Appetitive and Consummatory Male Sexual Behavior in Japanese Quail Are Differentially ...

16 Pages·1998·0.44 MB·English

TheJournalofNeuroscience,August15,1998,18(16):6512–6527
Appetitive and Consummatory Male Sexual Behavior in Japanese
Quail Are Differentially Regulated by Subregions of the Preoptic
Medial Nucleus
Jacques Balthazart,1 Philippe Absil,1 Martin Ge´rard,1 Didier Appeltants,1 and Gregory F. Ball2
1LaboratoiredeBiochimie,Unite´ deRecherchesenNeuroendocrinologieduComportement,Universite´ deLie`ge,
B-4020Lie`ge,Belgique,and2DepartmentofPsychology,BehavioralNeuroendocrinologyGroup,JohnsHopkins
University,Baltimore,Maryland21218
Centraltestosteronearomatizationisrequiredfortheactivation acterized. Lesions damaging the POM completely abolished
ofbothappetitive(ASB)andconsummatory(CSB)malesexual CSB and also significantly decreased ASB. Lesions of the
behavior in Japanese quail. There are two major clusters of rostral BST had no effect on ASB, but moderately decreased
aromatase immunoreactive (ARO-ir) cells in the rostral fore- CSB. Detailed anatomical analysis revealed that lesions of a
brain;theseoutlinethenucleuspreopticusmedialis(POM)and subdivisionofthePOMjustrostraltotheanteriorcommissure
thenucleusstriaeterminalis(BST).Weinvestigatedtheroleof specificallyimpairCSB,whereaslesionsthataremorerostralto
thesenucleiintheregulationofASBandCSB.Appetitivemale this subdivision induce a severe deficit in ASB. These data
sexualbehaviorwasmeasuredwiththeuseofalearnedsocial indicatethatdifferentsubregionsofthePOMregulateASBand
proximityprocedurethatquantifiedthetimespentbyamalein CSB in a somewhat independent manner, whereas the BST is
frontofawindowwithaviewofafemalewhowassubsequently onlyimportantintheregulationofCSB.
releasedintothecage,providinganopportunityforCSB.Males
firstacquiredtheresponseandthenreceivedbilateralelectro- Keywords:appetitivesexualbehavior;consummatorysexual
lyticlesionsaimedatthePOMorBST,followedbyretestingfor behavior; medial preoptic area; bed nucleus striae terminalis;
ASB and CSB. Brain sections were stained for ARO-ir, and testosterone aromatization; electrolytic lesions; learned social
lesionstothetwoARO-ircellgroupswerequantitativelychar- proximityresponse;preopticareasubdivisions
Male sexual behavior includes appetitive and consummatory rewarded with access to females (Everitt, 1990, 1995; Kondo et
components(Beach,1956;BalthazartandBall,1997,1998).Some al.,1997;Liuetal.,1997).ThesestudiessuggestthatthemPOA
stereotyped behaviors result in a functional outcome that is integratessensoryinputsneededfortheactivationofcopulation
associated with a reduction in motivation; these constitute con- butappetitivecomponentsofthemalesexualresponseareregu-
summatory behaviors such as copulation. Other more variable latedbypathwaysindependentofthemPOA(Everitt,1995;Liu
behaviors allow an individual to converge on this functional et al., 1997). There are some inconsistencies, in that there is
outcome; these are appetitive behaviors such as seeking out a evidence that lesions to the mPOA can impair measures of
female(TimberlakeandSilva,1995).Experimentalstudieshave appetitive male sexual behavior (Edwards and Einhorn, 1986;
revealed dissociations between aspects of the neural system reg- Paredes et al., 1993). An alternative hypothesis suggests that
ulatingappetitiveandconsummatorycomponentsofmalesexual dopamine acting in the mPOA enhances the processing of sen-
behavior. In rats, lesions to the medial preoptic area (mPOA) sations from the genitals as well as olfactory cues from estrous
eliminatemale-typicalcopulatorybehavior(HeimerandLarsson, females and redirects behavior in favor of sex-related activities
1966;MeiselandSachs,1994).Theseanimalsstillexhibitappet- (Hull,1995;Hulletal.,1995).
itive behaviors in that they pursue females and acquire learned In quail, appetitive and consummatory aspects of male sexual
responsestogainaccesstofemales(Everitt,1990,1995).Damage behaviorareactivatedbyestrogensproducedinthebrainviathe
tothemPOAdoesnotimpairotherappetitivemeasuressuchas local aromatization of testosterone (Balthazart and Surlemont,
penileerectionsinresponsetoremotecuesfromestrousfemales 1990b;Balthazartetal.,1995,1997a).Therefore,thelocalization
(Liu et al., 1997). Lesions to the medial amygdala or the bed of brain aromatase can guide one to components of the neural
nucleus of the stria terminalis (BST) decrease noncontact erec- circuitregulatingthesebehaviors.Thenucleuspreopticusmedi-
tionsortheabilityofmalestoacquirelearnedresponsesthatare alis (POM) and the BST contain a high number of aromatase-
immunoreactive cells (Panzica et al., 1996). Lesioning the POM
ReceivedMarch2,1998;revisedMay28,1998;acceptedJune1,1998. profoundlyimpairscopulatorybehavior(BalthazartandSurlem-
ThisworkwassupportedbyaNationalInstituteofMentalHealthgrant(R01 ont,1990a),butappetitivebehaviorhasnotbeeninvestigated.In
MH50388)toG.F.B.andJ.B.,andbygrantsfromtheBelgianFondsdelaRecherche thisstudy,electrolyticlesionsweredirectedatthePOMorBST,
FondamentaleCollective(Nbr.9.4565.96F),theUniversityofLi`ege(Cr´editssp´e-
andmalesexualbehaviorwasmeasured.Appetitivemalesexual
ciaux),andtheGovernmentoftheFrenchCommunityofBelgium(ActionCon-
cert´ee93/98–171)toJ.B.ThecollaborationbetweenJ.B.andG.F.B.wassupported behavior was assessed by a learned social proximity response
byaNATOcollaborativeresearchgrant(CRG910526). (Balthazart and Ball, 1997) and by measuring rhythmic cloacal
CorrespondenceshouldbeaddressedtoJacquesBalthazart,LaboratoryofBio-
chemistry,UniversityofLi`ege,17placeDelcour(Bat.L1),B-4020Li`ege,Belgium. sphincter muscle movements in reaction to the visual presenta-
Copyright©1998SocietyforNeuroscience 0270-6474/98/186512-16$05.00/0 tionofafemale(Seiwert,1994).Consummatorysexualbehavior
Balthazartetal.•PreopticSubdivisionsandMaleSexualBehavior J.Neurosci.,August15,1998,18(16):6512–6527 6513
was assessed by observing copulatory behavior. These studies introducedinourlaboratoryhavebeenvalidatedanddescribedindetail
demonstrate that the POM is involved in the activation of con- (Balthazartetal.,1995,1997a,b;BalthazartandBall,1997;Castagnaet
al.,1997).Theyareonlybrieflysummarizedbelow.
summatory and appetitive aspects of male sexual behavior. Le-
Two-compartmenttestcages.Four,two-compartmentcageswereused
sions to the BST also impaired copulatory behavior, but there inthisstudy.Thelargerofthetwocompartmentswas90cmwide390
werenoeffectsonappetitiveaspectsofmalesexualbehavior. cmdeep350cmhigh.Asmallercompartment,forstimulusfemales,was
20 cm wide 3 26 cm deep 3 24 cm high and was centered on the left
MATERIALS AND METHODS lateralwallofthemaincageandseparatedfromitbyaverticallysliding
door, 20 cm wide 3 20 cm high, that could be controlled remotely by
Subjects. Experiments described in this paper involved male Japanese
quail (Coturnix japonica), age ;3 weeks, bought from a local breeder s1trcimngwsiadned3pu1l5leycms.Ahigsmh,aclultnainrrtohwewpliynwdoowod()cownassisloticnagteodfianvtehreticmaildsdlilte,
(DujardinFarms,Liernu,Belgium).Aftertheirarrivalinthelaboratory,
of this door and provided the male with limited visual access to the
the young birds were housed in groups in brooders. All subjects were
female. This window could be closed by an opaque swinging plywood
alwaysprovidedwithfoodandwateradlibitumandweremaintainedon
panel attached by a hinge just above the door. The lower part of that
a photoperiod simulating long summer days (16 hr light/8 hr dark)
panel was attached to a string and pulley system that allowed remote
throughout the experiments. Sexually mature stimulus females used
duringthebehavioraltestswerepurchasedatthesamedealerandwere liftingofthepanel.Onesquareareaofthefloor(30330cm),locatedin
housedunderthesameconditionsasthemales. themiddleofthelateralleftwall(infrontofthedoor/window),repre-
AllexperimentalprocedureswereinagreementwiththeBelgianlaws sentedthetestareaforbirdposition.Whenthewindowwasopen,the
ontheProtectionandWelfareofAnimalsandtheProtectionofExper- male located in the main chamber could see the female located in the
imentalAnimalsandtheInternationalGuidingPrinciplesforBiomedical lateralchamberonlyifhestoodinfrontofthewindowinthisarea.This
Research involving animals published by the Council for International squareareawasmountedonfourspringsandfourmicroswitches(onein
OrganizationsofMedicalSciences.Theprotocolswereapprovedbythe each corner) wired in parallel and powered by a 4.5 V battery so that
SupervisorofAnimalCarefortheUniversityofLi`ege. depression of any of these switches generated a positive signal. The
Castration and endocrine treatments.All male subjects were castrated output signals were digitized and sent to a MacIntosh computer using
undercompleteanesthesia(15mg/kgHypnodil;JanssenPharmaceutica, commercially available hardware and software. A computer program
Beerse,Belgium)within1weekaftertheirarrivalinthelaboratory.The writteninBasicrecordedduringtheobservationperiods(5minperiods;
twotesteswereremovedthroughaunilateralincisionbehindthelastrib seebelow)thetotaltimespentbythebirdinthetestareaandthenumber
asdescribedpreviously(BalthazartandSchumacher,1984).Twoweeks oftimesthatthebirdenteredthisarea.Thepresenceofabirdinthetest
later,approximatelythree-quartersofthebirdsreceivedoneSILASTIC area(i.e.,microswitchesactivated)wassampledinthetestareaofeach
implant(catalog#602–252;DowCorning,Midland,MI)(1.57mminner cageonceeverysecond.
diameter;2.41mmouterdiameter;length520mm)filledwithcrystal- Behavioraltests:generalprocedure.Fourtestswerealwaysruninpar-
line testosterone. The rest of the subjects served as controls and were allelinthefourexperimentalcages.Eachlastedatotalof25min.Atthe
implanted with a similar capsule that was left empty. Capsules were beginning of the test, one male was introduced into the main chamber
implanted subcutaneously in the neck region. At that time, all subjects and one stimulus female was placed in the adjacent smaller cage. The
weretransferredtoindividualcageswheretheyremaineduntiltheendof window between the two compartments was closed at that time. Birds
the experiments. Throughout the experiment, birds were periodically weregiven5mintohabituatetothenewenvironment.Thepositionof
weighed to the nearest gram, and the size of their cloacal gland was themalewasthenrecordedcontinuouslyduringthenext5minperiod
measured with calipers (cloacal gland area 5 largest length 3 largest withthewindowstillclosed(pretest).Thewindowwasthenopened,and
widthinsquaremillimeters).Thisglandisanandrogen-sensitivestruc- the position of the male was again recorded for 5 min (time at the
ture(Sachs,1967),anditssurfacethereforeprovidesasensitivemeasure window).Duringthese5min,abeeperwasactivatedandemittedaweak
oftheendocrinestateofthebirds(FollettandMaung,1978;Delvilleet sound every 5 sec. At each beep, the observer recorded whether the
al.,1985).Thesedataconfirmedtheefficacyofthetestosteronereplace- subjectwasactuallylookingthroughthewindow.Lookingbehaviorwas
ment(thespecies-typicalenlargementofthecloacalglandwasregularly definedasastereotypedpositioningoftheheadthatallowsthesubjectto
observedinallsubjects)aswellastheabsenceofadverseeffectsofthe focus on the female through the window. This point sampling (Martin
experimentaltreatmentsonthegeneralhealthconditionofthesubjects
and Bateson, 1986) provided a score for the looking behavior ranging
(allbirdsslowlygainedweightwithtime,asnormallyobservedinthisage
from 0 (never observed) to 60 (behavior present at every beep). It has
range).Nofurthermentionofthesedataaremadeinthispaper.
been demonstrated previously that the social proximity response (time
Behavioral screening and acquisition of the social proximity response.
spentatwindowandfrequencyoflooksthroughthewindow)develops
TwoweeksaftertheimplantationoftheSILASTICcapsules,allbirds
onlyinbirdsthatarepermittedtocopulateduringthe5mininteraction
were tested four times for the presence of male-typical copulatory
with the female and that this response is steroid-dependent (Domjan,
behavior using behavioral procedures that have been described previ-
1987;Balthazartetal.,1995).Theseobservationssupportthenotionthat
ouslyindetail(BalthazartandSchumacher,1984).Briefly,eachmalewas
introduced into a small arena (50 3 60 cm) that contained a sexually thesocialproximityresponseisavalidmeasureofappetitivemalesexual
behaviorinquail.
maturefemale,andtheoccurrenceofcopulatorybehaviors[neckgrabs,
Attheendofthatperiod,thedoorseparatingthetwocompartments
mounts,andcloacalcontactmovements(fordescriptions,seeAdkinsand
waslifted,and thetwobirdswereallowedtointeractfreelyfor5min.
Adler,1972;Hutchison,1978)]wasrecorded.Testosterone-treatedbirds
During that time, the frequency and latency of the first occurrence of
that failed to exhibit mounts and cloacal contact movements were ex-
male sexual behaviors were recorded. The following behavior patterns
cludedfromthestudyatthispoint.Asexpected,mostofthecastrated
weresystematicallynoted:strut,neckgrab(NG),mountattempt(MA),
birds possessing empty implants remained behaviorally inactive during
mount (M), and cloacal contact movements (CCM) [for a detailed
these tests. The few subjects that exhibited low levels of copulatory
behavior,indicatinganincompletecastrationandgonadalregeneration, description,seeAdkinsandAdler(1972)andHutchison(1978)].These
werealsoexcluded. data provided a measure of the consummatory sexual behavior of the
Duringthenext10dperiod,allremainingsubjectsexperienced,ina birds. The female was then removed from the experimental chamber
two-compartmenttestcage,eightassociativelearningtrials,eachtaking wherethemalestayedforanother5minbeforehewasreturnedtohis
placeonadifferentday(tests1–8).Duringthesetrials,visualaccessto homecage.
asexuallymaturefemale,whowasinanadjacentchamberandcouldbe Stereotaxic brain lesions. As expected on the basis of our previous
seenthroughasmallwindow,waspairedwiththesubsequentfreeaccess studies,mostofthetestosterone-treatedmalesacquiredthesocialprox-
to that female, allowing sexual interactions and copulation. In these imityresponseduringtheseeighttests,whereascastratedcontrolsubjects
conditions,allmalesubjectswhoengagedincopulatorybehavioracquire wereneverobservedtospentasignificantamountoftimeinfrontofthe
thelearnedsocialproximityresponse(thisinvolvesstandinginanareain window. The few testosterone-treated birds that had not acquired the
frontofthewindowandlookingthoughitatthefemale)thatisusedin responsewerediscardedatthistime.Theremainingtestosterone-treated
ourexperimentstotestappetitivebehavior.Thisprocedureisbasedon birds were then randomly assigned to one of two groups that were
experimental protocols originally developed by Dr. M. Domjan at the balancedonthebasisofthecumulativetimethatbirdshadspendinfront
University of Texas (Domjan and Hall, 1986a,b; Domjan et al., 1986; ofthewindowduringthelasttwotrainingtests.Subjectsinoneofthese
Crawford et al., 1993; Domjan, 1994), and the specific modifications groupsreceivedinthefollowingdaysabilateralelectrolyticbrainlesion,
6514 J.Neurosci.,August15,1998,18(16):6512–6527 Balthazartetal.•PreopticSubdivisionsandMaleSexualBehavior
whereas the other testosterone-treated birds and the castrated controls thesemovementswhentheyareprovidedwithvisualaccesstoafemale.
weresubjectedtoashamoperation. Thesemovementsthusprovideanadditionalmeasureofappetitivemale
The experiment described here was performed in three replicates sexual behavior in quail. The testing procedure used to assess these
duringwhichtheelectrolyticlesionswereaimedatthreedistinctbrain movementsisasfollows.Eachmalewasplacedinanaquarium(20340
areas characterized by the presence of a dense group of aromatase- cm) located on a raised platform. A mirror was placed under the
immunoreactive(ARO-ir)cells:thenucleusPOM,therostralpartofthe aquariumata45°angleandprovidedanobserverwithanunobstructed
nucleus striae terminalis, where ARO-ir cells are clustered as a small view of the male’s cloacal area. Feathers of the experimental subjects
group dorsal to the anterior commissure (rostral BST), and the caudal werepluckedfromthecloacalareatofacilitatetheassessmentofcloacal
part of the nucleus striae terminalis, where ARO-ir cells that were movements.Atthebeginningofeachbehavioraltest,theaquariumwas
presentatmorerostrallevelsinPOMandrostralBSTmergetoforma divided into two chambers by an opaque partition, and RCSMs were
bilateralV-shapedstructure(caudalBST)[foradetaileddescription,see quantifiedfor2.5minduringwhichthemalecouldnotseethefemale.
Foidartetal.(1995);fordefinitionsofthePOMandBSTinquail,see Theopaquepartitionwasthenremovedsothatthemaleandthefemale
Panzicaetal.(1991)andAsteetal.(1998);alsoseeFig.1foraschematic wereseparatedonlybyawire-meshgridandthemalehadvisualaccess
illustrationofthesecellgroups),respectively.Aftertheactuallocations to the female, although he could not physically interact with her. The
of these lesions were analyzed by histological techniques, however, it RCSMs were then quantified for an additional 2.5 min under these
appearedthatbecauseofthecloseproximityofthesethreebrainareas, stimulusconditions.
therewasanextensiveamountofoverlapbetweenthelesionsaimedat Histological verification of the lesion sites and aromatase immunocyto-
the three theoretically distinct targets. All data were therefore reorga- chemistry.Atthecompletionofbehavioraltests,allbirdswereinjected
nizedandanalyzedonthebasisoftheactualpositionofthelesions(see intravenouslywith150mlofheparinsolution(SigmaH-7005,20mg/ml;
below). Sigma,St.Louis,MO)anddeeplyanesthetizedwithHypnodil(Janssen
To produce the desired electrolytic lesions, birds were anesthetized Pharmaceutica;50mg/kgbodyweight).Theywereperfusedthroughthe
with Hypnodil (15 mg/kg) and placed in a stereotaxic apparatus (with heartinitiallywithasalinesolution(9gm/l;0.15MNaCl)untilthereturn
pigeon head holder; Trent Wells, Inc. South Gate, CA), with the beak bloodwasclearandthenwith400mloffixative(4%paraformaldehyde
holderplaced45°belowthehorizontalaxisofthestereotaxicassembly. and0.1%glutaraldehydein0.1Mphosphatebuffer,pH7.2).Brainswere
BilaterallesionswereproducedusingelectrodesthatweremadeofNo. dissectedoutoftheskullandplacedovernightina20%sucrosesolution
00 steel insect pins insulated with Eukitt (O. Kindler, Freiburg, Ger- in0.1Mphosphatebuffer.Thenextdaytheywerefrozenonpowdered
many)exceptatthetip.Beforeuse,theinsulationoftheelectrodeswas dryiceandstoredinafreezerat275°Cuntiltheywereprocessed.Atthe
tested by passing current while the electrodes were immersed in egg timewhentheperfusionwasperformed,thebirdswerecheckedforthe
albumin,andthepresenceorabsenceofcoagulationcouldbechecked. completenessofcastrationandforthepresenceofsubcutaneoustestos-
Current was produced by a Grass S48 stimulator and passed simulta- teroneSILASTICimplantsinthelesionexperiments.Allbirdsshowing
neouslyinbothelectrodes(1.25mAfor10sec).Ametalclampwasfixed testicular remnants or who had lost their testosterone implant were
totheskinoftheheadandservedastheindifferentelectrode.Thesame discardedbeforeanydataanalysis.
manipulations, including the lowering of the electrodes to the desired Frozen brains were cut with a cryostat into 50-mm-thick coronal
brain target were performed in control birds (either treated or not sections,andsectionslocatedbetweenthelevelofthetractusseptomes-
treatedwithtestosterone),butnocurrentwaspassedthroughtheelec- encephalicusandthelevelofthebednucleusofthesupraopticdecussa-
trodesinthiscase.Electrodesweresubsequentlyremoved,theopening tion were collected in PBS (PBS, 0.01 M; NaCl, 0.125 M, pH 7.2). The
in the skull was closed with dental cement, and the skin was sutured. planeofsectionwasadjustedtomatchascloselyaspossibletheplaneof
Birds were then allowed to recover from the anesthesia in a warm thequailbrainatlas(Bayl´eetal.,1974).Alternatesectionsweredistrib-
environment and returned to their cage, where they usually started to utedintwoseriesthatwereeitherstainedbytoluidinebluefortheNissl
drinkandeatwithin1hr. substanceorbyimmunocytochemistryforaromatase.
Electrodecoordinateswere5.0mmanterior(x)and2.3mmabovethe Astandardperoxidase–antiperoxidase(PAP)procedureusingdiami-
zeroreferencepoint(centeroftheinterauralaxis,y)and0.4mmlateral nobenzidine as the chromogen was used to visualize immunoreactive
tothesagittalmidline(identifiedoneachbirdbythesutureofthefrontal aromataseasdescribedpreviously(Foidartetal.,1995).Briefly,sections
bones, z) for lesions aimed at the POM; x 5 5.0, y 5 3.5, z 5 1.6 for were rinsed three times in PBS containing 0.1% Triton X-100 (PBST)
lesionsaimedattherostralBST;andx54.9,y52.5,z50.5forlesions andthenin0.6%hydrogenperoxidePBSfor20mintoblockendogenous
aimedatthecaudalBST.Thesecoordinateshadbeenobtainedoriginally peroxidases.AfterthreeadditionalrinsesinPBST,theywereincubated
fromthequailbrainstereotaxicatlas(Bayl´eetal.,1974)andadjustedby for30mininnormalgoatserum(5%inPBST)andthenovernightat4°C
trialanderrorfortheheavierbodyweightofourbirds. in the primary polyclonal antibody against aromatase (1:200 in PBST).
Quantificationofbehavioraldeficits.Beginningonthedayaftersurgery, Thisantibodyhasbeenraisedinrabbitagainstapreparationofrecom-
allbirdsexperiencedninebehavioralteststoquantifytheeffectofthe binant quail aromatase and then purified by affinity chromatography.
experimentalproceduresofthemeasuresofappetitiveandconsumma- This antibody specifically recognizes aromatase-containing cells in the
torysexualbehavior(testsA–I).Thesetestingprocedureswereidentical quailbrain[fordetailsonpreparationandvalidationoftheantibody,see
tothetrainingtestsusedtoacquirethesocialproximityresponse(25min Foidartetal.(1995)].
total duration in the two-compartment test cage), and they were com- On the next day, sections were processed according to the PAP
pletedwithinthefirst15dafteroperation. technique. The goat anti-rabbit (dilution 1:100 for 120 min) and PAP
Birds from replicates 2 and 3 were subsequently submitted to two complex(1:500for120min)werebothdilutedinPBST.Extensiverinses
additional behavioral tests to control for both the specificity of the in PBST were made between each step. The peroxidase was finally
acquired proximity response and the potential effects of the lesions on revealedbyimmersingsectionsfor6–7mininasolutionofdiaminoben-
anothermeasureofappetitivesexualbehaviorinquail:theinductionof zidine(20mgin50mlofPBSTcontaining20mlofhydrogenperoxideat
rhythmiccloacalsphinctermovementsinthepresenceofafemale. 30%). Sections were then mounted on microscope slides and
Inthefirstofthesetests,eachmale(fromreplicates2and3)wastested coverslipped.
once in the two-compartment cage with the same protocol as before Data analysis. On the basis of sections stained by toluidine blue and
except that no female was present in the adjacent small compartment. sectionsstainedbyimmunocytochemistryforaromatase,theextentand
Thisprocedurewasrunafterthelesioninghadoccurredtoinsurethat location of each lesion was first drawn for each bird on a series of
thepresenceofamaleinfrontofthewindowprovidingvisualaccessto schematicdrawingsofthepreopticarea–anteriorhypothalamus.These
the female was still related to the actual presence of the female in the plots were made while paying maximal attention to the position of the
adjacentchamberratherthantheresultofsomeothernonspecificeffect lesionwithrespecttotheARO-ircellgroups.
ofthelesion. Sections stained by immunocytochemistry were then digitized by
Malequailfromreplicate3werealsotestedonceforrhythmiccloacal meansofaCCDcameraconnectedtoaMacintoshCIcomputer,andthe
sphinctermusclemovements(RCSMs)inreactiontothevisualpresen- lesionsandgroupsofARO-ircellsweredrawnwiththemouseoneach
tation of a female. These movements are used by males just before image. The corresponding areas were calculated by the program NIH
copulationtoproducethestiffmeringue-likefoamthatistransferredto Image(Version1.52,WayneRasband,Bethesda,MD),andvolumesof
femalesduringcopulation(Seiwert,1994).Ithasbeenshownpreviously the lesions and the remaining ARO-ir cell groups (POM, rostral and
(SeiwertandAdkins-Regan,1992;Seiwert,1994;Thompsonetal.,1998) caudalBST)weresubsequentlyreconstructedbymultiplyingareasbythe
that gonadally intact, sexually active males rapidly increase the rate of distancebetweenconsecutivesections(inthiscase100mm).Becausein
Balthazartetal.•PreopticSubdivisionsandMaleSexualBehavior J.Neurosci.,August15,1998,18(16):6512–6527 6515
mostcasesthelesionsdestroyedonlyaportionofthePOMorBSTand forthefivebehaviors)butindicatedthepresenceofasignificant
theshapeofthesenucleichangesonlygraduallyintherostrocaudalaxis, interactionbetweensubgroupsandrepeatedtestingforallbehav-
itwasoftenpossibletoreconstruct,withreasonableprecision,thecon-
iorsexceptMA.Additionalanalyseswereperformedtoidentify
tours of the nuclei as they would be in the absence of a lesion. These
putativeareasthatwouldhavebeenoccupiedbytheintactnucleiwere theoriginofthesedifferences.One-wayANOVAscomparingthe
thenusedtocalculatetheestimatedtotalvolumesofthenuclei. meanbehaviorofthethreesubgroupsduringthelasttwoacqui-
Five behavioral variables were selected for systematic analysis. They sitiontests(tests7and8)revealednogroupdifference(p.0.05
consisted of three measures of appetitive sexual behavior, namely, the
forthefivebehaviors).Similarly,thesameanalysisonthemean
timespentinfrontofthewindow(time),thefrequencyoflooksthrough
behaviorsexhibitedduringtheexperimentalphase(meanoftests
thewindow(looks),andthenumberoftimesthatbirdsenteredthetest
areainfrontofthewindow(entrances)duringthe5mintestperiod,and AthroughI)revealednogroupsdifferenceexceptinthecaseof
twomeasuresofconsummatorysexualbehavior,namely,thefrequencies LOOK, where larger behavioral scores in one of the three sub-
ofMAsandofCCMs.Otherbehavioralmeasuresofcopulatorybehav- groups produced a significant group difference in the ANOVA
ior(e.g.,frequenciesofneckgrabsormounts)arehighlycorrelatedwith (F 5 8.039; p 5 0.003). However, this difference occurred in
thesetwomeasuresandthereforeredundant.Thesebehavioraldatawere 2,19
analyzed by one- or two-way ANOVA, which were followed, when only one of many analyses and concerned only one subgroup.
appropriate,byposthocFisher’sprotectedleastsignificantdifferencetest Overall,itwasconcluded,therefore,thattheinteractionsidenti-
(PLSD).Analevelof0.05wasusedforallstatisticaltests. fiedpreviouslyinthetwo-wayANOVAresultedfromshort-term
random variations in the behavior of the three subgroups of
RESULTS CX1Tbirds,butthatingeneralthesethreegroupsbehavedina
similar manner. This was particularly the case at the end of the
A total of 73 birds completed the entire behavioral experiment
trainingperiodandduringtheexperimentalphase.Therefore,it
and had their brain lesions analyzed with enough detail so that
wasdecidedtopooldatafromthethreeCXandthethreeCX1T
theirbehaviorcouldberelatedtotheactualsiteandextentofthe
groupsinallsubsequentanalyses,andfromnowonthesegroups
lesion. Included in this total sample of 73 are 30 castrated,
arereferredtoastheCXandCX1Tbirds.
testosterone-treatedbirdswithalesionthatdestroyedtovarying
degreeseitherthePOM(n521),ortheBST(n59),aswellas
Qualitative and quantitative evaluation oflesions
22castrated,testosterone-treatedbirds(CX1T)and21castrated
Asalreadyindicatedabove,asubstantialnumberofsubjectswere
birds (CX) with empty implants that had no lesion (but had
found at the end of the experiment to have lesions overlapping
undergone the sham operation). In addition, 11 subjects had
extensively with the POM (n 5 21) or the rostral BST (n 5 9).
lesions that could not be fully reconstructed because of various
Theselesionstypicallydestroyedasignificantpartofthenucleus,
technicalproblemsandarethereforenotincludedintheanalyses.
as visible in a Nissl stain and more precisely in sections stained
ThreeotherbirdshadlesionsthatdidnotaffectPOMorBSTand
for aromatase. Photomicrographs of typical examples of these
were not considered further because they were not available in
twotypesoflesionsareshowninFigure2.
sufficientnumbertodrawnanyconclusion.Itmustbenotedhere
It has been demonstrated in previous work on the role of the
that although lesions had been specifically aimed at the caudal
preoptic area in the control of reproduction that only bilateral
portion of the BST (bilateral V-shaped structure caudal to the
lesions are able to produce significant deficits in male sexual
anterior commissure) during the third replicate of the experi-
behavior.Subjectsbearingaunilaterallesionusuallyshowlittleor
ment,histologicalexaminationoftheselesionsindicatedthatthis
no impairment (Numan, 1988; Yahr, 1995). Therefore, we first
partofthenucleushadneverbeentouchedexclusively;alllesions
quantified the degree of symmetry of the electrolytical lesions
aimed at the caudal BST were in reality placed in the caudal
that had been produced. Quantitative reconstructions indicated
POM.Thiscanbeexplainedbythefactthatthisstructureextends
onlyinaveryshortportionoftherostrocaudalaxis(100–150mm) that the volume of lesions was very similar on both sides of the
brain, for lesions targeting the POM (0.145 6 0.013 vs 0.144 6
so that it is very difficult to hit this structure specifically. All
0.008mm3;mean6SEM;t50.083;df518;p50.935)aswell
lesionstoBSTdescribedinthisstudythereforerefertotherostral
as for those targeting the BST (0.164 6 0.038 vs 0.161 6 0.024
and dorsal part of the nucleus, above the anterior commissure
mm3; mean 6 SEM; t 5 0.079; df 5 8; p 5 0.939). Because the
(Fig.1).Itmustbenotedthat,asillustratedinFigure2D,E,these
total volume that would have been occupied by the POM in the
lesionstothedorsorostralpartoftheBSTveryoftenaffectedthe
absence of a lesion could almost always be reconstructed, it was
adjacent nucleus accumbens, which also contains a scattered
possibletocalculateinalargenumberofsubjectsthepercentage
populationofARO-ircells.Withthisexception,lesionsaimedat
ofthenucleusthatremainedafterthelesion.Thispercentagewas
thegivennucleusalwaysaffectedthisnucleusspecifically.
also nearly identical on both sides of the brain (46.21 6 5.96 vs
Initial datareduction 46.7764.35%;mean6SEM;t50.144;df515;p50.887),and
SimilarnumbersoftheCXandCX1Tbirdswererunascontrols onanindividualbasisthevolumeofanucleusremainingonone
from the three replicates of the experiments (n 5 6–9 for each sidewashighlycorrelatedwiththevolumeremainingontheother
replicate).Thebehaviorofthesethreesubsetsofsubjectswasfirst side (r 5 0.759; n 5 16; p , 0.001). This analysis could not be
compared to test whether these data could be pooled in further performedinapertinentwayfortheBSTbecausethelesionand
analyses. Two-way ANOVAs with one independent variable nucleusvolumecouldbereconstructedonbothsidesofthebrain
(three subgroups of CX birds) and one repeated variable [17 in only a limited number of subjects. Given the high degree of
behavioral tests, i.e., eight learning trials (tests 1–8) plus nine symmetry of all lesions, all subsequent analyses considered only
post-surgery tests (tests A–I)] indicated no overall group differ- thetotalvolume(orsize)oflesionswithoutpayinganyattention
enceandnointeractionbetweenrepeatedtestingandgroupsfor totheirverylowdegreeofasymmetry.
thefivebehavior-dependentvariablesthatwereconsidered(time, The volume of lesions and of the ARO-ir cell groups (POM,
look,entrances,MA,andCCM;p.0.05ineachcase).Thesame rostralandcaudalBST)couldbereconstructedinmostofthe73
analysis performed to compare the three subgroups of CX1T experimental subjects in which the exact position of the lesions
birdssimilarlyfailedtodetectoverallgroupdifferences(p.0.05 hadbeenobserved,althoughquantitativestudieswereoccasion-
6516 J.Neurosci.,August15,1998,18(16):6512–6527 Balthazartetal.•PreopticSubdivisionsandMaleSexualBehavior
Figure1. Schematicdrawingsofcoronalsectionsthroughthequail
brain illustrating the distribution of aromatase-immunoreactive
cellsinthemedialpreopticnucleus(POM)andinthebednucleus
striaeterminalis(BST)/nucleusaccumbens(n.Ac.).Platesarear-
rangedinarostraltocaudalorderfromthetoptothebottom.
allyimpossibletocompletebecauseofaccidentallossofinterme- smaller in birds bearing a POM lesion than in CX1T or even
diatesections. inCXbirds.Thislatterdifferencewaspresentinthevolumeof
Aone-wayANOVAcomparingthetotallesionvolumesinthe the remaining POM (obviously decreased by the lesion) but
twoexperimentalgroupsthathadactuallybeenlesioned(Fig3A, also in the total extrapolated volume. Birds bearing a BST
POMandBST)indicatedthepresenceofnosignificantdifference lesionhadPOMvolumesthatwereintermediatebetweenthose
(F 5 30.651; p 5 0.427): lesions in POM or in BST were observed in CX and in CX1T birds, and they did not differ
1,26
overallverysimilarinsize(Fig.3A). statistically from these two groups.
The volumes of the POM, rostral BST, and caudal BST, as Incontrast,theone-wayANOVAscomparingtheremaining
measured based on the dense clusters of ARO-ir cells, are pre- orestimatedtotalvolumesoftheothertwoARO-ircellgroups
sentedinFigure3B–D.Whenalesionactuallydestroyedapartof (rostral and caudal BST) did not identify overall significant
a given nucleus, a double column is presented indicating the effectsofthetreatments(p.0.05ineachofthesefourcases).
remaining volume of the nucleus as well as the estimated total This lack of overall significant effect makes it statistically
volumethatwouldbeoccupiedbythenucleusifthelesionwere invalidtoconductanyfurtherposthoccomparisons.However,
notpresent(volumeextrapolatedfromtheremainingpartofthe a visual examination of Figure 3C,D suggests that the In BST
boundaries). groups are lower than some of the comparison groups, and it
The remaining and total volume of the POM was signifi- shouldbenotedthatFisher’sPLSDtestscomparingallgroups
cantlyaffectedbytheexperimentaltreatments(F 563.378, twobytwosuggestedthatthevolumeofremainingrostralBST
3,51
p , 0.001 and F 5 26.812, p , 0.001, respectively). As wassmallerintheInBSTgroupthanintheCX1Tgroup,and
3,48
indicated by Fisher’s PLSD tests presented in Figure 3B, the the caudal BST was smaller in the In BST group than in the
POMvolumewaslargerinCX1TthaninCXmales,anditwas CX1T group.
Balthazartetal.•PreopticSubdivisionsandMaleSexualBehavior J.Neurosci.,August15,1998,18(16):6512–6527 6517
Figure2. PhotomicrographsillustratingtheextentofatypicallesionofthePOM(A–C)andoftherostralpartoftheBST(D,E)asseeninNisslstain
(C,E)andinsectionsstainedbyimmunocytochemistryforaromatase(A,B,D).A,LesioninthedorsalpartoftherostralPOMasillustratedinsections
stainedforaromatase.B,HighermagnificationoftheboxinAshowingtheremainingaromatase-immunoreactivecellsintheventralpartofthePOM.
C,LesioninthecaudalpartofthePOMattheleveloftheanteriorcommissure(CA)fromaNissl-stainedsection.D,LesionoftherostralBSTasseen
inasectionstainedforaromatase.E,AdjacentsectionillustratingthesamelesionafterstainingforNisslmaterial.ThislesionoftheBSTpartlydestroys
thegroupofaromatase-immunoreactivecellslocateddorsaltothecommissure(BSTproper)butalsoextendstoacellgroupjustventraltothelateral
ventriclesthatisidentifiedasthenucleusaccumbensinthestereotaxicatlasofthechickenbrain(KuenzelandMasson,1988).Scalebar(showninE):
A,C,D,E,1mm;B,250mm.FPL,Fasciculusprosencephalilateralis(lateralforebrainbundle);LV,lateralventricle;VIII,thirdventricle.
Effect of POM and BST lesions on appetitive and CXbirdswiththethreeotherexperimentalgroupsthatwereall
consummatory sexualbehavior treatedwithtestosterone.Whenthesameanalyseswererepeated
Dataillustratingtheacquisitionandmaintenanceoftheresponse afterexclusionoftheCXgroup,allthegroupdifferencesdisap-
indicativeofappetitivesexualbehavior(timeatwindow)andof pearedintheANOVA(p.0.10),andallinteractionsofgroups
thefrequencyofconsummatorysexualbehavior(cloacalcontact by repeated testing became nonsignificant (p . 0.10 in each
movements) after stereotaxic surgery are presented in Figure 4 case).Significanteffectsoftherepeatedtestingconductedduring
forthefourexperimentalgroups(InPOM,InBST,CX1T,CX). theacquisitionphasewerestillobservedforthethreemeasuresof
The analysis by two-way ANOVA (independent variable 5 appetitivesexualbehavior(time,look,andentrances;p,0.0001
fourexperimentalgroups;repeatedvariable517behaviortests) ineachcase).ThefrequenciesofMAandCCMalsosignificantly
ofthesedataindicatedforthefivebehavioraldependentvariables changedduringtheacquisitionperiod(F 53.244,p50.002,
7,343
(Time,Look,Entrances,MA,andCCM)thepresenceofsignif- andF 53.683,p,0.001,respectively)becausetheeffectsof
7,343
icantgroupdifferences,ofsignificantchangesbetweenthediffer- the testosterone treatment were not yet fully established and
ent tests, and of significant interactions between the two factors birds were still developing their copulatory skills. Overall, how-
(p , 0.001 for each of these 15 F ratios, i.e., three tests for five ever, it can be concluded that the three groups of testosterone-
behaviors).Thesameanalyseswerethenrepeatedondataaccu- treated birds (In POM, In BST, and CX1T) behaved in a very
mulatedseparatelyduringtheeightacquisitiontrialsandduring similar manner during the acquisition phase (Pretests) as illus-
theninepostoperativeteststoidentifybettertheoriginofthese trated in Figure 4 for Time at Window and Cloacal Contact
experimentaleffects. Movements.
Duringtheeightacquisitiontrials(Fig.4,Pretests),thetwo-way During the experimental postoperative phase (Fig. 4, Tests),
ANOVAsindicatedtheexistenceofsignificantgroupdifference two-wayANOVAscomparingthefourgroups(independentvari-
andeffectsofrepeatedtestingforallvariables(p,0.001foreach able)acrosstheninebehaviortests(repeatedvariablelabeledby
ofthefivedependentvariables).Asignificantinteractionbetween letters A–I in Fig. 4) demonstrated the presence of significant
groupsandrepeatedtesting(p#0.05)wasalsoobservedfortwo groupdifferencesforeachdependentvariable(F .14.9;p,
3,69
measures of appetitive (Time, Look) and one measure of con- 0.001ineachcase).Significantchangesinbehaviorasafunction
summatory (CCM) sexual behavior. The group differences and of repeated testing was also observed for two of the dependent
interactionsoccurredprimarilybecauseofthecomparisonofthe variables (Look, F 5 6.793, p , 0.001; MA, F 5 2.699,
8,552 8,552
6518 J.Neurosci.,August15,1998,18(16):6512–6527 Balthazartetal.•PreopticSubdivisionsandMaleSexualBehavior
Figure3. Reconstructionofthevolumes(meansandSEM)oftheelectrolyticlesions(A)andofthevolumesofthePOM(B),rostralBST(C),and
caudalBST(D)inthefourexperimentalgroups.Whenthelesionactuallydestroyedapartofagivennucleus(POMintheInPOMgroupandRostral
BSTintheInBSTgroup),thecorrespondingbarhasbeendividedintoahatchedbarthatindicatesthevolumeofthenucleusremainingafterlesionand
anopenbarthatindicatesthetotalextrapolatedvolumethatwouldbeoccupiedbythenucleusifnolesionwerepresent.Experimentalgroupswere
comparedtwobytwobyFisher’sPLSDtestswhoseresultsareindicatedatthetopofthebarsasfollows:*p,0.05bycomparisonwiththeCX1Tgroup
and#p,0.05bycomparisonwiththeCXgroup.ParenthesesaroundasymbolindicatethatthecorrespondinggeneralANOVAcomparingthefour
groupsdidnotdetectasignificanteffect,sothatresultsofFisher’sPLSDtestscanonlybeconsideredasindicative(seeResultsformoredetail).
p 5 0.006) but not for Time at Window (F 5 1.818, p 5 behaviors (frequencies of mount attempts and cloacal contact
8,552
0.071), Entrances (F 5 0.890, p 5 0.525) or Clocal Contact movements were significantly lower in the In POM than in the
8,552
Movements (F 5 1.508, p 5 0.151). Interactions between CX1Tgroupsandarenotdifferentfromthesefrequenciesinthe
8,552
groups and repeated testing were present for Look (F 5 CX group). These same lesions also significantly decreased the
24,552
1.542, p 5 0.049) and Mount Attempts (F 5 1.801, p 5 measures of appetitive sexual behavior (time at window and
24,552
0.012) but not for the three other dependent variables (Time, lookingthroughwindow)incomparisonwiththeCX1Tgroup,
F 51.173,p50.260;Entrances:F 51.448,p50.078; buttheinhibitionwasonlypartialinthiscase(20–30%decrease)
24,552 24,552
CloacalContactMovements:F 50.706,p50.847). sothatbehavioralscoreswerestillsignificantlyhigherthaninthe
24,552
The origins of the group differences were then investigated CX group. Lesions destroying a part of the BST significantly
by comparing the behaviors of the five experimental groups, decreased,butdidnotabolish,measuresofconsummatorysexual
two by two, by means of Fisher’s PLSD tests. To facilitate the behavior(MAandCCMfrequencieslowerthaninCX1Tgroup
presentation, these comparisons of groups are illustrated in but higher than in the CX group), but these same lesions had
Figure 5, which represents the mean behavioral activity (time absolutely no effect on appetitive sexual behavior. However, the
in seconds or behavior frequency) observed across the nine numberofentrancesinthetestareainfrontofthewindowwas
postoperative tests. stronglydecreasedbybothtypesoflesions(fordetailofstatistical
Significantoveralleffectsoftheexperimentaltreatmentswere comparisons, see Fig. 5). It should also be pointed out that the
detected for each of the behaviors that were considered (p , behavioral deficits produced by the lesion became apparent im-
0.001fortheANOVAcomparingthefourexperimentalgroups; mediatelyafterthestereotaxicsurgery,andtherewasnoappar-
seedetailsconcerningtheresultsinFig.5).Fisher’sPLSDtests entrecoveryduringthedurationoftheexperiment(Fig.4).The
comparing all groups two by two indicated that lesions of the significant effects of repeated testing detected in the general
POMalmostcompletelyabolishedthetwoconsummatorysexual ANOVA of the data (see above) therefore appear to reflect
Balthazartetal.•PreopticSubdivisionsandMaleSexualBehavior J.Neurosci.,August15,1998,18(16):6512–6527 6519
with scores of nonlesioned birds). By contrast, there was a very
broad range of scores for the two measures of appetitive sexual
behavior,andthelimiteddecreasethatwasobservedinthemean
values (Figs. 4, 5) resulted from the averaging of scores that
differedwidely.InsomecasesindividualswithPOMlesionswere
not affected at all, whereas in other cases a nearly complete
inhibitionwasobserved.Wethereforedecidedtoinvestigatethe
origin of this marked variation in the effects of POM lesions on
appetitivesexualbehaviorwiththegoalbeingtheidentificationof
a brain area that would be specifically related to this behavioral
deficit.
Afirstqualitativeattempttosubdividethelesionsaccordingto
whethertheywereprimarilyaffectingtherostralversuscaudalor
thelateralversusmedialPOMdidnotprovidemeaningfulinsight
intothisquestion.Wethereforedecidedtoassesstheextentand
location of these lesions in a more sophisticated manner. Four
equally spaced levels in the rostrocaudal axis extension of the
POMwereselectedforanalysis.Themostcaudaloftheselevels
corresponds to the caudal end of the nucleus located under the
anterior commissure; the most rostral level is ;600 mm more
rostral (;200 mm between each level). At each of these levels,
each side of the POM was subdivided into four quadrants (dor-
somedial, dorsolateral, ventrolateral, and ventromedial), and in
eachofthesequadrantstheextensionofthelesionwasscoredon
a five-point ordinal scale ranging from 0 (quadrant totally de-
stroyedbythelesions)to4(quadrantintact,nolesionhittingthis
part of the nucleus). Intermediate values (1–3) corresponded to
Figure4. EffectsoflesionsofthePOMorBSTforoneofthemeasures decreasing lesions, so that ;25, 50, and 75% of this part of the
ofappetitive(TimeatWindow)andforoneofthemeasuresofconsum-
matory (Cloacal Contact Movements) male sexual behavior in castrated nucleus was still intact. Eight of these scores were therefore
male quail treated with exogenous testosterone. Data for CX birds not collectedateachofthefourrostrocaudallevelsofthePOM[four
treatedwithtestosteronearealsoillustrated.Thedatashown(means6 ontheleftside(a–d)andfourontherightside(e–h),alwaysinthe
SEs)representtheacquisitionofbehaviorsduringtheeightpreoperative
orderdorsomedial,dorsolateral,ventrolateral,andventromedial].
tests (Pretests) and then the effects of the experimental manipulations
Intheend,theoveralllesionofthenucleuscouldbedescribedby
observed during the nine postoperative tests (Tests) in the four experi-
mentalgroups. aseriesof32scores(fourrostrocaudallevels3twosides3four
quadrants)ona5-pointscale.Theaveragepostoperativebehav-
ioral scores [values that generated the means plotted in Fig. 5
randomfluctuationsinbehaviorratherthanaprogressivedecline (Time at Window, Looking through Window, Entrances in Test
orrecoveryafterlesion. Area,MountAttempts,andCloacalContactMovements)]were
addedtothisdatatabletoformamatrixof37columns(32lesions
Dissociation between lesions to different subregions
of the POM and appetitive and consummatory scoresplusfivebehavioralmeasures)by16rows(individualsthat
sexual behavior hadalesioninPOMthatcouldbeassessedentirelybythemethod
described above). A correlation analysis produced a 37 by 37
Theresultsdescribedaboveindicatedafirstlevelofdissociation
correlationmatrix,andthisentiresetofresultswassubmittedto
betweenthetwocomponentsofsexualbehaviormeasuredinthis
factoranalysisbytheprincipalcomponentmethod(Statview4.0
study. On the one hand, lesions of the POM almost completely
abolished our measures of consummatory sexual behavior, but program, Abacus Concepts, Calabasus, CA) to summarize the
theyonlypartlysuppressedourmeasuresofappetitiveaspectsof major relationships between these different variables (Fruchter,
thisbehavior.Ontheotherhand,lesionsoftheBSTsignificantly 1954).
decreasedthefrequencyofconsummatorysexualbehaviors(MA Thefactoranalysisextractedsixmeaningfulfactorsthathadan
and CCM), but they had absolutely no effect on the appetitive eigenvalue superior to 1. However, the magnitude of these eig-
component. A closer inspection of the experimental results sug- envalues decreased rapidly so that the first three factors alone
gestedadditionallevelsofdissociation. already explained .74% of the total variance. Therefore only
A qualitative analysis of the individual results indicated that these three factors are considered in the following data presen-
within the group of subjects that were bearing a lesion affecting tation. The unrotated factor loadings are plotted in a three-
the POM, behavioral deficits independently affected the appeti- dimensionalgraphinFigure7Aandastwocombinationsofthese
tive and consummatory aspects of male sexual behavior. This threefactors/axes(Level1vsLevel2andLevel1vsLevel3)in
notion is illustrated in Figure 6, which presents the individual Figure 7B,C. These plots all identified four discrete groups of
correlationbetweenthemeasuresofthesetwocomponentsofthe variables that were found to correspond precisely to the lesion
behavior in the CX1T and In POM groups. As can be readily scoresobtainedatthefourrostrocaudallevels.Thetwomeasures
observed,allbirdsoftheInPOMgrouphadsubstantialdeficitsin ofconsummatorysexualbehavior(MAandCCM)werefoundto
copulatory behavior by comparison with those of the CX1T be closely associated with lesion scores for Level 3, whereas the
group(MAandCCMfrequenciesarezeroorverylowcompared measures of appetitive sexual behavior (TIME, LOOK, and
6520 J.Neurosci.,August15,1998,18(16):6512–6527 Balthazartetal.•PreopticSubdivisionsandMaleSexualBehavior
Figure5. Meansofthebehavioralscoresforallthebehavioralmeasurestakenforbothappetitiveandconsummatorymalesexualbehaviorobserved
inthefourexperimentalgroupsduringthepostoperativephaseoftheexperiment.Datapresentedarethemeans6SEsofthemeanofthebehavioral
frequencies or of time spent in front of the window during the nine separate tests. For each dependent variable, results corresponding to the four
experimentalgroupswerecomparedbyaone-wayANOVA,andtheseresultsaresummarizedinthebottomrightpanel(Fvalues,df,andassociated
probabilities;***p,0.001).ExperimentalgroupswerethencomparedtwobytwobyFisher’sPLSDtestswhoseresultsareindicatedatthetopofthe
bars.
ENTR) were located in a position intermediate between lesion and an apparently normal appetitive sexual behavior (ASB1/
scoresatlevels2and3. CSB1; n 5 5), birds experiencing a complete suppression of
This analysis therefore suggested a differential relationship consummatory sexual behavior (postoperative CCM frequency
betweenlesionscoresandmeasuresofASBandCSB.Anaddi- equal to zero) but with an apparently normal appetitive sexual
tional characterization of these relationships was therefore per- behavior(ASB1/CSB2;n57),andfinallybirdswithacomplete
formed. Birds of the In POM group were subdivided into three suppression of consummatory sexual behavior (postoperative
subgroupsbasedonthebehavioraleffectsoftheirlesion,namely CCMfrequencyequaltozero)andaninhibitedappetitivesexual
agroupthathadretainedaweakconsummatorysexualbehavior behavior (i.e., Looking through Window frequency below 15;
Balthazartetal.•PreopticSubdivisionsandMaleSexualBehavior J.Neurosci.,August15,1998,18(16):6512–6527 6521
consummatory behavior (ASB1/CSB1) possessed a lesion that
destroyed primarily the rostral part of the POM (low scores at
levels 1 and 2). By contrast, birds in which CSB had been sup-
pressed completely (ASB1/CSB2 and ASB2/CSB2) had an
extensive lesion of the level 3 in POM. These last two groups,
however,wereclearlyseparable,anditappearedthattheinhibi-
tion of ASB (group ASB2/CSB2) was specifically associated
with a large lesion at level 2 that was not observed in the other
two groups [(Fig. 8B) compare lesion score for level 2 in the
ASB1/CSB2withASB2/CSB2].
These differences between subgroups were confirmed statisti-
cally. A general ANOVA comparing the four levels in the three
subgroups indicated no overall difference between the groups
(F 5 2.654, p 5 0.108) but indicated significant differences
2,13
betweenlevels(F 55.171,p50.004)andsignificantinterac-
3,39
tions between levels and subgroups (F 5 5.122, p , 0.001).
6,39
Furthermore,separateANOVAscomparingthethreesubgroups
ateachlevelseparatelyindicatedsignificantornearlysignificant
differencesinlesionsbetweenthethreegroupsateachlevel(level
1,F 53.726,p50.053;level2,F 53.042,p50.082;level
2,13 2,13
3, F 5 7.238, p 5 0.008; level 4, F 5 4.078, p 5 0.054).
2,13 2,13
Comparisons of the subgroups two by two at each rostrocaudal
levelbyFisher’stestsaresummarizedinFigure8B.Thespecific
inhibition of consummatory sexual behavior (compare ASB1/
CSB1 with ASB1/CSB2) was associated with significantly
larger lesions at levels 3 and 4 (and smaller lesions at level 1).
OnlyonesignificantdifferencewasobservedbetweentheASB1/
CSB2andASB2/CSB2groups:birdsinthelattercategoryhad
significantlylargerlesionsatlevel2.
Some additional analyses were also performed to investigate
Figure 6. Relations between the two measures of appetitive sexual whether the deficits in consummatory sexual behavior that were
behaviorandthetwomeasuresofconsummatorysexualbehaviorinbirds induced by lesions in the BST were associated with a specific
bearingalesionofthePOMandintheircontrolgroup(CX1T).Cor-
rostrocaudal position of these lesions. The smaller number of
relationcoefficientsassociatedwiththefourregressionlinesindicatedin
thefigurearenotsignificant(p.0.05)exceptforCCMFrequencyversus available subjects and more diffuse nature of the BST (ARO-ir
LookFrequencyintheInPOMgroup(r50.536;p50.012).Thesedata cells form a dense cluster at the caudal level but are more
clearlyillustratethenearlycompleteinhibitionofconsummatorysexual scattered at the rostral pole of the nucleus) prevented us from
behaviorbutthequitevariableinhibitionofappetitivesexualbehaviorin
establishinganyclear-cutanatomicalrelationships.
thelesionedgroup.
Tests in the absence of thefemale
ASB1/CSB2;n54).Aswouldbeexpectedonthebasisofthese It was noted during behavioral observations that some of the
definitions, all aspects of the ASB and CSB in these three sub- birdsbearinglesionsinthePOMenteredthetestareainfrontof
groups of subjects were significantly different (Time, F 5 the window less frequently during the post-lesion tests, and this
2, 13
16.843,p,0.001;LookingthroughWindow,F 517.693,p, effectofthelesionwasconfirmedintheanalysisdescribedabove.
2,13
0.001;MountAttempts,F 57.916,p50.006;CloacalContact Because this behavioral effect was not associated in all subjects
2,13
Movements,F 510.013,p50.002)exceptfortheEntrances withadecreaseinthetimespentinfrontofandlookingthrough
2,13
inTestArea(Number,F 50.071,p50.932).Comparisonsof thewindow,wewantedtoinvestigatewhetherthesocial proxim-
2,13
groups two by two by Fisher’s PLSD tests also indicated that a ity response displayed by these lesioned subjects still reflected
group with an inhibited component of behavior always signifi- appetitivesexualbehavior(i.e.,theanimalswerestillsensitiveto
cantly differed from the corresponding group(s) in which this the stimulus they were viewing through the window) or perhaps
component was not inhibited (for details of these two by two reflected only the stereotyped repetition of a learned response.
comparisons,seeFig.8A). Alternatively,itwasalsopossiblethatthedecreaseinthenumber
Themeanlesionscoresinthesethreesubgroupsofbirdswere of times that birds entered the test area reflected a general
firstcomputedforthe32subdivisionsofthePOMthathadbeen nonspecificeffectonthemobilityofthesubjects.
analyzedseparately.Thesescoreswerethenfurtheraveragedon Inanattempttodiscriminatebetweenthesepossibilities,birds
thebasisoftheeightsubdivisionsthatwerepresentateachofthe from replicates 2 and 3 of this experiment (In POM, n 5 8; In
four different rostrocaudal levels. This was valid because at a BST,n59;CX1T,n516;CX,n514)weresubmittedafterthe
given rostrocaudal level, the analysis indicated no obvious rela- ninth postoperative test (Fig. 4, test I) to an additional test
tionship between the position of the lesion in a specific subdivi- performedundertheexactsameconditions,exceptthatnofemale
sionandbehavior(ingeneral,theeightsubdivisionsaresimilarly was placed in the small lateral cage of the two-compartment
affectedatagivenlevel;datanotshown).Thedatabasedonthese chamber. The appetitive sexual behaviors (Time at Window,
analysesaredisplayedinFigure8B.Birdsthathadmaintainedan LookingthroughWindow,andEntrancesinTestArea)displayed
apparentlynormalappetitivesexualbehaviorandexhibitedweak during these tests without female and during the last test per-