Neuroanatomical plasticity in the gonadotropin-releasing hormone system of the ewe: Seasonal variation in glutamatergic and γ-aminobutyric acidergic afferents

Neuroanatomical Plasticity in the Gonadotropin-ReleasingHormone System of the Ewe: Seasonal Variation inGlutamatergic and �-Aminobutyric Acidergic Afferents

ANNA SERGEEVA AND HEIKO T. JANSEN*Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology (VCAPP), Washington State University,Pullman, Washington 99164-6520

ABSTRACTTemperate zone animals time the onset of reproductiveevents to coincide with specific portions of the siderealyear. Although the neural mechanisms involved remainpoorly understood, a marked annual variation in the brain’ssensitivity to estradiol negative feedback is thought to me-diate many of the changes in neuroendocrine hormone se-cretion, especially that of the gonadotropin-releasing hor-mone (GnRH) neurons, via neural afferents. The aim of thepresent study was to determine whether glutamatergic in-puts to GnRH neurons in sheep vary seasonally and toexpand our previous observations of seasonal changes in�-aminobutyric acid (GABA)-ergic inputs. Brains from adultsheep were collected during the breeding season (N � 8) orthe nonbreeding season (anestrus; N � 7). Confocal micros-copy and optical sectioning were used to quantify the den-sity of labeled VGLUT2 and VGAT immunoreactivity onto

GnRH neurons. The results reveal a significantly greaternumber of VGLUT2-ir inputs to GnRH dendrites during thebreeding season vs. the nonbreeding season but no sea-sonal changes on GnRH cell somas. The number of VGAT-irterminals onto GnRH dendrites was reduced in the breedingseason compared with the nonbreeding season. GnRH neu-rons were also found to receive dual-phenotype (VGLUT �VGAT) inputs; these varied with season in a manner similarto VGAT inputs. Morphologically, the numbers of branchesof proximal dendrites increased significantly in a subset ofGnRH neurons located near the midline. Together theseresults reveal a dynamic seasonal reorganization of identi-fied inputs onto GnRH neurons and lend additional supportto the overall hypothesis that seasonal modulation of GnRHneurons involves glutamatergic and GABAergic neural plas-ticity. J. Comp. Neurol. 515:615–628, 2009.© 2009 Wiley-Liss, Inc.

Indexing terms: sheep; vesicular glutamate transporter 2; vesicular GABA transporter;estradiol; negative feedback

Seasonal changes in day length provide a useful cue foranimals living in temperate climates, and these serve to timekey biological events such as reproduction (mating, birth),migration, pelage changes, and hibernation, to name just afew. Although the precise neural mechanisms involved formany seasonal events remain to be elucidated, the annualrhythm of reproductive cycles appears to be generated endo-genously by a neural process that is synchronized to thesidereal year (Gwinner, 1986; Jansen and Jackson, 1993a;Karsch et al., 1989; Zucker, 1988, 2001). The final commonneural pathway mediating this dramatic event is thegonadotropin-releasing hormone (GnRH) system and its pro-jections to the median eminence of the hypothalamus.

Although GnRH neurons appear to exhibit autonomous ac-tivity leading to episodic peptide release (Terasawa, 2001),both the annual variations in GnRH release and those ob-served during the estrous cycle are under neuromodulatorycontrol. The influences of photoperiod directly and the annualwaxing and waning of the brain’s sensitivity to steroid nega-tive feedback are two important mechanisms for the seasonal

control of reproduction (Goodman, 1996; Goodman et al.,1982; Legan et al., 1977; Sisk and Turek, 1983; Thiery et al.,2002). However, insofar as very few GnRH neurons containestrogen receptors (Lehman and Karsch, 1993; Shivers et al.,1983), the neuronal origin of estradiol negative feedback sig-naling has remained elusive. Both light microscopic and ul-

Additional Supporting Information may be found in the online version ofthis article.

Grant sponsor: National Research Initiative grant from the USDA Coop-erative State Research, Education, and Extension Service; Grant number:2005-35203-15848.

*Correspondence to: Heiko T. Jansen, PhD, Dept. VCAPP, 205 WegnerHall, P.O. Box 646520, Pullman, WA 99164-6520.E-mail: [email protected]

Received 3 October 2008; Revised 6 March 2009; Accepted 27 March2009

DOI 10.1002/cne.22087Published online April 3, 2009 in Wiley InterScience (www.interscience.wiley.

com).

The Journal of Comparative Neurology 515:615–628 (2009)

Research in Systems Neuroscience

© 2009 Wiley-Liss, Inc.

trastructural studies have identified seasonal changes in num-bers of GnRH neural inputs, supporting the hypothesis thatafferent regulation of GnRH neurons is seasonally modulated(Jansen et al., 2003; Lehman et al., 1997; Xiong et al., 1997).Supporting a functional role for these GnRH inputs is a vastbody of experimental evidence using pharmacological manip-ulations to modify reproductive neuroendocrine function(Goodman, 1996; Sagrillo et al., 1996; Smith and Jennes,2001).

By using confocal imaging and optical sectioning, our grouprecently confirmed earlier ultrastructural findings in sheep ofseasonal changes in the total number of GnRH inputs andexpanded these seasonal changes to include identified affer-ents such as neuropeptide-Y (NPY), tyrosine hydroxylase (TH),�-endorphin (�-EN), and glutamate decarboxylase (GAD) im-munoreactivity (Jansen et al., 2003; Xiong et al., 1997). Al-though NPY, TH, �-EN, and GAD accounted for virtually allinputs to GnRH somas, they were responsible for only a frac-tion of the total inputs onto GnRH dendrites. Thus, the largestnumber of unidentified inputs onto GnRH dendrites remainedunidentified. We had hypothesized, based on these observa-tions and our inability to visualize glutamatergic inputs directlyat the time, that these unidentified dendritic inputs were likelyto be glutamatergic (Jansen et al., 2003).

Glutamate agonists and antagonists are potent modulatorsof GnRH secretion in seasonally breeding mammals (Ebling etal., 1995a; Hileman et al., 1992; Jansen et al., 1991; Urbanskiet al., 1994). Unfortunately, although glutamate is the predom-inant excitatory neurotransmitter in the brain, its potential rolein seasonal anatomical plasticity is not completely under-stood. A previous study by Pompolo et al. (2003) reported thatVGLUT-ir terminals made close contacts in sheep GnRH neu-rons. However, only a few animals were examined, and sea-sonal comparisons were not made.

As with glutamate, �-aminobutyric acid (GABA) has beenheavily implicated in many aspects of the reproductive neu-roendocrine control of reproduction (Clarkson and Herbison,2006; DeFazio et al., 2002). GABAergic neurons in the sheepbrain have also been shown to express estrogen receptors(Herbison et al., 1993) and thus are likely to serve as keymediators of estradiol negative feedback to GnRH neurons.The purpose of the present study was to test a number ofhypotheses related to seasonal plasticity in the GnRH system,using the sheep, a model seasonal breeder. First, we testedthe hypothesis that the numbers of both glutamatergic andGABAergic inputs to GnRH neurons in the sheep vary withseason. To this end, identification of amino acid transporters(VAAT) was used. VAATs are heavily implicated in modulatingglutamatergic and GABAergic neurotransmission in the cen-tral nervous system (Erickson et al., 2006; Hisano, 2003). Theavailability of antibodies against numerous transporters al-lows direct examination of the relationships between pre- andpostsynaptic elements and quantitative analysis. Second, wetested the hypothesis, based on a long-standing questionregarding morphological plasticity of GnRH neurons, thatGnRH dendrites exhibit seasonal variation in their degree ofbranching. Lehman et al. (1986) previously provided anecdotalevidence in support of this notion, but a quantitative analysishad not been performed. Third, based on recent evidence inrats (Ottem et al., 2004), we tested the hypothesis that GnRH

neurons receive dual-phenotype inputs (i.e., glutamatergicand GABAergic) and that these exhibit seasonal variation.

MATERIALS AND METHODSAnimals

Adult North Country Cheviot ewes (2–4 years old) weremaintained on pasture at the University of Idaho Sheep Re-search Facility (47°N latitude) with food and water available adlibitum. Animals were cared for in accordance with the Guidefor the care and use of laboratory animals (National ResearchCouncil, National Academy Press, 1996). The WashingtonState University and the University of Idaho Institutional Ani-mal Care and Use Committees approved all procedures in-volving animals.

OvariectomyAnimals were ovariectomized and 2–3 weeks later received

two 1-cm estradiol silastic implants (ID 3.35 mm, OD 4.55 mm;Dow, Midland, MI) containing crystalline estradiol-17� (E;Sigma, St. Louis, MO) into the axial region of the left and rightforelimbs. This implant size is sufficient to produce lutealphase levels (<2 pg/ml) of the steroid in the circulation (Leganand Karsch, 1979; Legan et al., 1977). Eleven and nine ewesoriginally received E implants. However, at the time of perfu-sion (see below), three nonbreeding season and two breedingseason animals, respectively, were found to have lost one orboth implants. The brains collected from these ewes were notused in the subsequent analyses.

Blood sampling and radioimmunoassayTwo or three days prior to death, blood samples (5 ml) were

collected for 4 hours at 12-minute intervals. Blood was col-lected via jugular venipuncture into heparinized tubes. Tubeswere centrifuged at 300g for 20 minutes, and the plasma wasstored at –20°C until assayed for luteinizing hormone (LH) asdescribed previously (Jansen et al., 1991). Pulse amplitudeand frequency were determined by using the PULSAR algo-rithm as described previously (Jansen and Jackson, 1993b). Gvalues used were G1 2.9, G2 2.0, G3 1.6, G4 1.4, and G5 1.1.

Tissue collectionGroups of ewes were moved indoors for 2–3 days either in

June (N � 8; nonbreeding season) or in December (N � 7;breeding season) and provided food and water ad libitum.Approximately 15 minutes prior to death, each animal re-ceived 25,000 U heparin (iv; Sigma). Animals were killed withpentobarbital and the heads perfused with 4 liters of fixative(4% paraformaldehyde in 0.1 M phosphate-buffered saline,pH 7.4) as previously described (Jansen et al., 2003). Afterperfusion, the brains were removed, the hypothalamus wasblocked (1 cm rostral to optic chiasm, 2–3 mm caudal tomammillary bodies, 2 cm lateral, just dorsal to septal nuclei),and the tissue blocks were placed into fresh fixative for anadditional 24 hours at 4°C. On the following day, brains weretransferred to 25% sucrose (4°C) until they were infiltrated(sunk). Frozen hypothalamic blocks were sectioned at a thick-ness of 55 �m on a sliding microtome (Leica, Bannockburn, IL)equipped with a freezing stage (Physitemp, Clifton, NJ). Sec-tions extending from the preoptic area (POA) to the mammil-lary bodies were collected into six equally spaced series (330�m between sections), and each series was stored in cryo-

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616 A. SERGEEVA AND H.T. JANSEN

preservative (Watson et al., 1986) at –20°C until processed forimmunocytochemistry.

ImmunocytochemistryDetails of the antisera used can be found in Table 1. All

incubation and wash steps were performed on a shaker tableunless otherwise indicated. After removal from cryopreserva-tive solution, the tissue sections were washed at least fourtimes for 5 minutes each in 0.1 M phosphate buffer (PB; pH7.4) containing 0.1% Triton X-100 (TX). Sections were thenincubated in 1% blocking serum in PBTX appropriate for thesecondary antibody used (i.e., donkey or goat; Jackson Im-munoresearch Laboratories, West Grove, PA) for 1 hour. Sec-tions were incubated for 48 hours at 4°C with shaking in acocktail of primary antibodies against GnRH, VGAT, andVGLUT2 in PBTX containing 15% blocking sera. On the thirdday, sections were washed three times in buffer and thenincubated in fluorescent secondary antibodies (diluted 1:200,1 hour at room temperature) appropriate for the specific pri-mary antibody used (see Table 1). After a final series ofwashes, the tissue sections were mounted on SuperFrost-Plus slides (Fisher Scientific, Pittsburgh, PA), dried in the darkovernight, briefly dipped in distilled water, and coverslippedwith ProLong Gold antifade reagent (Invitrogen, Carlsbad,CA). Because of the large amount of tissue processed, immu-nocytochemistry was completed in several runs with an inves-tigator (H.T.J.) not performing the immnocytochemistry as-signing equal numbers of breeding season and anestrousanimals to each run.

Antibody description and immunocytochemistrycontrols

Three primary antibodies were used to process tissue forquantitative analysis. 1) The vesicular GABA transporter(VGAT; dilution 1:50) monoclonal antibody from Synaptic Sys-tems, Gottingen, Germany; catalog No. 131 011) was raisedagainst amino acids 75–87 from the N-terminus of mammalianVGAT. According to manufacturer’s product information, thisantibody recognizes a protein of 50–65 kDa. Preabsorptionwas carried out with 2 ml of antiserum (dilution 1:50) to which100 �g of immunogenic peptide (catalog No. 131 0P, 100 �g;Synaptic Systems) was added and the solution incubatedovernight at 4°C with gentle mixing. On the following day, theantibody plus peptide solution was centrifuged at 10,000g for30 minutes and the supernatant used for tissue incubation asdescribed above. All specific labeling was abolished by prea-sorption with peptide (Supp. Info. Fig. 1A,B). 2) The VGLUT2(Chemicon, Temecula, CA; dilution 1:1,000) guinea pig anti-

body on Western blots of brain extract from sheep recognizesa single band 55–60 kDa corresponding to the expected mo-lecular mass of VGLUT2 (Supp. Info. Fig. 2). Preincubation of2 ml antibody solution (dilution 1:1,000) with 50 �g immuniza-tion peptide (catalog No. AG209; Chemicon International/Millipore, Billerica, MA), as described above, eliminated allimmunostaining of axonal terminals; however, some immuno-reactivity within GnRH somas with this antibody was not com-pletely abolished by preabsorption with the control peptide,so this GnRH soma labeling with the guinea pig anti-VGLUT2was considered to be nonspecific (Supp. Info. Fig. 1C,D).Because we were interested only in identifying VGLUT2 ter-minal appositions onto GnRH neurons (not whether GnRHneurons were glutamatergic), we performed an additionalcontrol experiment with triple-immunofluorescent labelingwith guinea pig anti-VGLUT2 polyclonal antibody (Chemicon),rabbit anti-VGLUT2 polyclonal antibody (Synaptic Systems),and mouse anti-GnRH monoclonal antibody (SternbergerMonoclonals/Covance, Princeton, NJ). In this case, confocalmicroscopy confirmed that both VGLUT2 antibodies exhibitedcomplete overlap of the same terminals, including those con-tacting the GnRH neurons (data not shown). 3) The GnRHantibody LR-5 (generous gift of Dr. Robert Benoit, McGillUniversity) was diluted 1:10,000. Incubation of 1 ml antiserumwith 50 �g GnRH (Sigma-Aldrich) overnight eliminated all celland fiber labeling (Supp. Info. Fig. 1E,F). Finally, experimentsin which one or more primary and/or secondary antibodieswere omitted resulted in no additional effect on the specificityof labeling of the other antibodies (data not shown).

Western blot analysisGuinea pig (Chemicon) and rabbit (Synaptic Systems) anti-

VGLUT2 antibodies (for details see Table 1) were also testedby Western blot analysis, essentially as described previously(Jansen et al., 1997b). Briefly, sheep hypothalamic tissue washomogenized by about 50 strokes of a glass and Teflon ho-mogenizer in a volume of 2 ml ice-cold homogenization buffer(Pierce, Rockford, IL). Samples were centrifuged at 10,000g,and the supernatant (cytosolic fraction) was stored frozen at–80°C. The concentration of proteins in the cytosolic fractionwas estimated with the BCA Protein Assay Kit (Pierce). Equalamounts of homogenate proteins (20 �g) were then separatedby electrophoresis using a Bio-Rad Criterion apparatus (Bio-Rad, Hercules, CA) and SDS-PAGE (12% gel; Bio-Rad). Pro-teins were resolved at 60 mA for 60 minutes and then weretransferred onto nitrocellulose membranes at 150 mA for 1hour. Nonspecific binding sites were blocked by incubation ofthe membranes in 10% low-fat milk and 5% BSA in Tris-

TABLE 1. Details of the Antibodies Used To Examine Seasonal Changes in Glutamatergic and GABAergic Inputs to GnRH Neurons in Sheep

Antibody

Immunogenic sequence(primary antibody) or

fluorochrome(secondary antibody) Host species1 Source Dilution

GnRH GnRH aa 7–10 Rbt polyclonal Dr. Robert Benoit (LR5) 1:10,000GnRH GnRH aa 6–10 Mo monoclonal Sternberger Monoclonals, Inc. (SMI-41) 1:500VGLUT2 VGLUT2 aa 565–585 GP polyclonal Chemicon, Inc. (AB5907) 1:1,000VGLUT2 VGLUT2 aa 510–582 Rbt polyclonal Synaptic Systems, GmbH (135 403) 1:1,000VGAT VGAT aa 75–87 Mo monoclonal Synaptic Systems (131 011) 1:50Rbt IgG Cy3 Dky Jackson Immunoresearch Laboratories, Inc. 1:200Mo IgG Cy2, Alexa 488 Dky Jackson Immunoresearch Laboratories, Inc.; Invitrogen 1:200GP IgG Cy3, Cy5 Gt Jackson Immunoresearch Laboratories, Inc. 1:200

1Dky, donkey; Gt, goat; GP, guinea pig; Mo, mouse; Rbt, rabbit.

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617SEASONAL PLASTICITY IN OVINE GnRH SYSTEM

buffered saline (TBS) for 1 hour at room temperature. Themembranes were then incubated overnight with guinea piganti-VGLUT2 antibody (1:2,000; Chemicon) or rabbit anti-VGLUT2 antibody (1:1,000; Synaptic Systems) diluted in TBScontaining 0.1% Tween-20 (TBS-T). After being washed inTBS-T, the membranes were incubated with horseradish per-oxidase (HRP)-conjugated donkey anti-guinea pig or donkeyanti-rabbit secondary antibodies diluted 1:25,000 (JacksonImmunoresearch) in TBS-T for 1 hour at room temperature.After further washing in TBS-T, the membranes were incu-bated in developer solution (ECL Plus system; GE Healthcare,Piscataway, NJ) for 30 minutes in the dark. Immunoreactivebands were visualized by exposure of the membranes to X-rayfilm for 2 minutes. A single major protein band of approxi-mately 62 kDa corresponding to the expected mass of theVGLUT2 (Morris et al., 2005) was observed with both antibod-ies (Supp. Info. Fig. 2).

Electron microscopyA separate group of animals (n � 3) was perfused during the

breeding season essentially as described above but with 4%paraformaldehyde and 0.5% glutaraldehyde in PB. Tissue waspostfixed overnight in the same fixative and sectioned on avibratome at 55 �m thickness into six series. Sections werestored in cryopreservative as described above until process-ing for dual-label preimbedding immunocytochemistry to vi-sualize GnRH and VGLUT2 immunoreactivity. The dual-labelpreembedding immunocytochemical method of Norgren andLehman (1989) was used, with several minor modifications asdescribed below. Free-floating sections were treated with 0.1M glycine to reduce aldehyde-related background and thenincubated in 0.5% H2O2 to block endogenous peroxidaseactivity. After incubation with 10% normal donkey serum,sections were incubated overnight with rabbit GnRH antibodyand guinea pig VGLUT2 antibody (for antibody details seeTable 1) in 0.1 PB (no Triton X-100) at room temperature.VGLUT2 immunoreactivity was visualized as follows. Sectionswere washed in PB, incubated in biotinylated secondary an-tibody (guinea pig), washed again, then incubated in avidin-HRP complex as described above. Sections were then rinsedin 0.1 M PB with molybdic acid (pH 6.0) three times, followedby incubation in cold tetramethyl benzidine (TMB) solution ona shaker table at 4°C. The TMB solution was prepared imme-diately before use by mixing solution A (0.24 g molybdic acidin 97.5 ml of 0.1 M PB, pH 6) with solution B (0.005 g TMB in2.5 ml of 100% ethanol). Then, 10 �l of 30% H2O2 was addedto the solution, after which the sections were added andexamined every 5 minutes until dark blue labeling appeared.The sections were then removed from the TMB solution andquickly rinsed in 0.05 M Tris buffer (pH 7.4) containing 1.5%NaCl (1.5 T) and placed for a short time in a diaminobenzidine(DAB)-cobalt solution. The DAB-cobalt solution was made bydissolving 0.05 g DAB in 100 ml 1.5 T, adding 200 �l 1% cobaltchloride and 10 �l 30% H2O2 to activate the reaction. Sectionscontaining GnRH-positive neurons were rinsed overnight inPB and then processed again with biotinylated secondaryantibody (rabbit), followed by the avidin-HRP complex. GnRHneurons were visualized by using DAB alone (unenhanced)and the sections washed as described above. An area ap-proximately of 2 mm2 containing individual GnRH neuronswas then trimmed from each section with a dissecting micro-scope, and the tissue fragments were postfixed in 2% osmium

tetroxide with 1.5 mg potassium ferrocyanide for 1 hour. Tis-sue was then dehydrated in alcohol and incubated in a 50%solution of propylene oxide in Epon resin for 1 hour. Finally,tissue blocks were embedded in Epon and cured overnight at60°C. Thin sections were cut from the tissue block, placed onformvar-coated nickel grids, and stained with uranyl acetateand lead citrate. Images were acquired with a JEOL 1200electron microscope and Soft Imaging System software(Olympus, Tokyo, Japan).

Confocal imagingGnRH neurons were identified at �20 magnification under

epifluorescent illumination with a Zeiss Axiovert inverted mi-croscope. Once identified, the imaging modality was switchedto confocal mode on a Zeiss LSM 510 Meta confocal scanhead (Zeiss, Inc., Thornwood, NY). All z-stacks for quantitativeanalysis were obtained with identical confocal settings. Im-ages were captured with a �63/1.4 NA water immersion ob-jective. AlexaFluor 488 and Cy2 were excited with the 488-nmargon laser line at 50% laser power and combined with a505–570 nm BP filter. Alexa 594 and Cy3 were excited with the594-nm HeNe laser line combined with a 560-nm LP filter. Cy5was excited with 633 nm HeNe laser line combined with a575-nm LP filter. All images were captured in the multitrackmode to eliminate bleed-through of fluorescent signals. De-tector gain, amplitude offset, and amplifier gain were normal-ized for each channel on each z-stack to a relative fluores-cence intensity range of 0–255 with a small percentage ofdarkest and brightest pixels (1–2%). Pinhole diameters werefirst set to 1 Airy Disk and then adjusted to match the thick-ness of optical sections for all channels. Eight-bit confocalimages were acquired with a frame size of 1,024 � 1,024pixels and a scan speed of 6 �sec, �1 line averaging, and �1zoom mode. No additional filtering was applied. All serialZ-sections were taken at a thickness of 0.38 �m.

Sampling of neurons for analysisFor each tissue section, the location of every GnRH neuron

(analyzed and not analyzed) was recorded and plotted (Supp.Info. Fig. 3A). By using these data, a cumulative frequencydistribution for analyzed and total numbers of GnRH neuronsin sections from the rostral hypothalamus was produced(Supp. Info. Fig. 3B). A Pearson correlation coefficient for XYcoordinates between the two cumulative frequency lines wasthen computed. The results of the analysis revealed a corre-lation value of 0.99 (P > 0.05 for slope � 1) indicating unity.Based on the finding that the anatomical location of sampledGnRH neurons was similar to the total population of GnRHneurons within the POA, our sampling was deemed random. Incontrast to the rostral population of GnRH neurons, the num-ber of GnRH neurons in the caudal population was muchsmaller, and virtually every GnRH neuron was sampled.

Approximately 15 GnRH neurons were acquired for eachewe (10 rostral, 5 caudal). Anatomical landmarks and locationof each neuron were identified according to our previousmethod (Jansen et al., 1997a). Rostral GnRH neurons werelocated primarily in the vicinity of the POA, whereas the caudalneurons were located predominantly in the medial basal hy-pothalamus (MBH). The rostral population of GnRH neuronsalso was subdivided into medial and lateral groups basedon their distance from the midline and anatomical location.The medial group included neurons located in the medial



portions of the medial preoptic area (MPOAm), MNPO, MS,AVPV, and OVLT; generally, these neurons were locatedapproximately <0.75 mm of the midline. The lateral groupof GnRH neurons was located predominantly in the lateralportions of the MPOAl, LPOA, and SON; these neurons werelocated at a distance approximately >0.75 mm from themidline.

Image analysisImage analysis was performed by an investigator (A.S.)

blind to the seasonal status of the animal from which thesections were obtained. Image stacks were first prepro-cessed to remove excess background by using the Back-ground subtraction plugin (scaling factor 3) of NIH ImageJ.This was followed by automated brightness and contrastadjustment applied separately to each channel prior toanalysis. Next, the soma and dendrites of GnRH neuronswere manually separated (soma identified as within 5 �mfrom the nuclear envelope). The Colocalization plugin ofImageJ was then used to exclude from the analysis anyVGLUT2 and VGAT pixels located within GnRH neurons.Quantification of VGLUT2 or VGAT contacts onto GnRHneurons was performed manually on each optical sectionby using the Point selection tool of ImageJ. Only varicosi-ties that were in direct contact with the GnRH neuron mem-brane and without any interposed (black) pixels were con-sidered bona fide contacts (terminals). Surface area andvolumes of GnRH somas and dendrites were calculated bythe 3D-object counter plugin of ImageJ. The density ofcontacts onto each GnRH neuron was calculated sepa-rately for somas and dendrites as the number contacts perunit of GnRH surface area (�m2). In addition, the density ofVGLUT2 and VGAT terminals in the vicinity of GnRH neu-rons, but not directly apposed to them, was estimated byusing three randomly assigned 10-�m2 boxes each com-prised of 10 z-sections (0.38 �m thickness). For quantifica-tion, the volume of VGAT and VGLUT2 immunoreactivity (invoxels) was determined with the 3D-object counter pluginof ImageJ.

Photomicrographs for publication were generated in Can-vas for the Macintosh (version X.0.2; ACD Systems, Inter-national Inc., Victoria, British Columbia, Canada). Raw tifffiles were imported into Canvas without any further adjust-ments in color quality (tone, hue, or saturation) orbrightness/contrast (brightness and contrast were adjustedpreviously in ImageJ; see above). Images were then resizedand grouped as appropriate, and text was added. Fileswere saved in tiff format without compression at a resolu-tion of 300 dpi or higher.

Statistical analysisMeans � SEM for each parameter were determined for each

ewe, and then the group data were analyzed by t-tests (sea-son) or two-way ANOVAs (season, location, and interactions).Bonferroni post hoc analyses were performed when appropri-ate. Data were analyzed in Prism for the Macintosh (version5.0a; GraphPad Software, Inc., San Diego, CA). Differenceswere considered significant at P < 0.05.

Figure 1.Three-dimensional reconstructions of GnRH neurons obtained from abreeding season ewe (A) and a nonbreeding season ewe (B). SeeSupporting Information Figure 4 for magenta-blue version. Scalebar � 10 �m.

TABLE 2. Reproductive Neuroendocrine Status Based on Plasma LHConcentrations (Mean � SEM) in Ewes Whose Brains Were Collected

During the Nonbreeding Season or the Breeding Season

ParameterBreeding season

(N � 7)Nonbreeding season

(N � 8) P value

LH: Mean (ng/ml) 6.34 � 1.20 1.19 � 0.76 <0.01LH: Pulse amplitude (ng) 7.95 � 1.58 8.30 � 2.11 nsLH: Pulse frequency

(pulses/4 hours) 5.75 � 0.25 2.25 � 0.67 <0.01

TABLE 3. Morphological Characteristics of GnRH Neurons (Mean � SEM)From Ewes in the Nonbreeding Season or Breeding Season

Parameter

Breedingseason(N � 7)

Nonbreedingseason(N � 8) P value

GnRH dendrite volume (�m3)1 701 � 76 561 � 53 nsGnRH soma volume (�m3) 918 � 83 970 � 42 nsNo. of primary branches 2.4 � 0.9 2.0 � 1.1 nsN. of secondary branches 1.5 � 0.6 0.9 � 0.5 <0.05

1Only the first 200 �m (from soma) were examined.



RESULTSEndocrine status

Examination of the plasma LH profiles in breeding seasonand nonbreeding season ewes confirmed seasonal differ-ences in reproductive neuroendocrine activity. Specifically,breeding season ewes exhibited significantly more LH pulsesand greater mean LH levels compared with nonbreeding sea-son ewes; however, pulse amplitude was not significantlydifferent (Table 2).

GnRH neuron morphologyNo seasonal changes were detected for either GnRH soma

or dendrite volumes (Table 3). Most GnRH neurons examinedwere bipolar in both seasons, although most GnRH neuronsexhibited numerous filopodia extending from the soma, ascan be seen in 3D reconstructions of representative GnRHneurons from a breeding season and a nonbreeding seasonewe (Fig. 1). The numbers of primary branches did not differbetween seasons, but the number of secondary branches wassignificantly greater in GnRH neurons of breeding seasoncompared with nonbreeding season ewes (Table 3).

VGLUT2 associationsGnRH neurons. Electron microscopy confirmed that ter-

minals immunoreactive for VGLUT2 made synaptic contactwith GnRH neurons (Fig. 2). Similar to the ultrastructural ob-servations, large numbers of VGLUT2 terminals were ob-served apposed to GnRH neurons and in their vicinity at thelight microscopic level (Fig. 3A,B). At the EM level, manyVGLUT2 terminals associated with GnRH neurons containedasymmetric synapses, whereas others contained both sym-metric and asymmetric synapses (Fig. 2), suggesting thatVGLUT2-ir terminals might also contain other transmitters(see below). Analysis of confocal optical sections revealed asignificant effect of location (F1,22 � 5.87, P < 0.05) onVGLUT2 appositions onto GnRH somas; however, no sea-sonal difference was observed (Fig. 4A). In contrast, two-wayANOVA revealed a significant effect of both season (F1,22 �5.19, P < 0.05) and location (F1,22 � 6.05, P < 0.05) onnumbers of GnRH dendrite contacts, but no interaction be-tween the two was observed (Fig. 4B). Student’s t-test re-vealed that the number of VGLUT2-ir contacts onto GnRHdendrites within the medial population was significantly lowerin neurons from nonbreeding season ewes compared withneurons from breeding season ewes (t11 � 2.61, P < 0.05).

Non-GnRH regions. Analysis of VGLUT2 immunoreactiv-ity in the vicinity of GnRH neurons revealed a significant effectof both season (F1,8 � 18.97, P < 0.01) and location (F1,8 �6.46, P < 0.05) but no interaction. Overall, the density ofVGLUT2 immunoreactivity was significantly greater in breed-ing season compared with nonbreeding season samples(Figs. 4C, 5, upper panels).

Figure 2.A–C: Electron micrographs demonstrating VGLUT2-immunoreactivesynaptic contacts onto GnRH neurons in the sheep brain. Note thepresence of both symmetric and asymmetric synapses and small andlarge vesicles within single VGLUT2 terminals. v, Vesicles; arrows,postsynaptic density; **, unidentified neuron. Scale bar � 0.2 �m.



VGAT associationsGnRH neurons. Similarly to VGLUT2, VGAT-ir terminals

onto GnRH somas and dendrites and in the vicinity of GnRHneurons were also very extensive (Fig. 3C,D). Two-wayANOVA revealed significant effects of season for both den-drite (F1,24 � 13.71, P < 0.01) and soma (F1,24 � 7.1, P < 0.01)contacts. In addition, a significant interaction between seasonand location, but no effect of location alone, was observed(F1,24 � 18.00, P < 0.001). For the medial group of GnRHneurons, the number of VGAT appositions on both GnRHdendrites (t � 5.62, P < 0.001) and somas (t � 3.39, P < 0.05)was greater in nonbreeding season vs. breeding season ewes(Fig. 6A,B). In contrast, no seasonal differences were ob-

served for contacts onto the lateral group of GnRH neurons(Fig. 6A,B).

Non-GnRH regions. Analysis of VGAT-ir in the vicinity ofGnRH neurons revealed a significant effect of season (F1,24 �

5.0, P < 0.05). The direction of change was the same as forappositions onto GnRH neurons (i.e., nonbreeding season >breeding season; Fig. 6C; see also Fig. 5, lower panels).

Caudal GnRH neuronsWithin the caudal population of GnRH neurons, the num-

bers of VGAT-, VGLUT2-ir contacts showed tendenciessimilar to those of the rostral population of GnRH neurons;however, these differences never attained statistical signif-

Figure 3.A–D: Single confocal optical sections demonstrating VGLUT2 (upper panels) and VGAT associations (lower panels) with GnRH neurons (arrows)during the breeding season (left panels) and nonbreeding season (right panels). Asterisk indicates intracellular labeling of identified andunidentified neurons. See Supporting Information Figure 5 for magenta-green version. Scale bar � 10 �m.



icance (data not shown). Medial and lateral distinctionswere not made because of the location and size of the thirdventricle.

Dual-phenotype inputsContacts that colocalized VGLUT2 and VGAT immunore-

activity were also found apposed to GnRH neurons (Fig. 7).

Two-way ANOVA revealed a significant effect of both sea-son (F1,22 � 5.87, P < 0.05) and location (F1,22 � 5.87, P <0.05) on the number of dual-phenotype contacts onto GnRHdendrites (Fig. 8A). The number of dual-phenotype inputsonto GnRH dendrites was significantly greater in nonbreed-ing season vs. breeding season ewes for the medial groupof GnRH neurons (t8 � 3.91, P < 0.01; Fig. 8A). However, noseasonal changes in dual-phenotype inputs onto GnRHsomas or within the lateral population of GnRH neuronswere observed (data not shown). The percentage of dual-labeled VGLUT2 contacts, but not VGAT contacts, changedsignificantly between seasons (Table 4; P < 0.05). No sig-nificant differences between seasons were observed in thedensity of dual-phenotype terminals located in the vicinityof GnRH neurons, although the overall tendency was similarto that observed for VGAT contacts (compare Figs. 8B and6C).

DISCUSSIONMorphology of GnRH neurons

Sheep GnRH neurons, as described previously for this andother species (Campbell et al., 2005; Clarkson and Herbison,2006; Jansen et al., 2003; Witkin and Romero, 1995), exhibiteda predominantly bipolar morphology. However, we observedthat the number of secondary branches (defined as branchesfrom the primary dendrite) varied significantly with season.Based on the sections used, we were able to examine only alimited portion of each GnRH neuron’s entire dendritic tree(extending to a maximum of approximately 200 �m from thesoma). Thus, it is likely that significant portions of each den-drite, which presumably could extend for several millimeters,as shown in biocytin-filled mouse GnRH neurons (Campbell etal., 2005), were not analyzed. Although it is tempting to spec-ulate that a causal relationsip exists between our observedseasonal changes in GnRH inputs and dendritic branchingcombined with evidence that glutamate and its receptors areinvolved in growth of dendritic arbors (Kalb, 1994; Sin et al.,2002), caution is required because our observations can berelated only to changes occurring within the proximal portionsof GnRH dendrites. Nevertheless, observations in the rat thatthe proximal portion of GnRH dendrites contains twice thedensity of spines and GABAergic synapses compared withdistal dendrites (Cottrell et al., 2006) suggest that the activeregions of the GnRH dendrites are located proximally. Further-more, we are unaware of any evidence on the GnRH systemindicating that responses to the same stimulus by proximaldendrites can be neutralized or reversed by modulations oc-curring at more distal sites.

In the current study, changes in dendrite volume had thesame tendency as the number of branches, although with Pvalues above the level of significance (P � 0.1). However,when examined closely, several of the data points were out-side of the 95% confidence range. Reanalysis without thoseoutliers resulted in the differences between seasons reachingsignificance (P � 0.02). The lack of a tight relationship be-tween dendrite volume and branching could be due to limita-tions of the immunocytochemical methods used. For exam-ple, immunocytochemical staining does not always produceentirely precise and smooth labeling of the cell membrane.The method we used for volume calculations was based on

Figure 4.Summary of VGLUT2 associations with GnRH somas (A), dendrites(B), and in the vicinity of GnRH neurons (C) during the breeding andnonbreeding seasons. Different from breeding season at *P < 0.05.



the number of voxels occupied by specific labeling on eachdata set recorded in a series of discrete optical sections, andthe result is therefore only approximate under these condi-tions. Increasing the sample size could help to resolve uncer-tainties, making our conclusions about seasonal changes indendrite volume more definite, and we leave this for futureinvestigations.

Our analysis revealed a strong tendency for GnRH neuronslocated medially to differ from their counterparts in morelateral locations. The existence of distinct subgroups of GnRHneurons and projections had been proposed earlier for the rat(Bennett-Clarke and Joseph, 1982; Hoffman and Gibbs, 1982)and sheep (Jackson et al., 1978; Whisnant and Goodman,1994). In addition, a functional distinction among subgroupshas repeatedly been suggested based on the physiologicalresponses to gonadectomy (Hiatt et al., 1992), neonatal hypo-thyroidism (Jansen et al., 2007), and hypothalamic deafferen-tation (Hoffman and Gibbs, 1982; Jackson et al., 1978). Pre-viously, it was shown in rats that predominantly the medialsubset of GnRH neurons contains N-methyl-D-aspartate(NMDA) receptors (Ottem et al., 2002) and progesterone re-ceptors (King et al., 1995). The importance of a medial sub-population of GnRH neurons for estrogen positive feedback inrodents has also been suggested from studies examining thenumbers of VGAT and dual-phenotype (VGAT/VGLUT) con-

tacts (Ottem et al., 2004), GABA postsynaptic currents (Chris-tian and Moenter, 2007), GnRH gene expression (Porkka-Heiskanen et al., 1994), and Fos expression (Lee et al., 1990).Our findings in the sheep now support an additional role,namely, for the medial subpopulation of GnRH neurons in thePOA during the annual (seasonal) reproductive cycle to me-diate changes in estradiol negative feedback senstivity. How-ever, some caution should be used when interpreting theseresults, insofar as it is clear that from our findings that termi-nals in the vicinity of GnRH neurons also exhibit seasonalvariability. Therefore, the medial preoptic area, including, butnot necessarily limited to the GnRH neurons, may hold manyadditional and important clues regarding the seasonal brain.

VGLUT2 and VGAT contactsGlutamate and GABA are the two major excitatory and

inhibitory neurotransmitters, respectively, in the mammalianbrain (Petroff, 2002; van den Pol and Trombley, 1993). How-ever, the roles they play in mediating estradiol negative feed-back to the GnRH system remain controversial. Glutamate issuggested to be involved in the regulation of seasonalchanges predominantly through NMDA receptors (Ebling etal., 1995b; Ebling and Cronin, 1998). The actions of NMDA aremost profound under conditions of elevated estradiol negativefeedback (Estienne et al., 1990), although central administra-

Figure 5.Volume renderings of VGLUT2-immunoreactive (green, upper) and VGAT-immunoreactive (white, lower) terminals in areas adjacent to GnRHneurons, but not directly associated with them (non-GnRH), during the breeding and nonbreeding seasons. Each box represents the compositeof 10 optical sections within a 10-�m � 10-�m area. Scale bar � �2 �m.



tion of the NMDA antagonist AP5 can suppress LH in ovari-ectomized (OVX) lambs (Hileman et al., 1992). While it is notclear whether glutamate effects occur directly on GnRH neu-rons, numerous studies have confirmed that GnRH neuronsexpress NMDA receptor subunits (Gore et al., 1996; Miller andGore, 2002; Ottem et al., 2002; Purnelle et al., 1997; Simonianand Herbison, 2001; Spergel et al., 1999).

In the context of seasonal plasticity, our results reveal that,regardless of season, the density of VGLUT2 contacts is

slightly (yet significantly) higher in laterally located GnRH neu-rons. This may correspond to the greater concentration ofglutamate receptors found in lateral portions of the hypothal-amus compared with the POA (Meeker et al., 1994). Further-more, we can speculate that this difference reflects greaternumbers of non-NMDA receptors given that NMDA receptorsare expressed predominantly by the medial group of GnRHneurons compared with lateral neurons in the rat (Ottem et al.,2002). Finally, because the changes that we observed oc-

Figure 7.A: Single confocal optical sections demonstrating dual-phenotypeVGAT (red) � VGLUT2 (green) contacts (arrows) onto a GnRH dendrite(blue). B: Orthogonal projection of dendrite shown in A. See Support-ing Information Figure 6 for magenta-green version. Scale bar � 5 �m.

Figure 6.Summary of VGAT associations with GnRH somas (A), dendrites (B),and in the vicinity of GnRH neurons (C) during the breeding andnonbreeding seasons. Different from nonbreeding season at *P < 0.05or ***P < 0.001.



curred in the absence of changes in circulating estradiol con-centrations, we can conclude that they are manifest as theresult of a combination of direct photoperiodic modulationand the expression of an endogenous rhythm of sensitivity toestradiol negative feedback (Karsch et al., 1989). Given thatour ewes were housed under natural photoperiodic condi-tions, we cannot discriminate between these two possibilities.

Whereas glutamate’s effect on GnRH neurons is most likelyexcitatory, GABA influences on GnRH neurons are variable.For example, GABA can produce both excitatory (DeFazio etal., 2002) and inhibitory (Han et al., 2004) effects on GnRHneurons in vitro. In general, however, our findings contributeto the bulk of evidence suggesting that the role of GABA isenhanced during periods of elevated negative feedback(Tomaszewska-Zaremba and Przekop, 2006). Although wealso observed that the density of both VGLUT2 and VGATcontacts in the vicinity of GnRH neurons changed seasonally,their identity remains unknown. Indeed, some of these couldbe estrogen-responsive neurons that are activated duringanestrus (Stefanovic et al., 2000) presynpatically by VGLUT2terminals.

Seasonal comparisons of GnRH neurons in sheep demon-strated in one study that GAD-ir contacts were greater in thenonbreeding season compared with the breeding season(Pompolo et al., 2003). In contrast, we previously showed thatthe number of GAD-ir inputs to GnRH neurons decreased inthe nonbreeding season (Jansen et al., 2003). We offer severalexplanations for the differences between our two studies. Onepossibility is related to the different antibodies used. Specif-ically, the different functional role and subcellular location ofthese two markers might have produced different outcomes.For example, VGAT is localized in presynaptic boutons(Chaudhry et al., 1998; Dumoulin et al., 1999; Takamori et al.,2000), making it a robust and reliable marker for synapticcontacts (Telfeian et al., 2003). In contrast, GAD is more widelydistributed in the brain, and both isoforms (GAD65 and GAD67)are also expressed by some excitatory neurons that do notrelease GABA (Cao et al., 1996; Sloviter et al., 1996; Telfeian etal., 2003). It is well established that GAD65 and GAD67 havedistinct functions and distribution in brain cells. GAD67 isfound primarily in the cytosol, is not associated with synapticvesicles and, under normal conditions and of importance, isnot involved in GABA transmission (Kaufman et al., 1991;Owens and Kriegstein, 2002). GAD65, on the other hand, isdirectly anchored to synaptic vesicles and is preferentiallylocalized in axon terminals (Fenalti et al., 2007; Tian et al.,1999). Only GAD65 is associated with the vesicular pool ofGABA (Erlander et al., 1991; Jin et al., 2003; Petroff, 2002;Soghomonian and Martin, 1998; Solimena et al., 1993), andimmunocytochemical studies have shown that the VGAT im-munoreactivity is paralleled specifically by GAD65 but notGAD67 immnunoreactivity (Chessler et al., 2002). Finally, quan-titative analysis has revealed that VGAT/GAD labeling doesnot overlap completely, and about 60% of VGAT-containingterminals lacked GAD immunoreactivity (Dumoulin et al.,1999). Our own analysis using triple-label immunocytochem-istry for GnRH/VGAT/GAD65/67 led to a similar conclusion andrevealed the presence of a much greater number of VGATcontacts compared with the number of GAD-IR contactsfound in our previous study (Supp. Info. Fig. 7).

An additional complication for the interpretation of ourVGAT results relates to the observation that VGAT can also befound in glycinergic terminals (Burger et al., 1991; Chaudhry etal., 1998; Jonas et al., 1998; Wojcik et al., 2006). Although therole of glycine in GnRH regulation is not yet firmly established,the POA receives glycinergic innervation and contains glycin-ergic neurons (Zeilhofer et al., 2005). Furthermore, all preopticneurons appear to respond to glycine (Karlsson et al., 1997).Glycine injected directly into the MPOA in rats induced pro-lactin release, indicating that glycine may play a physiologicalrole in this brain region (Banzan and Donoso, 1983). Never-theless, where it has been examined, glycine either failed toevoke any response of GnRH neurons (Sullivan et al., 2003) or,as was observed in immature rat GnRH neurons, caused anincrease in GnRH pulse frequency (Bourguignon et al., 1997).

Dual-phenotype contactsOur current working hypothesis is that the balance be-

tween inhibitory and excitatory afferents mediates the sea-sonal changes in GnRH neurosecretory activity, pre-sumably via separate terminals. However, a possible phys-iological role for dual-phenotype afferents is now sup-ported, for example, by the finding that stimulation of glu-

Figure 8.Summary of dual-phenotype (VGLUT2 � VGAT) associations withGnRH dendrites (A) and in the vicinity of GnRH neurons (B) during thebreeding and nonbreeding seasons. Different from nonbreeding sea-son at *P < 0.05.



tamate receptors decreases GABA-mediated currents inGnRH neurons through a presynaptic mechanism (Chu andMoenter, 2005). In the context of our present findings, thecorelease of GABA and Glu from the same terminal may bebeneficial in controlling this seasonal process by conserv-ing metabolic energy and possibly by reducing signalingerror, given that only a single terminal would be involved(Somogyi, 2006). Dual-phenotype GABA/glutamatergic ter-minals exist in many different brain regions (Davanger et al.,1991; Gutierrez and Heinemann, 2006; Kao et al., 2004;Ornung et al., 1998; Somogyi, 2002; Somogyi and Llewellyn-Smith, 2001; Wellman, 2001) and thus may represent arelatively widespread, yet little understood, mechanism forneuronal plasticity, possibly also in a seasonal context.

Although this was not confirmed in the present study,VGLUT and VGAT colocalization in nerve terminals has re-cently been confirmed at the ultrastructural level (Boulland etal., 2009). The amount of VGAT and VGLUT in dual-phenotypeterminals reportedly shifts in an opposite direction in re-sponse to the same treatment (De Gois et al., 2005); thus, dualphenotype terminals could be ideally situated to regulate thebalance between glutamatergic and GABAergic inputs and tomediate steroid negative feedback seasonally. Indeed, theexistence of dual-phenotype VGLUT2/VGAT inputs to GnRHneurons originating in AVPV estrogenic neurons was demon-strated previously (Ottem et al., 2004). Although this paperdescribes the regulation of the GnRH/LH surge (via estradiolpostive feedback), VGAT immunoreactivity decreased beforethe preovulatory surge, whereas VGLUT immunoreactivity in-creased. This complements our observation that the numbersof dual-labeled contacts and VGAT levels in these terminalsare greater during anestrus and make dual-phenotype GABA/glutamate neurons a potential target for steroid negative feed-back. We found that, on average, about 30% of VGAT orVGLUT2 contacts are of dual phenotype, which may be suffi-cient to mediate steroid negative feedback signaling of GnRHneurons, although this remains to be determined. A final ca-veat to our interpretations is that we may be underestimatingthis population greatly, because under different physiologicalconditions the amount of VGAT or VGLUT can be reduced toundetectable levels in dual-phenotype terminals (Boulland etal., 2009). Taken together, although many uncertainties re-main, dual-phenotype afferents may play an important role inregulating the balance of excitatory/inhibitory inputs in theseasonal regulation of GnRH neurons in sheep.

ACKNOWLEDGMENTSWe are grateful to Dr. Abdur Rehman for his excellent tech-

nical assistance and to Mr. David Casebolt at the University of

Idaho Sheep Research Facility for his outstanding husbandry.We are indebted to Dr. Bryan K. Slinker for statistical adviceand to the anonymous reviewers for their thoughtful com-ments and suggestions.

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TABLE 4. Mean (� SEM) Percentage of GnRH Afferents Exhibiting a Dual (VGLUT2 Plus VGAT) Phenotype

Total percentage of contacts that are ofdual phenotype (VGLUT2 � VGAT)

Percentage of VGLUT2 contactscolocalizing VGAT

Percentage of VGAT contactscolocalizing VGLUT2

Breeding season Nonbreeding season Breeding season Nonbreeding season Breeding season Nonbreeding season

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Neuroanatomical plasticity in the gonadotropin-releasing hormone system of the ewe: Seasonal variation in glutamatergic and γ-aminobutyric acidergic afferents