been found in the human placenta (1) and rat ovary (18), suggesting local actions of kisspeptin in these organs. In addition, our results from BRET analysis showed that GnRH-R and GPR54 can form heterooligomers when expressed in HEK-293 cells, in which constitutive and GnRH agonist-induced BRET signals were observed. In kinetic studies, constitutive heterooligomerization showed a monotonic increase in the BRET signal. In contrast to GnRH-R-GPR54 heterooligomers, GnRH-R homooligomers did not show constitutive activity, and their formation occurred after receptor activation (19, 20). The temporal increase in BRET signal was also elicited by GnRH-R activation and showed marked dose dependence. Similar to other G protein-coupled receptors in which formation of heterooligomers influences binding and/or signaling properties (21, 22), the formation of GnRH-R-GPR54 heterooligomers may provide for integrated cellular signaling that regulates GnRH and kisspeptin secretion from hypothalamic GnRH neurons.

Kisspeptin treatment of GT1–7 cells, which have a similar GnRH secretory profile similar to that of cultured hypothalamic cells and hypothalamic tissue slices (23), stimulates pulsatile GnRH release with increased GnRH-peak amplitude and duration. The maintenance of pulsatile GnRH release during continuous kisspeptin treatment indicates that activation of GPR54 does not interrupt the signaling pathway that drives pulsatile GnRH release, in which activation of Gs and Gq is necessary to initiate pulse formation, and activation of Gi is necessary for its termination (24, 25, 26). Coupling of GPR54 to Gq/11 has been found in hippocampal neurons and GPR54-transfected COS-7 cells (27, 28). In addition, an estimated prediction of GPR54 coupling using Hidden Markov models (29) indicates that the receptor could also couple to Gi/o and Gs. The inhibition of kisspeptin secretion by GnRH suggests that GnRH-induced activation of the heterodimeric GnRH-R/GPR54 receptor complex favors coupling to the Gi/o signaling pathway and promotes inhibition of kisspeptin secretion. In such heterodimeric organizations, positive cooperation may increase responsiveness of a receptor system, whereas negative cooperativity can inhibit responsiveness of a receptor system and prevent its overactivation (30). Thus, cross talk between receptors in a dimeric or oligomeric complex during ligand binding may give rise to a cascade of interconnected signaling events and maintain the pulsatile GnRH release that is necessary for normal reproductive processes in mammals.

Kisspeptin immunoreactive neurons are found in several hypothalamic nuclei including the preoptic nucleus, the dorsomedial nucleus, the anteroventral periventricular nucleus, and the arcuate nucleus (3, 31). Close appositions of kisspeptin fibers with GnRH neuron cell bodies are found in the diagonal band of Broca, and preoptic nucleus (32, 33). These findings, together with the idiopathic hypogonadotropic hypogonadism caused by a homozygous 155-bp deletion in GPR54 (7), and the delayed puberty caused by site-directed mutagenesis of GPR54 in animal models (6), are defining new components in the regulatory system that controls the hypothalamo-pituitary-gonadal axis.

In electrophysiological studies, kisspeptin-induced activation of GPR54 in identified hypothalamic GnRH neurons significantly increased the frequency of AP firing. This was associated with membrane depolarization and increased AP firing. Similarly, kisspeptin-induced membrane depolarization was observed during activation of GPR54 in transgenic mice expressing GFP-tagged GnRH neurons (10). These data are consistent with functional and biochemical evidence that kisspeptin activates the phospholipase C/inositol-1-4-5-trisphosphate pathway and mediates membrane depolarization in both cultured hypothalamic neurons and GnRH neurons in situ (10, 34).

The expression of kisspeptin and GPR54 mRNAs in identified hypothalamic GnRH neurons, and the constitutive and agonist-induced heterooligomerization of GnRH-R and GPR54, indicates that both receptors are involved in the regulation of hypothalamic GnRH neurons. Moreover, stimulation of GnRH secretion by kisspeptin, and inhibition of kisspeptin secretion by GnRH, indicate that GnRH-receptor-GnRH and GPR54-kisspeptin autoregulatory systems are integrated by negative feedback action to control GnRH and kisspeptin secretion from cultured hypothalamic GnRH neurons (Fig. 5Go).


Figure 5
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  		Fig. 5. GnRH and Kisspeptin Secretion from Cultured Hypothalamic Cells and GT1–7 Neurons

A and B, Prominent increase in GnRH peak amplitude and duration during treatment with KiSS-10. C, Lack of stimulation of GnRH release by metastin in perifused GnRH neurons. D, Basal kisspeptin production and GnRH-induced inhibition of KiSS-10 secretion in static cultures of GT1–7 neurons and hypothalamic cells. Data are means ± SEs of four experiments.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Tissue and Cell Culture
Hypothalamic tissue was removed from fetuses of 18-d pregnant Sprague Dawley rats as previously described (35). The tissue was dispersed in 0.2% collagenase containing 0.4% BSA, 0.2% glucose, and 0.05% DNase I. Hypothalamic cells were cultured in medium consisting of 500 ml DMEM containing 0.584 g/liter L-glutamate and 4.5 g/liter glucose, mixed with 500 ml F-12 medium containing 0.146 g/liter L-glutamine, 1.8 g/liter glucose, 100 µg/ml gentamicin, 2.5 g/liter sodium bicarbonate, and 10% heat-inactivated fetal bovine serum. Immortalized GnRH neurons (GT1–7 cells) were provided by Dr. Richard Weiner (University of California at San Francisco) (36) and were cultured under the same conditions as primary hypothalamic cells. Both hypothalamic and GT1–7 cells were cultured for 7 d before use in experiments. Our animal studies protocol was approved by National Institutes of Health (NIH) Office of Animal Care and Use.

Cell Perifusion Procedure and Hormone Measurement
Bead-attached GT1–7 cells were perifused at a flow rate of 0.15 ml/min at 37 C. Fractions were collected at 5-min intervals and stored at –20 C before RIA. GnRH was measured using [125I]GnRH (Amersham Pharmacia Biotech, Piscataway, NJ), GnRH standards (Peninsula Laboratories, Inc., Division of Bachem, San Carlos, CA), and primary antibody donated by Dr. V. D. Ramirez (University of Illinois, Urbana, IL). The intra- and interassay coefficients of variation at 50% binding in standard samples (15 pg/ml) were 5% and 7%, respectively. The sensitivity of the assay, defined as twice the SD at zero dose, was 0.2 pg/tube. Kisspeptin was measured using human kisspeptin-13 (4–13) RIA kits from Peninsula Laboratories, Inc. The sensitivity of assay, defined as twice the SD at zero dose, was 0.2 pg/tube with 100% cross-reactivity for kisspeptin-13 and kisspeptin-54 (17–54).

Whole-Cell Recording of GnRH Neurons
For whole-cell recording, hypothalamic cells were cultured on collagen-coated coverslips and continuously perifused with artificial extracellular solution at a rate of 0.6 ml/min. The extracellular solution contained (in millimolar concentration): 140 NaCl, 5 KCl, 10 HEPES, 10 D-Glucose, 2 CaCl2, 1 MgCl2. pH was adjusted to 7.4 with NaOH. The cells were viewed under an inverted microscope (Olympus IX70, Olympus Corp., Lake Success, NY) with a x40, long working distance objective. All recordings were done at room temperature (23–25 C). Patch pipettes (3–5 M{Omega}) were pulled from thick-wall borosilicate capillary glass (1.5 mm OD and 0.86 mm ID, World Precision Instruments, Inc., Sarasota, FL) on a Flaming/Brown puller model P-87 (Sutter Instruments Co., Novato, CA). The pipette solution was prepared containing (in millimolar concentration): 70 KCl, 70 K gluconate 0.1 CaCl2, 2 MgCl2, 10 HEPES, 2 KATP, 0.1 Na2GTP, 5 EGTA, with pH adjusted to 7.2 with KOH. An Ag/AgCl pellet was used as the reference electrode. Spontaneous activities were recorded under I-clamp mode with a Multi-Clamp700A amplifier (Axon Instruments, Foster City, CA), filtered at 2 KHz, and digitized at 10 KHz through Digidata 1320A (Axon Instruments). Firing of APs in identified cultured E-18 hypothalamic neurons was analyzed in single isolated GnRH neurons to eliminate the influences of electrical and synaptic coupling between cells. Acquisition and subsequent analysis of the experimental data were performed using Clampex 9.0 software (Axon Instruments). Traces and voltage-current curves were plotted using Origin 7 computer software (MicroCal Software, Northampton, MA). Individual hypothalamic GnRH neurons were selected by differential interference contrast microscopy, which permits their morphological identification in cultured hypothalamic cells with an accuracy of more than 95%. After recording, the cytoplasmic contents of each neuron were harvested under visual control and subjected to single-cell RT-PCR to confirm the presence of GnRH transcripts as previously reported (37). As controls, the contents of hypothalamic cells that did not show typical GnRH neuronal morphology were also analyzed by RT-PCR.

Single-Cell RNA Harvesting and cDNA Conversion
Dispersed fetal hypothalamic cells were maintained in culture for up to 2 wk. Before cell harvesting the cultures were washed with ice-cold PBS and GnRH neurons identified by their morphological characteristics were individually harvested with a polished glass pipette. A modification of the protocol described by (38) was used to complete cDNA conversion. Cellular contents were expelled into 8.5 µl of a cDNA conversion mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 20 mM dithiothreitol, 0.5 mM deoxynucleotide triphosphates, 100 ng random hexamer primers, 200 ng oligo (dT)12–15, and 20 U RNaseOUT [hexamers, oligo(dT), and RNaseOUT; Invitrogen, Carlsbad, CA]. Contents were collected by brief centrifugation, heated to 65 C for 5 min and placed on ice for 1 min, followed by brief centrifugation. SuperScript II RNA H– (Invitrogen) and RNaseOUT were mixed in equal amounts and 2 µl was added to each single cell-cDNA mixture. cDNA synthesis was performed at room temperature for 5 min and then at 42 C for 1 h. Contents were collected by centrifugation, frozen on dry ice, and stored at –70 C before multiplex RT-PCR.

Single Cell Multiplex RT-PCR for GnRH, GPR54, and Mouse Kisspeptin Transcripts
First-round PCR was performed on 10 µl of reverse transcriptase product from each cell using oligonucleotide pairs in a 100-µl reaction containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 2.5 U DNA Platinum Taq polymerase (Invitrogen) and optimized primer concentrations of GnRH, GPR54, and kisspeptin. Pooled fetal hypothalamic cDNA prepared from tRNA extraction was used as a positive control, and water was used as a negative control in each reaction.

First-round amplification (36 cycles) was performed using a MyCycler Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA) in thin-walled 0.2 ml PCR tubes according to the following protocol: first cycle at 95 C (3 min), 58 C (2 min), and 72 C (3 min) followed by 35 cycles at 95 C (40 sec), 58 C (85 sec), and 72 C (1 min). A final 5-min incubation was performed at 72 C. Second-round nested PCR was performed using 10 µl aliquots of the first-round amplified products. PCR for GnRH (GenBank accession no. M31670), GPR54 (GenBank accession no. NM 023992), and kisspeptin (accession no. AY196983) was performed in 20 µl reaction capillaries with their respective secondary nested primer sequences utilizing FastStart DNA Master SYBR Green 1 Kit according to manufacturer’s instructions and the LightCycler (Roche Applied Sciences, Indianapolis, IN): first cycle 95 C (10 min), followed by 40 cycles of 95 C (6 sec), 50 C (10 sec), and 72 C (9 sec). Melting curve analysis and cooling cycle were performed. The resulting products were resolved on 2.0% Tris-buffered EDTA-agarose gels stained with ethidium bromide.

Plasmid Construction
The codon humanized pRluc(h)-N and pGFP2-N terminal expression vectors were purchased from PerkinElmer Life Sciences (Waltham, MA). To generate the fusion proteins, restriction enzyme sites were introduced into the full-length GnRH-R and GPR54 cDNA by PCR using sense and antisense primers harboring unique KpnI and SacII sites for GnRH-R and EcoRV and BamHI for GPR54 at their 5'- and 3'-ends, whereas the stop codons of the cDNA fragments were removed in the antisense primers. The cDNA fragments then were subcloned to be in frame into the KpnI and SacII restriction site of the pRluc(h)-N vector and into the EcoRV and BamHI restriction site of the pGFP2-N vectors. All constructs were verified by direct DNA sequencing.

BRET2 Measurement
The use of BRET assay to monitor molecular interaction depends on the transfer of energy from one fluorophore (the donor) to another fluorophore (the acceptor). Energy transfer occurs when the proteins of interest bring the donor and acceptor into close proximity (10–100 Å), a distance generally indicative of protein-protein interactions, either directly or as part of a complex. Recent structural data on the rhodopsin receptor have shown that the monomeric unit has a diameter of 43 Å. This suggests that BRET can occur between receptors that are more than one receptor apart (39). HEK-293 cells stably expressing GnRH-R-Rluc (HEK-GnRH-R-Rluc) were plated in 60-mm dishes (1.2 x 106 cells per dish) and incubated in culture medium overnight. Cells were transfected with GPR54-GFP2 using Polyfect transfection reagent (QIAGEN, Valencia, CA). Forty-eight hours after transfection, the cells were washed twice in PBS, harvested with PBS/EDTA solution, and resuspended in BRET buffer (PBS/glucose 0.1%). Cells (100,000) were incubated with vehicle or a GnRH agonist analog ([D-Ala6]Ag) (Peninsula Laboratories) in 96-well white Optiplates (PerkinElmer Life Sciences). After addition of 5 µM DeepBlueC substrate, luminescence and fluorescence emissions were measured immediately on the Mithras LB 940 (Berthold Technologies, Oak Ridge, TN). After catalytic degradation of the substrate DeepBlueC by the energy donor GnRH-R-Rluc, light is emitted with a peak at 400 nm. The energy acceptor green GFP54-GPF2 is excited by nonradiative energy transfer if GFP2 is located within a distance of less than 100 Å from the energy donor. The net BRET signal was calculated by dividing the 530-nm emission of GPR54-GFP2 by the 475-nm emission of the GnRH-R-Rluc after subtracting the background signal determined from cells transfected with the GnRH-Rluc construct alone using the formula [(Em530 – Em475) x CF]/EM475, where CF represents Em530/Em475, the ratio of the signals from cells expressing GnRH-R-Rluc alone. For time course studies BRET2 measurements were collected every 1 sec for up to 40 sec.

Materials
Oligonucleotides were obtained from Gene Probe Technologies (Gaithersburg MD). The Absolutely RNA RT-PCR Miniprep Kit was purchased from Stratagene (La Jolla, CA). SuperScript III RNase H– radical extender, Reverse Transcriptase, Platinum Taq DNA polymerase, pCR2.1 vector, and TOPO TA cloning kit were purchased from Invitrogen. Wizard plus Minipreps DNA purification system was purchased from Promega Corp. Kisspeptin-1 [KiSS-10; (110–119)] metastin-54 (1–54; human) and GnRH agonist (des-Gly10-[D-Ala6]GnRH N-ethylamide were from Phoenix Pharmaceutical (Belmont, CA). The potent GnRH antagonist, acyline [CDB 3883H (Acetyl-D-Nal-D-4-Cl-Phe-D-Pal-Ser-Aph(Ac)-D-Aph(Ac)-Leu-Lys(Ipr)-Pro-DAlaNH2)] was provided by Dr. Hyun Kim (NIH, Bethesda, MD).

Data Analysis
GnRH pulses were identified and their parameters determined by computerized cluster analysis (40). Individual point SDs were calculated using a power function variance model from the experimental duplicates. A 2 x 2 cluster configuration and a t statistic of 2 for the upstroke and downstroke were used to maintain false-positive and false-negative error rates below 10%. The pulse parameters were analyzed by ANOVA and results were expressed as mean ± SEM. Statistical comparisons were performed using the Kruskal-Wallis test followed by the Mann-Whitney U test. BRETmax and BRET50 were derived from fitting data to a logit function [logit(p) = ln(p/1 – p)].


   FOOTNOTES
 
This work was supported by the Intramural Research Program of the NIH, National Institute of Child Health and Human Development.

Present address for N.M.: Department of Pharmacology, Catholic University of the Sacred Heart, 00168 Rome, Italy.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 14, 2007

1 S.Q., L.H., and P.K.L. contributed equally to this paper. Back

Abbreviations: Acyline, [CDB 3883H (acetyl-D-Nal-D-4-Cl-Phe-D-Pal-Ser-Aph(Ac)-D-Aph(Ac)-Leu-Lys(Ipr)-Pro-DAlaNH2)]; AP, action potential; BRET, bioluminescence resonance energy transfer; GFP, green fluorescent protein; GnRH-R, GnRH receptor; GPR54, G protein-coupled receptor 54; HEK, human embryonic kidney; KiSS-10, kisspeptin-10; Rluc, Renilla luciferase.

Received for publication April 23, 2007. Accepted for publication August 7, 2007.


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 ABSTRACT
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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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