Photoperiodic and pineal influences on estrogen-stimulated behaviors in female Syrian hamsters

Physwlogy&Behavtor,Vol. 54, pp 19-28, 1993

0031-9384/93$6 00 + 00 Copyright© 1993PergamonPressLtd

Pnntedm the USA

Photoperiodic and Pineal Influences on EstrogenStimulated Behaviors in Female Syrian Hamsters J O N A T H A N D. K A R W A N D J. B R A D L E Y P O W E R S 2

Department of Psychology, Vanderbilt Umversity, Nashville, TAr 37240 Received 28 July 1992 KARP, J. D. AND J. B POWERS. Photopertodlc and pmeal mfluences on estrogen-sttmulated behavtors m female Syrian hamsters PHYSIOL BEHAV 54(1) 19-28, 1993.--Three experiments investigatedthe effects of short photoperiod exposure on the estrogemc facilitationof locomotor actwlty and lordosis. In Experiment I, ovarlectom~zedfemale hamsters were administered exogenous estrogen to stimulate locomotor actwity m running wheels Estrogen was effectwe m the long photoperiod group but &d not stimulate running-wheelactivity m the short photopenod group. In Experiment 2, the role of the pineal gland in mediating photopeno&c influences on female hamster behawor was examined. Both estrogen-reduced locomotor activity and estrogen + progesterone-stimulated lordosis behawor were s~gmficantlyreduced m short photopenod females. Both these photopenodlc effects were absent m pinealectomizedhamsters. Sham-pmealectomized,short photopenod femalesexpressed behaworal deficits; pmealectom~zedhamsters in the short photopenod did not Expenment 3 investigatedlordosis only and used hormone reJections rather than stlast~cimplants to administer estrogen. The photopenodic and pineal effectsobservedm Experiment 2 were replicated m Experiment 3. Additionally, the suppression of lordosis responsweness by short photopenod exposure was estrogen dose dependent. Photopeno&c effects were present when 2 ug estra&ol cypionate was used but absent when higher estrogen doses were used. These findings are discussed m the context of other results that suggested photopenodlc effects on hamster lordosis were pineal independent. Female hamsters

Photopenod

Pineal

Locomotoractivity

SEASONAL changes in day length (photopenod) are important environmental cues for regulating a variety of endocrine, metabohc, and behaworal rhythms (6,34,43,55,56). For example, when adult Syrian hamsters (Mesocrwetus auratus) are exposed to long days (typically 12.5 or more h light/day), they are reproductively competent. If animals are transferred from long to short days, gonadal function is suppressed and reproductive capabihty dechnes. These photoperiodic influences on reproductive physiology require an intact pineal gland and its secreUon of melatonin; removal of the pineal or its denervation prevents the reproductive system of hamsters from responding to changes in day length (46,56). Melatonin is the primary hormonal signal used in the photoperiodic control of reproduction among many mammals (20). Its synthesis and secretion are regulated by the circadian system (28,56); in constant darkness, melatonin levels in the systemic circulation are elevated during a single, time-limited episode, once each orcadian cycle. Under conditions of alternating light and dark, melatonin ~ssecreted only during the dark for a period that is proportional to the duration of darkness (23,24), and most evidence suggests that the interval of uninterrupted melatomn secretion each night is the critical parameter in conveying

Lordosis

Estra&ol

photoperiodic reformation to the reproductive and other systems (21). Most studies that have investigated the role of pineal melatonin in conveying photoperlodic information have used endocrine or morphological indices of reproductive function. The few reports that have examined photoperiodic influences on behavior (43) indicate that in the majority of cases the pineal gland is also ~mportant. For example, we demonstrated that the inhibition of copulatory behavior among male Syrian hamsters exposed to a short photoperiod (39) requires an intact pineal gland (38). Similarly, photoperiodic influences on female hamster aggressive behaviors presumably require a functional pineal gland because appropriate administration of melatonin to long photopenod-exposed animals can produce short photoperiod behavioral effects (16). However, some behaviors influenced by changes in photoperiod duration may be mediated by mechanisms that do not involve the pineal gland or melatonin. For example, pinealectomy or pineal gland denervation did not prevent short days from antagonizing the expressxon of hormoneinduced lordosis In female hamsters (2,3). In addition, melatonin mj.ecUons that induced anestrus in long-day females did not impair lordosis m response to exogenous estradiol (2). These results

Requests for repnnts should be addressed to J Bradley Powers, Department of Psychology, Tobln Hall, Umverslty of Massachusetts, Amherst, MA 01003 2 Present address. Department of Psychiatry, Umverslty of Rochester School of Me&cme and Dentistry, 300 Crlttenden Boulevard, Rochester, NY 14642

19

20

N4RP

suggest that the photopenodic control of reproductive behavior may be regulated ~a separate mechamsms m male and female hamsters, ff confirmed, they raise ~mportant questions concernmg the development of this sexual dimorphlsm. The experiments reported here examined whether pineal mdependent behavioral effects of short-day exposure among female hamsters were hm~ted to lordosis or involved the estrogenic facilitation of locomotor acn~tty as well Our results replicate earher reports that the stlmulatory effects of estrogen on both these behaviors are &mmlshed m females exposed to short days In addmon, we found that both effects reqmre the pineal gland for their expression GENERAL METHOD

Animals Female Synan hamsters (Mesocrwetm attratus, aged 8-10 weeks, Harlan Sprague-Dawley, Indianapolis, IN) were group housed (four to five/cage) with food and water continuously available. Body weights were recorded at several times dunng each expenment.

Acm, ttv Records

\"~1) P()W[ Rq

placed m a stereotaxtc device The skull ~as exposed and a small hole made over the confluence of the saglttal and lmnsversc sinuses "I he pineal gland was then grasped and removed using micro forceps After allowing for a small amount of bleedmg, the bone piece was replaced and the skin sutured Sham pmealectom,, was performed by exposing the saglttal sinus and puncturing it to permit a small ~olume of blood loss, but the pineal was otherwise not mampulated The efli~chveness of p~nealectomy was assessed b.~ determining if this procedure ehmmated c~ther the dlsruptmn of estrous cycles or lhc uterine regression that normally results when pineal lntakI females arc exposed to a short photopenod ttotmone

['tealmolls

Ovarian hormones were admlmstered to facflnate locomotor acu~lt~ and to induce sexual receptwity This was accomplished either by the subcutaneous (SC) lmplantanon of capsules conraining estra&ol (Expenments 1, 2) or by SC mjectmns of estrogen and progesterone (Expenment 3). Estrogen (E2) capsules were made by packing B-estradiol (Sigma, St Louis, MO) into a 6 mm length of Sflastic tubing (1.57 mm Ld, 2 41 mm o d ) and seahng each end w~th Sflastlc adhesive: they were nnsed in 70% ethanol then incubated m 0 9% saline for at least 24 h before use Blank capsules (BL) containing no hormone were used for control purposes; they were the same smze as the E2 capsules but were filled with Sllastlc adhesive. Hormone mjecnons consisted of estradiol cypionate and progesterone administered SC m 0 1 ml sesame oil. Details concerning the doses used, the sequence of administration, and the procedures tbr assessing lordosis are provided under the relevant experiments belo~

Locomotor actlvlty was monitored for each hamster housed individually in a plastic cage with access to a running wheel Plastic cages with running wheels (17 × 8 5 cm) were located in hght-tlght, ventilated chambers (61 × 63 × 245 cm) in which photopenod duration was controlled by a programmable timer; a Commodore 64 computer counted the number of wheel revolutions in successive 5-mln time bins. These counts were recorded each hour and transferred to MS-DOS compatible files as previously described (30). Two measures of locomotor activity were evaluated One was the total number of wheel revolutions exhibited per 24 h. Typically, a mean for each animal was denved by averaging over a block of days that vaned according to the specific experiment and treatment condition A second measure was the duration of the activity penod each day (alpha) Alpha was determined from actograph plots by measunng the interval between the beginning and end of locomotor actlvlty for each ofa senes of days and averaging over these intervals. Actographs were constructed in one of two ways; either a) a pen excursion of constant height was generated for each successive 5-mln time bin in which the number of wheel revolutions exceeded some cntenon, or b) the height of the pen excursion for each time bin was proportional to the number of wheel revolutions counted. Additional plots to visually depict the amplitude and timing of activity throughout the day-night cycle were denved by using appropnate averages for a specified number of days over 72 successive 20-rain time bins representing a 24-h penod. In this way, the temporal distnbutlon of activity throughout the day and night could be depicted, averaged over a group of animals for a specified period of time.

The purpose of this expenment was to evaluate photopeno&c influences on the ability of estrogen to facilitate locomotor activity Our approach was to quantify wheel-running behavior while female hamsters were exposed to long or short photopenods, then determine whether the effects of ovarieetomy and subsequent estrogen replacement would differ as a function of photoperlodic treatment. Part of our intent was to replicate earher findings (58) using different experimental protocols, quantlficauon procedures, and methods of data analysis

Surgical Ptocedures

Speclhc Methods'

Bilateral ovanectomy (OVX) was performed through a single abdominal mxdhne incision while hamsters were anesthetized with sodium pentobarbital (Nembutal, 75 mg/kg, IP). In some circumstances, uterine horns were ligated at the cervix, removed, and weighed so that the extent of utenne regression occumng after short photoperiodlc exposure could be evaluated Plnealectomms (PX) and sham pinealectomles (SPX) were performed by a modification of the Hoffman and Reiter method (26) Each hamster was anesthetized with either Nembutal or Avertin (2.2% tnbromoethanol in 0.9% saline, 1 ml/100 g. IP) before being

Females were initially exposed to a long photopenod (16L 8D; lights on at 0300 h) for 2 weeks, then housed individually with access to running wheels for an additional 2 weeks. The average number of wheel revolutions per day was determined for each hamster over the final 7 days: based on this pretreatment measure, animals were divided into two equivalent groups Subsequently, activity wheels were removed and the hamsters either remained m the long photoperiod (n = 10) or were transferred to a short photoperiod (8L: 16D; lights on at 0700 h, n = 10) for 6 weeks (weeks 1-6), Locomotor activity was again measured

Stall sltcal Proc edm e.s

Treatment effects on measures of locomotor activity and lordosis were evaluated by analyses of vanance (ANOVA) and Duncan's multiple-range tests for post hoc compansons using SPSS/PC+ Additional within-groups t-tests and between-groups chl square analyses were used when appropnate. Two-tailed probabilities equal to or less than 0.05 were considered significant EXPERIMENT 1

DAY LENGTH AND FEMALE HAMSTER BEHAVIORS

Results

Total Daily Locomotor Activity

10000

/

8000

Long Photopenod

/ /

-H 0

/

6~0

0

"~

4000

t t

/

/

r-

Short Photoperiod

20oo

Before Ovx

After Ovx

21

After Estradiol

Experimental Conditions

FIG. 1 Wheel-running actiwty of female hamsters housed m a long (16L:8D) or short (8L:16D) photoperlod. Values plotted represent 12day averages for three treatment condmons administered to the same hamsters at &fferenttimes of photopenod exposure. Before ovanectomy (OVX): weeks9-10; after OVX: weeks 13-14, after Estradxol:weeks 1516. n = 10/group Estrad~ol faclhtated locomotor activity in the long, but not the short photoperlod group

Exposure of pineal intact females to a short photoperiod for 11 weeks caused uterine regression; uterine horn weights were slgmficantly lower among short, compared to long photopenod females. Mean values (mg _+ SEM) were 177.9 _+ 24 vs. 400.2 _+ 31, t(16.9) = 5.7, p < 0 001. Although estrous cycles were not monitored m these animals, the significant reduction of uterine weights among the short photoperiod females indicates they were anestrus at the time of ovariectomy. The mean number of wheel revolutions per day was lower among the short photoperlod females in all three phases of the experiment (Fig. I ) and these differences were significant during the first and last [before OVX: t(17.1) = 2.1, p = 0.05; after estradiol: t(15.9) = 3 8, p = 0.002]. Estradiol administration following ovariectomy had no facilitatory effect on locomotor activity m the short photopenod group, but did stimulate this behavior in long photoperiod females [after OVX vs. after estradiol: t(9) = 3.9, p = 0.004]. This influence of day length on locomotor actwity was not an artifact of&fferences in alpha. In fact, alpha was longer among the short photopenod hamsters during each of the three experimental phases (Fig. 2), but because variabihty m alpha was high, &fferences between the two photopenod conditions were not statistically significant. The temporal &stributlon of locomotor activity over an average day dunng the after OVX and after estradiol phases is depicted m Fig 3. These plots emphasize that the facihtatory effects of estrogen among the long photoperlod females were maximal dunng the first half of the night, although modest effects were ewdent throughout the remaining portions of the acttve period. In contrast, estrogen did not facilitate activity in the

Activity Period Duration

for 11 consecutive weeks beginning on week 7. Dunng week 11, females were ovariectomized and their uterine horns removed and weighed; 2 weeks later (week 13), all females received an estradiol implant. Photoperiodic and hormonal effects on locomotor actwity were assessed over three experimental phases; these consisted of 12 day blocks that 1. ended 1 day before ovanectomy (before OVX), 2. began 2 days following ovariectomy (after OVX), and 3. began 2 days following estradiol administration (after estradiol). The calculation of alpha during these three phases was based on an esUmate of each day's onset and terminatton of actwity, derived from visual mspection of actographs that required at least 25 wheel revolutions/5 min to generate a pen excursion. During week 17, a brief test to determine whether the estrogen-treated females would exhibit lordosis was performed. A sexually experienced male was placed in the female's home cage and the behavior of the pair was observed for 10 min beginning when the male's nose came within 1 cm of the female. The female was judged to be m lordosis when she became immobile for at least 5 s with a slight but rigid dOrSlflexlon of the vertebral column and elevatton of the perineum. These tests occurred during the subjective day (1300-1500 h), because at this time the nocturnal hamster is least sensitive to the activational effects of gonadal steroids, thus potentially most responsive to photoperiodic and pineal manipulations.

12

9

Short Photopenod

o~ -H ~3 O "1-

Long Photopenod s

Before Ovx

After Ovx

After Estrad,ol

Experimental Conditions

FIG 2. Averageduration of the actwe penod (alpha)during which wheel running was expressed. Alpha was longer for the short photopenod females but neither group differencesnor treatment effectswere significant. See Fig. l legend for procedural detaals

22

b,.\RP

TemporalPatternof DaffyLocomotorActw~ty 700

600

.~~

500

LongPhotopenod

400

300

200

+t

_.9.

100

0

I

I

0 >

700

e-

I

o

After

•

A f t e r Estradtol

Ovarlectom¥ .

.

.

.

600

¢o

500

ShortPhotopenod

400

300

200

100

0

lILt

,

I IL,

,

,

i

i

i

1

i

)

i

i

i

i

i

i

i

i

, -.

Hours FIG 3 Plots of the average distnbunon of wheel running dunng the 72 consecutive 20-ram time bins compnsmg a 24 h day. Panels depict long and short photopenod groups, showing the acnvit~ patterns of the same group of hamsters both after ovanectomy and after estradlol treatment Periods of hght and dark are shown under each plot See Fig 1 legend for procedural details

short photopenod group even though alpha was longer for these females (Fig 2) In the sexual receptwlty tests performed 3 weeks after females received estra&ol implants (week 17 of photoperiod treatment), 82% and 100% of the long and short photopenod hamsters displayed lordosis, respecnvely FXPERIMENT 2 The results of Experiment 1 demonstrated that exposure of female hamsters to a short photopenod significantly lmpmred the snmulatory effects of estrogen on locomotor activity. This replicates earlier results from another laboratory (58), although we used a d~fferent experimental protocol to evaluate photopeno&c influences on running-wheel activity. However, photopenod did not affect the expressmn of lordosis. The purpose of the second experiment was to determine whether the activity effect was dependent on an intact pineal gland and to further explore photoperiodic and pineal influences on the lordosis response. Spectfic Method~

Female hamsters were group housed for 6 weeks in 16L:8D (hghts on at 0500 h). They were then housed individually in the

\Ni)

I'()W[RS

same photopenod with access to running wheels h)r 2 ~cek~ After this they. were assigned to one of three groups, each eqm~ alent on ax erage dalb locomotor actl~ It3 The groups were long photopenod-pmealectomlzed (LP-PX) n - 16, ~hort photopenod-pmeatectomlzed (SP-PX), n 14, and short photopenodsham pmealectomlzed (SP-SPX), n 22 Three days after pineal surgery, running wheels ,~erc removed and the SP-PX and SPSPX hamsters were transferred to g l , 16D (hghts on at 0900 h, week I) During weeks 7-10, estrous cycles were monitored by daft3 inspection of vaginal &scharges to evaluate the effectiveness of pineal surgery. Females in the short photopenod were ehmmated from the experiment if they were pmealectomtzed but had disrupted estrous c~cles, or ffthe?y v~ere sham pmealectomtzed but continued to display normal estrous cycles At the start of week I 1, hamsters were ovanectom~zed and thmr uterine horns rcmo~ed and weighed, at the same lime they recmvcd rather an estradml or blank Sflasnc capsule The number of hamsters m each of the treatment groups lbr the estradml and blank condmons, respecnveb, was nme and sc~en (LP-PX). mght and s~x (SP-PX), and nine and seven (SP-SPX) Locomotor acUv~D measurements then resumed for the next 30 da~s {weeks I114) Treatment effects on actlvlt~ levels were e~aluated usmg a~erages derived from a 5-day period that began 20 days alter hamsters received thmr implants (~eek 14) Photopeno&c and hormonal effects on sexual recepUvny were assessed in two Afferent contexts Ftrst. lordosis was tested 3, 7, and 11 days after estradlol admmlstranon {week 11) After 30 days, the females were housed without access to acn~lty wheels and their estradlol capsules were removed The subsequent dechne m sexual responsiveness ~as assessed 1.2. 4, and 5 days later as the priming effects of estrogen &SSlpated. On the seventh da~, each hamster recmved an mjecnon of progesterone {500 ,zg SC), and lordosis was tested 3-4 h later (week 16) In each case, the lordoMs response was exaluated as described m Experiment I R e3 ult s

Pmealectom~zed hamsters m both photopenods (LP-PX; SPPX) continued to &splay 4-day estrous cycles from week 7 until the time of ovanectomy. Most pineal intact hamsters m the short photoperlod (SP-SPX) had either disturbed or absent estrous cycles, the s=x hamsters that continued to cycle normally were excluded from analysis. This photoperiodm disruption of ovarian function m 16 of 22 females was also evidenced by their atrophic uteri At the rime of ovanectomy, their uterine horn wexghts (mg -+ SEM) were significantly lower (258.8 _ 23) compared to pmealectomized hamsters m either the long (571.9 _+ 4 l ) or short (588 0 +- 39) photopenod [SP-SPX vs LP-PX. t( 18 7) = 6,7, p < 0.001; SP-SPX vs. SP-PX t(21) = 7 5, p < 0.001]. [reatment effects on average daily actlvny levels (days 2024 after hormone administration) were first evaluated by a 3 × 2 ANOVA, using photopenod/pmeal treatment and hormone c o n d m o n as the two variables A main effect of hormone was significant, F(1, 32) = 10.5, p = 0.003, and the interaction between photopenod/pmeal and hormone conditions was nearly so, F(2, 32) = 3.1, p = 0.057. Estrogen increased locomotor activity among pmealectomized females within each photoperiod [LP-PX: t(10 8) = 3.6, p = 0.004: SP-PX" t(11) = 2 4, p = 0.04]. In contrast, th~s estrogenic effect was absent among the pineal intact, short photopenod hamsters (SP-SPX; Fig. 4). The influence of treatment conditmns on alpha was determined by similar statlsncal analyses In this case, there was a significant main effect of photopenod/pmeal treatment, F(2, 32) = 12.7, p <

DAY LENGTH AND FEMALE HAMSTER BEHAVIORS

23

Total Dmly Locomotor Actwlty

BB

Estrad/ol Capsule

E m -H o _=

>o

EXPERIMENT 3

Experiment 2 provided evidence that photopenodic influences on estrogen facihtation of locomotor activity and lordosis were dependent on an intact pineal gland. The purpose of the present expenment was to investigate further the photoperio&c effect on lordosis. Hormones were admlmstered to long-term ovariectomized females by acute injections rather than chronic ~mplants because in Experiments 1 and 2, the potent estrogenic sumulatlon provided by Sllastic capsules may have minimized the expression of photopenodic effects on sexual receptivity.

Specific Methods .¢:

4000

2000

LP-PX

SP-PX

SP-SPX

Experimental Cond=tions FIG 4 Wheel-running activity shown by six groups of female hamsters housed m a long(LP, 16L8D) or short (SP, 8L. 16D)photopenod;ammals were either pmealectomlzed(PX) or sham-operated (SPX) dunng week 0 and ovariectormzed and given an estradlol or blank Sdastic implant SC during week I 1 Values represent 5-day averages taken dunng week 14 of photopenod exposure. Locomotor activity was sigmficantlyfacilitated by estradml in the pmealectomlzedgroups (LP-PX; SP-PX), but not m the females with intact pineal glands (SP-SPX).

Female hamsters were group housed upon arrival in 16L:8D (lights on at 0500 h) and after 9 weeks were divided into two equal groups based upon body weights; one-half of each group was pinealectomlzed (n = 26) and the other half sham pinealectomized (n = 24). Following a recovery penod of 7 days, 12 of the sham plnealectomized animals and 14 of the pinealectomized animals were transferred to 8L:16D (hghts on at 0900 h). Thus, four groups were formed dunng week 0: long and short photopenod conditions with pinealectomlzed and sham pinealectomxzed subgroups within each During week 2, all hamsters were ovanectomized; 6 weeks later (week 8), they were hormonally primed by an injection of 1 #g estradiol cyplonate (EC) and 250 #g progesterone 72 h later. Females were exposed for 2-3 mm to a sexually experienced male approxlmately 3 h after the progesterone mject~on; this accustomed females to our lor-

A c t i v i t y Period Duration 0.001, but neither the hormone condition alone nor its interaction with the photoperiod/pineal treatment was significant. Alpha was longer among short, compared to long photoperiod females, independent of pineal condition (Fig. 5; all relevant comparisons: p < 0.03). The temporal distribution of locomotor activity over an average 24-h period is shown in Fig. 6. These data confirm the findings of Experiment 1; the facditating effects of estradiol were expressed throughout the entire active period among the pinealectomlzed, long, and short photoperlod groups (Fig. 6; top and middle panels). This was not true for the pineal intact, short photoperiod females. In this condition, females with blank implants were on average more active than females treated with estradlol during many of the 20-min time bins throughout the night (Fig. 6; bottom panel). Ovariectomized females that received blank implants never exhibited lordosis on any of their sexual receptivity tests. Estrogen-stimulated females were unresponsive during their first receptivity test (day 3 after implantation) but lordosis was exhibited by some hamsters after 7 days. After 11 days, more than half the hamsters in each group d~splayed lordosis; neither photoperiodic nor pineal effects were present (Table 1). Estrogen implants were removed after they had been m place for 30 days; 5 days later, fewer than 25% of the females in any of the treatment conditions &splayed lordosis (Table 1). Progesterone was given after 2 more days; this increased the proportmn of females exhibiting lordosis among the pinealectomized females in both long and short photopenods but not among the pineal intact females in the short photoperiod. None of the latter group displayed lordosis, whereas half or more of the plnealectomized females became sexually receptive. The percentage of short photoperiod females exhibiting lordosis was significantly lower in the pineal intact group (Fisher's exact test, p = 0.029; Table l)

m

EstradlolCapsule

-~

BlankCapsule

12

=." -H

,= O "1" t¢o

LP-PX

SP-PX

SP-SPX

Experimental Conditions FIG. 5. Averageduration of the active period (alpha)during which wheel running was expressed. All short photopenod groups has alphas that were sigmficantlylonger than the alphas of the two long photopenod groups See Fig 4 legend for procedural detmls

KARP ~ N I ) P()WERS

24

800

T e m p o r a l Pattern of Dail, L o c o m o t o r A c t w ~ t y

600

400

~1~0 ~

[

BlankCapsule

0

200

0

I

I

I

)

I

I

I

,

.

E

.

.

.

.

,

,

,

I 800

~9

44 600

Short Photopenod-PX

0

_= 0 >

400

n-

IIII 800

600

elapsed or the lordosis score became 2, alter which the male ~as removed When the lordosis score was 2 dunng week l I tests (5 #g EC), the female was left in the testing chamber t\)~ another 90 s in the absence of the male, afte~ which brush stimulation alone was used to determine ~f the lordosis response could be relnltlated using onl> th~s suboptimal stimulus, rhls second phase of lordosts testing lasted for 90 s or until a lordosis response occurred and ~ts latency recorded Dunng week 13 tests (10 #g EC), brush stimulation was applied contmuall> for 60 s after lordosis had been induced in phase 2 to determine how long the lordosis response could be maintained

Short Photopenod-SPX

400

200

0

II FIG 6. Plots of the average dlstnbutlon of wheel-running behavior dunng the 72 consecuhve 20-ram Ume bins compnsmg a 24 h day Separate groups wtthm each treatment condition recewed either estradlol or blank capsules See Fsg 4 legend for procedural details

dosis testing procedures. On week 10, the mjectton sequence was repeated usmg a higher dose of EC (2 #g) but the same progesterone dose, followed by a lordosis test. Thts sequence was repeated on later weeks, again using the same dose of progesterone but increasing doses of EC (week 11. 5 ug; week 13 10 #g). Lordosts tests began by placmg the female mto a small Plextglas arena (30 × 36 × 30 cm) for a 5-min adaptation period before a stimulus male was introduced. When the male's nose came to within one cm of the female, timing began (time 0) and the female was lightly stimulated on the hind limbs and genttal region by an eyebrow brush when such stimulation was not prowded by the male. This lasted for 90 s or unttl lordosis was observed and the lordosis latency recorded. A latency of 90 s was asstgned when the female was unreceptive. The presence of lordosss was judged as m the precedmg expenments. In additaon, a lordosis quality score was assigned whtch reflected the rigldtty of the lordosis posture ( 0 - - n o lordosis: l--lordosis wtth some m o v e m e n t of the vibnssae, 2--lordosis with no m o v e m e n t of the vtbnssae). If the lordosis score was 2, brush sUmulation was terminated and the male removed; if the lordosis score was 1, brush sttmulatton continued either until the testing period

Results Photopenodlc condttlons had a slgmficant effect on the abthty of ovarian hormones to facihtate lordosis, but this effect was both dose and pmeal dependent On week 10 tests (2 #g EC), the percentage ofpmeal intact females exhibiting lordosis (quality score = I or 2) was slgntficantly lower in the short photopenod group [ 1/11 vs 7/12, ×z(i ) = 4 2, p = 0 04] This effect of photopenod was not slgmficant between the plnealectomlzed groups (5/12 vs 3/12; Table 2). Additional analyses on lordosis latencles and lordosis quahty scores were performed by a 2 × 2 A N O V A (photopenod by pineal condition) m which the data from both responders and nonresponders were included. These analyses Indicated that for both dependent vanables, mam effects were not stgmficant but the interaction between photoperiod and ptneal condition was, lordosis latency F(1, 43) = 5 5, p = 0.02, lordosis score. F(1, 43) = 5 1, p = 0 03. Post hoc tests showed that photoperlodtc condlttons were sxgntficant among the pineal intact females, for the short photoperlod hamsters, lordosis latencles were stgmficantly longer, t( 18 3) = 2 7, p : 0 02, and lordosis scores significantly lower, t(18) = 2 8, p ~ 0 01, than the> were for females in the long photoperiod. Stgnlficant dtfferences on these measures were not present among the plnealectomtzed females in the two photoperiods (Table 2) When the estrogen dose was increased to 5 #g EC (week 11), photopenodlc effects on lordosts were no longer apparent among the pineal intact antmals, even though the proportton of females respondmg was lower in the short (8/I 2) than in the long (12/ 12) photoperlod group. The proportion of plnealectomtzed females responding was generally high and not stgmficantly different between the long (9/11) and short (11/14) photopertods There were no significant effects on lordosis latenctes or quality scores, hkewlse, the more sensmve measure utthzed in phase 2 of testmg also failed to detect a photoperiodtc influence on lor-

TABLE l PROPORTION OF OVARIECTOMIZED FEMALE HAMSTERS DISPLAYING LORDOSIS Days at Estrogen SUmulatmn

Days Alter Estrogen Removed

Group

7

11

1

2

4

~

7 (+Prog)

LP-PX SP-PX SP-SPX

5/9 6/8 3/9

5/9 6/8 6/9

4/9 8/8 7/9

4/9 7/8 7/9

3/9 4/8 1/9

1/9 2/8 1/9

5/9 4/8* 0/9"

LP-PX. Long photopenod, pmealectomlzed SP-PX: Short photopenod, plnealectomlzed SP-SPX Short photopenod, sham pmealectomlzed * p = 0 029

DAY LENGTH AND FEMALE HAMSTER BEHAVIORS

25

TABLE 2 LORDOSIS RESPONSIVENESSOF OVARIECTOMIZEDFEMALE HAMSTERS AFTER ESTROGENAND PROGESTERONETREATMENT

differences in the amount of daily locomotor activity, it is surprising that long photoperiod activity levels were not reduced following ovanectomy (Fig. l; after OVX). Such reductions have been observed in hamsters after removing their ovaries (58), but this has not been a consistent finding (7). Interestingly, Pieper and colleagues (45) have shown that when females housed in a long photopenod are given daily melatonin injections to induce a short photopenod reproductive state, the expected disruption of estrous cycles eventually develops in sedentary hamsters, but estrous cycles persist If hamsters are given access to running wheels during the course of melatonin treatment. Of relevance to the present discussion is that even though both the vehicle and melatonln injected groups had normal estrous cycles and presumably equivalent average estradiol levels, the melatonm injected females were still significantly less active. Thus, under some circumstances, short photoperiod females exhibit reduced locomotor activity for reasons that do not depend on altered secretion of ovarian hormones. However, in addition to this steroid-independent reduction in behavioral responsiveness, such females are also clearly insensitive to the effects of exogenous estradlol. Estradlol treatment significantly facilitated wheel running in long photopenod females but was completely without effect among short photoperiod females (Figs. l, 3). Although the dose of estradlol we used most likely exceeded the physiological range, this in no way diminishes the significance of the complete estradlol insensitivity exhibited by the short photoperiod females. Experiment 2 replicated photoperiodic effects on locomotor activity using a between- rather than a within-groups design. Estrogen-treated females exposed to the long photoperlod had significantly higher activity levels compared to control hamsters receiving blank implants In contrast, estrogen-treated and control females exposed to the short photoperlod did not differ on this measure. However, the most important result was that this Influence of short photoperiod was eliminated by pinealectomy. That is, without their pineal glands, short photoperiod females regained their sensitivity to estradlol (Figs. 4, 6). Because we did not include a long photoperiod, sham pinealectomized group in Experiment 2, it could be argued that plnealectomy simply removed some inhibitory influence on the mechanisms affected by estradiol in stimulating locomotor activity, rather than functioning more generally to disrupt the process of photoperiodic time measurement. However, a comparison of results from Experiments 1 and 2 makes this possibility seem unlikely. The stImulatory effects of estrogen in Experiment 1 were demonstrated by comparing the same females, before and after estrogen treatment. Among these long photoperiod, pineal intact hamsters, the magnitude of the estradiol effect, as evidenced by both the increased number of wheel revolutions per 24 h (Fig. l) and by the altered pattern of nocturnal activity (Fig. 3) appeared nearly identical to the analogous effects observed among the long photopenod, pinealectomlzed females in Experiment 2 (Figs. 4, 6). This suggests that estrogen was functioning comparably among long photoperiod females, whether or not their pineal glands were intact. Thus, pineal gland secretions do not specifically modulate the effectiveness of estrogen in stimulating activity, but rather they mediate the discnminatlon of long from short days, and events subsequent to this process participate more directly in modifying the behavioral effects of estrogen. In summary, our findings indicate that exposure to a short photoperiod significantly diminishes the facllitatory effects of estrogen on locomotor activity and that these effects can be restored by removing the pineal gland. The ability of testosterone to facilitate locomotor activity in male hamsters is also Influenced by photoperiodic conditions (13), but whether

PinealIntact LongPP

PinealRemoved

Sho~ PP

LongPP

Sho~ PP

3/12 724_+93 0.5_+03

5/12 63.7_+102 0.7_+ 03

9/11 402_+9.6 15_+02

I 1/14 410_+ 9.5 1 4 _ + 07

10/12 25.4_+89 18_+02 218_+68

12/14 28.6_+ 84 1.8_+ 0.2 30.5_ 66

2 #g Estradlol Percent Resp 7/12 Lordlat 443_+120 Lordscore 12_+ 03

l/l l* 825_+ 75* 0.2_+ 02* 5 #g Estradlol

Percent Resp 12/12 Lordlat 24.7_+ 78 Lordscore 19_+ 0.1

8/12 40.2_+ ll 1 1.3_+ 02 10 #g Estradml

Percent Resp 11/11 Lordlat 11.5_+ 63 Lordscore 2.0_+ 00 Lorddur 42.3_+ 67

11/12 192_+ 7.8 1 9 + 0.1 37.4_+ 75

Long PP-16L:8D; Short PP-8L:I6D. Lordosis latencles and durations are m seconds, mean + SEM. See text for lordosis score definmon; values are mean _+SEM. * Significantly&fferent from long PP, pineal intact; p < 0 05.

dosis; the percentage of females in which lordosis could be reinitlated with brush stimulation alone was low (27%-43%) and no significant between-groups differences were present. This lack of photoperiodic or pineal effect was also characteristic of week 13 tests (10 #g EC); over 90% of the females in all four groups exhibited lordosis. The maximal lordosis duration elicited by brush stimulation alone did not differentiate between experimental treatments (Table 2). GENERAL DISCUSSION Our results confirm and extend earlier reports that short photoperiodic conditions modify the behavioral effects of ovarian hormones among female hamsters (2-4,8,12,16,27,58). Both the increase in wheel running following estrogen and the facilitation of lordosis following estrogen and progesterone were significantly reduced among pineal intact females housed in a short photoperiod. The new findings presented here demonstrate that photopenodic effects on these two different behaviors are eliminated or substantially reduced by removing the pineal gland. In Experiment l, gonadally intact females were less active if housed in a short, compared to a long, photopenod (Fig. l; Before OVX). Many investigators have demonstrated that running-wheel activity during the hamster estrous cycle is positively correlated vath estradiol levels (15,47,53). Although estrous cycles were not directly assessed in the present experiment, differences observed in utenne weights at the time of ovariectomy are consistent with the inference that the short photoperiod females had become anestrus. Thus, the less active females in the short photoperiod most likely had decreased systemic estradiol levels, evidenced not only by their reduced uterine weights, but also by other reports in which systemic estrogen levels were measured under these conditions (1,59). However, if differences in endogenous hormone levels were Important for producing

26

the reduced hormonal sensitivity of short photoperlod males would be eliminated by plnealectomy is not known. Our results may not be in complete agreement w~th other findings (53), These investigators studied the hormonal control of locomotor activity cycles in female hamsters, focusing on clrcadmn regulation and the interactive effects of estradlol and progesterone. Estradiol influences the circadian system of female hamsters in a vanety of ways (41 ). Takahashl and Menaker (53) confirmed that estradiol shortened the period of the locomotor activity rhythm and decreased the vanabllity in activity onset times, but also demonstrated that this hormone increased the amount of activity expressed within each cycle It is important to note that the females used were blinded and that hormone effects were evaluated 1 to 3 months later Dunng much of the t~me that the presence or absence of estradlol was modulating these vanous features of the actlVlt~y cycle, the hamsters were functionally in short photoperlodlc conditions That is, if these females had been gonadally intact, ovanan cychclty would have ceased because they had been without photic input tbr up to 16 weeks Because estrogen significantly increased locomotor activity levels among these bhnded females, this result seems mconsistent with ours It is possible that differences in the way that locomotor activity was quantified between the two experiments might be relevant. We counted each wheel revolution by computer, whereas Takahashi and Menaker (53) analyzed actograph plots and determined the number of time penods dunng which some acnvlt~ was present Estradlol may have different effects on activity, depending on whether females are entrained to a short photopenod as in the present expenments or are allowed to free run in the absence of any photlc input. Another factor to consider is the dose of estradlol administered, if we had provided h~gher circulating levels of estradlol, some snmulatory effects on activity might have been observed among our short photopenod hamsters These caveats make ~t clear that the way locomotor acUvlty is evaluated and quantified can influence the amplitude of any photopenodlc and/or estrogenic effect obtained. However, our demonstration that short photopenod exposure modulates the facflltatory effects of estrogen, based on actual counts of daily wheel revolutions, and that this photopenodic influence is ehmmated by pinealectomy (Figs 1, 4) reinforces the general hypothesis that responsxvity to gonadal hormones can be significantly affected by photopenod duration Hormonal facilitation of sexual recephvlty was influenced by photopenod, but this effect was variable In Experiment 1, lordosis was tested after Sflast~c capsules of estradiol had been in place for 30 days: the majonty of females m both the long and short photopenods became sexually receptive, thus providing no evidence that hormonal responsiveness had been reduced among the short photopenod females However, these tests took place dunng week 17 of photoperiod exposure so the development of photorefractorlness might have contributed to this lack of effect In Experiment 2, lordosis was tested during earher weeks to reduce the potential effects of photorefractorlness and much sooner after estradiol was administered (days 3, 7, and 11 ). Th~s shorter time interval between estrogen treatment and subsequent testing resulted m a smaller percentage of females responding (Table 1) compared to the longer interval that we used in Expenment 1. Although we reasoned that the use of suboptimal conditmns for inducing lordosis (37) would increase the probability of observing photopenodxc effects, this was not the case. Photopenodac condmons still had no significant influence on the percentage of animals that became sexually receptive. However, when the estradiol stimulus was later removed and ItS facilitating effects on lordosis allowed to d~sslpate over the following week, the ability of progesterone to synerglze with this

KARP , \ \ 1 ) P~)~vERS

mm,malb estrogen-primed lordosis mechanism was mlluenced by pholoperlodlc conditions Progesterone was completely ineffective among the short photopenod females with functional pineal glands, whereas over half the hamsters without their plneals m both the short and long photopenod displa.~ed lordosis (Table 11 This shows that the hormonal mducnon of lordosis can be reduced b.~ short photopenod treatment and that th~s altered responsiveness is influenced by the pineal gland l'h~s same conclusion is also supported by the results of Experiment 3 in which estrogen injections rather than Silastlc ~mplants were used to stimulate receptlvlt.~ The findings of Experiment 3 mdlcate that the photopenodic suppression of hormone-stimulated lordosis was both dose and pineal dependent. When females received 2 p.g of estrogen, photopenod effects were present among pineal intact but absent among pInealectomized hamsters ITable 2) On later tests with higher estrogen doses, all groups responded equivalently It could be argued that the loss of photopenodlc effects with the 5 and 10 #g doses of estradlol cyplonatc was due more to the onset of photorefractorIness than to the masking effects of increased hormone dose These two higher doses were given on weeks 1 I and 13, respectively, when photopenodic effects could have been diminished because of the length of time that females had been contmuousb exposed to the 8L 16D photoperiod However, in Experiment 2, photopenodlc effects on lordosis occurred dunng week 16, and effects on estrogen-stimulated locomotor activity were evident during week 14 These findings argue against the possiblht) that the loss of photopenodlc effects m Experiment 3 with the higher doses of estrogen was due to photorefractonhess

Two features of our results are inconsistent with earlier findlngs Badura and her colleagues (4) reported that photoperlodlc effects on female hamster lordosis were evident when estrogen alone was used but that progesterone treatment of estrogenpnmed females overcame this photopenodlc suppression of lordosis Addmonally, the pineal gland was not required for these behavioral effects of short photopenod exposure (2). The discrepancy with progesterone treatment is probably not significant and may represent differences in hormone responsiveness between groups of hamsters Other investigators have observed photopenodlc effects when both estrogen and progesterone have been used to stimulate lordosis (8,27) However, the discrepanc~ in the role of the pineal is a more substantml difference The fact that photopenodlC conditions can have effects on mammahan neuroendocnne regulation m the absence of the pineal is certainly not without precedent, this has been reported for a variety of endocnne and metabolic responses For mstance, pinealectomy does not completely ehmlnate photoperxod-lnduced changes In energy metabolism (5,17,25), prolactln secretion (9), pelage charactenstxcs (50), or neurotransmltter metabolism (52) Although retinal lnnervatmn of the suprachmsmatac nuclei of the hypothalamus is essential for the photlc modulation of pineal secretory activity, retinal efferents to other dlencephahc and basal forebraln areas in hamsters have been described (29,44,61) and could be involved in mediating these pineal independent effects of photoperiodlc change. Nevertheless, the existence of pineal independent, photopenodlc effects on behaviors functionally hnked to reproductive competence appears to constitute an exceptmn to the general mammalian pattern. There were numerous procedural differences between our experiments and those reported by Badura and Nunez (2), but attributing our dxffenng results to such factors provides little satisfaction One variable that may be Important concerns the absence an their study of a long photoperiod, pmealectomized group We have found that pinealectomy in long photopenod

DAY LENGTH AND FEMALE HAMSTER BEHAVIORS

27

hamsters is occasionally associated with lordosis deficits (see Table 2); this perhaps is caused by damage to underlying sites in the dorsal midbrain that have been implicated in lordosis control mechanisms (48). Such damage could have contributed to the lordosis impairments of the pinealectomized, short photoperiod females reported by Badura and Nunez (2). How short photoperiods attenuate the effects of ovanan hormones on female hamster behavior is unknown. To the extent that the participation of the pineal is required, its secretion of melatonin is probably involved, although this remains to be shown directly for the behaviors investigated here. There has been considerable interest in locating melatonm binding sites m the CNS (22,40,51) in order to understand better how melatonin can affect response systems influenced by photoperiodic change. In Syrian hamsters (60), melatonin binding sites overlap with areas known to contam steroid-concentrating neurons (11,18,31,42) and it has been tempting to speculate that photoperiod and/or melatonin might affect the funcUonal capacity of steroid receptors. The sites where estrad~ol facilitates locomotor activity are not known in hamsters, but estradiol implants in the medml preopt~c area of rats increase running-wheel actlwty (10,14,57). Estrogen and progesterone facihtate lordosis m hamsters by acting in the hypothalamus and mldbrain (19,35,36,48,49,54). Photoperiodic conditions might influence the functioning of either one or both of these hormone response systems. Bittman and colleagues (8) examined photoperiodlc effects on the con-

centration of nuclear estradiol receptors and cytoplasmic progesterone receptors in a block of tissue containing the hypothalamus and preoptic area after 2 days of estradiol treatment. They found no effects on these measures even though their short photoperiodic treatment did reduce lordosis responsiveness. Because photoperiodic effects on steroid response systems, if present, most likely are restricted to regaonally specific subsets of hormone-concentrating neurons, techniques affording greater anatomical resoluUon will be necessary to address this issue. Lawson et al. reported that reduction of short photopeno&c conditions m ovanectomized hamsters by appropriate melatonin injections significantly reduced the number of cells in the medml preoptic area expressing immunoreactive estradiol receptors (32) and significantly decreased the steady state levels of estradiol receptor m R N A (33). This would be consistent with a diminished influence of estradiol in facihtatlng locomotor activity among females housed in a short photoperiod, but whether these estrogen receptor effects were restricted to the medial preoptic area or might have occurred in other regions as well was not reported. It should be emphasized, however, that photoperiodic effects could be exerted on mechamsms that in no way change the functional characteristics of hormone receptors, but alter m some other manner the responsiveness of neuroendocrine mechanisms that subserve the behaviors in question. ACKNOWLEDGEMENTS This study was supported by HD 14535 to J.B P. J D.K was supported by predoctoral training grant HD 07043.

REFERENCES 1. Albers, H. E.; Moline, M. L.; Moore-Ede, M. C. Sex differences m orca&an control of LH secretion. J Endocnnol. 100:101-105; 1984 2. Badura, L. L., Nunez, A. A Photoperiodlc modulation of sexual and aggressive behavior m female golden hamsters (Mesocrtcetus auratus): Role of the pineal gland. Horm Behav 23:27-42; 1989. 3. Badura, L. L.; Slsk, C. L.; Nunez, A. A. Neural pathways revolved m photopenod~c control of reproductwe physiology and behavior in female hamsters (Mesocrwetus auratus) Neuroendocnnology 46: 339-344; 1987 4 Badura, L. L.; Yant, W. R.; Nunez, A. A. Photopenodlc modulation of steroid-induced lordosls in golden hamsters. Physiol. Behav 40: 551-554; 1987. 5. Bartness, T. J ; Wade, G N. Photopeno&c control of body weight and energy metabolism in Synan hamsters (Mesocrtcetus auratus): Role of pineal gland, melatonm, gonads and diet Endocrinology 114:492--498; 1984. 6. Bartness, T. J., Wade, G. N. Photopenodic control of seasonal body weight cycles. Neurosci. Biobehav. Rev. 9:599-612; 1985. 7 Bhatia, A. J.; Wade, G N. Energy balance in pregnant hamsters. No role for voluntary exerose9 Am. J Physiol (in press) 8 Blttman, E. L.; Hegarty, C. M; Layden, M Q.; Jonassen, J. A. Influences ofphotopenod on sexual behaviour, neuroendocnne steroid receptors and adenohypophysial hormone secretion and gene expression m female golden hamsters J. Mol Endocnnol. 5:15-25; 1990. 9 Blask, D. E.; Leadem, C. A ; Orstead, M.; Larson, B. R. Prolactln cell activity m female and male Syrian hamsters: An apparent sexually dimorphlc response to light deprivation and pmealectomy Neuroendocnnology 42:15-20; 1986. 10. Colvm, G. B.; Sawyer, C. H. Induction of running activity by lntracerebral implants of estrogen in ovariectomlzed rats Neuroendocnnology 4.309-320, 1969. 11. Doherty, P. C.; Sheridan, P J. Uptake and retention of androgen in neurons m the brain of the golden hamster Brain Res 219:327334; 1981. 12. Elliott, A S., Nunez, A. A Photoperiod modulates the effects of steroids on soclosexuai behaviors of hamsters. Physiol. Behav 5 I. 1189-1193; 1992.

13. Elhs, G. B ; Turek, F W. Testosterone and photoperiod interact to regulate locomotor activity in male hamsters. Horm Behav. 17:6675; 1983 14 Fahrbach, S. E.; Metsel, R. L ; Pfaff, D W. Preoptlc implants of estradlol increase wheel runmng but not the open field activity of female rats. Physiol. Behav 35:985-992; 1985. 15 Flnkelstem, J. S.; Baum, F. R., Campbell, C. S. Entrainment of the female hamster to reversed photoperiod: Role of the pineal. Physiol. Behav. 21:105-11 l; 1978 16. Fleming, A. S.; Phllhps, A., Rydall, A., Levesque, L. Effects ofphotoperiod, the pineal gland and the gonads on agonistic behavior in female golden hamsters (Mesocrwetus auratus). Physiol. Behav. 44: 227-234; 1988. 17. Fleming, A. S ; Scardlcchlo, D.; Scar&cchlo, L. Photopenodic and pineal effects on food intake, food hoarding, and body weight m female Syrian hamsters. J. Biol. Rhythms 1:285-301; 1986. 18. Fralle, I. G ; Pfaff, D. W.; McEwen, B. S. Progestm receptors with and without estrogen induction in male and female hamster brain. Neuroendocnnology 45:487-491; 1987. 19. Frye, C. A ; Mermelstein, P G., DeBold, J. F Evidence for a nongenomlc action of progestms on sexual receptw~ty an hamster ventral tegmental area but not hypothalamus Brain. Res. 578:87-93; 1992. 20. Goldman, B. D The physiology of melatonm in mammals Pineal Res Rev 1:145-182; 1983. 21. Hastings, M H Neuroendocnne rhythms. Pharmacol. Ther. 50: 35-71; 1991. 22. Hastings, M. H., Maywood, E S., Ebllng, F J. P ; Wilhams, L. M ; Tltchener, L. Sites and mechanism of action of melatomn m the photopenodic control of reproduction. Adv. Pineal Res. 5:147-157; 1991 23 Hastings, M. H., Walker, A P ; Herbert, J. Effect of asymmetncal reductions of photopenod on pineal melatonm, locomotor activity and gonadal condition of male Syrian hamsters. J. Endocnnol 114" 221-229; 1987 24 Hastings, M. H.; Walker, A. P.; Powers, J B.; Hutchlson, J ; Steel, E. A ; Herbert, J. Differential effects of photopenochc history on the responses of gonadotrophlns and prolactin to mtermechate daylengths in the male Syrian hamster. J. Biol. Rhythms 4:335-350, 1989.

28

25 Hoffman. R A, Davldson. K , Steinberg, K. Influences of photopenod and temperature on weight gain, food consumption, fat pads, and thyroxine m male golden hamsters Growth 46 150-162, 1982 26 Hoffman, R. A, Rmter. R J Rapid plnealectom} m hamsters and othersmaU rodents Anat Rec 153 19-22, 1965 27 Honrado, G I, Pacik, L, Fleming, A S The effects of short day exposure on seasonal and circadian reproductive rhythms of female golden hamsters Physml Behav 50 357-364. 1991 28 Illnerova, H Entrainment ofmammahan c~rcadmn rhythms m melatonln production by light Pineal Res Rev 6 173-217. 1988 29 Johnson, R F , Monn, L P , Moore, R Y Retmohypothalamm projections m the hamster and rat demonstrated using cholera toxin Brain Res 462 301-312, 1988 30 Karp, J D , Dixon, M E, Powers, J B Photopenod history, melatonm, and reproducUve responses of male S?man hamsters J Pineal Res 8 137-152, 1990 31 Kneger, M S, Morrell, J I, Pfaff, D W Autoradlographlc locallzaUon of estradlol-concentratmg cells m the female hamster brain Neuroendocnno[ogy 22 193-205, 1976 32 Lawson, N O , Wee, B E F , Blask, D E, Castles, C G , Sprlggs, k L, Hill, S M Melatonln decreases estrogen receptor expressmn in the medial preoptlc area of inbred (LSH/SsLak) golden hamsters Bml Reprod 47 1082-1090, 1992 33 Lawson, N O,Wee, B E F,Drane, J G,HIII, S M Melatonm suppression of estrogen receptor protein and messenger RNA m ovanectomlzed outbred LVG Synan hamsters Soc Neuroscl Abstr 18 112, 1992 34 Lincoln, G A , Short, R Seasonal breeding Nature's contraceptive Rec Prog Horm Res. 36 1-52, 1980 35 LlSClOttO,C A., DeBold, J. F Ventral tegmental lesmns impair sexual receptivity m female hamsters Brain Res. Bull 26'877-883, 1991 36 Measel, R L, Fralle, I G , Pfaff, D W Hypothalamlc sites of progestm actmn on aggression and sexual behavmr in female Synan hamsters Physml Behav 47 219-223, 1990. 37 Melsel,R k , Sterner, M R , Dmkman, M A DlfferenUal hormonal control of aggression and sexual behavior m female Syrian hamsters. Horm Beha~ 22453-466, 1988 38 Mmrmckl, M , Karp, J D , Powers, J B Pmealectomy prevents short photopenod lnhlbmon of male hamster sexual behavior Physlol Behav 47 293-299. 1990 39 Mmrmckl, M , Posplchal, M W , Powers, J B Short photopenods affect male hamster sociosexual behaviors m the presence and absence of testosterone Physlol Behav 47 95-106, 1990 40 Morgan, P J , Williams, L M Central melatonln receptors. Implications for a mode of achon Expenenua 45:955-965, 1989 41 Monn, L P Blologmal Rhythms In: Siegel, H. I., ed The hamster Reproduction and behavior New York Plenum, 1985.323-361 42 Munn, A R , Sar, M., Stumpf, W E Topographic dlstnbuuon of progesterone target cells m hamster brain and pituitary after injection of[~H]R5020 Brain Res 274 1-10, 1983 43 Nelson, R J , Badura, L L, Goldman, B D Mechanisms of seasonal cycles of behavior Annu Re~ Psychol 41 81-108:1990 44 Plckard, G, E, Sfl~erman, A J Direct retinal projectmns to the hypothalamus, preform cortex and accessory optic nuclet m the

K~kRP ANI) I'()WFR ~,

45 46

47

48

49

50

51 52

53

54 55

56

57

58.

59

60

61

golden hamster as demonstrated b~¢ a sensitive anterograde horseradish peroxldase techmque J Comp Neurol 196 155--172, I t)81 Pleper, D R , Borer, K. T , l.obock~, ( A, Samuel. 1) Exercise inhibits reproductive quiescence induced by exogenous melatonm m hamsters Am J Physlol 255 R718-R723, 1988 Relter, R J Mechamsms of control of reproductive physlolog~ bx the pineal gland and its hormones A.dv Pineal Res 2 100-12~ 1087 Rlchards, M P M AChVlty measured by running wheels and observatmns dunng the estrous cycle, pregnancy, and pseudopregnancy in the golden hamster Atom Behav 14 450-458, 1066 Rose, J D Bralnstem influences on sexual behavior In Klemm, W R , Vertes, R P, eds Bramstem mechanisms of behavior New York Wlle~-Llss, 1990'407-464 Siegel, H 1, Senatore, A, Rogers. S, Ahdmh, H B Sexual receptlvlti~ m hamsters Brain nuclear estrogen and cytosohc progestm receptors after single and multiple steroid treatments and during the estrous cycle Horm Behav 23 173-184, 1989 Smale, L, Dark, J , Zucker. I Pineal and photopenodlc influences on fat deposition, pelage, and tesucular activity m male meadow voles J Blol Rhythms 3 349-345 1988 Stankov, B, Fraschlnl, F, Rmter, R J Melatonm binding sites in the central nervous system Brain Res Rev 16 245-256, 1991 Steger. R W , Rmter, R J , Sller-Khodr, T M. Interactions of p~nealectoms, and short-photopenod exposure on the neuroendocnne axis of male Syrian hamsters Neumendocnnology 38' 158-163, 1984 Takahashi, J S, Menaker, M InteracUon ofestradtol and progesterone Effects on mrcadmn locomotor rhythm of female golden hamsters ~m J Physlol 239 R497-RS04, 1980 TakahashL L K Hormonal regulation of SOClOSexual behavmr m female mammals Neuroscl Bmbeha~ Rev 14 403-413, 1990 ]-amarkm, L, Bmrd, C J , a.lmmda, O F X Melatonm A coordlnatmg signal for mam mallan reproduction? Science 227 714-720, 1985 Underwood, H, Goldman. B D Vertebrate clrcadmn and photoperiodic systems Role of the pineal and melatonm J Blol Rhythms 2 279-3t5:1987 Wade, G N , Zucker, I Modulation of food intake and locomotor actl~lt) )n female rats by diencephahc hormone implants J Comp Ph~rslol Psychol 72 328-336, 1970 Wldmamr, E P, Campbell, C S Interactmn ofestradml and photopenod on acUvlty patterns In the female hamster Physml Beha~ 24 923-930, 1980 Wldmamr, E P, Campbell, C S Fhe interaction of estradml and daylength in modifying serum prolactln secretion m female hamsters EndocrlnologT~ 108 371-376, 198t Williams, k M , Morgan, P J , Hastings, M H , Lawson, W , Davldson, G , Howell, H, E Melatonm receptor sites in the Synan hamster brain and pituitary Locahzatlon and charactenzatmn using [~-'~l]lodomelatomn J Neuroendocnnol 1 315-320, 1989 ~oungstrom, T G , Wmss, M L, Nunez, A A Reunofugal projecuons to the hypothalamus, anterior thalamus and basal forebraln m hamsters Brain Res. Bull 26 403-411, 1991