Proc. Nati. Acad. Sci. USAVol. 88, pp. 4508-4512, May 1991Physiology/Pharmacology

Galanin: A hypothalamic-hypophysiotropic hormone modulatingreproductive functions

(luteinizing hormone-releasing hormone/portal blood/neurosecretion)

FRANCISCO Jost L6PEZ*, ISTVAN MERCHENTHALERt, MELVIN CHING*t, MIRIAM G. WISNIEWSKI*,AND ANDRtS NEGRO-VILAR**Reproductive Neuroendocrinology and tFunctional Morphology Sections, Laboratory of Molecular and Integrative Neuroscience, National Institute ofEnvironmental Health Sciences, Research Triangle Park, NC 27709

Communicated by S. M. McCann, February 25, 1991 (received for review September 20, 1990)

ABSTRACT Galanin (GAL) is widely distributed in theperipheral and the central nervous systems. In the brain, thehighest GAL concentrations are observed within the hypothal-amus and, particularly, in nerve terminals of the medianeminence. This location, as well as GAL actions on prolactin,growth hormone, luteinizing hormone (LH), and LH-releasinghormone (LHRH) secretion, suggest the possibility that GALmay act as a putative hypothalamic-hypophysiotropic hor-mone. To establish this, GAL and LEIRH levels were measuredin hypophyseal portal plasma samples using specific radioim-munoassays. Rat galanin (rGAL) concentrations in portalblood were -7-fold higher than those observed in peripheralplasma in male and female (estrus, diestrus) rats, indicating anactive secretory process of rGAL into the portal vasculature.Frequent (10 min) sampling revealed that rGAL and LHRHwere secreted into the portal circulation in a pulsatile mannerwith a pulse frequency of one pulse per hour. Interestingly,both hormone series depicted a high degree of coincidentepisodes. In fact, the probability of random coincidence,calculated by the algorithm HYPERGEO, was <0.01. Moreover,the retrograde tracer Fluoro-Gold, when given systemically,was taken up by GAL neurons in the hypothalamus, includinga subset of neurons expressing rGAL and LHRH, strengthen-ing the notion of the existence of a GAL neuronal systemconnected to the hypophyseal portal circulation. These obser-vations reinforce the concept that GAL regulates pituitaryhormone secretion. To analyze this in further detail, the effectsof rGAL on LH secretion were evaluated under basal andstimulated conditions. rGAL induced a small but dose-dependent increase in LH secretion from cultured, dispersedpituitary cells. Interestingly, rGAL enhanced the ability ofLHRH to stimulate LH release. The tight link between GALand LHRH neuronal systems is strengthened by the observa-Ition that during the estrous cycle of the rat, rGAL and LHRHcontents in the median eminence show an identical profile (r =1.00). These data indicate that GAL should be considered as ahypothalamic-hypophysiotropic hormone and as an importantneuromodulator of LHRH secretion and action. The colocal-ization and cosecretion ofGAL and LHRH and the cooperativeaction at the level of the anterior pituitary afford importantevidence for the functional significance of coexistence of neu-rotransmitters in neurons of the central nervous system.

Galanin (GAL) is a 29-amino acid peptide originally isolatedfrom porcine intestine (1) and subsequently reported to beubiquitously distributed in many different tissues. GAL im-munoreactivity is abundant in the central and peripheralnervous systems, and high concentrations are reported in thehypothalamus (2, 3). In fact, the highest levels of GAL

mRNA (4) and receptor sites (5) are located in this specificarea of the brain. Within the hypothalamus, the medianeminence (ME) contains the highest levels of GAL (3, 6, 7).This particular location and the observation that GAL mayaffect the release of several pituitary hormones, includinggrowth hormone (GH), prolactin (PRL), and luteinizing hor-mone (LH) (8-15), suggest that this peptide may be a memberof the hypothalamic-hypophysiotropic hormone family.

Since the establishment ofthe neurovascular concept in theregulation of the hypothalamus-pituitary axis (16), it is wellknown that the ME represents a key area for neuroendocrineregulation. Hypothalamic releasing and inhibiting factors aresecreted from ME terminals into the portal circulation toreach the adenohypophyseal cells where they exert specificactions. The following studies were aimed at clarifyingwhether GAL could be considered as a member of thehypothalamic hormone family. We report here that GALmeets the criteria to be considered as a hypothalamic-hypophysiotropic hormone, since (i) GAL levels are higherin hypophyseal portal than in the peripheral circulation; (it)GAL neurons can be labeled from the ME using Fluoro-Gold,a retrogradely transported tracer; and (iii) GAL release intothe portal blood occurs in a pulsatile fashion, showing a pulsefrequency similar to that of LH-releasing hormone (LHRH).Thus, GAL shares with other hormones the current conceptthat hormone signals are codified in a pulsatile manner. Inaddition, GAL stimulates LH secretion and enhancesLHRH-induced LH release from dispersed anterior pituitarycells in vitro. An intriguing colocalization (ref. 7; presentstudy) and cosecretion (demonstrated by the finding of co-incident secretory episodes) of GAL and LHRH are alsoreported.

MATERIALS AND METHODSAnimals. Male and female Sprague-Dawley rats (CD;

Charles River Breeding Laboratories) were used for theseexperiments. For the estrous cycle studies, only those ani-mals showing at least two consecutive 4-day cycles wereused.

Materials. The following compounds and materials wereused: rat GAL (rGAL) and LHRH (Peninsula Laboratories);sheep anti-rabbit IgG, rabbit anti-sheep IgG, and their re-spective peroxidase-antiperoxidase (PAP) complexes (ArnelProducts, New York); fluorescein isothyocyanate-labeleddonkey anti-rabbit IgG and rhodamine-labeled donkey anti-sheep IgG (Chemicon); 3,3'-diaminobenzidine (Aldrich);

Abbreviations: LH, luteinizing hormone; LHRH, LH-releasing hor-mone; GAL, galanin; rGAL, rat GAL; ME, median eminence; GH,growth hormone; PRL, prolactin; PAP, peroxidase-antiperoxidase.tPresent address: Department of Veterinary Anatomy, Ohio StateUniversity, A100 Sisson Hall, 1900 Coffey Road, Columbus, OH43210-1092.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Tmax 3200 black-and-white and Ektachrome P 800/1600films (Kodak); and Fluoro-Gold (Fluorochrome, Englewood,CO).

Portal and Peripheral Blood Sample Collection. Portal andperipheral blood samples were collected as described (17).Either a single (30 min) or multiple (10-min intervals for up to2 hr) portal blood samples were collected.Immunocytochemical Procedures. Neurons connected to

fenestrated capillaries were detected by retrograde labelingwith Fluoro-Gold as described (18). The procedures used foranimal preparation and tissue fixation have been described indetail elsewhere (7, 19). Paraffin sections (5 Arm) were simul-taneously stained for rGAL and LHRH immunoreactivitiesusing specific rabbit (anti-rGAL serum FJL 7-3, 1:4000; ref.20) and sheep (anti-LHRH serum A772, 1:6000; ref. 21)antisera and the indirect immunofluorescence method ofCoons (7, 22) or the PAP technique (19).

Dispersed Pituitary Cell Cultures. Dispersed anterior pitu-itary cells were obtained as described (23) except that acollagenase/DNase solution was used for dispersion andcells were plated and incubated for 3-5 days. On the day ofthe experiment, test substances were added followed by a3-hr incubation period. Media were collected and assayed forLH.rGAL and LHRH Content in ME. Animals were sacrificed

by decapitation before noon in each of the estrous cyclestages. Brains were rapidly removed from the skull and MEswere dissected under a stereomicroscope using a fine pair ofscissors as reported (24). ME fragments were homogenized in0.1 ml of ice-cold 0.1 M acetic acid using glass microhomog-enizers. Tissue homogenates were prepared as described forpeptide (24) and protein (25) assays.RIAs. GAL levels were measured in duplicate as described

(20). Complete parallel displacement with serum or tissueextracts has been demonstrated using this assay (20). Screen-ing with our FJL 7-3 antiserum of fractions from SephadexG-50 chromatography of arcuate nucleus-ME extracts re-vealed that =93% of all immunoreactive materials eluted inthe same position as synthetic rGAL (data not shown).LHRH concentrations were measured in duplicate withoutextraction using anti-LHRH serum Rice no. 5. A completecharacterization of this antiserum has been reported else-where (26, 27). Medium samples were assayed for LH usingmaterials kindly supplied by the National Institute of Diabe-tes, Digestive and Kidney Diseases. To avoid interassayvariability, all samples from the same experiment weremeasured in a single assay. Intraassay variabilities were 8%,7%, and 9o for GAL, LHRH, and LH, respectively.

Pulse Analysis and Statistics. Pulsatility patterns were eval-uated using the algorithm DETECT (28). Significance thresh-olds for detecting a pulse were routinely set at P < 0.05. Todefine a secretory episode, we utilized the criterion that anypulse must consist of an ascending phase followed by adescending phase as previously reported for PRL pulsatility(29). The analysis of coincidence between rGAL and LHRHpulses was performed by calculating the probability of ran-dom coincidence using the program HYPERGEO (30).Data were routinely analyzed for normality and variance

homogeneity using the Shapiro-Wilk's test and Barttlet'stest, respectively. Some data sets did not accomplish thenormality and variance homogeneity criteria required forapplying parametric statistics. When possible, a logarithmictransformation of the data was performed to satisfy thesecriteria; if so, parametric statistics were utilized (31). Other-wise, data were analyzed using nonparametric statistics (31).One-way analysis of variance or the equivalent in nonpara-metric statistics, Kruskal-Wallis' test, was used when com-paring more than two experimental groups. When any of theabove-mentioned tests resulted in statistically significantdifferences, either the parametric or nonparametric Student-

Newman-Keul's test was used to obtain specific groupdifferences. Comparisons between portal and peripheral lev-els ofrGAL in the same animals were assessed using Wilcox-on's test (31). Linear regression was utilized to correlaterGAL and LHRH content in the ME (31).

RESULTSGAL as a Hypophysiotropic Peptide. As shown in Fig. 1

Upper, A, abundant GAL immunoreactivity can be found inthe external layer of the ME. To assess whether GAL axonterminals establish functional connections with capillaries ofthe portal vasculature, we conducted a series of studiestaking advantage of a recently developed technique of ret-rograde labeling that uses Fluoro-Gold as a tracer (18, 32, 33).

I

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FIG. 1. rGAL meets all of the criteria to be considered ahypothalamic-hypophysiotropic hormone. (Upper) (A) GAL-immunoreactive nerve terminals in the ME evenly occupy theexternal zone; however, many can be observed in the internal zoneas well. (B) Fluoro-Gold accumulating. (C) GAL-immunoreactiveperikarya in the same tissue section of the arcuate nucleus. Smallarrows and arrowheads in B and C label identical perikarya. Notethat cell bodies labeled by larger arrows, an asterisk, and a star in Bcontain Fluoro-Gold but they are not immunoreactive for GAL.(Lower) rGAL levels in portal (30-min collection, open bars) andperipheral plasma (before hypophysectomy, filled bars) in male andfemale rats [diestrus (D) and estrus (E)]. Results are expressed asmeans ± SEM of five or six animals per group. Asterisks denotestatistically significant differences (P< 0.05, Wilcoxon's test; ref. 31)against the respective level in portal plasma. (x50.)

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Fluoro-Gold, a retrogradely transported dye that does notcross the blood-brain barrier, when injected systemically,apparently labels neurons that have access to the vasculature(33, 34). Combining specific immunostaining for rGAL withFluoro-Gold uptake, we observed that GAL neurons in thefollowing hypothalamic structures took up Fluoro-Gold whenthe marker was injected intraperitoneally (Fig. 1 Upper, Band C): anterior periventricular nucleus, paraventricularnucleus in the parvocellular and magnocellular subdivisions,arcuate nucleus, and scattered cells in the dorsal hypotha-lamic area and the accessory magnocellular nuclei. Retro-gradely labeled neurons in the diagonal band of Broca andmedial preoptic nucleus resembled the morphological fea-tures of LHRH neurons. These observations indeed suggestthat many GAL neurons are functionally linked to the portalvasculature.The evaluation of GAL concentrations in portal and pe-

ripheral blood samples is depicted in Fig. 1 Lower. rGALconcentrations in portal plasma were higher than in periph-eral plasma regardless of the animals' endocrine situation. Infact, rGAL levels in portal blood ranged from =3.8 to =5.3ng/ml, whereas levels observed in peripheral blood rangedfrom 0.6 to 0.7 ng/ml in female rats during diestrus and estrusas well as in male rats (Fig. 1 Lower). It is noteworthy thatthere was a high variability in rGAL levels measured in portalblood, suggesting that GAL may be released episodically.Experiments to evaluate this possibility are described below.rGAL was evaluated for its ability to affect anterior pitu-

itary hormone secretion. As depicted in Fig. 2 Upper, rGALstimulated LH release from dispersed pituitary cells in vitroin concentrations similar to those observed in the portal

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circulation. In addition, GAL secreted into the portal vas-culature was able to interact with pituitary cells to potentiateLHRH-induced LH release from dispersed pituitary cells inculture (Fig. 2 Lower). Taken together, the above-mentionedobservations clearly indicate that GAL meets the criteria tobe considered a hypothalamic-hypophysiotropic hormone.GAL-Immunoreactive Neurons that Coexpress LHRH Proj-

ect to the ME. Because of our earlier observations that GALand LHRH are colocalized in a subset ofLHRH neurons (7),we evaluated whether these neurons coexpressing the twopeptides are also functionally linked to the portal vasculature.In Fig. 3, two neurons presenting triple labeling (rGAL,LHRH, and Fluoro-Gold) are shown. These results suggestthat neurons coexpressing GAL and LHRH project to theME and may, therefore, contribute to the presence of GALand LHRH in hypophyseal portal vasculature.

Evaluation of GAL and LHRH Pulsatile Patterns in PortalBlood. rGAL and LHRH were secreted into the portalcirculation in a pulsatile manner (Fig. 4 Upper). Interestingly,rGAL pulses either were coincident with those of LHRH orpreceded them by 10-20 min (Fig. 4 Upper). Data wereanalyzed by calculating the exact possibility of having coin-cident pulses at random using the program HYPERGEO (30).Table 1 depicts the results of this analysis. Thirty-threepercent of all observed pulses displayed a perfect coinci-dence, defined as the pulses in both series that have the peakvalue in the same time point (lag = 0). The exact probabilityof coincidence occurrence at this level did not indicate thatthe null hypothesis of having those coincident pulses atrandom could be rejected (Table 1). In contrast, whencoincident pulses were defined as those coinciding exactly inthe peak (lag = 0), in addition to those presenting a 10-mindelay (lag = +1), a probability <0.01 was obtained, indicat-ing that the number of coinciding pulses could not be ran-domly explained. Moreover, the inclusion of those pulses

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LHRH 1 nM +rGAL (nM)

FIG. 2. GAL stimulates LH secretion and potentiates LHRH-induced LH secretion from anterior pituitary cells in vitro. Resultsare expressed as means ± SEM of three independent experimentswith a total of 16 or 17 wells per group. Basal LH secretion fromanterior pituitary cells was 626.89 ± 42.48 (17) ng/ml. *, P < 0.05 vs.control levels. **, P < 0.05 vs. all groups. The Kruskal-Wallis' testwas followed by the nonparametric Student-Newman-Keul's test(31).

FIG. 3. Two neurons (Left and Right) immunoreactive for LHRH(Top) and GAL (Middle) and retrogradely labeled with Fluoro-Gold(Bottom). Asterisks denote the same blood vessel. (x230.)

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FIG. 4. (Upper) rGAL (filled circles) and LHRH (open circles)levels in portal blood. Asterisks denote statistically defined secretoryevents using the program DETECT (28). Profiles correspond to tworepresentative animals out of eight. (Lower) rGAL (filled circles) andLHRH (open circles) concentrations in the ME during the estrouscycle. Results are expressed as means ± SEM of 6-17 animals pergroup. E, estrus; M, metestrus; D, diestrus; P, proestrus. *, P < 0.05vs. estrous and metestrous levels (Kruskal-Wallis' test followed bythe nonparametric Student-Newman-Keul's test; ref. 31). (Inset)Linear regression analysis ofrGAL and LHRH concentrations in theME during the estrous cycle.

coinciding with a 20-min delay (lag = +2) reduced theprobability of random coincidence to <0.001 (Table 1). Thedata strongly suggest that rGAL and LHRH pulses are

related and/or synchronized.GAL and LHRH Content in the ME During the Estrous

Cycle. LHRH content in the ME reached the highest levels(about 100 ng/mg of protein) during diestrus and proestrus,abruptly decreasing to minimum values during estrus (Fig. 4Lower). rGAL content in the ME was highest (around700-800 ng/mg of protein) during diestrus and proestrus,reaching minimum levels (about 500 ng/mg of protein) duringestrus (Fig. 4 Lower). On a molar basis, rGAL content was2.5- to 3-fold higher than that of LHRH without any remark-

Table 1. Evaluation of random coincidence between rGAL andLHRH pulses

Coinciding pulses RandomCoincidence coincidence

definition (lag) Yes:no % probability0 2:4 33.33 0.3418660 and +1 4:2 66.67 0.0097590 and +1 and +2 6:0 100.00 <10-4

Probability of random coincidence was calculated using the pro-gram HYPERGEO, as described (30). Random coincidence representsthe exact probability of rejecting the null hypothesis that coincidenceis a random phenomenon and, therefore, both series are not related.

able change in this ratio during the estrous cycle (data notshown). Linear regression analysis revealed a perfect corre-lation (r = 1.00, P < 0.001) between the content of bothpeptides (Fig. 4 Lower, Inset) indicating a close relationshipbetween rGAL and LHRH during physiological events re-lated to the estrous cycle.

DISCUSSIONIn general, a key series of requirements to ascribe a hypo-physiotropic role to a hypothalamic peptide include (i) pres-ence in the hypothalamus, (it) projections to the ME andcontacts with portal capillaries, (iil) presence in portal bloodin higher concentrations than in the peripheral circulation,(iv) an action at the pituitary level at concentrations similarto those observed in portal circulation, and (v) presence ofreceptors on pituitary cells. Our data reinforce the existenceof a neuronal system that secretes rGAL specifically intoportal circulation and provide support for most of the abovecriteria: (i) GAL immunoreactivity is present in the hypo-thalamus (refs. 2 and 3; present results); (ii) GAL-producingneurons can be retrogradely labeled from the ME (presentstudy), indicating a direct access of GAL terminals to theportal vasculature; (iii) GAL concentrations in portal plasmaare higher than in the peripheral circulation depicting apulsatile secretory pattern (ref. 20; present study); (iv) GALis able to stimulate and potentiate LHRH-induced LH se-cretion at the pituitary level (present study). Together, theseobservations indicate that GAL should be considered as animportant component of the hypothalamic-hypophysiotropichormone family.Our present studies indicate that rGAL and LHRH were

secreted into the portal circulation in a pulsatile manner.Moreover, rGAL pulses either were coincident with those ofLHRH or preceded them by 10-20 min. Since none of theLHRH pulses preceded those of rGAL and, in addition, wehave observed a stimulatory effect of rGAL on LHRHrelease from ME nerve terminals in vitro (35), we could inferthat rGAL/LHRH pulse coincidence may be related to anaction ofrGAL enhancing LHRH release and supporting theidea that rGAL release is the initial event in this cosecretion.The presence of a triple labeling for rGAL, LHRH, and

Fluoro-Gold in the same neuron indicates that rGAL andLHRH neuronal systems have a subpopulation of neuronsthat contain both peptides and, further, that these neuronsare connected with fenestrated capillaries (7). The distribu-tion of Fluoro-Gold-labeled GAL-immunoreactive neuronsindicates that they form axon terminals on portal capillariesof the ME. In addition, some of them may have taken upFluoro-Gold from the organum vasculosum of the laminaterminalis (OVLT) but, since in male rats the number ofGAL-immunoreactive terminals in OVLT is very low (I.M.and A.N.-V., unpublished observation), it appears that GAL-and Fluoro-Gold-positive neurons are linked to the ME. Thefinding of a relationship between rGAL and LHRH secretioninto the portal circulation is intriguing. The colocalization ofrGAL and LHRH in the same neurons (ref. 7; present study)may be responsible for the relationship between rGAL andLHRH secretion. Moreover, it is tempting to speculate thatthose pulses coinciding with a lag = 0 may represent thesecretion of those neurons that display rGAL and LHRHimmunoreactivity. On the other hand, those pulses thatcoincide with a lag > 0 (GAL pulse preceding that of LHRH)may represent LHRH secretion induced by the GAL input tothe LHRH neurons and/or terminals. The former idea issupported by the observation that immunoreactive rGALsynaptic-like buttons seem to be present on LHRH neurons(7) and by the finding that pulse frequency for rGAL andLHRH series was identical (approximately one pulse everyhour).

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The colocalization and apparent cosecretion of rGAL andLHRH suggest that, in physiological reproductive situations,both peptides should display very similar changes. Indeed,rGAL and LHRH levels in the ME during the estrous cyclereach the highest values during diestrus and proestrus,abruptly decreasing to minimum values during estrus. Thetemporal changes of LHRH during the estrous cycle are ingood agreement with those reported by others (36, 37) andindicate a close relationship between rGAL and LHRHduring physiological events.

In addition to GAL/LHRH interactions at the hypotha-lamic level, GAL stimulates and enhances LHRH-inducedLH secretion by acting directly on the pituitary. AlthoughGAL receptors have not yet been identified and/or charac-terized in the anterior pituitary gland, GAL effects on LHsecretion are exerted at concentrations ofGAL ranging in anorder of magnitude similar to those observed in portal blood.In addition to brain GAL, it is also possible that intrapituitaryGAL may contribute to the spectrum of GAL actions ongonadotropin secretion. Recent studies have shown thatmRNA coding for rGAL exists in the pituitary gland and thatestrogens dramatically increase GAL message (38). Eventhough the actions of pituitary GAL are not yet known, theseobservations suggest that GAL may modulate pituitary func-tion by acting locally and/or be released to the peripheralcirculation exhibiting its actions at a remote location. Thefact that the pituitary gland contributes to some degree toGAL levels in peripheral blood (=30%; ref. 25) supports thenotion that pituitary GAL may act not only as a paracrineregulator of pituitary hormone secretion but also as anendocrine signal for peripheral tissues.The present studies clearly establish GAL as an important

member of the hypothalamic-hypophysiotropic hormonefamily of peptides, indicating that GAL terminals in the MEare functionally linked to the portal capillaries; that thepeptide is secreted in a pulsatile fashion into the portalvasculature; and that it acts, alone or in combination withother hypophysiotropic peptides, to modulate anterior pitu-itary hormone secretion. The colocalization and cosecretionofGAL and LHRH and the cooperative action at the level ofthe anterior pituitary afford important evidence for the func-tional significance of coexistence of neurotransmitters inneurons of the central nervous system.

We thank David Lennard and Edwin H. Meade for their help inconducting these studies. F.J.L. acknowledges the continuous sup-port of the Department of Physiology of C6rdoba University and Dr.Amador Jover Moyano, President of C6rdoba University, over theyears.

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