Modification of Gonadotropin Releasing Hormone (GnRH) mRNA Expression in the Retinal-Recipient Thalamus

Modification of Gonadotropin Releasing Hormone (GnRH)mRNA Expression in the Retinal-Recipient Thalamus

Christy M. Foran,* Dean A. Myers,†,1 and Andrew H. Bass*,‡*Section of Neurobiology and Behavior, Cornell University, Ithaca, New York 14853; †Department of Physiology, NewYork State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; and ‡University of CaliforniaBodega Marine Laboratory, Bodega Bay, California 94923

Received August 21, 1996

Although the environmental cues that trigger reproduc-tive behaviors are known for many species, the mecha-nisms through which these signals influence the neuro-chemistry of the brain to produce behavior have beenelusive. In this study, we describe a retinally modulatedsystem of gonadotropin releasing hormone (GnRH) pro-ducing neurons in the thalamus of the plainfin midship-man fish, Porichthys notatus. Previously, we cloned andsequenced the cDNA for prepro-GnRH in midshipman.Here, using in situ hybridization, we localized prepro-GnRH mRNA to the ventrolateral nucleus of the thala-mus, three divisions of the preoptic area, the ganglion ofthe terminal nerve, and the olfactory bulb. Since thethalamus, terminal nerve ganglion, and preoptic areahave been associated with visual functions, we investi-gated the retinal connections in midshipman. In particu-lar, biocytin tract tracing delineated a reciprocal connec-tion between the ventrolateral nucleus of the thalamusand the retina. Retinofugal projections are exclusivelycontralateral. Experimental manipulation of this retinal-thalamic loop through complete optic nerve transectionshows that GnRH mRNA expression in the contralateralventrolateral nucleus may be influenced by the retina.We hypothesize that a reciprocal retinothalamic GnRHcircuit is important in modulating the expression ofseasonal reproductive behaviors. r 1997 Academic Press

Recent studies of gonadotropin releasing hormone(GnRH) focus on the neuroanatomical localization ofdifferent forms of GnRH peptides and mRNA. Mul-tiple forms of the GnRH peptide have been described,and in some cases several forms have been foundwithin a species (review: Grober et al., 1995). Together,these studies imply the existence of multiple GnRHsystems in the brain, each playing a specialized role inthemodification of brain function and behavior (Muske,1992; see below). Despite GnRH localization in severalvertebrate species, the mechanisms through whichidentified GnRH systems are activated by externalcues remain unknown. For example, although a rolefor GnRH in controlling the expression of reproductivebehavior has been documented (Mason et al., 1986), amechanistic linkage between GnRH release and theenvironmental cues that trigger reproduction remainselusive.Neurons in the terminal nerve ganglion (TN) con-

tain GnRH in many vertebrate species, as shown bothby the immunocytochemical localization of the GnRHpeptide and through the use of in situ hybridization todetect GnRH mRNA (reviews: Muske, 1992; Grober etal., 1995). Demski and Northcutt (1982) used horserad-ish peroxidase labeling of the olfactory epithelium toshow that TN neurons link the olfactory epithelium tothe forebrain in goldfish. GnRH-immunoreactive fi-bers have also been identified in the olfactory epithe-lium of eels (Grober et al., 1987). However, whether ornot the TN is involved in the transmission of sensory

1 Present address: Department of Physiology, College of Medicine,University of Oklahoma, Health Science Center, Oklahoma City, OK73190.

General and Comparative Endocrinology 106, 251–264 (1997)Article No. GC976875

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0016-6480/97 $25.00Copyright r 1997 by Academic PressAll rights of reproduction in any form reserved.

signals from the olfactory system remains unknown(see Fujita et al., 1991). In fact, the morphology andphysiology of the TN cells suggest that they mainlyplay a neuromodulatory role through the release ofGnRH throughout the forebrain (Oka and Mat-sushima, 1993).In teleost fishes, GnRH neurons in the preoptic area

(POA) directly innervate the pituitary gland (Oka andIchikawa, 1990). Hence, it is assumed that changes inthe activity of GnRH neurons in the POAdirectly affectthe release of gonadotropins from the pituitary. Fewstudies in fishes have explored either the sensitivity ofthese neurons to sensory signals or the pathways thatare involved in the relay of sensory signals to the POA(Davis and Fernald, 1990; Amano et al., 1995). Al-though environmental cues are known to be essentialto the timing of reproduction in many species, it isunclear how those cues influence brain areas that areknown to be involved in the production of reproduc-tive behavior.We previously used a monoclonal antibody to the

conserved region of the GnRH decapeptide to demon-strate GnRH peptide expression in the TN and POAofplainfin midshipman, Porichthys notatus, a marine te-leost which reproduces seasonally along the intertidalzone of the Pacific coast (Grober et al., 1994; Bass, 1996).In order to study GnRHmRNAexpression in midship-man, the cDNA for the salmon form of GnRH (aminoacid sequence: pGluHisTrpSerTyrGlyTrpLeuProGly-NH2) was cloned and sequenced and is the only formfound in this species (Grober et al., 1995). Radiolabeled(33P) oligonucleotide probes complementary to theknown GnRH cDNA sequence were then synthesizedand hybridization signals were identified over neuronsin the TN and the anterior parvocellular division of thePOA (Grober et al., 1995).This study had three main goals. The first goal was

to delineate the entire position and extent of GnRHmRNA localization in midshipman forebrain. Besidesthe TN and anterior POA, mRNAtranscripts have nowbeen identified in the olfactory bulb, magnocellularand suprachiasmatic nuclei of the POA, and theventrolateral nucleus of the thalamus. A midbrainpopulation of GnRH mRNA-containing cells was notdetected.A second goal of this study was to identify possible

retinal influence on forebrain GnRH mRNA expres-sion. In particular, we first identified a reciprocalconnection between the ventrolateral nucleus of thethalamus and the retina using biocytin tract-tracingmethods. We then used unilateral optic nerve transec-tions and tetrodotoxin injections into a single eye tofurther investigate the possible role of the retina inmodifying levels of GnRH mRNA expression in theretinal-recipient thalamus.Midshipman have been the subject of a number of

neurobiological studies because they have two adultmale reproductive morphs with alternative matingtactics and distinct, nonoverlapping developmentaltrajectories (Brantley and Bass, 1994; Bass et al., 1996;Bass, 1996). Type I, or parental, males are larger,mature later, excavate nests under rocks, produce amate call to attract females, and care for the eggslaid by females in their nest. Type II, or sneaker, malesare small, early maturing males that hide in or lieoutside the nests of Type I males and shed their spermwhen a female comes to mate with the residentmale. Hence, a third goal of this study was to identifyany morph-specific patterns of GnRH mRNA expres-sion; the only difference noted was an absence ofGnRH transcripts in the magnocellular POA in Type IImales.Portions of these results have appeared earlier (Fo-

ran et al., 1993, 1994; Bass and Foran, 1995).

METHODS

Localization of Prepro-GnRH in Adult Midshipman

This portion of the study includes the results from insitu hybridization in 33 adult plainfin midshipmancategorized on the basis of gonadal morphology andsize, sonic muscle size, and coloration as Type I orparental males, Type II or sneaker males, or females(Bass and Marchaterre, 1989): 13 Type I males (stan-dard length (STL) from 11.8 to 16.0 cm, body weight(WT) from 18.1 to 39.0 g); 15 females (STL 9.3–15.2 cm,WT 9.1–48.6 g); 5 Type II males (STL 8.5–11.0 cm, WT9.1–15.1 g).

252 Foran, Myers, and Bass

Copyright r 1997 by Academic PressAll rights of reproduction in any form reserved.

All fish were deeply anesthetized with MS 222(tricaine methansulfonate, Sigma Chemical, St. Louis,MO), perfused transcardially withmarine teleost Ring-er’s solution until blood was cleared, followed by50–75 ml of 4% paraformaldehyde in 0.1M phosphatebuffer (PB). Brains were removed from the skull,postfixed for 1 hr in 4% paraformaldehyde in PB, andthen stored in PB. Prior to sectioning, brains were sunkovernight in 30% sucrose–PB for cryoprotection. Allbrains were mounted in Cryo-M-Bed (Bright Instru-ments, Huntingdon, England), sectioned frozen in thetransverse plane at 30 µm on a cryostat, and mountedon chrom–alum-coated slides or Superfrost Plus slides(Erie Scientific, Portsmouth, NH).

In Situ Hybridization (ISH)

The procedure for ISH was adopted from Grober etal. (1995). Briefly, hybridization was performed using acocktail of two oligonucleotide probes (40-mers) withsimilar G/C content, antisense to nucleotides 112–152(spanning the decapeptide coding region) and 169–209(spanning a portion of the GnRH-associated peptide)of midshipman prepro-GnRH (see Grober et al., 1995).Oligonucleotides were labeled with a terminal deoxy-nucleotidyl transferase reaction using [a-33P]d-ATP(SA, 1000–3000 Ci/mmole; New England Nuclear,Dupont, Boston, MA).Hybridization was carried out by placing 300 µl of

hybridization solution [43 SSC (13 SSC 5 0.15 Msodium chloride; 0.015M sodium citrate, pH 7.2), 40%deionized formamide, 10% (wt/vol) dextran sulfate,500 µg/ml of denatured calf thymus DNA, 250 µg/mltransfer RNA, 43 Denhardt’s (13 Denhardt’s 5 1%BSA, Ficoll, and polyvinylpyrolidone), 4 mM EDTA,0.1% pyrophosphate, 1.54 mg/ml dithiothreitol, and3 3 106 cpm 33P-radiolabeled GnRH probe] on eachslide. Slides were coverslipped with parafilm andincubated overnight (at least 15 hr) in a 37°, humidifiedchamber. Following hybridization, slides were twicewashed briefly in 13 SSC at 23°, twice washed for 30min in 13 SSC at 55°, washed for 30 min in 13 SSC,and 0.1% Triton X-100 at 23°, immersed in water,immersed in 70% ethanol, and air dried.Specificity of hybridization was determined both by

the elimination of mRNA signal with RNase [RNase-A

(10 µg/ml) and RNase-T1 (1 µg/ml)] pretreatment ofthe tissue and with competition using a 1000-foldmolar excess of denatured midshipman GnRH cDNAadded to the hybridization solution.Slides were exposed to X-Omat AR film (Eastman

Kodak, Rochester, NY) overnight (at least 10 hr) at220°. If a hybridization signal was detected, all slideswere coated in nuclear emulsion (NTB 2, Kodak) andexposed for 10 or 28 days. Slides were developed inKodak D-19 (4 min at 14°), rinsed in distilled water (10sec at 14°), fixed (Kodak GBX Fixer; 5 min at 14°), andrinsed in distilled water (5 min). Sections were thendehydrated, counterstained in cresyl violet, and cover-slipped with Permount.

Optic Nerve Transection

Complete unilateral optic nerve transection wasperformed on four animals (two females and two TypeI males; STL 12.3–14.5 cm, WT 22.4–40.3 g). Threeanimals were used as unoperated controls (two fe-males and one Type I male: STL 10.5–13.5 cm, WT14.8–28.4 g). To perform the transection, animals wereanesthetized with MS 222, and the optic nerve wasexposed surgically and bisected just behind the eye.The wound was then sutured shut and sealed withVetbond (3M, St. Paul, MN). Five days after thesurgery, the animals were again anesthetized inMS 222and perfused, and the brains processed for ISH asdescribed above.

TTX Injections

Seven animals received tetrodotoxin (TTX) injec-tions into one eye (one female: STL 11.6 cm, WT 21.8 g;two Type II males: STL 8.8–9.8 cm,WT 12.0–13.2 g; fourType I males: STL 10.9–12.7 cm, WT 18.6–29.7 g).Animals receiving injections were anesthetized withMS 222, the medial half of the eye was exposedsurgically, and the injection was made with a 10-µlHamilton syringe into the medial portion of the eye. A2-µl injection, containing 0.1 µg of TTX, was initiallygiven. The wound was then sutured shut and sealedwith Vetbond. After 2.5 days, the medial portion of theeye was again exposed surgically, an identical injection

GnRH Expression in the Thalamus 253


was given, and the wound was resealed. Studies ofother teleosts using TTX injections have determinedthat a comparable protocol can lead to a block of actionpotentials in the optic nerve (Schmidt et al., 1983; alsoEdwards and Grafstein, 1983). Five days after the

initial injection, the animals were again anesthetized inMS 222 and perfused, and the brains processed for ISH.One animal was considered a surgical control. The

eyeof thisanimalwasexposedsurgicallyandanunsuccess-ful attemptwasmade to penetrate it with the syringe.

FIG. 1. Shown are transverse, Nissl-stained sections (A–D) through the preoptic area. The preoptic area includes the anterior parvocellular(PPa), magnocellular (PM) [which includes gigantocellular (g) and magnocellular (m) regions], and posterior parvocellular (PPp) divisionsalong with the diffusely organized suprachiasmatic nucleus (SCN). Also shown (D) is the ventrolateral nucleus of the thalamus (VL). Otherabbreviations: AC, anterior commissure; D, area dorsalis of the telencephalon; V, area ventralis of the telencephalon; HoCo, horizontalcommissure; OpTr, optic tract; VP, ventral posterior nucleus of the telencephalon. Scale bar represents 500 µm.



Quantification of ISH

The silver grains over the retinal-recipient ventrolat-eral nucleus of the thalamus (VL), indicating thepresence of prepro-GnRHmRNA, were counted in thebrains of all experimental animals. Grain counting wasaccomplished with the assistance of Cue-2 ImagingSoftware (Olympus). Images (403) from each brainsection containing VL were acquired by a computer(VGA Plus frame grabbing card) using a video attach-ment (CUECCD video camera) to amicroscope (Olym-pus BH2). Using this software, section images wereenhanced to clarify grains out of the plane of focus.Grain densities were counted manually in three areas:a 14,000-µm2 area containing VL on each side of thebrain and a third 14,000-µm2 area, just below theventral extent of VL on one side of the brain, to assessbackground levels. As shown, each retina projects onlycontralaterally to the thalamus. Thus, for all sectionscontaining VL, the number of grains minus back-ground label was calculated for each side of the brain.Then, the counts above background for each side of thebrain were summed across all sections in the braincontaining VL, and the ratio of ipsilateral (unmanipu-lated) to contralateral (nerve transected or eye in-jected) grain total was calculated.

Tract Tracing

Juvenile and adult midshipmanwere used for biocy-tin tract tracing (methods after Bass et al., 1994).Animals were anesthetized in MS 222, and the medialhalf of the eye was surgically exposed. The optic nervewas cut, and a crystal of biocytin (Sigma Chemical, St.Louis, MO), or dextran biotin (3KD, Molecular Probes,Eugene, OR), was placed on the distal end. After thecrystal dissolved, the eye was sutured shut and sealedwith Vetbond, and the animal was allowed to survive

for 5 to 7 days. Animals were then perfused and thetissue treated as in the ISH procedure described above,with one addition: the fixative also included 1% glutar-aldehyde. Sections were cut frozen at 50 µm on asliding microtome and stored in phosphate-bufferedsaline (PBS) at 4° for 1–5 hr. The following procedurewas used for the detection of biocytin and dextranbiotin: (1) 30min incubation in 0.4% Triton X in PBS, (2)3 hr incubation in avidin:biotinylated horseradishperoxidase complex (Elite kit, Vector Laboratories,Burlingame, CA), (3) two 10-min washes in 0.1 Mphosphate buffer, (4) 1–2 min incubation in 0.05%diaminobenzidine and 0.01% hydrogen peroxide in 0.1M phosphate buffer, and (5) two 10-min washes in 0.1M phosphate buffer. Sections were stored in phosphatebuffer until they were mounted on chrom–alum slides.Slides were dried overnight in a 37° incubator, stainedin cresyl violet, dehydrated in a series of alcohols, andcoverslipped.

RESULTS

Cytoarchitecture of the Diencephalon

For comparison with other species, we include herea brief description of the preoptic area (POA) and theventrolateral nucleus of the thalamus (VL) of midship-man (Fig. 1). The description generally follows that ofBraford and Northcutt (1983) for goldfish and Striedter(1990 a,b) for catfish.The POA contains four major divisions, the anterior

parvocellular preoptic nucleus (PPa), the magnocellu-lar preoptic nucleus (PM), the posterior parvocellularpreoptic nucleus (PPp), and the suprachiasmaticnucleus (SCN) (Fig. 1). The POA begins at the level ofthe anterior commisure (AC, Fig. 1A) and extends

FIG. 2. (A) A transverse, Nissl-stained section shows the olfactory bulbs (OB) and the optic tract (OpTr). Scale bar represents 1 mm. (B)Autoradiographic grains located over a GnRH synthesizing neuron at the base of the olfactory bulb. Scale bar represents 10 µm.FIG. 3. (A) Autoradiographic grains located over the terminal nerve ganglion (TN) indicating the location of GnRH mRNA. Scale barrepresents 10 µm. (A8) Shown in a Nissl-stained, transverse section is the location of TN at the base of the telencephalon. (B) Biocytin tract tracingfrom the optic nerve retrogradely labels cells of the TN. Scale bar represents 20 µm. (C, D) Autoradiographic grains indicating the expression ofGnRH in the preoptic area (POA). Cells containing low levels of GnRH in the anterior preoptic (PPa) division, just lateral to the ventricle (C), andnear the ventricle in the magnocellular division (PMm) of the POA (D). Scale bars represent 50 µm. Other abbreviations: D, area dorsalis of thetelencephalon; V, area ventralis of the telencephalon; v, ventricle.





caudally along the ventricular wall of the diencepha-lon. The PPa begins ventral to the anterior commisureand extends caudally to the posterior pole of thetelencephalon. PPa contains small neurons clusteredalong the ventricle with cells sparsely distributed inthe lateral neuropil. The PM nucleus, which containsmagno- and gigantocellular neurons (PMm, PMg; Figs.1B–1D), begins ventral to PPa. The magnocellularneurons in PM line the ventricle and extend furtherlaterally into the neuropil than the gigantocellularneurons, which are confined to the dorsal and caudalregion of the nucleus. As PM extends caudally, thesmaller cells of PPp appear ventral to the nucleus (Fig.1C). The cells of PPp occur along the ventricle and theborder of the optic tract. The SCN is found ventral tothe caudal pole of PPp (Fig. 1D). The scattered, largerneurons of the SCN occur laterally along the border of,and into, the optic tract.VL (Fig. 1D) begins at the level of the horizontal

commisure (HoCo, Fig. 1D). The cells of VL are slightlylarger and more clustered than those of surroundingnuclei. Just caudal to the beginning of VL, a cluster oflarger cells form a ventral extension from the nucleus(see below). VL extends to the level of the habenula.

In Situ Hybridization (ISH)

GnRH mRNA was localized to several areas of theforebrain. Although the extent of labeling varied witheach area, label was always concentrated over somata.GnRH mRNA was detected in the olfactory bulb, TN,POA, and ventrolateral nucleus of the thalamus (VL).Both controls verified the specificity of hybridization(see Methods).The most rostral cells expressing GnRH mRNA in

the forebrain are contained in the olfactory bulb (Fig.2A). GnRH mRNA was localized along the ventral,outer margin of the olfactory bulb, and cells weretypically densely labeled (Fig. 2B). Although there isindividual variation in expression in this area, labeledcells were frequently found. Of the 33 midshipman

included in this study, all but 8 from across all morphscontained labeled cells in the olfactory bulb.The TN is a sphere of cells located ventrally at the

transition between the olfactory bulb and area ventra-lis of the telencephalon (Fig. 3A8; also see Grober et al.,1994, for cellular detail). The cells of the TN ganglionwere found to express GnRH mRNA consistently andbilaterally in all animals. Label over the terminal nerveganglion was always dense and concentrated over thecytoplasm of the cells in all animals (Fig. 3A).Within the POA, three nuclei contained GnRH

mRNA: the PPa, the magnocellular division of the PM,and the SCN (Figs. 3 and 4). Label was not consistentlylocated bilaterally in any of the POA regions.In PPa, GnRH mRNA expression varied among

individuals from no label to several lightly labeledcells (Fig. 3C). Sixteen of 33 individuals (48%) con-tained label in the PPa, with nearly equal distributionamong the three reproductive morphs (Type I andType II males and females).Cells in PMm were typically more robustly labeled

than those in PPa (Fig. 3D). The only morph-specificdifference in the location of GnRH mRNA expressionfor any brain region was in PMm. Although 11 of 28(39%) of the brains of Type I males and femalescontained detectable GnRHmRNA in PMm, no detect-able label was seen in Type II males.Some cells in the diffusely organized SCN express

low, but clearly above background, levels of GnRHmRNA (Fig. 4B). GnRH mRNA was detected in theSCN in 23 of 33 individuals (70%) in nearly equaldistribution across all morphs.Prepro-GnRH mRNA expression in VL was densely

clustered over both the dorsal cells of the nucleus andthe larger cells found in its ventral extension (Figs. 5Cand 5D). Expression was consistently found bilaterallyand in all animals, regardless of morph.In sum, GnRH mRNA expression was observed in

several forebrain nuclei but was consistently foundbilaterally in all individuals in only the TN and VL.

FIG. 4. (A) Labeling the optic nerve with biocytin reveals terminals, dark biocytin-filled punctate endings (e.g., arrows), in the suprachiamaticdivision of the preoptic area (SCN). The ventricle (v) can be seen on the left. (B) Low levels of GnRH mRNA are detected in the SCN. Grains,clearly above the background label, are seen clustered over cells (arrows). The optic tract runs below the row of dark Nissl-stained cells and theventricle is to the left. Scale bars represent 50 µm.



Retinal Tract Tracing

Biocytin label from the optic nerve experimentsdelineated retinofugal and retinopetal nuclei.All biocy-tin-labeled fibers, terminal fields, and filled somatawere contralateral to the labeled optic nerve except inthe TN, where filled cells were found bilaterally, and inthe SCN, where terminals were found bilaterally.Retinal-recipient nuclei are found within the ventraland dorsal thalamus and several divisions of the POA,as has been reported in other teleosts (Northcutt andButler, 1993, and references within). Only those regionswhich exhibit GnRH mRNA transcripts are described.Retrogradely filled neurons mainly were located in

TN and in the larger neurons in the ventral extensionof VL (Figs. 3B, 5A, and 5B). Isolated biocytin-filledcells were also located in the telencephalon just lateralto the anterior commisure (at the level of Fig. 1A). Thepopulations of retinopetal neurons in TN and thetelencephalon were not continuous with those in VL.Scattered, retrogradely filled neurons are not seen inthe rest of the thalamus as has been described in otherteleosts (Northcutt and Butler, 1991). In TN and theventral extension of VL, biocytin-filled neurons occurin the same location as GnRH mRNA (Figs. 3 and 5).Among those nuclei expressing GnRHmRNA, a denseterminal field was only found over VL (Figs. 5A and5B). Only sparse fibers and terminals were seen in thePPa and SCN (Fig. 4A).In sum, VL was both the densest source and target

of, respectively, retinal afferents and efferents amongthe nuclei expressing GnRH mRNA. Because of itsreciprocal and exclusively contralateral connectionswith the retina and its consistent expression of mRNA,we decided to quanitatively assess the potential influ-ence of the retina on GnRHmRNAexpression in VL.

Optic Nerve Transection

Optic nerve transections had a significant effect onthe expression of GnRH mRNA in VL. In comparing

the amount of GnRHmRNA(number of grains locatedin VL) in the two halves of the brain from nonsurgicalcontrol animals (n 5 3), the amount of mRNA wasvery similar; the mean ratio of grain counts in the twohalves of the brain (with the higher side as thenumerator of the ratio) was 1.066. By contrast, in thebrains from experimental animals, the mean ratio ofipsilateral (unmanipulated) to contralateral (nerve tran-sected) grain counts in animals that received opticnerve transection was 1.584 (n 5 4). The ratio in con-trol animals is significantly lower than that in experi-mental animals (Mann–Whitney U; U 5 0, P 5 0.034),indicating lower expression of GnRH mRNA in thathalf of the brain without direct retinal input (Fig. 6).Additionally, we note that other label continued to

be seen in experimental animals. Label was seen in thePPa of all three control animals and two of fourexperimental animals. Although the SCN containedGnRHmRNA in one of the three control animals, noneof the experimental animals had obvious label in theSCN. All experimental animals continued to showrobust label in the TN, which projects to the retina. Onecaveat to these results is that we did not performsham-operated surgical controls.

TTX Injections

The mean ratio of ipsilateral (unmanipulated) tocontralateral (nerve-transected) grain counts in ani-mals that received TTX injections into one eye was notstatistically different from that of control animals(Mann–Whitney U; U 5 10, P 5 0.45) (Fig. 6). How-ever, TTX treatment did induce a significant differencein the variance of the ratio of grains between thecontrol and the treated groups (F test, n 5 4, 7, F 5 131,P , 0.001).The grain ratio was 0.936 for the one animal that was

operated on and an unsuccessful attempt was made toinject its eye with TTX; this ratio is within the normalvariation of other control animals. We considered this

FIG. 5. Several views of the ventrolateral nucleus of the thalamus (VL) are shown. Following labeling of the optic nerve with biocytin (A, B),filled cells and terminals are found in VL. (A) The ventral extension of VL contains dark, biocytin-filled neurons that project to the retina. Thebiocytin-filled fibers of the optic tract (OT) can be seen lateral to the cells. (B) Many biocytin-filled terminals, dark punctate endings, can be seenin VL (e.g., arrows). (C, D) GnRH in situ hybridization detects mRNA in VL. (C) VLwith its ventral extension is located just below the optic tract(OT). (D) Higher power photograph shows silver grains concentrated over the cells in VL. Other abbreviations: Tel, telencephalon. Scale barsrepresent 50 µm.




animal as serving as a surgical control for both the TTXand transection experiments, although we realize thelimitation in using a single animal in this controlgroup.Additionally, two of the seven TTX-treated animals

showed label in the SCN but not PPa or PMm. Alltreated animals showed robust TN label.

DISCUSSION

Function of GnRH in the Terminal Nerve andPreoptic Area

GnRH-containing neurons of the TN in teleosts havea variable pattern of organization ranging from a

dense sphere of cells, as in midshipman (this study,Grober et al., 1994), to cell clusters extending from theolfactory bulb–telencephalon border to the base of theolfactory epithelium, as in salmonids (e.g., Nevitt et al.,1995). The salmon form of GnRH mRNA expressingneurons described here for the olfactory bulb maysimply represent a rostral extension of the TN. Intracel-lular staining and recording studies in gouramis showthat individual TN neurons can have an extensivearborization throughout the forebrain (Oka and Mat-sushima, 1993). The morphology and oscillatory-likefiring properties of TN neurons in gouramis suggest itplays a neuromodulatory role by releasing GnRHthroughout the forebrain.Cells of the preoptic area directly innervate the

pituitary gland in teleost fishes (for review of thestructure and innervation of the pituitary gland, seePeter and Fryer, 1983). Therefore, the output of thesecells translates into changes in the activity of gonado-tropes. Since the preoptic area directly influences thefunction of the pituitary, variation in the GnRH-preoptic phenotype has been suggested to contributeto the proximate mechanisms underlying individualvariance in reproductive behavior. For example,changes in POA GnRH-ir cell number and/or sizehave been implicated in the social mediation of adultreproductive behavior in cichlids (Davis and Fernald,1990), sex and role change in blueheadwrasses (Groberand Bass, 1991; Grober, Jackson and Bass, 1991), andalternative mating strategies in midshipman (Grober etal., 1994) and platyfish (Halpern-Sebold et al., 1986).However, the lack of any robust morph-specific differ-ences in GnRHmRNAexpression in midshipman mayreflect the similar reproductive states of all three adultmorphs, namely, sexual maturity.

GnRH Expression in the Suprachiasmatic Nucleusand Ventrolateral Nucleus of the Thalamus

We hypothesize that the levels of expression of thesalmon-form of GnRHmRNAby neurons in ventrolat-eral and suprachiasmatic nuclei may be modulated bysignals received directly from the retina. Since VLconsistently expresses GnRHmRNAin all animals andhas strictly contralateral, reciprocal connections to theretina in midshipman, we were able to compare theexpression of GnRH in VL across the two halves of thebrain in animals with manipulations done to one eye.

FIG. 6. To determine the influence of retinal signals on GnRHexpression in the thalamus several animals received either complete,unilateral optic nerve transections or TTX injections into one eye.The ratio of grains counted on the unmanipulated side of the brain tothe number on the manipulated side is shown for each animal. Themean ratio is significantly different for the control group and thegroup with the optic nerve transections (Mann–Whitney U test,U 5 0, P 5 0.034). There is no significant difference in the meanratios of the control group and the TTX-treated group. However, theTTX treatment does produce a significant increase in the variance ofthe ratio (F test, F 5 131, P , 0.001). The asterisk indicates a surgicalcontrol. The arrow (,) indicates two overlapping data points.



Results from in situ hybridization for GnRH on ani-mals with unilateral optic nerve transection or tetrodo-toxin injections suggest that expression in this areacould be modulated by the retina.Changes in GnRHmRNAexpressionmay arise from

either severing the axons of retinopetal GnRH neuronsand/or loss of retinofugal input to GnRH neurons.Studies of the effects of axotomy on mRNA expressionhave mainly been done in peripheral neurons ofprimary sensory ganglia (Hokfelt et al., 1994). Theresults suggest that while the production of mRNA ofsome neurotransmitters decreases, the synthesis ofothers increases. Although it is difficult to specificallydefine the impact of axotomy of retinopetal or retinofu-gal neurons on GnRH synthesis in the thalamus, TTXeye injections suggest a definite retinofugal contribu-tion. TTX injections would likely have their greatesteffect on GnRH thalamic neurons via an inhibition ofthe activity of retinal ganglion cell axons (Schmidt etal., 1983). Although TTX injections did not induce asignificant change in the mean GnRH mRNA expres-sion, they did produce significant variation in the levelof expression.The GnRH peptide has been located in the TN and

POA of many species (review: Grober et al., 1995). Toour knowledge there have been no other reports ofGnRH in the SCN. However, if the SCN is a diffusecollection of neurons, as it is in midshipman, localiza-tion of GnRH-containing neurons to this region of thePOAmay be difficult in some species. GnRH (LHRH)-immunoreactive neurons have been found in thalamicnuclei in the brain of a stickleback fish (Borg et al.,1982).There have been no reports, other than the present

one, of GnRH mRNA localization to either the thala-mus or SCN. However, GnRH mRNA has only beenlocalized in a few species (midshipman, this study;masu salmon, Suzuki et al., 1992; atlantic salmon,Bailhache et al., 1994; cichlid, White et al., 1995; sea-bream, Gothilf et al., 1996), and some studies of GnRHexpression use a nonradioactive detection methodwhich may not be as sensitive as the method describedhere. Perhaps the concentration of mRNA in theseareas are low enough that autoradiographic detectionis necessary to visualize the signal.Our previous immunocytochemical study in mid-

shipman reported GnRH-containing neurons in the

TN and POA (Grober et al., 1994). A reexamination ofthat material identified GnRH-immunoreactive neu-rons in VL, but not the SCN (C. Foran and A. Bass,unpublished observations).Arecent study of neuropep-tide Y expression in goldfish (Vecino, Perez, andEkstrom, 1994) localized mRNA in areas where thepeptide had not been localized by immunocytochemis-try, demonstrating the increased sensitivity of in situhybridization in this system. In some neuropeptidergicsystems with high secretory rates and low biosyntheticrates, peptide may only be detectable after blockingtransport with colchicine (Alonso et al., 1986). How-ever, in the absence of immunocytochemical localiza-tion of the GnRH peptide in these areas, we cannot becertain that mRNA is always processed into GnRH.Alternatively, the mRNA might be processed intoother bioactive peptides contained within the prepro-GnRHmRNA.We report that GnRH mRNA expression is associ-

ated with neuronal somata. An alternative hypothesisis that GnRH mRNA is localized to the terminals ofneurons projecting, for example, to the thalamus. Thisseems unlikely; a recent review of mRNA localizationconcludes that mRNA encoding neurotransmitter isprimarily found in somata and dendrites (Steward andBanker, 1992).In sum, the consistent localization of GnRH mRNA

in the ventrolateral nucleus presents a unique opportu-nity to assess the expression of a behaviorally relevantneurochemical in response to the direct influence ofsensory signals, in this case retinal, received by ananimal. Visual inputsmay also influence GnRH expres-sion in mammals, although via a more indirect path-way. In hamsters, projections from the SCN contactGnRH-ir neurons in the hypothalamus and POA (de laIglesia et al., 1995). Therefore, GnRH expression inthese regions may be indirectly influenced by retinalsignals through changes in the activity of the SCN.

Reproductive Significance of Retinal Influence onGnRH Expression

Changes in environmental light cues may be impor-tant in the timing of reproduction for midshipman andother teleosts. The influence of the visual system onGnRH mRNA expression is consistent with recentstudies in masu salmon (Amano et al., 1995). Gonadalmaturation in masu salmon can be accelerated by



shortening the light cycle to which animals are ex-posed. Correlated with accelerated gonadal develop-ment seen under shortened light cycles is an increasein the number of cells in the POA and ventral telen-cephalon expressing GnRHmRNA.Traditionally, GnRH function has been interpreted

in the context of regulation of the hypothalamic–pituitary–gonadal axis. Since the ventrolateral nucleusforms a reciprocal connection with the retina, environ-mental light may influence GnRH mRNA productionin the ventrolateral nucleus which, in turn, may effectGnRH modulation of retinal activity (also see Demski,1991). Hence, GnRH has the potential to modify retinalfunction through input from the neurons of the ventro-lateral nucleus. In fact, GnRH has been shown to play arole in the dark adaptation of the retina in goldfish(Behrens et al., 1993, and references within). The resultsof this study support the hypothesis that expression ofthe GnRH gene by central neurons, usually associatedwith neuroendocrine regulation of pituitary function,is also likely to be involved in the mediation of visualsensory events directing seasonal reproductive behav-iors.

ACKNOWLEDGMENTS

The authors acknowledge the experimental assistance of MargaretMarchaterre and Robert Baker for suggesting the TTX experiment.This study was supported in part by NIMH Training Grant Fellow-ship 5T32MH15793 to C.M.F. and by NSF Grants IBN9021563 and9421319 and New York State Hatch Grant NYC191423 to A.H.B.

REFERENCES

Alonso, G., Szafarczyk, A., andAssenmacher, I. (1986). Immunoreac-tivity of hypothalamo-neurohypophysial neurons which secretecorticotropin-releasing hormone and vasopressin immunocyto-chemical evidence for a correlation with their functional state incolchicine-treated rats. Exp. Brain Res. 61, 497–505.

Amano, M., Hyodo, S., Kitamura, S., Ikuta, K., Suzuki, Y., Urano, A.,and Aida, K. (1995). Short photoperiod accelerates preoptic andventral telencephalic salmon GnRH synthesis and precociousmaturation in underyearling male masu salmon. Gen. Comp.Endocrinol. 99, 22–27.

Bailhache, T., Arazam, A., Klungland, H., Alestrom, P., Breton, B.,and Jego, P. (1994). Localization of salmon gonadotropin releasing

hormone mRNA and peptide in the brain of Atlantic salmon andrainbow trout. J. Comp. Neurol. 347, 444–454.

Bass, A. H. (1996). Shaping brain sexuality. Am. Sci. 84, 352–363.Bass, A. H., and Foran, C. M. (1994). GnRH mRNA expression in theforebrain of a teleost fishwith alternativemorphs.Aquaculture 135,73–74.

Bass, A. H., Horvath, B. J., and Brothers, E. B. (1996). Non-sequentialdevelopmental trajectories lead to dimorphic vocal circuitry formales with alternative reproductive tactics. J. Neurobiol., 30,493–504.

Bass, A. H., and Marchaterre, M. A. (1989). Sound-generating (sonic)motor system in the teleost fish (Porichthys notatus): sexual polymor-phisms in the ultrastructure of myofibrils. J. Comp. Neurol. 286,141–153.

Bass, A. H., Marchaterre, M. A., and Baker, R. (1994). Vocal-acousticpathways in a teleost fish. J. Neurosci. 14, 4025–4039.

Behrens, U. D., Douglas, R. H., and Wagner, H. J. (1993) Gonadotro-pin-releasing hormone, a neuropeptide of efferent projections tothe teleost retina induces light-adaptive spindule formation onhorizontal cell dendrites in dark-adapted preparations kept invitro.Neurosci. Letters 164, 59–62.

Borg, B., Goos, H. J. Th., and Terlou, M. (1982). LHRH-immunoreac-tive cells in the brain of the three-spined sticleback, Gasterosteusaculeatus L. (Gasterosteidae). Cell Tissue Res. 226, 695–699.

Braford, M. R., and Northcutt, R. G. (1983). Organization of thediencephalon and pretectum in ray-finned fishes. In ‘‘Fish Neuro-biology’’ (R. E. Davis and R. G. Northcutt, Eds.), Vol. II pp.117–164. Univ. of Michigan Press, AnnArbor.

Brantley, R. K., and Bass, A. H. (1994). Alternative spawning tacticsand acoustic signals in the plainfin midshipman fish, Porichthysnotatus (Teleostei, Batrachodidae). Ethology 96, 213–232.

Davis, M. R., and Fernald, R. D. (1990). Social control of neuronalsoma size. J. Neurobiol. 21, 118–1188.

de la Iglesia, H. O., Blaustein, J. D., and Bittman, E. L. (1995). Thesuprachiasmatic area in the female hamster projects to neuronscontaining estrogen receptors and GnRH. NeuroReport 6, 1715–1722.

Demski, L. S. (1991). Neural substrates for photic control of elasmo-branch sexual development and behavior. J. Exp. Zool. 5, 121–129.

Demski, L. S., and Northcutt, R. G. (1982). The terminal nerve: a newchemosensory system in vertebrates? Science 220, 435–436.

Edwards, D. L., and Grafstein, B. (1983) Intraocular tetrodotoxin ingoldfish hinders optic nerve regeneration. Brain Res. 269, 1–14.

Ekstrom, P. (1984). Central neural connections of the pineal organand the retina in the teleostGasterosteus aculeatus L. J. Comp. Neurol.226, 321–335.

Foran, C. M., Marchaterre, M. A., Myers, T. R., Bass, A. H., andMyers, D. A. (1993). Gonadotropin releasing hormone (GnRH)messenger RNA (mRNA) expression in a fish with two classes ofadult males. Soc. Neurosci. Abstr. 19, 1454.

Foran, C. F., Myers, D. A., and Bass, A. H. (1994). Retinal modulationof GnRH mRNA expression in the thalamus of a teleost fish. Soc.Neurosci. Abstr. 20, 372.

Fujita, I., Sorensen, P. W., Stacey, N. E., and Hara, T. J. (1991). Theolfactory pathway and not the terminal nerve functions as the



primary chemosensory pathwaymediating responses to sex phero-mones in male goldfish. Brain Behav. Evol. 38, 313–321.

Gothilf, Y., Elizur, A., and Zohar, Y. (1995). Three forms of gonadotro-pin-releasing hormone in the gilthead seabream and striped bass:Physiological andmolecular studies. In ‘‘Reproductive Physiologyof Fish,’’ Fish Symposium 95, Austin, Texas.

Grober, M. S., Bass, A. H., Burd, G., Marchaterre, M. A., Segil, N.,Scholz, K., andHodgson, T. (1987). The nervus terminalis ganglioninAnguilla rostrata: an immunocytochemical and HRP histochemi-cal analysis. Brain Res. 436, 148–152.

Grober, M. S., and Bass, A. H. (1991). Neuronal correlates of sex/rolechange in Labrid fishes: LHRH-like immunoreactivity. Brain Be-hav. Evol. 38, 302–312.

Grober, M. S., Jackson, I. M. D., and Bass, A. H. (1991). Gonadalsteroids affect LHRH preoptic cell number in a sex/role changingfish. J. Neurobiol. 22, 734–741.

Grober, M. S., Fox, S. H., Laughlin, C., and Bass, A. H. (1994). GnRHcell size and number in a teleost fish with two male reproductivemorphs: sexual maturation, final sexual status and body sizeallometry. Brain Behav. Evol. 43, 61–78.

Grober, M. S., Myers, T. R., Marchaterre, M. A., Bass, A. H., andMyers, D. A. (1995). Structure, localization, and molecular phylog-eny of a GnRH cDNA from a parachanthoptergyian fish, theplainfin midshipman (Porichthys notatus). Gen. Comp. Endocrinol.99, 85–99.

Halpern-Sebold, L., Schreibman, M. P., and Margolis-Nunno, H.(1986). Differences between early- and late-maturing genotypes ofthe platyfish (Xiphophorus maculatus) in the morphometry of theirimmunoreactive luteinizing hormone releasing hormone-contain-ing cells: a developmental study. J. Exp. Zool. 240, 245–257.

Hokfelt, T., Zhang, X., and Wiesenfeld-Hallin, Z. (1994). MessengerRNA plasticity in primary sensory neurons following axotomyand its functional implications. Trends Neurosci. 17, 22–30.

Mason, A. J., Hayflick, J. S., Zoeller, R. T., Young, W. S., Phillips, H. S.,Nikolics, K., Seeburg, P. H. (1986). A deletion truncating thegonadotropin-releasing hormone gene is responsible for hypogo-nadism in the hpgmouse. Science 234, 1366–1371.

Muske, L. E. (1992). Evolution of gonadotropin-releasing hormone(GnRH) neuronal systems. Brain Behav. Evol. 42, 215–230.

Nevitt, G. A., Grober, M. S., Marchaterre, M. A., and Bass, A. H.(1995). GnRH-like immunoreactivity in the peripheral olfactorysystem and forebrain of Atlantic salmon (Salmo salar): A reassess-ment of multiple life history stages. Brain Behav. Evol. 45, 350–358.

Northcutt, R. G., and Butler, A. B. (1993). The diencephalon of thepacific herring Clupea harengus: Retinofugal projections to thediencephalon and optic tectum. J. Comp. Neurol. 328, 547–561.

Northcutt, R. G., and Butler, A. B. (1991). Retinfugal and retinopetalprojections in the green sunfish, Lepomis cyanellus. Brain Behav.Evol. 37, 333–354.

Oka, Y., and Ichikawa, M. (1990). Gonadotropin-releasing hormone(GnRH) immunoreactive system in the brain of the dwarf gourami(Colisa lalia) as revealed by light microscopic immunocytochemis-try using a monoclonal antibody to common amino acid sequenceof GnRH. J. Comp. Neurol. 300, 511–522.

Oka, Y., and Matsushima, T. (1993). Gonadotropin-releasing hor-mone (GnRH)-immunoreactive terminal nerve cells have intrinsicrhythmicity and project widely in the brain. J. Neurosci. 13,2161–2176.

Peter, R. E., and Fryer, J. N. (1983). Endocrine functions of thehypothalamus of actinopterygians. In ‘‘Fish Neurobiology’’ (R. E.Davis and R. G. Northcutt, Eds.), Vol. II pp. 165–201. Univ. ofMichigan Press, AnnArbor.

Schmidt, J. T., Edwards, D. L., and Stuermer, C. (1983). There-establishment of synaptic transmission by regenerating opticaxons in goldfish: Time course and effects of blocking activity byintraocular injection of tetrodotoxin. Brain Res. 269, 15–27.

Steward, O., and Banker, G. A. (1992). Getting the message from thegene to the synapse: sorting and intracellular transport of RNA inneurons. Trends Neurosci. 15, 180–186.

Striedter, G. F. (1990a). The diencephalon of the channel catfish,Ictalurus punctatus. I. Nuclear organization. Brain Behav. Evol. 36,329–354.

Striedter, G. F. (1990b). The diencephalon of the channel catfish,Ictalurus punctatus. II. Retinal, tectal, cerebellar and telencephalicconnections. Brain Behav. Evol. 36, 355–377.

Suzuki, M., Hyodo, S., Kobayashi, M.,Aida, K., and Urano,A. (1992).Characterization and localization of the mRNA encoding thesalmon-type gonadotropin-releasing hormone precursor of themasu salmon. J. Mol. Endocrinol. 9, 73–82.

Vecino, E., Perez, M. T. R., and Ekstrom, P. (1994). In situ hybridiza-tion of neuropeptide Y (NPY) mRNA in the goldfish brain.NeuroReport 6, 127–131.

White, S. A., Kasten, T. L., Bond, C. T., Adelman, J. P., and Fernald, R.D. (1995). Three gonadotropin-releasing hormone genes in oneorganism suggest novel roles for an ancient peptide. Proc. Natl.Acad. Sci. USA 92, 8363–8367.



Modification of Gonadotropin Releasing Hormone (GnRH) mRNA Expression in the Retinal-Recipient Thalamus

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