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General and Comparative Endocrinology 117, 8–19 (2000)doi:10.1006/gcen.1999.7361, available online at http://www.idealibrary.com on

hanges in Brain Gonadotropin-Releasing Hormone- andasoactive Intestinal Polypeptide-like Immunoreactivityccompanying Reestablishment of Photosensitivity inale Dark-Eyed Juncos (Junco hyemalis)

ierre Deviche,*,1 Colin J. Saldanha,† and Rae Silver‡Institute of Arctic Biology, University of Alaska–Fairbanks, Fairbanks, Alaska 99775; †Department of Physiologicalciences, University of California–Los Angeles, Los Angeles, California 90095; and ‡Department of Psychology,epartment of Anatomy and Cell Biology, Barnard College and Columbia University, New York, New York 10027

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n seasonally breeding, photoperiodic birds, the develop-ent of photorefractoriness is associated with decreased

rain expression of gonadotropin-releasing hormone-likemmunoreactivity (GnRH-li ir) and increased expressionf vasoactive intestinal polypeptide-like immunoreactiv-ty (VIP-li ir). Dissipation of photorefractoriness andeestablishment of photosensitivity are associated withncreased GnRH-li ir brain production, but concurrenthanges in VIP-li ir expression have not been investi-ated. To address this question, we compared the expres-ion of VIP-li ir in the infundibulum (INF) of adult maleark-eyed juncos (Junco hyemalis) that were made pho-orefractory (PR) by prolonged exposure to long daysith that of birds that were not photostimulated (PS),ut had regained photosensitivity by exposure to shortays for 5 (short-term-PS, ST-PS) or 13 (long-term-PS,T-PS) consecutive months. Photosensitive males hadmaller INF VIP-li ir cell bodies than PR males, but theumbers of INF VIP-li ir cells were independent ofhotoperiodic condition. Changes in infundibular VIP-lir were correlated with changes in preoptic area (POA)nRH-li expression. Specifically, photosensitive malesad more and larger POA GnRH-li ir cells and more

1 To whom correspondence and reprint requests should be ad-ressed at Department of Biology, Arizona State University, Tempe,

LZ 85287-1501.

8

nRH-li ir fibers in this region than PR males. Further,T-PS males had more GnRH-li ir POA fibers and largerestes than ST-PS juncos. Thus, induction of photorefrac-oriness is associated with increased VIP and decreasednRH brain expression whereas dissipation of photore-

ractoriness concurs with decreased VIP and increasednRH brain expression. These results suggest a physi-logical role for VIP in the control of changes in GnRHxpression as a function of the photosensitiveondition. r 2000 Academic Press

Key Words: GnRH; VIP; reproduction; prolactin; season-lity; photoperiodism; photosensitivity; photorefractori-ess; immunocytochemistry; preoptic area; infundibu-um.

Most birds breeding at middle and high latitudeseproduce only during spring and early summer,hen environmental conditions are the most favorable

o successful raising of young (Lack, 1968; Perrins,970). In these species and as originally demonstratedxperimentally in dark-eyed juncos (Junco hyemalis;owan, 1925), photoperiod plays an essential role in

he control of seasonal changes of reproductive systemctivity. Exposing photosensitive birds to daylengthonger than approximately 12 h (defined as long days,
D), as is naturally the case during the spring, stimu-
0016-6480/00 $35.00Copyright r 2000 by Academic Press

All rights of reproduction in any form reserved.

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Brain GnRH, VIP, and Photosensitive Condition in Dark-Eyed Juncos 9

ates the release of hypothalamic gonadotropin-eleasing hormone (GnRH) and pituitary-luteinizingormone (LH) and follicle-stimulating hormone (FSH;ewis and Farner, 1973; Wingfield et al., 1980; Dawsonnd Goldsmith, 1983; Fehrer et al., 1985; Wilson, 1985;oster et al., 1987; McNaughton et al., 1995; Meddle andollett, 1997). These hormones in turn induce gonadalevelopment and the secretion of gonadal hormones

hat control many behavioral changes associated withhe breeding period as well as the development ofecondary sexual characteristics (Balthazart et al., 1979;ilverin and Viebke, 1994).In most photoperiodic species, breeding is followed

y a photorefractory period. At this time, reproductivectivities are curtailed although photoperiod remainsell in excess of the vernal threshold for stimulation.he insensitivity to previously stimulating photope-iod that defines photorefractoriness is independent ofcular and pineal inputs (Wilson, 1990, 1991) and iselieved to originate centrally (for review, see Nichollst al., 1988; Juss, 1993). Photorefractoriness is associ-ted with decreased GnRH production and circulatingH levels, rapid gonadal involution, and molt (Wing-eld et al., 1980; Foster et al., 1987; Kubokawa et al.,994; Hahn and Ball, 1995; Parry et al., 1997; Cho et al.,998). In European starlings (Sturnus vulgaris), thenset of photorefractoriness is accompanied with de-reased hypothalamic expression of the GnRH precur-or peptide, proGnRH-GAP (Parry et al., 1997). Thisecrease, which precedes a change in hypothalamicnRH immunostaining, indicates that photorefractori-ess results from a reduction in GnRH synthesis rather

han release of this peptide at the median eminenceevel. In most species, photorefractoriness can be allevi-ted, i.e., photosensitivity can be restored, only byxposure to short days (SD) as would naturally occururing the fall and winter (Nicholls et al., 1988; Wilson,992). Restoration of photosensitivity in adults (Daw-on et al., 1986; Dawson and Goldsmith, 1997), as wells acquisition of photosensitivity in juvenile birdsGoldsmith et al., 1989), is associated with an increasedroduction of hypothalamic GnRH.Photoinduced changes in the activity of the hypothal-

mo–pituitary–gonadal axis are accompanied by alter-tions of the secretion of the anterior pituitary hor-one, prolactin. As is the case for LH, the secretion of

rolactin is photoinduced (Dawson and Goldsmith, 1

983; Mauro et al., 1992; Silverin and Goldsmith, 1997;reekumar and Sharp, 1998; for review, see Sharp et al.,998). Photoinduced high circulating prolactin concen-rations are reached during the development of photore-ractoriness, when gonads are regressing (Dawson andoldsmith, 1983; Dawson, 1997; Dawson and Sharp,

998). Prolactin is not thought to control the transitionrom photosensitivity to photorefractoriness (Nichollst al., 1988; Juss, 1993; Dawson and Sharp, 1998), but itarticipates in the rapid collapse of the reproductiveystem that characterizes photorefractoriness by inhib-ting the hypothalamo–pituitary–gonadal axis at mul-iple levels (Buntin and Tesch, 1985; Janik and Buntin,985; Sharp et al., 1998).Pituitary prolactin synthesis and release in birds are

egulated primarily by vasoactive intestinal polypep-ide (VIP; Cloues et al., 1990; Mauro et al., 1992; El

alawani et al., 1995; Youngren et al., 1994). A role forIP in the reproductive system regression that occurst the onset of photorefractoriness is suggested by theact that male European starlings that have beenctively immunized against VIP exhibit slower photo-nduced regression than control males and do not moltDawson and Sharp, 1998). In addition, brain expres-ion of GnRH and VIP change in opposite directionshen birds become photorefractory. Specifically, preop-

ic area (POA) GnRH and infundibular VIP are lower andigher, respectively, in photorefractory than in photosensi-ive male dark-eyed juncos (Saldanha et al., 1994).

The present investigation determined whether annverse relationship between brain GnRH and VIPxpressions also exists as birds regain photosensitivity.e hypothesized that initially photorefractory male

uncos exposed to SD would regain photosensitivitynd restoration of photosensitivity would concur withncreased brain GnRH expression and with decreasedIP expression. Our results support the idea that VIParticipates in the neuroendocrine changes that takelace when birds become photorefractory as well ashen photorefractoriness is dissipated.

ATERIALS AND METHODS

Hatching-year male dark-eyed juncos were caughtrom the local population in Fairbanks, Alaska (65° N,
48° W) in the fall of 1993 and transferred to the
Copyright r 2000 by Academic PressAll rights of reproduction in any form reserved.

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aboratory where they were housed in individualages and exposed to SD (10 L:14 D) until January 14,994. At this time, birds were randomly divided intoour groups. Three groups were transferred to one ofhe following light regimes until sacrificed on October0, 1994: short-term photosensitive group (ST-PS; n 5 5):D (15 L:9 D) until May 6, 1994, then SD (8 L:16 D);

ong-term photosensitive group (LT-PS; n 5 5): SD (10:14 D) until May 6, 1994, then SD (8 L:16 D); andhotorefractory group (PR; n 5 5): LD (15 L:9 D).To confirm that the ST-PS group was photosensitivehen sacrificed, a fourth group (photostimulated

roup; CONT-PS; n 5 7) was treated as the ST-PSroup until October 20, 1994. On this date (D0),ONT-PS males were transferred to LD (20 L:4 D) for2 days and then sacrificed. One day before thisransfer (D 2 1) as well as approximately every 4 dayshereafter, we measured the cloacal protuberance (CP,n androgen-sensitive secondary sexual characteristic:chwabl and Farner, 1989; Deviche, 1995) width ofhese males to the nearest 0.1 mm with calipers. Inree-living male juncos, seasonal changes in CP sizeorrelate with changes in testis mass (Deviche et al.,

IG. 1. Schematic representation of the experimental design. Maleransferred to long days (15 L:9 D; LD) or exposed to SD (10 L:14 D, tmonths of LD exposure, LD males either remained exposed to LD f:16 D) for the same period (short-term photosensitive, ST-PS).

998) and we, therefore, predicted an increase in CP p

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idth following transfer from SD to LD. To determinehether the ST-PS, CONT-PS, and PR groups becamehotorefractory after exposure to LD starting in Janu-ry, we examined them weekly for signs of body molt,tarting March 3, 1994. Brains from the CONT-PSuncos are not included in the present study. While inaptivity, all birds were at room temperature andeceived ad libitum food (Purina finch chow) and tapater supplemented with soluble vitamin (Avicon,et-A-Mix). The experimental design is summarized inig. 1.At the time of sacrifice, all subjects were euthanizedith an overdose of anesthetic (ketamine/xylazine

olution) and perfused transcardially with 0.3 ml ofeparin solution (1000 IU/ml) immediately followedith 25 ml of 0.1 M phosphate buffer (PB) and 30 ml of

reshly prepared 4% paraformaldehyde solution in PB.estes were excised and placed into 0.9% saline solu-ion. The testicular sizes of ST-PS, LT-PS, and PR males

ere estimated visually without knowledge of thexperimental treatment. Testes collected from CONT-PSuncos were weighed to the nearest milligram. The topf the cranium was removed and the heads were

were exposed to short days (10 L:14 D; SD) for 4 months, then either:16 D) for another 9 months (long-term photosensitive, LT-PS). Afterher 5 months (photorefractory, PR) or were transferred back to SD (8

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ostfixed in situ for 24 h at 4°C. Brains were then

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issected out and stored at 4°C in 0.1 M PB containing.1% sodium azide until further processed. All experi-ental procedures were approved by The University

f Alaska Fairbanks Institutional Animal Care and Useommittee.Tissue preparation and immunocytochemistry.

rains were individually embedded in a block ofelatin (8% gelatin following a coat of 4% gelatin).locks were allowed to harden at 4°C for 2–4 h and

hen immersed in 4% paraformaldehyde solution for8 h at 4°C in preparation for sectioning. Coronalections (50 µm) were cut on a vibratome from the levelf the anterior parolfactory lobe to the appearance ofhe fourth nerve. Alternate sections were processedmmunocytochemically with polyclonal antibodiesaised against either GnRH (LR-1; gift of R. Benoit) orIP (Incstar, Stillwater, MN) following a previouslyublished and validated protocol (Saldanha et al.,994). Studies using avian species other than juncosound that the LR-1 anti-GnRH antibody recognizesrain GnRH-I, but not GnRH-II (Silver et al., 1992;ilverman et al., 1994). Specifically, preoptic area GnRHeurons did not stain following preabsorption withnRH-I, but were unaffected by preabsorption withnRH-II. Furthermore, omission of the primary antise-

um resulted in the absence of any reaction product.Sections were serially exposed to 0.05% H2O2, 10%

ormal goat serum, primary antibody (dilution: 1:40,000anti-GnRH) or 1:10,000 (anti-VIP); 48 h at 4°C), 1:250iotinylated goat anti-rabbit IgG, 1:200 avidin–biotinVectastain), and 0.04% diaminobenzidine activated

ith 0.001% H2O2. All sera were prepared in 0.3%riton X-100 in 0.1 M PB, and three 15-min washes in.1% Triton X-100 in PB were performed between allncubations except immediately prior to incubation inhe primary antibody solution. Following processing,ections were mounted onto gelatin-coated micro-cope slides, dried, dehydrated by immersion intolcohol solutions (70, 95, 95, and 100%), cleared, andoverslipped in preparation for study with a lighticroscope. To control for variations in processing,

ach immunocytochemical run included sections fromne junco belonging to each experimental group.Data collection and statistical analysis. Expres-

ion of GnRH in the POA was ascertained via theeasurement of (a) GnRH soma number, (b) GnRH

omal area, and (c) GnRH fiber number. The total l

umber of GnRH-positive soma was counted in fivelternate sections through the extent of the POA, fromts rostral aspect, where the tractus septomesencephali-us extends to the ventral surface of the brain, andxtending caudally to the region where the thirdentricle is enlarged (Stokes et al., 1974; Saldanha et al.,994). Only soma with detectable nuclei were includedn the analysis. The somal area of 50–75 neuronsapproximately 10–15 per section) was measured usingIH Image Analysis software. The number of immuno-

eactive fibers in a section in the middle of the POAas quantified using the method of Shivers et al.

1983). Briefly, the section was aligned under highagnification and overlaid with an ocular grid and the

umber of intersections of immunoreactive fibers withrid segments was used as an estimate of immunoreac-ive fiber numbers.

VIP expression in the infundibulum (INF) was alsovaluated by measurement of the number of immuno-eactive soma and somal area, using the methodsescribed for GnRH (vide supra). The number of VIP-ositive soma was counted in six to eight alternateections through the entire INF. All the neuroanatomi-al data were collected by an investigator who was notware of the experimental condition of the animals toinimize potential biases in data analysis.Neuron numbers and sizes and fiber numbers were

ompared across groups using analysis of varianceANOVA) with photosensitive condition as the mainariable. Post hoc Fisher least significance differenceests were used to determine specific differences be-ween groups. For the neuron size measures, the meanell area was computed for each bird and individualeans were compared across groups. Changes in CP

izes over time in CONT-PS birds were analyzed usingne-way ANOVA for repeated measures. CP sizes afterransfer of these birds to LD were compared to pre-timulation sizes using Dunnett’s method tests. Statis-ical significance level was in all cases set at a 5 0.05.

ESULTS

Verification of photosensitivity and photorefractoryondition. LT-PS males showed no sign of body moltt any time. Fifteen of 17 males that were photostimu-
ated starting in January 1994 were molting on March

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, 1994, and the remaining 2 males started to moltithin the following few weeks, suggesting that they

ecame photorefractory. Thirteen males had com-leted molt 1 day before transfer of ST-PS and CONT-PSroups from LD to SD on May 6.The CP widths of CONT-PS juncos gradually in-

reased after transfer from 8 L:16 D to 20 L:4 D. Atatistical difference between pre- and poststimulationizes was reached after 14 days of exposure to LD (Fig.). When sacrificed, these males also had partiallyeveloped testes (mean 6 sd: 112 6 75 mg) whereasirds belonging to the three other groups had smallestis sizes (median testis length: ,0.5 mm; maximum:

mm). Thus, ST-PS (and, by extension, also LT-PS)roups were photosensitive when sacrificed. Althoughestis lengths were small in PR, ST-PS, and LT-PSuncos, these groups differed (Kruskall–Wallis one-

ay ANOVA on ranks: P , 0.001). All PR and ST-PSales had testis lengths not exceeding 0.5 mm, but

T-PS males had slightly larger testes (median andnterquartile interval: 1 mm (1–1.38 mm); Student–

ewman–Keuls test comparisons with PR and ST-PSroups: P , 0.05).GnRH expression. The junco hypothalamus con-

ains numerous GnRH-expressing neurons and fiberscattered through the anterior–posterior extent of the

IG. 2. Changes in cloacal protuberance width (mean 6 standard do 15 L:9 D on Day 0. *P , 0.05, comparison with prestimulation size

iencephalon. Most of the neuronal staining is ob- i


erved close to the midline, but some dispersed peri-arya are seen more laterally. GnRH-like immunoreac-ive (GnRH-li ir) somas were observed in the preopticrea, nucleus of the lateral hypothalamus, paraventricu-ar nucleus of the hypothalamus, periventricularucleus, and the nucleus of the pallial commissure.nRH-li ir fibers were observed at all the above loci asell as in the lateral septum (SL), infundibulum, andedian eminence (ME). Since previous work on juncos

ad identified changes in GnRH-li ir in the POA as aunction of photoperiodic condition (Saldanha et al.,994), we quantified immunoproduct only in this area.GnRH expression across photosensitive states. Ex-

erimental groups differed with respect to POAnRH-li ir neuron (ANOVA: F2,14 5 10.06, P 5 0.003)

nd fiber (id: P 5 0.0001) numbers as well as neuronalreas (id: P 5 0.0001; Fig. 3). PR males had fewermmunostained perikarya and fibers and smaller neu-on areas than ST- and LT-PS males (Fig. 4). Althoughot quantified, the intensity of staining was noticeably

ighter in cells and fibers of PR than in those ofhotosensitive birds. The two photosensitive groupsad similar numbers of POA GnRH-li ir neurons andoma sizes. However, LT-PS males had more immu-opositive fibers than ST-PS males.VIP expression. The expression of VIP is surpris-

n) of adult male dark-eyed juncos (n 5 7) transferred from 10 L:14 D-1; Dunnett’s method test).

eviatios (Day

ngly sparse in the junco diencephalon. Although ir

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bers course along the midline toward the ME, VIP-lir soma are observed only in the infundibulum (Fig. 5).hese cells are numerous and clustered around the

hird ventricle. Another group of VIP-li ir cells is seenore rostrally within the telencephalic SL. Since

hanges in VIP-li ir have been described across photo-ensitive states in the infundibular, but not the septal,roup of VIP neurons (Saldanha et al., 1994), weeasured ir signal only in the infundibulum.VIP expression across photosensitive states. The

umbers of INF VIP-li ir cells did not differ amongroups (Fig. 6), but there were differences in soma sizesANOVA: F2,14 5 28.00, P 5 0.0001). PR birds had largerIP-li ir soma than ST- or LT-PS groups. There was noifference between ST- and LT-PS males (Fig. 6).

ISCUSSION

Previous studies on birds identified marked changes

IG. 3. Gonadotropin-releasing hormone-like immunoreactive neueviations) in the preoptic area of photorefractory (PR), short-termark-eyed juncos (n 5 5/group). For a given parameter, columns wignificant difference test).

n POA GnRH synthesis (Parry et al., 1997) and content c

Dawson et al., 1986; Millam et al., 1995; Dawson andoldsmith, 1997) as a function of the reproductive

ondition. Generally, birds that are undergoing go-adal regression or are photorefractory have less hypo-

halamic GnRH than do photosensitive subjects (Fos-er et al., 1987; Saldanha et al., 1994; Cho et al., 1998).he present investigation confirms and extends thesendings. Photosensitive males were exposed to LDntil they became photorefractory, as indicated byody molt (Nicholls et al., 1988; Dawson, 1997). At thisoint, they were either held on LD to maintain refracto-iness or transferred to SD to restore photosensitivitynd then sacrificed. Restoration of photosensitive con-ition in ST-PS males at the time of sacrifice wasonfirmed by the results obtained for CONT-PS males.s expected, the reproductive system (testis sizes,

loacal protuberance widths) of these males recru-esced within weeks of photostimulation. The resultsemonstrate that restoration of photosensitivity corre-

ates with an increased number and size of GnRH-ontaining neurons as well as with increased GnRH-

mbers, neuronal soma areas, and fiber numbers (means 1 standardsensitive (ST-PS), and long-term photosensitive (LT-PS) adult maledifferent superscript letter differ statistically (P , 0.05, Fisher least

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IG. 4. Photomicrographs depicting the distribution of gonadothotorefractory (A), short-term photosensitive (B), and long-term p

ropin-releasing hormone-like immunoreactivity in the preoptic area ofhotosensitive (C) adult male dark-eyed juncos. All photomicrographs are

aken at the same magnification. V, third ventricle.


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hanges took place in birds held on SD and, therefore,id not require photostimulation. Similarly, Dawsonnd Goldsmith (1997) found that POA GnRH concen-ration increases after transfer of LD-exposed photore-ractory European starlings to SD. It is unknown howong photorefractory juncos must be exposed to SD forypothalamic GnRH expression to increase. It is likely

IG. 5. Low- (top) and high- (bottom) power magnification photomike immunoreactive cell bodies and fibers in the infundibulum anentricle.

hat considerably less than the 5 months used in this g

tudy is necessary because GnRH levels in hypo-halami of starlings were elevated after transfer fromD to SD for as little as 10 days (Dawson andoldsmith, 1997).Photorefractory and photosensitive SD-exposed jun-

os have an inactive pituitary–gonadal axis (but seeelow) as assessed by plasma LH concentrations and

aphs showing the distribution of vasoactive intestinal polypeptide-edian eminence (ME) of an adult male dark-eyed junco. V, third

icrogrd the m

onadal sizes (Saldanha et al., 1994). These birds,


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owever, differ in that photosensitive birds have moreypothalamic GnRH than photorefractory birds. Inac-

ivity of the pituitary–gonadal axis in photosensitive,ut nonphotostimulated, birds presumably results fromlack of release rather than synthesis of the peptide

nd dissipation of photorefractoriness in juncos, as inther species (Dawson and Goldsmith, 1997), is there-ore probably associated with increased production

ithout concurrent release of GnRH. It should, how-ver, be noted that photosensitive males that werexposed to SD for 5 months had similar POA GnRH-ontaining neuron numbers and sizes, but fewernRH-li ir nerve fibers and smaller testes than males

IG. 6. Vasoactive intestinal polypeptide-like immunoreactive neu-on numbers and neuronal soma areas (means 1 standard devia-ions) in the infundibulum of photorefractory (PR), short-termhotosensitive (ST-PS), and long-term photosensitive (LT-PS) adultale dark-eyed juncos (n 5 5/group). For a given parameter, col-

mns with a different superscript letter differ statistically (P , 0.05,isher least significant difference test).

xposed to SD for 13 consecutive months. Thus, e


rolonged (i.e., more than 5 months) exposure to SDay have been associated with some release of GnRH

hat induced a modest, but detectable activation of theituitary–gonadal axis. Other investigations support

he hypothesis that changes in hypothalamo–pituitary–onadal axis activity can occur in SD-exposed birdshat are regaining or have regained photosensitivity.or example, pituitary stores of FSH in starlings

ncrease after prolonged exposure to SD, indicating aelease of GnRH from the median eminence (Dawsont al., 1986). Further, several species show a subtle, buteal pituitary–gonadal axis stimulation when regain-ng photosensitivity under SD (canary, Serinus ca-arius, Nicholls and Storey, 1976; rook, Corvus frugile-us, Lincoln et al., 1980; European starling, Dawson,983). Finally, dissipation of photorefractoriness is arogressive rather than an all-or-none process (Boulak-ud and Goldsmith, 1995; Dawson and Goldsmith,997). For example, initially photorefractory Harris’parrows (Zonotrichia querula) require 13 weeks of SDxposure to achieve full photosensitivity (Wilson, 1992)s measured by testicular growth rate after transfer toD. Similarly, in castrated male Japanese quail (Cotur-ix coturnix japonica), 5 weeks of SD exposure areecessary to fully restore LH response to photostimula-

ion (Follett and Pearce-Kelly, 1990). Although juncosxposed to SD for 5 months were photosensitive,omplete dissipation of photorefractoriness in thispecies may require additional time.

In turkeys (Meleagris gallopavo; Mauro et al., 1992)nd dark-eyed juncos (Saldanha et al., 1994) develop-ent of photorefractoriness is associated with high

ypothalamic VIP expression. The present study ex-ends these findings, demonstrating that reestablish-

ent of photosensitivity is associated with decreasedrea of infundibular VIP-containing neurons, but nohange in numbers of detectable neurons. In contrast,uncos that were refractory as a result of prolonged LDxposure had more VIP-li ir neurons than photosensi-ive birds that were or were not photostimulatedSaldanha et al., 1994). Further research is necessaryefore it can be concluded with certainty that thehanges in VIP-containing neuron characteristics thatccur as a function of the photoperiodic conditionepresent alterations of the peptide synthesis. Neverthe-ess, the results suggest that brain GnRH and VIP
xpressions change in opposite directions when birds

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ecome photorefractory as well as when they regainhotosensitivity.VIP is the main hypothalamic neuropeptide control-

ing prolactin release in birds (Lea et al., 1991; Elalawani et al., 1995). Plasma concentrations of prolac-

in are low in photosensitive birds kept on SD, increaseuring photostimulation, and are highest at the onsetf photorefractoriness (Goldsmith, 1985; Nicholls et al.,988; Dawson, 1997). This hormone inhibits the repro-uctive system (Buntin and Tesch, 1985; Janik anduntin, 1985; Juss, 1993). Thus, elevated circulatingrolactin concentrations when birds become photore-

ractory and shortly thereafter probably contribute tohe rapid involution of reproductive tissues that takeslace at this time (Dawson and Sharp, 1998). VIP may,

herefore, participate in the inactivation of the hypothal-mo–pituitary–gonadal axis that characterizes the on-et of photorefractoriness indirectly by stimulatingrolactin secretion.VIP receptors are present in various diencephalic

egions of the pigeon (Columba livia) brain includingreas rich in GnRH cells and fibers such as the lateraleptum, POA, and median eminence (Hof et al., 1991).urther, there is a close association between VIP-xpressing terminals and GnRH-expressing dendritesn the lateral septum of this species (Kiyoshi et al.,998). A similar association between VIP-li ir axons,IP receptors, and GnRH-li ir dendrites may exist in

he junco as suggested by previous studies on thispecies showing an overlap between VIP- and GnRH-xpressing elements in the septum and POA (Saldanhat al., 1994). This association may underlie a mecha-ism whereby elevated VIP may inhibit GnRH duringhotorefractoriness, and a decrease in this inhibitionay permit hypothalamo–pituitary–gonadal axis activ-

ty during the reestablishment of photosensitivity.Low infundibular VIP content in birds that have

egained photosensitivity as a result of SD exposureay also be of physiological significance when these

irds are exposed to LD. When transferred from SD toD, photosensitive birds can respond by a rapid

within 1 day) activation of their hypothalamo–ypophyseal system including GnRH release (Pererand Follett, 1992) and elevated plasma LH levelsFollett et al., 1975; Meddle and Follett, 1997). The rapid
ransduction of photic information into a neuroendo- D
rine response may be facilitated by the fact thatypothalamic VIP content is then relatively low.In conclusion, it appears that VIP may have an

mportant role in the regulation of seasonal cycles anday be part of the neuroendocrine processes thatediate transition from one condition of photosensitiv-

ty to another. Further research is warranted to identifynd localize avian brain VIP receptors in photoperi-dic species and to define the mechanism responsibleor the anti-gonadotropic activity of this neuropeptiden birds. Additional research is also necessary tovaluate the functional relationships between GnRHnd VIP at different stages of the reproductive cycle.

CKNOWLEDGMENTS

The authors thank Dr. Cynthia Gulledge for assistance and Reneerain for comments on an early draft of the manuscript. This workas partly supported by National Scientific Foundation AwardNS-9121258 (P.D.) and IBN 9511300 (R.S.).

EFERENCES

althazart, J., Massa, R., and Negri-Cesi, P. (1979). Photoperiodiccontrol of testosterone metabolism, plasma gonadotrophins, cloa-cal gland growth, and reproductive behavior in the JapaneseQuail. Gen. Comp. Endocrinol. 39, 222–235.

oulakoud, M. S., and Goldsmith, A. R. (1995). The effect of durationof exposure to short days on the gonadal response to long days inmale starlings (Sturnus vulgaris). J. Reprod. Fertil. 104, 215–217.

untin, J. D., and Tesch, D. (1985). Effects of intracranial prolactinadministration on maintenance of incubation readiness, ingestivebehavior, and gonadal condition in Ring Doves. Horm. Behav. 19,188–203.

ho, R. N., Hahn, T. P., MacDougall-Shackleton, S., and Ball, G. F.(1998). Seasonal variation in brain GnRH in free-living breedingand photorefractory house finches (Carpodacus mexicanus). Gen.Comp. Endocrinol. 109, 244–250.

loues, R., Ramos, C., and Silver, R. (1990). VIP-like immunoreactiv-ity during reproduction in doves: Influence of experience andnumber of offspring. Horm. Behav. 24, 215–231.awson, A. (1983). Plasma gonadal steroid levels in wild starlings(Sturnus vulgaris) during the annual cycle and in relation to thestages of breeding. Gen. Comp. Endocrinol. 49, 286–294.awson, A. (1997). Plasma-luteinizing hormone and prolactin dur-ing circannual rhythms of gonadal maturation and molt in maleand female European starlings. J. Biol. Rhythms 12, 371–377.
awson, A., and Goldsmith, A. R. (1983). Plasma prolactin and

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D

E

F

F

F

F

G

G

H

H

J

J

K

K

L

L

L

L

M

M

M

M

N

N

P


CA

gonadotrophins during gonadal development and the onset ofphotorefractoriness in male and female starlings (Sturnus vulgaris)on artificial photoperiods. J. Endocrinol. 97, 253–260.awson, A., and Goldsmith, A. R. (1997). Changes in gonadotrophin-releasing hormone (GnRH-I) in the pre-optic area and medianeminence of starlings (Sturnus vulgaris) during the recovery ofphotosensitivity and during photostimulation. J. Reprod. Fertil.111, 1–6.awson, A., and Sharp, P. J. (1998). The role of prolactin in thedevelopment of reproductive photorefractoriness and postnuptialmolt in the European starling (Sturnus vulgaris). Endocrinology 139,485–490.awson, A., Goldsmith, A. R., Nicholls, T. J., and Follett, B. K. (1986).Endocrine changes associated with the termination of photorefrac-toriness by short daylengths and thyroidectomy in starlings(Sturnus vulgaris). J. Endocrinol. 110, 73–79.eviche, P. (1995). Androgen regulation of avian premigratoryhyperphagia and fattening: From ecophysiology to neuroendocri-nology. Am. Zool. 35, 234–245.eviche, P., Meddle, S. L., and Wingfield, J. (1998). Age-relateddifferences in reproductive physiology of adult male dark-eyedjuncos (Junco hyemalis) during the breeding period. Abstract, West.Reg. Meeting on Comp. Endocrinol., Flagstaff, AZ.

l Halawani, M. E., Youngren, O. M., Rozenboim, I., Pitts, G. R.,Silsby, J. L., and Phillips, R. E. (1995). Serotonergic stimulation ofprolactin secretion is inhibited by vasoactive intestinal peptideimmunoneutralization in the turkey. Gen. Comp. Endocrinol. 99,69–74.

ehrer, S. C., Silsby, J. L., Behnke, E. J., and El Halawani, M. E. (1985).Hypothalamic and serum factors influence on prolactin andluteinizing hormone release by the pituitary gland of the youngturkey (Meleagris gallopavo). Gen. Comp. Endocrinol. 59, 73–81.

ollett, B. K., Farner, D. S., and Mattocks, P. W. (1975). Luteinizinghormone in the plasma of white-crowned sparrows (Zonotrichialeucophrys gambelii) during artificial photostimulation. Gen. Comp.Endocrinol. 26, 126–134.

ollett, B. K., and Pearce-Kelly, A. (1990). Photoperiodic control ofthe termination of reproduction in Japanese quail (Coturnix cotur-nix japonica). Proc. R. Soc. London. B Biol. Sci. 242, 225–230.

oster, R. G., Plowman, G., Goldsmith, A. R., and Follett, B. K. (1987).Immunohistochemical demonstration of marked changes in theLHRH system of photosensitive and photorefractory Europeanstarlings (Sturnus vulgaris). J. Endocrinol. 115, 211–220.oldsmith, A. R. (1985). Prolactin in avian reproduction: Incubationand the control of seasonal breeding. In ‘‘Prolactin: Basic andClinical Correlates’’ (R. M. MacLeod, U. Scapagnini, and M. O.Thorner, Eds.), pp. 411–425. Springer-Verlag, Padua, Italy.oldsmith, A. R., Ivings, W. E., Pearce-Kelly, A. S., Parry, D. M.,Plowman, G., Nicholls, T. J., and Follett, B. K. (1989). Photoperi-odic control of the development of the LHRH neurosecretorysystem of European starlings (Sturnus vulgaris) during pubertyand the onset of photorefractoriness. J. Endocrinol. 122, 255–268.ahn, T. P., and Ball, G. F. (1995). Changes in brain GnRH associatedwith photorefractoriness in house sparrows (Passer domesticus).Gen. Comp. Endocrinol. 99, 349–363.
of, P. R., Dietl, M. M., Martin, J.-L., Bouras, C., Palacious, M., and

Magistretti, P. J. (1991). Vasoactive intestinal polypeptide bindingsites and fibers in the brain of the pigeon Columbia livia: Anautoradiographic and immunohistochemical study. J. Comp. Neu-rol. 305, 393–411.

anik, D. S., and Buntin, J. D. (1985). Behavioural and physiologicaleffects of prolactin in incubating ring doves. J. Endocrinol. 105,201–209.

uss, T. S. (1993). Neuroendocrine and neural changes associatedwith the photoperiodic control of reproduction. In ‘‘Avian Endocri-nology’’ (P. J. Sharp, Ed.), pp. 47–60. Society for Endocrinology,Bristol, UK.

iyoshi, K., Kondoh, M., Hirunagi, K., and Korf, H. (1998). Confocallaser scanning and electron-microscopic analyses of the relation-ship between VIP-like and GnRH-like immunoreactive neurons inthe lateral septal–preoptic area of the pigeon. Cell Tissue Res. 293,39–46.

ubokawa, K., Ishii, S., and Wingfield, J. C. (1994). Effect of daylength in luteinizing hormone b-subunit mRNA and subsequentgonadal growth in the white-crowned sparrow, Zonotrichia leucoph-rys gambelii. Gen. Comp. Endocrinol. 95, 42–51.

ack, D. (1968). ‘‘Ecological Adaptations for Breeding in Birds.’’Methuen, London.

ea, R. W., Talbot, R. T., and Sharp, P. J. (1991). Passive immunizationagainst chicken vasoactive intestinal polypeptide suppressesplasma prolactin and crop sac development in incubating ringdoves. Horm. Behav. 25, 283–294.

ewis, R. A., and Farner, D. S. (1973). Temperature modulation ofphotoperiodically induced vernal phenomena in white-crownedsparrows (Zonotrichia leucophrys). Condor 75, 279–286.

incoln, G. A., Racey, P. A., Sharp, P. J., and Klandorf, H. (1980).Endocrine changes associated with spring and autumn territorial-ity of the rook, Corvus frugilegus. J. Zool. 190, 137–153.auro, L. J., Youngren, O. M., Proudman, J. A., Phillips, R. E., and ElHalawani, M. E. (1992). Effects of reproductive status, ovariec-tomy, and photoperiod on vasoactive intestinal peptide in thefemale turkey hypothalamus. Gen. Comp. Endocrinol. 87, 481–493.cNaughton, F. J., Dawson, A., and Goldsmith, A. R. (1995). Acomparison of the responses to gonadotrophin-releasing hormoneof adult and juvenile, and photosensitive and photorefractoryEuropean starlings, Sturnus vulgaris. Gen. Comp. Endocrinol. 97,135–144.eddle, S. L., and Follett, B. K. (1997). Photoperiodically drivenchanges in Fos expression within the basal tuberal hypothalamusand median eminence of Japanese quail. J. Neurosci. 17, 8909–8918.illam, J. R., Craig-Veit, C. B., and Faris, P. L. (1995). Concentrationof chicken gonadotropin-releasing hormones I and II in microdis-sected areas of turkey hen brain during the reproductive cycle.Domestic Animal Endocrinol. 12, 1–11.icholls, T. J., Goldsmith, A. R., and Dawson, A. (1988). Photorefrac-toriness in birds and comparison with mammals. Physiol. Rev. 68,133–176.icholls, T. J., and Storey, C. R. (1976). The effects of castration onplasma LH levels in photosensitive and photorefractory canaries(Serinus canarius). Gen. Comp. Endocrinol. 29, 170–174.

arry, D. M., Goldsmith, A. R., Millar, R. P., and Glennie, L. M. (1997).
Immunocytochemical localization of GnRH precursor in the hypo-

P

P

R

S

S

S

S

S

S

S

S

S

S

W

W

W

W

W

Y


thalamus of European starlings during sexual maturation andphotorefractoriness. J. Neuroendocrinol. 9, 235–243.

erera, A. D., and Follett, B. K. (1992). Photoperiodic induction invitro: The dynamics of gonadotropin-releasing hormone releasefrom hypothalamic explants of the Japanese quail. Endocrinology131, 2898–2908.

errins, C. M. (1970). The timing of birds’ breeding seasons. Ibis 112,242–255.

owan, W. (1925). Relation of light to bird migration and develop-mental changes. Nature 115, 494–495.

aldanha, C. J., Deviche, P. J., and Silver, R. (1994). Increased VIP anddecreased GnRH expression in photorefractory dark-eyed juncos(Junco hyemalis). Gen. Comp. Endocrinol. 93, 128–136.

chwabl, H., and Farner, D. S. (1989). Endocrine and environmentalcontrol of vernal migration in male white-crowned sparrows,Zonotrichia leucophrys gambelii. Physiol. Zool. 62, 1–10.

harp, P. J., Dawson, A., and Lea, R. W. (1998). Control of luteinizinghormone and prolactin secretion in birds. Comp. Biochem. Physiol. C119C, 275–282.

hivers, B. D., Harlan, R. E., Morrell, J. I., and Pfaff, D. W. (1983).Immunocytochemical localization of luteinizing hormone-releas-ing hormone in male and female brains. Quantitative studies onthe effect of gonadal steroids. Neuroendocrinology 36, 1–12.

ilver, R., Ramos, C. L., and Silverman, A-J. (1992). Sex behaviortriggers appearance of non-neural cells containing gonadotropin-releasing hormone in doves. J. Neuroendocrinol. 4, 207–210.

ilverin, B., and Goldsmith, A. (1997). Natural and photoperiodicallyinduced changes in plasma prolactin levels in male great tits. Gen.Comp. Endocrinol. 105, 145–154.

ilverin, B., and Viebke, P. A. (1994). Low temperatures affect thephotoperiodically induced LH and testicular cycles differently inclosely related species of tits (Parus spp.). Horm. Behav. 28, 199–206.

ilverman, A. J., Millar, R. P., King, J. A., Zhuang, X., and Silver, R.(1994). Mast cells with gonadotropin-releasing hormone-like im-munoreactivity in the brain of doves. Proc. Natl. Acad. Sci. USA 26,3695–3699.

reekumar, K. P., and Sharp, P. J. (1998). Effect of photostimulationon concentrations of plasma prolactin in castrated bantams (Gallusdomesticus). J. Neuroendocrinol. 10, 147–154.

tokes, T. M., Leonard, C. M., and Nottebohm, F. (1974). Thetelencephalon, diencephalon, and mesencephalon of the canary,Serinus canaria, in stereotaxic coordinates. J. Comp. Neurol. 156,337–374.ilson, F. E. (1985). Androgen feedback-dependent and -indepen-dent control of photoinduced LH secretion in male tree sparrows(Spizella arborea). J. Endocrinol. 105, 141–152.ilson, F. E. (1990). Extraocular control of seasonal reproduction infemale tree sparrows (Spizella arborea). Gen. Comp. Endocrinol. 77,397–402.ilson, F. E. (1991). Neither retinal nor pineal photoreceptorsmediate photoperiodic control of seasonal reproduction in Ameri-can tree sparrows (Spizella arborea). J. Exp. Zool. 259, 117–127.ilson, F. E. (1992). Photorefractory Harris’ sparrows (Zonotrichiaquerula) exposed to a winter-like daylength gradually regainphotosensitivity after a lag. Gen. Comp. Endocrinol. 87, 402–409.ingfield, J. C., Follett, B. K., Matt, K. S., and Farner, D. S. (1980).Effect of day length on plasma FSH and LH in castrated and intactwhite-crowned sparrows. Gen. Comp. Endocrinol. 42, 464–470.

oungren, O. M., Silsby, J. L., Rozenboim, I., Phillips, R. E., and ElHalawani, M. E. (1994). Active immunization with vasoactiveintestinal peptide prevents the secretion of prolactin induced byelectrical stimulation of the turkey hypothalamus. Gen. Comp.Endocrinol. 95, 330–336.


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