Cells Expressing RFamide-Related Peptide-1/3, theMammalian Gonadotropin-Inhibitory HormoneOrthologs, Are Not Hypophysiotropic NeuroendocrineNeurons in the Rat

Mohammed Z. Rizwan, Robert Porteous, Allan E. Herbison, and Greg M. Anderson

Centre for Neuroendocrinology and Departments of Anatomy and Structural Biology (M.Z.R., G.M.A.) and Physiology(R.P., A.E.H.), University of Otago School of Medical Sciences, Dunedin 9054, New Zealand

An RFamide peptide named gonadotropin-inhibitory hormone, which directly inhibits gonado-tropin synthesis and secretion from the anterior pituitary gland, has recently been discovered in theavian hypothalamus. It is not known whether the mammalian orthologs of gonadotropin-inhib-itory hormone and RFamide-related peptide (RFRP)-1 and -3 act in the same way. We used a newlygenerated antibody against the rat RFRP precursor combined with retrograde tract tracing tocharacterize the cell body distribution and fiber projections of RFRP-1 and -3 neurons in rats.RFRP-1/3-immunoreactive cell bodies were found exclusively within the dorsomedial hypothala-mus. Immunoreactive fibers were observed in the septal-preoptic area, hypothalamus, midbrain,brainstem, and hippocampus but not in the external zone of the median eminence. Intraperitonealinjection of the retrograde tracer Fluoro-Gold in rats resulted in the labeling of the majority ofGnRH neurons but essentially no RFRP-1/3 neurons. In contrast, intracerebral injections of Fluoro-Gold into the rostral preoptic area and CA2/CA3 hippocampus resulted in the labeling of 75 � 5%and 21 � 8% of RFRP-1/3 cell bodies, respectively. To assess actions at the pituitary in vivo, RFRP-3was administered as an iv bolus to ovariectomized rats and plasma LH concentration measured at0, 2.5, 5, 10, and 30 min. RFRP-3 had no effects on basal secretion, but GnRH-stimulated LH releasewas reduced by about 25% at 5 min. Together these observations suggest that RFRP-3 is not ahypophysiotropic neuroendocrine hormone in rats. (Endocrinology 150: 1413–1420, 2009)

The GnRH neurons are described as the final common path-way regulating fertility because they are generally thought to

be the sole neuroendocrine neurons responsible for controllinggonadotropin secretion (1, 2). The idea of direct inhibition ofgonadotrope activity by a release inhibiting factor has been pos-tulated on several occasions (3, 4) but not proven until recentlywhen an RFamide peptide located in hypothalamic neurons withfiber projections to the external zone of the median eminence wasshown to act in this manner in the quail (5, 6). This novel peptidedecreased gonadotropin release from cultured quail anterior pitu-itaries in a dose-dependent manner and was termed gonadotropin-inhibitory hormone (GnIH) (5). GnIH may also influence the avianGnRHneuronal systemasdouble-label immunohistochemistryhasshown GnIH fibers in close contact with GnRH cell bodies andfibers in sparrows (7).

The GnIH precursor protein produces three mature neu-ropeptide variants of GnIH [GnIH, GnIH-related peptide(GnIH-RP)-1, and GnIH-RP2], each with a Leu-Pro-Leu-Arg-Phe-amide or Leu-Pro-Gln-Arg-Phe-amide C-terminal sequence.The orthologous mammalian precursor protein, the RFamide-related peptide (RFRP) precursor, produces two Leu-Pro-Leu-Arg-Phe-amide or Leu-Pro-Gln-Arg-Phe-amide neuropeptides(RFRP-1 and RFRP-3, although mature RFRP-1 has not yet beenidentified by purification in rodents). When avian GnIH andmammalian RFRP precursor proteins are aligned, avian GnIH-RP1 aligns with RFRP-1, whereas GnIH falls between RFRP-1and RFRP-3 (8–10). Evidence suggests that these RFamide mol-ecules can influence neuroendocrine function in rodents. Treat-ment with sparrow GnIH either ip or intracerebroventricular hasbeen reported to decrease LH concentrations in hamsters, rats,

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2009 by The Endocrine Societydoi: 10.1210/en.2008-1287 Received September 3, 2008. Accepted October 31, 2008.First Published Online November 13, 2008

Abbreviations: GnIH, Gonadotropin-inhibitory hormone; GnIH-RP, GnIH-related peptide;RFRP, RFamide-related peptide; TBS, Tris-buffered saline.

N E U R O E N D O C R I N O L O G Y

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and mice (11). RFRP neurons may inhibit the preovulatory surgeand mediate seasonal rhythms because their expression and ac-tivity is modulated during the course of the surge (12) and inresponse to melatonin treatment (13) in seasonally breedinghamsters. RFRP-1 has also been shown to act centrally to stim-ulate prolactin release in the rat (8).

However, direct evidence for mammalian RFRP-1 and -3 ashypophysiotropic neuroendocrine regulators of pituitary functionis lacking inanymammalianspecies.Asa first step toward thisgoal,we sought to characterize the distribution and fiber projections ofRFRP-1 and -3 neurons in the rat brain using a new antiserumdirected against the RFRP precursor. Using retrograde tracing tech-niques, we examined the projections of these neurons in the ratbrain and, finally, tested whether peripherally administeredRFRP-3 is able to suppress LH secretion in ovariectomized rats.

Materials and Methods

AnimalsMale and female Sprague Dawley rats, aged 9–12 wk and weighing

250–300 g, were obtained from the University of Otago animal breedingfacility. Rats were grouped housed under conditions of controlled light-ing (lights on from 0500 to 1900 h) and temperature (22 � 1 C) and hadfree access to food and water. All animal experimental protocols wereapproved by the University of Otago Committee on Ethics in the Careand Use of Laboratory Animals.

Generation of RFRP precursor peptide polyclonalantibody

RFRP precursor peptide antiserum was generated to a synthetic se-quence corresponding to amino acids 119-132 of the rat RFRP precursorpeptide sequence (PSLPQRFGRTTARR) (Fig. 1) with Cys added to theN terminus and keyhole limpet hemocyanin as the conjugated carrierprotein. This sequence, which overlaps with the sequence that encodesmature RFRP-3, was chosen based on predicted antigenicity. Because theRFRP precursor peptide produces both RFRP-1 and RFRP-3 (14), la-beling the precursor effectively defines RFRP-1/RFRP-3-expressing neu-rons (hereafter termed RFRP-1/3). Antigen (200 �g/rabbit) solution wasmixed with Freund’s complete adjuvant (Sigma-Aldrich, Inc., St Louis,MO) and injected into New Zealand white rabbits. Five booster injec-tions (100 �g in Freund’s incomplete adjuvant; Sigma) were adminis-tered at 2-wk intervals. Two weeks after the final booster injection, therabbits were exsanguinated and the serum antibodies were affinity pu-rified on a C-18, 250- � 4.6-mm, 5-�m column (Sigma) and sterilefiltered through a 0.45 �m cellulose acetate membrane. Sodium azide(0.1%) was added as a preservative.

Western blottingTwo female rats were decapitated and hypothalami isolated. The

total protein was extracted and measured by a Bradford protein assay(Bio-Rad Laboratories, Inc., Hercules, CA) to determine the protein con-centration in each sample. Samples (50 �g) were separated through 15%SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher andSchuell, Keene, NH). After blocking with 3% nonfat dried milk powderin PBS containing 0.1% Tween 20 (1 h at room temperature), membraneswere incubated overnight at 4 C in RFRP precursor peptide antibody(GA197; 1:5000 dilution). Unbound antibody was rinsed from the mem-branes before incubation with horseradish peroxidase-conjugated goatantirabbit secondary antibody (Sigma; 1:20,000). Immunoreactivity wasdetected using an enhanced chemiluminescence substrate (AmershamBiosciences, Arlington Heights, IL).

Tissue preparation and immunohistochemistryRats were anesthetized with 240 mg/kg sodium pentobarbital and

perfused through the heart with a 4% paraformaldehyde/0.1 M PBSsolution (pH 7.4). Brains were removed, postfixed in paraformaldehyde,and cryoprotected in 30% sucrose solution as previously described (15).Coronal (30 �m thick) sections throughout the whole of the forebrainand brain stem were cut from each brain on a sliding microtome with afreezing stage to provide three sets of consecutive sections (90 �m apart);these were used immediately for immunocytochemistry or stored in cryo-protectant at �20 C.

Single-label chromagen immunocytochemistryAll steps were performed at room temperature (unless noted other-

wise) and were separated by four to six washes in 0.05 M Tris-bufferedsaline (TBS). One set of sections was incubated in 1% hydrogen peroxideand 40% methanol to quench endogenous peroxidases and then blockedin 0.25% BSA made up in TBS containing 0.5% Triton X-100 for 30 min.After the blocking step, the sections were then incubated for 24 h inpolyclonal rabbit anti-RFRP precursor peptide (GA197; 1:5000 dilu-tion) in 0.25% BSA made up in TBS containing 0.5% Triton X-100containing 2% normal goat serum at 4 C. Sections were then incubatedin biotinylated goat antirabbit IgGs (1:250; Vector Laboratories Inc.,Burlingame, CA) for 90 min followed by a 90 min incubation in VectorElite ABC (1:100, Vector Laboratories) and a 5- to 7-min incubation indiaminobenzidine solution (Sigma). Controls consisted of the following:1) preadsorption controls in which the RFRP precursor peptide antibodywas incubated at working dilution with the RFRP precursor sequence(used to generate the antibody), mature RFRP-3 peptide, or rat prolactin-releasing peptide-31 (Phoenix Pharmaceuticals, Burlingame, CA; an-other RFamide, which shares the three C-terminal amino acids withRFRP-3) at a concentration of 20 �g/ml overnight at 4 C before use, and 2)immunocytochemistry experiments in which the primary or secondary an-tibodies were removed. Sections were mounted on (3-aminopropyl)-tri-thoxyl-silane-coated slides, dried, hydrated in water, dehydrated in gradedseries of alcohols, and then cleared in xylene before coverslipping with dibu-tylphthalate polystyrene xylene (DPX). RFRP-1/3 staining was observedusing an Olympus AX70 Provis light microscope at �40 and �200.

ImmunofluorescenceFor simultaneous visualization of RFRP-1/3 neurons and the retro-

grade tract tracer Fluoro-Gold (Biotium, Hayward, CA), sections con-taining the dorsomedial and ventromedial hypothalamic nuclei fromFluoro-Gold-injected rats (see below) were incubated in RFRP precursorpeptide antisera as above, followed by 90 min in Cy-3 goat antirabbit(Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Fluoro-Gold itself is a fluorophore and has absorption/emission wavelengths of361/536 nm. This fluorescence was visualized without additional stain-ing in the 330–385 nm excitation wavelength range. Sections weremounted on slides and coverslipped using VectaShield mounting me-dium (Vector Laboratories). Cy-3 was examined in the 540- to 580-nmexcitation wavelength range. As a positive control for uptake of Fluoro-

FIG. 1. Partial amino acid sequence of the rat precursor protein that encodesRFRP-1 and RFRP-3, showing the sequence against which the polyclonal antibodyGA197 was targeted (boxed region). The amino acids G and R after the C-terminal RF motif act as an amide donors and proteolytic cleavage sites,respectively. The mature form of RFRP-1 has not been identified by purificationbut is predicted to be either 12 or 37 residues long, depending on the N-terminalcleavage site. When aligned with orthologous precursor proteins from avianspecies, avian GnIH-RP1 aligns with RFRP-1 and GnIH falls between RFRP-1 andRFRP-3. Amino acid sequence numbers are indicated.

1414 Rizwan et al. RFRP-1/3 Neurons Are Not Hypophysiotropic Endocrinology, March 2009, 150(3):1413–1420

Gold by hypophysiotropic neuroendocrine neurons, one set of sectionscontaining the medial septum and preoptic area was immunostained forGnRH using the same protocol as that outlined above, except that apolyclonal rabbit antibody raised against GnRH (LR5, 1:20,000; gift ofR. Benoit, Montreal, Canada) was used instead of the RFRP precursorpeptide antiserum. All RFRP-1/3 and GnRH neurons were analyzed forcoexpression of Fluoro-Gold. A primary control (no RFRP precursorpeptide or GnRH primary antibody) and an animal not treated withFluoro-Gold were also stained to confirm that no false-positive colocal-izations resulted from Cy-3 bleed-through into the 330- to 385-nmrange. RFRP-1/3 or GnRH neurons expressing Fluoro-Gold werecounted as colocalized cells. The total number of RFRP-1/3 or GnRHneurons and the total number of colocalized neurons were recorded fromeach animal and the percentage of RFRP-1/3 or GnRH neurons coex-pressing Fluoro-Gold calculated.

Retrograde tract-tracing experiments

Intraperitoneal injectionsHypophysiotropic hormones are released by their nerve terminals in

the external zone of the median eminence, a region outside the bloodbrain barrier, from which they enter the fenestrated capillaries of thepituitary portal blood system to regulate anterior pituitary gland func-tion. To determine whether RFRP-1/3 neurons project outside the bloodbrain barrier, three male and three female adult rats were each given anip injection of the retrograde tract tracer Fluoro-Gold (Biotium), dis-solved in 0.9% saline, at a dose of 15 mg/kg body weight. Fluoro-Goldis well established as a retrograde tracer that does not penetrate theblood-brain barrier but is taken up by nerve terminals that project toareas supplied by the fenestrated capillaries or to the periphery (16). Oneweek after Fluoro-Gold injections, animals were perfused for RFRP-1/3and GnRH immunofluorescence as described above. The duration of 1wk was chosen to encompass at least one estrous cycle in females.

Intracerebral injectionsMale and female adult rats were anesthetized with 2% halothane and

positioned in a stereotaxic frame for central, unilateral injection ofFluoro-Gold (Biotium) as described previously (17). The head of theanimal was shaved and prepared for aseptic surgery. A 0.5-�l Hamiltonmicrosyringe, filled with Fluoro-Gold (1% solution diluted in saline) waspositioned stereotaxically at the following coordinates; CA2/CA3 hip-pocampus: 2.76 mm posterior to bregma, 1.9 mm lateral to midline, 3.80mm below the skull surface; rostral preoptic area: 0.00 mm posterior tobregma, 0.5 mm lateral to midline, 8.00 mm below the skull surface. Thesyringe was left in situ for 10 min and then Fluoro-Gold (50 nl) wasinjected over a period of 2 min. The syringe was left in situ for a further10 min before being slowly withdrawn. Rats were perfused for RFRP-1/3immunofluorescence 1 wk after surgery.

Effect of iv RFRP-3 treatment on plasma LHconcentrations

Adult female rats were anesthetized with 2% halothane and ovari-ectomized. One week later they were anesthetized as before fitted withan atrial cannula chronically implanted via the right jugular vein toenable repeated blood sampling. To determine the effect of peripherallyadministered RFRP-3 on plasma LH levels, ovariectomized rats (n � 6)were injected iv with RFRP-3 at two different doses (1 and 10 �g/rat) orsaline vehicle, either alone or in combination with GnRH (0.1 �g/rat) ina randomized order. After each treatment, blood samples were collectedat 0, 2.5, 5, 10, and 30 min. The plasma was collected, stored at �20 Cand then assayed for LH as described below. Red blood cells were re-suspended in physiological saline and replaced after the subsequentsample.

LH RIAPlasma LH concentrations were measured in 50-�l sample volumes

by RIA. Values are expressed in terms of the rat standard NIDDK-rat

LH-RP-3. The tracer used was NIDDK-rat LH-I-10 (iodinated hormone)and the primary antiserum was NIDDK-rabbit antirat LH-S11 (finaldilution 1:500,000). The secondary antiserum used was sheep antirabbitat 1:50 dilution. The sensitivity of the LH assay (95% confidence intervalat 0 ng/ml on the standard curve) was 0.3 ng/ml. The intraassay coeffi-cient of variation for a serum pool falling in the middle of the standardcurve was 15%. All samples were run in a single assay.

Statistical analysisSex differences in RFRP-1/3 and Fluoro-Gold colocalization were

analyzed using Student’s t test. To determine significant effects of treat-ment on plasma LH levels, two-way ANOVA with repeated measureswas performed, with time and RFRP-3 treatment as the factors (GnRHtreated and nontreated experiments were analyzed separately). This wasfollowed by post hoc Bonferroni t tests to determine where significanteffects occurred. Results are presented as mean � SEM, and differenceswere considered significant at P � 0.05.

Results

Western blottingA single band was observed for rat tissue at the predicted

molecular mass for the RFRP precursor peptide (21 kDa) after 1min of exposure (Fig. 2D), confirming the specificity of the an-tibody for this peptide.

Immunohistochemical distribution of RFRP-1/3 cellbodies and fibers

Cell bodiesRFRP-1/3-ir cell bodies were observed only within the dor-

somedial hypothalamus (Fig. 2A). The cells were scattered

FIG. 2. A–C, Coronal brain sections showing distribution of RFRP-1/3immunoreactive cell bodies within the rat dorsomedial hypothalamus (DMH).VMH, Ventromedial hypothalamus. B, Enlargements of A. C, Control section inwhich the RFRP precursor peptide antibody was preabsorbed with mature RFRP-3. Scale bars, 200 �m. D, Western blot using the novel RFRP precursor peptidepolyclonal antibody. Rat hypothalamic tissue revealed a single band in themolecular mass range expected for the RFRP precursor peptide (21 kDa; leftlane). The molecular mass standard is shown in the right lane.

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throughout this regionandexhibitedaneuronal,multipolarmorphol-ogy. The distribution and number of RFRP-1/3-ir neurons was notdifferentbetweenmaleanddiestrousfemaleanimals (23�5and26�

7 cells/section, respectively). Preabsorption of the primary antiserumwith either the RFRP precursor peptide sequence or mature RFRP-3peptide resulted in a complete absence of cytoplasmic staining (Fig.2C), whereas preabsorption with prolactin-releasing peptide had no

effect on RFRP-1/3 immunoreactivity (notshown). Similarly, removal or either the RFRPprecursor peptide antisera or the secondary an-tibody resulted in an absence of staining.

FibersThe distribution of RFRP-1/3 fibers is

shown in Figs. 3 and 4. Within the telen-cephalon, the most dense accumulation ofRFRP-1/3 fibers was observed within theCA2/CA3 hippocampus (Figs. 3, E and G,and 4). Elsewhere in the forebrain, scatteredfibers were detected in the horizontal andvertical limbs of the diagonal band of Broca,lateral septum, accumbens nucleus, preopticarea (including the region around the orga-num vasculosum of the lamina terminalis;Fig. 3A), periventricular nucleus, anterior hy-pothalamic area, and rostral aspects of the lat-eral hypothalamus. In the medial hypothala-mus, fibers were observed only in thedorsomedialhypothalamusandrostralpartofthe ventromedial hypothalamus. Only a veryfew fibers were seen in the arcuate nucleus andinternal layer of the median eminence. Fiberswere notably absent in the external zone of themedian eminence (Fig. 3, B and D).

Brainstem fibers were evident throughout allaspects of the periaqueductal gray (Fig. 3C) andsuperior and inferior colliculus and around thetegmentaldecussationandraphenucleus.Fiberswere also present in the ventral cochlear and su-perior vestibular nuclei and laterally in the ret-rotubral, reticular,andfacialnucleiaswellasthetrigeminal nerve sensory root. Ventrally, a smallnumber of fibers were seen around the mediallemniscus and ventral tegmental area. A few fi-bers were present as far caudally as the tectospi-nal and ventral spinocerebellar tract (Fig. 4).

Retrograde tract tracing experiments

Intraperitoneal injectionsThe distribution and number of RFRP-

1/3 cell bodies detected in all immunofluo-rescence experiments (males: 20 � 4 cells/section; females: 19 � 5 cells/section) wasthe same as that observed previously for sin-gle-labeling chromagen staining (males:23 � 5 cells/section; females: 26 � 7 cells/

section). After ip injection, neurons containing Fluoro-Gold (Bi-otium) were identified throughout the hypothalamus but wereparticularly prominent in the magnocellular nuclei and arcuatenucleus. With respect to RFRP-1/3 neurons, only three of a totalof 234 RFRP-1/3-ir cells counted in three male and three femalerats contained Fluoro-Gold (Fig. 5, A–C). In contrast, in rostralpreoptic area tissue from the same animals, 91 � 2 and 95 � 1%

FIG. 3. RFRP-1/3-immunoreactive fiber staining in the rat brain. A, Medium-power view of the rostralpreoptic area at the level of the organum vasculosum of the lamina terminalis (OVLT) showingnumerous immunoreactive fibers. B, High-power view of the median eminence (ME) showing a singlefiber (arrowhead) in the internal zone. Note that the asterisk indicates nonspecific staining alsopresent in controls. C, Single fiber (arrowhead) in the periaqueductal gray (PAG) of the midbrain. IV,Fourth ventrical. D, High-power view of the median eminence (ME) showing a single fiber (arrowhead)in the internal zone. E, Low-power view of the hippocampus showing dense fiber staining in the CA3pyramidal layer (inset is expanded in G). DG, Dentate gyrus. F, Immunostaining in hippocampus usingRFRP-3 precursor peptide antisera preadsorbed with mature RFRP-3 (inset is expanded in H). Scalebars, 25 �m in all plates except E and F in which they represent 125 �m.

1416 Rizwan et al. RFRP-1/3 Neurons Are Not Hypophysiotropic Endocrinology, March 2009, 150(3):1413–1420

of male and female GnRH neurons contained Fluoro-Gold, re-spectively (Fig. 5, D–F).

Intracerebral injectionsUnilateral injections of Fluoro-Gold (Biotium) into the CA2/

CA3 hippocampus resulted in numerous Fluoro-Gold-labeledcells being detected within the medial hypothalamus. Dual-la-beling immunofluorescence showed that 24 � 9 and 16 � 8% ofmale and female RFRP-1/3 neurons coexpressed Fluoro-Gold,respectively (Figs. 5, G–I, and 6A, n � 3/sex). Dual-labeled cellswere found predominantly ipsilateral to the injection site andmostly within the more dorsal aspect of the RFRP-1/3 distribu-tion. Unilateral injections of Fluoro-Gold into the rostral pre-optic area also resulted in numerous Fluoro-Gold-labeled cellsbeing detected within the medial hypothalamus. Dual-labelingimmunofluorescence showed that 74 � 7 and 75 � 3% of maleand female RFRP-1/3 neurons coexpressed Fluoro-Gold, respec-tively (Figs. 5, G–I, and 6B, n � 3/sex). Dual-labeled cells werefound predominantly ipsilateral to the injection site and, in thiscase, were found mostly within the more ventral aspect of theRFRP-1/3 distribution.

Effect of iv RFRP-3 treatment on plasma LHconcentrations

In ovariectomized rats with elevated LH secretion, there wasno effect of either dose of RFRP-3 on basal LH concentrations(Fig. 7). As expected, when GnRH (0.1 �g/rat) was administered,there was a marked increase in LH concentration, peaking at 5min after injection. When simultaneously injected with RFRP-3(1 or 10 �g/rat), a significant interaction between time andRFRP-3 treatment was observed (two way, repeated-measures

ANOVA; F � 2.16, P � 0.05). Post hocanalysis showed that there was a small butsignificant (P � 0.05) reduction in peak LHconcentration after 5 min but not at 2.5, 10,or 30 min at both RFRP-3 doses comparedwith control animals (Fig. 7).

Discussion

The aim of the present study was to testwhether RFRP-1/3 neurons are a hypophy-siotropic neuroendocrine population in rats.We show here that, as in birds, RFRP-1/3-immunoreactive neurons are scatteredwithin the dorsomedial hypothalamus of themale and female rat. However, RFRP-1/3-immunoreactive fibers were not found in themedian eminence and peritoneal Fluoro-Gold administration did not result in the la-beling of RFRP-1/3 neurons, suggesting thatthese cells do not project to the median em-inence. Furthermore, iv RFRP-3 was unableto influence LH release in ovariectomizedrats and had a small effect on GnRH-stim-ulated LH secretion at only the 5 min time

point. These observations suggest that RFRP-1/3 neurons are nothypophysiotropic neuroendocrine neurons. They may, however,be involved in neuroendocrine circuits within the limbic systembecause we found that mediobasal hypothalamic RFRP-1/3 neu-rons project to the rostral preoptic nucleus, in which GnRHneuron cell bodies are located, as well as to the CA2/CA3hippocampus.

Using a new antibody specific for the rat RFRP precursorpeptide, we show here that RFRP-1/3-immunoreactive neuronsare scattered within the dorsomedial hypothalamus of rats, withno evidence of cell bodies in other brain regions. The RFRPprecursor peptide antisera detected an approximately 21-kDaprotein, predicted for the RFRP precursor (http://ca.expasy.org/uniprot/Q9ESQ9), and staining was eliminated when the anti-serum was preadsorbed with RFRP-3 or the RFRP precursorpeptide sequence, but not another RFamide, prolactin-releasingpeptide. The distribution of RFRP-1/3 neurons reported here isconsistent with previous studies, which showed that RFRP-1 andRFRP-3 are expressed in the same perikarya around the dorso-medial hypothalamus of rats (14, 18–20), and with the study ofKriegsfeld et al. (11), which investigated the location of white-crowned sparrow GnIH-immunoreactive neurons in hamsters,rats, and mice. That study reported the presence of GnIH-im-munoreactive cell bodies in the region of the dorsomedial hypo-thalamus with no fibers identified in the external median emi-nence in any of the three mammalian species. This suggests thatthe antisera to white-crowned sparrow GnIH, which has lessthan 30% sequence identity to mammalian RFRP-1 or RFRP-3(10), nevertheless detects mammalian RFRP-1/3 neurons in therodent brain. In situ hybridization studies further confirmed thislocation of cell bodies in rats (8, 14), mice (10), and hamsters (11,

FIG. 4. Schematic coronal sections through the rat forebrain and brain stem showing distribution ofimmunoreactive RFRP-1/3 cell bodies (F) and fibers (�). Brain section figures are adapted from the atlas ofPaxinos and Watson.

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21). One early study using the GnIH antibody reported perikaryaexpression in the mouse brain stem (nucleus of the solitary tractand lateral superior olive) (22), but this has not been supportedby in situ hybridization experiments (10) or our own immuno-histochemical studies in mice using the RFRP-1/3 antibody de-scribed in this paper.

RFRP-1/3 immunoreactive fibers were readily detectedthroughout the rat hypothalamus and brainstem. The key find-ing was that no RFRP-1/3-immunoreactive fibers were identifiedwithin the external zone of the median eminence. Whereas al-most all studies using other antibodies have also noted an ab-sence of fibers in this region (11, 14, 19, 22), Kriegsfeld and

coworkers (12) recently reported fiber immunoreactivity in themedian eminence external zone of hamsters using an immuno-histochemistry amplification protocol. Because of the importantimplication of this issue in regard to the possible hypophysio-tropic nature of RFRP-1 and -3, we sought to confirm our an-atomical observation by testing whether RFRP-1/3 neurons wereable to take up peripherally administered Fluoro-Gold (Bi-otium), a retrograde tract tracer that does not cross the blood-brain barrier. Whereas about 95% of GnRH neurons were foundto contain Fluoro-Gold after ip administration, only 1% (threeof 234 neurons) of RFRP-1/3 neurons expressed Fluoro-Gold.This makes it very unlikely that RFRP-1/3 neurons in the rat areable to regulate gonadotropin secretion at the level of the anteriorpituitary gland. This conclusion is further supported by lack ofthe putative RFRP-3 receptor OT7T022 (23) in the pituitarygland (8). In contrast, GnIH fibers have been shown to extend tothe external layer of the median eminence in birds (5, 6), al-though there is evidence from one irregularly breeding sparrowspecies that this might not always be the case (24). Furthermore,the putative GnIH receptor mRNA appears to be relatively abun-dant in the avian pituitary gland (25). These findings are in keep-ing with a neuroendocrine role for GnIH in avian species.

FIG. 5. Examples of colocalization of Fluoro-Gold with RFRP-1/3 and GnRHneurons in rat brain sections. Left panels show Fluoro-Gold fluorescence, middlepanels show RFRP-1/3 or GnRH immunoreactivity in the same sections, and rightpanels show merged images. There was almost no Fluoro-Gold uptake by RFRP-1/3 neurons after ip injection of Fluoro-Gold (A–C). In contrast, almost all GnRHneurons contained Fluoro-Gold after ip injection (D–F). G–I, RFRP-1/3 neuronsexpress Fluoro-Gold after unilateral injection into the hippocampus and are alsorepresentative of animals injected unilaterally into the rostral preoptic area. Filledarrows denote colocalization; open arrows show neurons that do not containFluoro-Gold, and arrowheads show Fluoro-Gold in unidentified neurons. Scalebars, 10 �m.

FIG. 6. Percentage of all RFRP-1/3 neurons containing Fluoro-Gold in male andfemale rats after unilateral injection of Fluoro-Gold into the hippocampus (A) orrostral preoptic area (B).

FIG. 7. Plasma LH concentration after iv injections of RFRP-3 in combinationwith 0.1 �g GnRH (triangles) or alone (circles). RFRP-3 was given at two differentdoses: 1 �g/rat (gray symbols) and 10 �g/rat (black symbols) or vehicle only(white symbols). *, Significant effect (P � 0.05) of both RFRP-3 doses at 5 min inpresence of GnRH vs. vehicle-treated controls.

1418 Rizwan et al. RFRP-1/3 Neurons Are Not Hypophysiotropic Endocrinology, March 2009, 150(3):1413–1420

If RFRP-1/3 does not regulate gonadotropin secretion at thepituitary in rodents, peripheral administration of these peptideswould not be expected to suppress LH secretion in the way thathas been demonstrated in two experiments conducted on white-crowned sparrow (26, 27). In these experiments, iv GnIH treat-ment caused a rapid and transient (2–5 min after injection) sup-pression of plasma LH concentration. In contrast, under basalconditions in our ovariectomized rats, iv administration ofRFRP-3 had no effect on plasma LH levels. This lack of responsewas not due to inefficacy of our RFRP-3 preparation because wehave shown in other experiments that the same peptide can actvia central mechanisms at nanomolar concentrations to inhibitGnRH neuron firing (37) and GnRH neuronal activation duringthe preovulatory surge (28). The lack of response to an iv RFRP-3injection supports the conclusion from our anatomical findingsthat RFRP-1/3 neurons do not appear to act outside the blood-brain barrier. However, we did observe a small but significanteffect of iv RFRP-3 when coadministered with GnRH at the5-min postinjection time point but not at other times.

This may represent the ability of RFRP-3 to activate RFamide-relatedreceptorsat the levelof thepituitaryandinteractwithGnRHreceptor-mediated stimulation of gonadotrophin release. In thisregard, one previous study reported a more robust effect of pe-ripheral GnIH treatment in mammals. In ovariectomized Syrianhamsters, ip administration of a low dose (600 ng) of sparrowGnIH markedly reduced the plasma LH concentration (�70%suppression) at 30 min after injection (11). This more robusteffect may be due to species, dose, or hormone treatment(RFRP-3 vs. GnIH) differences. It is also possible that the effectof the peptide is influenced by duration of exposure such that amore prolonged treatment (likely to have been achieved by ipadministration compared with iv in the present study) is requiredfor effects to be seen (29).

Yet another recent study reported an effect of an iv RFRP-3injection (�40% suppression), surprisingly 2 h after injection(30). Such a delayed effect would not have been seen in thepresent study, in which samples were collected only up to 30 minafter injection. By comparison, in two separate studies conductedin white-crowned sparrows, iv GnIH treatment caused a rapidand transient (2–5 min after injection) suppression of plasma LHconcentration (26, 27). To specifically address the issue ofwhether RFRP-3 can act on pituitary cells to modulate LH se-cretion, our group has treated primary anterior pituitary cellcultures with RFRP-3 for 3 h at doses of up to 10�7 M. In agree-ment with the conclusions from the present in vivo study in whichRFRP-3 was given iv, no significant effects of RFRP-3 were seen(28). Very recently, however, RFRP-3 has been shown to be ableto block LH release from whole pituitary cell cultures from sheep(29) and rats when given over a 24-h period (30). The apparentdiscrepancy between the cell culture results of our study and theaforementioned reports (29, 30) is not easily explainable at thecurrent time. Pituitary level effects of another RFamide neu-ropeptide, kisspeptin, have been reported in primary cell culturesat doses thought to be of little physiological relevance whencompared with the concentrations measured in hypophysial por-tal blood (31). In the case of RFRP-3, hypophysial portal bloodmeasurements have not been reported to date.

RFRP-1/3 fibers were observed in the preoptic area of rats. As inbirds (6, 7), rats and Syrian hamsters both exhibit avian GnIH-immunoreactive fibers that make apparent contacts with GnRHneurons (11, 19, 32). This suggests that GnRH neurons are regu-lated by GnIH and RFRP-1/3 in birds and mammals, respectively.Furthermore,80%ofGnRH-1neurons in the starlinghaverecentlybeen shown to coexpress GnIH receptor mRNA (33). WhereasGnRH and RFRP-3 receptor colocalization has not yet been as-sessed in mammalian species, the available data suggest thatRFRP-3 may control mammalian gonadotropin secretion by reg-ulating GnRH neuronal activity. Intracerebroventricular adminis-tration of RFRP-3 (19, 28) or sparrow GnIH (11) has been shownto suppress plasma LH concentrations under a variety of reproduc-tiveconditions. Inthepresentstudy,weshowthat thegreatmajority(�75%)ofRFRP-1/3neuronssendprojections tothepreopticarea,thus supporting the hypothesis that RFRP-1/3 neurons may inner-vate the GnRH neuron cell bodies in the rat.

The observation here of RFRP-1/3 fibers in many brain regionsincluding some in thebrain stemsuggest that theRFRP-1/3neuronsof the hypothalamus project widely within the brain. The extensivefiber distribution we report closely mirrors that reported by othersusing different antibodies (11, 14, 19, 22). Hence, as with mostneuropeptidergic neurons, RFRP-1/3 peptides will be involved inmultiple neuronal networks regulating diverse functions. We wereintrigued tonote that themostdenseRFRP-1/3 fiberprojectionwasfound in the pyramidal layer of the CA2/CA3 hippocampus, whichhas not been described previously. Furthermore, using a retrogradetract tracing approach, we have been able to confirm that this inputderives from hypothalamic RFRP-1/3 neurons. This unexpectedfinding suggests that RFRP peptides are involved in modulatinghippocampal activity and is supported by the presence of RFRP-3receptors in the rodent hippocampus (8). Whereas there is a grow-ing appreciation of the role of the dorsomedial nucleus in the inte-gration of circadian rhythms within neuroendocrine circuits (34),nothing is known about the physiology of projections from thishypothalamic nucleus to the hippocampus.

It is worth noting that in all descriptions of GnIH (5, 6, 35, 36)and RFRP-1 or -3 (8, 11, 19, 23 and our present data) publishedto date, GnIH cell bodies in birds appear to be many times morenumerous than RFRP-1/3 cell bodies in mammals. This mayreflect the dual (neuropeptide and neuroendocrine) mechanismsof LH regulation in birds, whereas in mammals we postulate thatonly central regulation exists.

In summary, RFRP-1/3 neurons are localized to the dorso-medial hypothalamus of rats. Although RFRP-1/3 fibers are dis-tributed throughout the brain, suggesting diverse roles for thisneuropeptide, they do not project to the median eminence. Assuch, RFRP-1/3 is unlikely to fulfill the role of a gonadotropinrelease-inhibiting hormone in rats.

Acknowledgments

Addressall correspondenceandrequests for reprints to:Dr.GregAnderson,Centre for Neuroendocrinology and Department of Anatomy and Struc-tural Biology, University of Otago School of Medical Sciences, P.O. Box913, Dunedin 9054, New Zealand. E-mail: greg.anderson@anatomy.otago.ac.nz.

Endocrinology, March 2009, 150(3):1413–1420 endo.endojournals.org 1419

This work was supported by The Royal Society of New ZealandMarsden Fund (to G.M.A. and A.E.H.).

Disclosure Statement: The authors have nothing to declare.

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