The Condor91:966-978 Q The CooperOrnithological Society 1989

BRAIN, AND BEHAVIOR INTERACTIONS IN AVIAN REPRODUCTION: STATUS AND PROSPECTUS

RAE SILVER Department of Psychology,Barnard Collegeof ,3009 Broadway, New York, NY 10027

GREGORY F. BALL Department of PsychologyBoston College,Chestnut Hill, MA 02167

Abstract. The centralnervous system transduces behaviorally significant environmental informationnecessary for successfulreproduction. Convergent lines of researchsuggest that a numberof questionsregarding the natureof speciesdifferences, and how thesedifferences havebeen implemented over evolutionary time, canbe approachedthrough the clarification of underlyingbrain mechanisms.The last 15 yearshave yieldeda host of techniquesfor studyingthe integrationby the nervoussystem of sensory,neural, and endocrinefactors mediatingspecies typical reproductivebehaviors. In this paper,we review major issues relevantto our presentunderstanding of environmental-brain-endocrinebehavior relation- shipsin avian breedingsystems, and indicatethe techniques that are now available to explore neuralmechanisms. Specifically, we discussrecent evidence on thelocalization of encephalic photoreceptors,the biologicalclock and its putativerole in timing breedingcycles, pepti- dergic(gonadotropic hormone releasing hormone, GnRH, and vasoactiveintestinal poly- peptide,VIP) influenceson the pituitary-gonadalaxis, and the neurochemicalconsequences of hormoneaction in the avian brain.

INTRODUCTION Lehrman 196 1, Mm-ton and Westwood 1977, It has been known for centuries that stimuli from Wingfield and Farner 1980, Follett 1984). the environment profoundly influence reproduc- In the decadessince Harris ’s discovery,we have tive physiology and behavior. Though many learned a great deal about environmental factors studies have documented the link between en- regulating reproduction. A complete understand- vironmental changesand endocrine physiology, ing of the ultimate causesof diverse reproductive the mechanisms underlying these changeswere strategies and of the evolution of associated not understood until well into the 20th century. mechanisms still eludes us. The classicissues of A major advance in our understandingof brain- why and when animals do as they do, however, hormone-behavior-environment interactions in terms of underlying hormonal mechanisms rested on the establishment of a route whereby have been successfullyaddressed in both labo- the CNS could mediate the complex interaction ratory and field studieson behavioral, endocrine, between endocrine functioning and behavioral and neural mechanisms of reproduction (Cheng and environmental signals. In 1955, Geoffrey 1979; Silver 1978; Silver and Cooper 1983; Harris demonstrated conclusively the existence Wingfield and Famer 1980; Silverin 1983, 1988; of a humoral link between the brain and the pi- Wingfield 1983; Wingfield et al. 1987). tuitary gland via the pituitary portal system.This Prominent featuresof the basic anatomical or- vascular connection is the route whereby signals ganization of the HPG are shared by all major from the brain reach the pituitary gland and vertebrate groups.Secretory neurons in the brain thereby have accessto the general blood supply synthesize a hormone-gonadotropic hormone of the body. We now know that such responses releasing hormone (GnRH)-which communi- are mediated by the hypothalamo-hypophyseal- cates with the hypophysis via a vascular con- gonadal (HPG) axis. In particular, it now appears nection-the pituitary portal system. Stimula- that these environmentally influenced reproduc- tory glycoprotein -gonadotropins- tive phenomena are regulated by changesin hor- are secretedby the pituitary gland, which in turn mones circulating in the blood (Marshall 1959, initiate hormone secretion by the gonads. Go- nadal steroidsmay stimulate or inhibit the func- tional status of the neural and/or pituitary com- LReceived 25 May 1989. Final acceptance26 July ponents. 1989. There is a distinct pattern of changes in

[9661 REVIEW 967 steroid and pituitary hormones correlated with mechanisms underlying speciesdifferences and behavioral and physiological events of the re- individual differencesin reproductive cyclesand productive cycle (Silver 1978, Cheng 1979, Far- reproductive behavior in birds may well be elu- ner and Follett 1979, Wingfield and Farner 1980, cidated by examining the target sites for hor- Follett 1984, Wingfield et al. 1987). The tem- mones in the brain. poral pattern ofhormone secretioninfluences the progression from one stage of the reproductive NEURAL PROCESSES IN AVIAN cycle to the next. The onset ofbreeding is marked REPRODUCTION by an increase in the secretion of pituitary go- At the level of the brain one can study the in- nadotropins (-LH, and fol- tegration of external environmental stimuli and licle-stimulating hormone-FSH) and gonadal internal bodily signals,and how these signals in- steroids,androgens (such as testosteroneand 50(- fluence the neural substrate.A cascadeof events dihydrotestosterone)in males and estrogens(such are involved in this process:(1) specialized re- as 17p-estradiol) in females. These hormones re- ceptors for the external signals transduce the main above nonbreeding baseline levels physical stimuli into neural events, (2) pathways throughout the breeding season. In all species from various receptorsreach target areas in the there is a rise in secretion associated brain, including the biological clock and neurons with incubation and/or brooding (Goldsmith elaborating releasing hormones in the preoptic 1983). area and hypothalamus,(3) neuromodulatory and Even though these hormone-behavior rela- enzymatic factors regulate the releasing hor- tionships have been established,the causeof the mones, and (4) peripheral steroid hormones hormone changesand consequencesof these en- themselves act back on the brain to alter its re- docrine changes remain to be fully explained. sponsiveness.To illustrate both the current sta- Also, knowledgeof endocrine changesalone leave tus of our understanding, and the methods used several other issues unresolved. Thus, surpris- to address these issues, we will discuss each of ingly, significant differences in social behavior these events in turn. among speciesare associatedwith relatively small differences in hormone secretion. For example, RECEPTORS FOR ENVIRONMENTAL STIMULI two specieswith natural history patternsthat vary Temperate zone birds limit their reproductive from the typical avian pattern, the brood-para- activity to a particular time of the year. This sitic Brown-headedCowbird (Molothrusater) and involves responsesto both photoperiodic infor- the polyandrous Spotted Sandpiper (Actitisma- mation and to supplementary cues such as be- cularia),have endocrine profiles of gonadotro- havioral interactions, the availability of nest sites, pins, sex steroids, and prolactin that are similar nest material, and food (Wingfield 1983, Famer in many ways to those previously identified for 1986). If we could identify the sensoryreceptors monogamous biparental species. The Spotted that transduce environmental cues this would Sandpipers exhibit reversed behavioral sex roles represent an important first step in understand- that are not based on a total reversal of the nor- ing how changesin hormone secretion are pro- ma1 male to female ratio of androgens to estro- duced by the brain. This in turn would set the gens (Fivizzani and Oring 1986). Brown-headed stagefor understanding speciesdifferences in re- Cowbirds which remain sexually active through- sponsivenessto environmental factors, and the out the breeding season show hormone profiles way in which mechanisms constrain the evolu- quite similar to parental species in the sexual tionary lability of reproductive systemsin birds. phase of the annual cycle (see Dufty and Wing- Of the relevant environmental stimuli, photic field 1986 for review). In a similar vein, indi- cues have been the most intensively studied. vidual differences in behavior within a given Photoperiodic cues provided by daylength de- population are generally not reflected by differ- termine whether the reproductive system is in ences in hormone secretion in mammals (Da- an active or inactive state, while supplementary massa et al. 1977, Harding and Feder 1976) cues accelerateor retard this process(Wingfield though at least one mammalian study has found 1983, Follett 1984, Famer 1986). In birds, pho- such individual differencesto be correlated with toperiodic information is detectedby an extraret- brain differences in hormone receptor levels inal encephalic photoreceptor while the supple- (Clark et al. 1985). These issues related to the mentary information is processedby the ears and 968 RAE SILVER ANDGREGORY F. BALL eyes. Benoit (1935) demonstrated that blinded in the septal and tuberal hypothalamic regions Mallard drakes (Anus platyrhynchos L.) contin- of the dove and duck brain (see Fig. 1). These ued to recrudescetheir testes in responseto ar- opsin positive perikarya also express another tificial photostimulation. The existence of en- peptide, namely vasoactive intestinal polypep- cephalic photoreceptors has been well tide (VIP). Thus, for the first time, we have a documented in a variety of avian species(Oliver potential anatomical marker for this brain pho- and BaylC 1980). One of the most recent analyses toreceptor. It should be noted that although this of the encephalic photoreceptor established an result is highly suggestive,it does not constitute action spectrum for the photoperiodic response definitive proof that thesecells are the long sought in Japanese Quail (Coturnix japonica) by pre- encephalic photoreceptors. Ideally, an indepen- senting light at different wavelengths and mea- dent line of evidence indicating that the antigen suring its effect on plasma luteinizing hormone stained is indeed opsin is desirable. For example, (LH) levels. This work establishedthat the pho- we are now using another anti-opsin antibody topigment mediating the LH response is very that recognizesa different epitope (antigenic site) similar to rhodopsin (Foster and Follett 1985). on the opsin molecule. Another important step Though the existenceof thesereceptors had been is to establish that the opsin-bearing cells can known for decades, available neuroanatomical respond to light. techniques were inadequate to determine the lo- The use of anti-opsin antibodies can guide us cation and morphological features of these cells. to circumscribed neuroanatomical locations of For example, a searchusing electron microscopic encephalic photoreceptors.Once the location of methods for retina-like photoreceptorswould in photoreceptor(s)is (are) establishedfurther func- theory have been possible, but as this technique tional investigations can indicate how the signal entails the analysis of almost microscopic blocks produced by this photoreceptor in response to of tissue,it is unsuitable for surveysof large brain light is conveyed to GnRH neurons and to the regions. A methodical search for brain photo- biological clock that measures daylength. Sub- receptorsbecame feasible with the application of sequently,one can determine whether speciesdif- the method of immunohistochemistry. ferencesin responseto photoperiod are reflected in the anatomy and/or physiology of the pho- Immunohistochemicaltechniques have been applied toreceptor and its efferent connections. It will be to the brain since the late 1960s.These permit the fascinating to compare the neural circuitry me- localizationof minute quantitiesof antigens,whether structuralproteins or peptidesand providea newclass diating the environmental responseof the repro- of informationabout function and physiology.In this ductive system in temperate zone photoperiodic procedure,antibodies are usedto localizea naturally specieswith speciesthat breed opportunistically occurringantigen present in appropriately fixed tissue. in responseto other cues such as rainfall. That is, antibodiesare usedas markersto label antigens naturally found in neurons. First, specific antibodies THE BIOLOGICAL CLOCK IN BIRDS to the peptide or transmitter of interest are prepared. Changes in daylength, constituting one of the The antibody is then bound to the antigensin the tissue primary environmental factors regulating sea- ofinterest. Next, a marker that can be visualized under sonal cycles of gonadal growth and regression, the microscopeis bound to the primary antibody. Fi- appear to be measured in birds by means of a nally the tissue is processedfor light or electron mi- biological clock. As illustrated first by Hamner croscopy.Methodological and interpretive aspectsare reviewed in Sternberger(1979). (1963), birds seem to use the phase of an en- dogenouscircadian oscillator rather than the ab- All known vertebrate photoreceptorscontain solute duration of light to measure photoperiod. the protein opsin as part of the photopigment In experiments of this design the amount of light that transducesthe photic signal.Antibodies have is held constant, while the length of the dark been developed against opsin allowing the lo- period is varied among the experimental groups. calization of this protein in the retina and other The results indicate that there is a circadian light sensitive structures such as the pineal. Us- rhythm in sensitivity to light, such that birds ing a monoclonal antibody that recognizes rod receiving light in phase with the hypothesized photoreceptorsin a variety of vertebrates, Silver 24-hr cycle in photosensitivity show gonadal et al. (1988) described opsin-like immunoreac- growth while birds receiving an equally long light tivity in cerebrospinal fluid-contacting neurons signal out of synchrony with this sensitive phase REVIEW 969

FIGURE 1. A drawing of a “generic” avian brain illustrating some of the neurochemical systemsdiscussed in the text. The box outlining a subset of the three-dimensional schematic brain on the left denotes the area enlarged and shown in detail on the right. This schematic presentsthe hypothalamic and infundibular area of a “generic” avian brain cut in the sagittalplane. The dark neuron-like cells illustrate opsin-like immunoreactive cellsin the lateral septum (SL) and infundibular region (IN). Note the dark lines running alongthe lateral ventricle in the lateral septalregion. This denotesthe fact that the cells containingopsin-like immunoreactivity are located in the lateral ventricular wall of the lateral septum. These cells are candidatesfor the cells that may contain the extra-retinal photoreceptor. These data are based on information contained in Silver et al. 1988. The open neuron-like cells that are scatteredrostrally from the POA and run in a column caudo-dorsallyuntil the medial septum illustrate the localization of immunoreactive perikarya containing the pituitary regulatingpeptide go- nadotropin-releasinghormone (GnRH). The dashedlines running ventrally in two columns representthe major fiber pathways that contain this peptide and innervate the median eminence and ultimately are released into the portal systemand regulatepituitary functioning. This distribution pattern is basedprimarily on information contained in Foster et al. 1987. The open circles that are scatteredthroughout the POA, PVN, SL, as well as ventrally in the IN and caudallynear the OMd representthe distribution ofneurons containingestradiol receptors. This information is based primarily on data contained in Martinez-Vargas et al. 1976. Abbreviations listed in the figure are as follows: CB: Cerebellum; CoA: Commissura anterior; CO: Chiasma opticum; FLM: Fasciculus longitudinalis medialis; IN: Nucleus infundibuli hypothalami; ME: Eminentia mediana;nIV: Nucleusnervi trochlearis;OMd: Nucleusnervi oculomotorii,pars dorsalis;Pit: pituitary gland; POA: Nucleuspreopticus anterioris;PVN: Nucleusparaventricularis magnocellularis; SL: Nucleusseptalis lateralis; SM: Nucleusseptalis medialis;TrSM: Tractusseptomesencephalicus; V: Ventriculus lateralis.

show no gonadal development (Hamner 1963, lamic suprachiasmatic nucleus (SCN) have all Follett et al. 1974, Turek 1974, Tewary and Ku- been suggestedas sites for the biological clock mar 1981). The results suggestthat a biological (Cassoneand Menaker 1985). However, in con- clock or oscillator is necessaryfor the measure- trast to the situation in mammals, most data ment of daylength (however, see Saiovici et al. suggesta minor role for the pineal gland in the 1987 for an alternative view). Little is known mediation of photoperiodic effectsin birds (Fol- about the physiological basis of such an entity. lett 1984, Gwinner 1986). Also, as noted pre- The pineal gland, the eyes, and the hypotha- viously, it has been shown in severalavian species 970 RAE SILVER AND GREGORY F. BALL that the eyes are not necessaryfor the exhibition nobenzidine(DAB) or tetramethylbenzidine(TMB). of photoperiodic responses (Follett 1984, Un- When hydrogenperoxide is addedto the mediumcon- derwood 1975). In mammals the existence of a taining floating tissuesections, the liberatedoxygen biological clock in a hypothalamic SCN is sup- combineswith the benzidineto makea pigmentwhich can easilybe visualizedunder the microscope.(Other ported by several independent lines of evidence sensitivetracers are available,see Nauta and Feirtag (RusakandZuker 1979; Moore 1982,1983; Leh- 1986 for discussion.) man et al. 1987). These consist of: (1) lesions of the SCN abolish circadian rhythms in physio- Application of HRP tract-tracing techniques logical and behavioral activity and result in the in a number of avian species has identified a loss of photoperiodic responsesto daylength, (2) retino-recipient region in the lateral hypothala- direct retinal input to the SCN mediates the en- mus in many avian species(Norgren and Silver, trainment (synchronization) of reproductive and in press). Only one behavioral study has been daily activity cycles produced by light cues, (3) directed specifically to this nucleus. Lesions of circadian cycles of multi-unit electrical activity the lateral hypothalamic retino-recipient nucleus within the SCN both in vivo and in vitro, (4) do not affect circadian rhythmicity in pigeons daily cycles of glucose metabolic activity in the (Ebihara et al. 1987). It is possible that the large SCN in the absence of photic cues, and (5) res- hypothalamic lesions of Takahashi and Menaker toration of circadian rhythmicity by SCN trans- (1982) in House Sparrows included this region plants of fetal donor tissue to an arrhythmic (Takahashi, pers. comm. with RS). The absence animal. of the several lines of evidence necessaryto es- Initial studies of the avian circadian system tablish the existenceand location of a neural os- were directed at a medial hypothalamic nucleus cillator in the hypothalamus of birds (as are lying in the same region as the mammalian SCN available in mammals) represents a significant (overlying the optic chiasm and adjacent to the hiatus in our knowledge. third ventricle). The results in Japanese Quail (Simpson and Follett 1981) House Sparrows HYPOTHALAMIC RELEASING FACTORS (Passer domesticus) (Takahashi and Menaker 1982), and Java Sparrows (Padda oryzivora) Once environmental stimuli are transduced by (Ebihara and Kawamura 1981) suggestedthat the appropriate receptor, they influence the se- lesions of this nucleus resulted in the loss of lo- cretion of the peptide GnRH located in neurons comotor rhythmicity but did not disrupt pho- of the preoptic and hypothalamic regions (see toperiodic responses(Simpson and Follett 198 1). Fig. 1 for the distribution of these cells). The However, at least in the caseof House Sparrows, avian brain contains two forms of this peptide hypothalamic lesions were large and resulted in (Millar and King 1984, Miyamoto et al. 1984, a decrease in overall activity level (Takahashi Sherwood et al. 1988) known as avian GnRH I 198 l), raising the possibility that the effectswere and II; however, all the available evidence sug- not specific. geststhat only GnRH I is involved in the control One feature of the mammalian SCN is direct of pituitary hormone secretion(Sharp et al. 1988). input from the retina (Moore 1973). A clue to GnRH regulatesthe activity of the two pituitary the location of the SCN in birds might be to gonadotropins, LH and follicle-stimulating hor- identify the retino-recipient nucleus. Older mone (FSH). methods for such an identification involved the StudiesofGnRH positive neurons by radioim- tracing of degenerating fibers following the tran- munoassay (Dawson et al. 1985) and by im- section of nerves of tracts. While this technique munohistochemistry (Foster et al. 1987) reveal was adequate for large fiber bundles, degenera- striking differencesin the activity of this peptide tion of very fine fiber bundles is harder to iden- in reproductively active vs. inactive European tify. The development of sensitive tracers such Starlings (Sturnus vulgaris). Radioimmunoassay as horseradishperoxidase (HRP) has allowed the allows the determination of the amount of a hor- resolution of fine neural connections. mone presentin a given piece of tissueor volume HRP is an enzyme taken up by neuronsand trans- of plasma. From these data it appears that the ported(200-300 mm/day) in the retrogradeand an- GnRH content of the POA-hypothalamus is 1O- terogradedirection (Mesulum 1983). Appropriately fold higher in photosensitive (i.e., capable of re- fixedtissue is visualizedusing the chromogendiami- sponding to long daylengths) compared to pho- REVIEW 971 torefractory (i.e., incapable of responding to long 1988, Mauro et al. 1988). In Ringed Turtle-Doves days) birds. (Streptopelia risoriu), Cloues et al. (1988) mea- Immunohistochemical evidence indicates the sured changesin VIP cell diameter throughout location of the GnRH cell bodies in the preoptic the reproductive cycle. The results indicate that and septal regions of the brain and confirms a VIP cell size starts to increase during courtship, seasonal change in the intensity of staining. In- peaks around the ninth day of incubation, re- dividual neuronal perikarya in the preoptic area mains elevated until day 14 of squab rearing, and increasein diameter and in the intensity of GnRH then slowly declines as the squabs become in- staining density in photosensitive animals. Thus dependent. Elevation of plasma prolactin in the seasonal cycles of sensitivity to daylength are Ringed Turtle-Dove are detected only at mid- accompanied by changes in the synthesis of incubation (Goldsmith et al. 1981, Cheng and GnRH. The induction of gonadal growth asso- Burke 1983) indicating that VIP immunoreac- ciated with photosensitivity (or the breaking of tivity precedesthese plasma hormone changes. photorefractoriness)is preceded by an increase Furthermore, parent doves rearing two squabs in hypothalamic GnRH content (Goldsmith et have a more prolonged period of enlarged VIP al. 1989). Similarly, gonadal regression is pre- immunoreactive cells compared to parents rear- ceded by a decline in GnRH (Goldsmith et al. ing a single squab. This parallels differences in 1989). These results indicate that the factors reg- crop development between these two groups. ulating this neuropeptide are the key to under- Similarly, inexperienced birds, who feed their standing the control of seasonal processes.Ad- young and secreteprolactin for longer periods of ditional studies employing tract tracing to time than experienced birds, have a prolonged determine how environmental information period of enlarged VIP immunoreactive cells reachesthe GnRH neurons, in combination with compared to parents with previous experience of chemical neuroanatomical methods, will pro- a reproductive cycle, again paralleling differences vide information on neuroendocrine integration. in crop growth between these two groups. It has VIP is another releasing factor that has re- long been known that inexperienced birds are less cently been implicated in the control of a pitu- efficient at rearing their young than are experi- itary hormone, prolactin. This hormone is mod- enced birds. To our knowledge, this is the first ulated by stimuli from the environment and plays demonstration that such experiential effectsare an important role in the control of reproduction. reflected in neuroendocrine cells. Control ani- VIP stimulates the secretion of prolactin by the mals paired with the same sex partners or with- anterior pituitary (Macnamee et al. 1986). Ex- out accessto nest bowl and straw showed no ogenously administered VIP releases prolactin changesin VIP immunoreactivity. when administered to intact birds (Lea and The timing of prolactin secretionvaries among Vowles 1986). In vitro studies also point to VIP speciesand even within speciesas a function of as directly stimulating the release of prolactin the pattern of parental care (Goldsmith 1983). from the anterior pituitary (Macnamee et al. It will be useful to assesswhether speciesdiffer- 1986). Incubation is associated with elevated ences and sex differencesin temporal aspectsof plasma prolactin in birds (Goldsmith 1983, Lea prolactin secretion are accompanied by differ- 1987). In order to establish whether VIP me- ences in the activity of VIP. If this proves to be diates normally occurring changes in prolactin the case,studies of VIP immunoreactivity could secretion it is necessary to demonstrate that direct us to higher neural processesunderlying changesin VIP precedechanges in prolactin. As speciesdifferences in behavior. noted earlier, VIP also is co-expressedin cells As discussedin the introductory paragraphs, that contain opsin-like immunoreactivity. How- the brain integrates sensory and endocrine in- ever, VIP is widely distributed throughout the formation to regulate the secretion of pituitary hypothalamus of birds and we do not at present and gonadal hormones. In this section we have know if these VIP-positive cells are involved in concentrated on three neural components of re- the regulation of prolactin. productive processesin birds: the encephalic Differences between incubating and nonincu- photoreceptor, the biological clock, and neural bating birds in the intensity of VIP staining in peptidergic and protein hormones. Much work the hypothalamus have been described in pi- on the anatomy and physiology of each com- geons, turkeys, and doves (Peczeley and Kiss ponent remains to be done. Also, connections 972 RAE SILVER AND GREGORY F. BALL among these neural entities are very poorly et al. 1976). However, consistent with the ver- understood. In the next section, we will review tebrate pattern is the presence of steroid recep- the ways in which steroid hormones act on the tors in the septum, archistriatum, preoptic re- neural substrate. gion, tuberal hypothalamus, specificsubtectal loci We will concentrate on the sex steroid hor- (e.g., the nucleus intercollicularis), and pituitary mones, testosterone, 17&estradiol and proges- gland. Many of these regions contain both an- terone, as they have been the most extensively drogen and estrogen receptors. More recently, studied. However, it should be noted that pro- antibodies to steroid receptor proteins have been gressis being made in understanding where and developed permitting the use of immunohisto- how other hormones such as the pituitary hor- chemical techniques (Gahr et al. 1987, Ball et al. mone prolactin act in the brain (Buntin and Walsh 1981a, Balthazart et al. 1989). To date, these 1988). We will further limit the discussion to studies have been in agreement with results influences on the adult brain. stemming from the autoradiographic work. Im- But, it should be recognized that the develop- munohistochemical techniques provide the ad- mental action of steroids in organizing neural vantage of identification of cellular components circuits is an important factor underlying inter- (nucleus vs. cytoplasm) in which steroid recep- specific differences in brain and behavior (see tors are located. Immunohistochemical tech- Arnold et al. 1986 for discussion). niques also allow double labeling of cells for the presence of both steroid receptors and neuro- NEURAL SITES OF STEROID ACTION peptides or transmitters, permitting analysis of Steroid hormones exert their effects in discrete how these chemical messengersinteract. areasof the brain where specializedreceptors are located. The development of autoradiographic STEROID-INDUCED NEUROCHEMICAL CHANGES methods in the late 1960s allowed for the iden- tification of these hormone sensitive sites (see The identification of steroid-concentrating brain Fig. 1). regions guides us to the analysis of how and where steroids act. Lesion and steroid implant studies Autoradiographicmethods were first widely applied to to steroid-concentrating brain regions indicate steroid hormones, such as the gonadal sex steroids. that these areas are necessary for the perfor- Autoradiography requires the removal of endogenous mance of reproductive behaviors such as copu- sourcesof the steroid (usually by gonadectomy) fol- lowed by the injection of the radioactively labeled hor- lation, certain courtship displays, and song mone of interest. The brain tissue is then placed on (Hutchison 1978, Nottebohm 1980). With re- emulsion coated slides. After exposure to the film (4 spect to parental care, little work on its neural to 12 months) and development (similar to photo- basis has been done in birds. graphic development) one can visualize the location in One way that sex steroidsaffect behavior is by the brain of the radiolabeled hormones (Morrell and modifying neurotransmission in steroid-concen- Pfaff 1981). trating brain regions (Dohanich et al. 1985, In birds some of the first studies of this sort McEwen et al. 1987). The actions of steroids were conducted by Martinez-Vargas et al. (1974, have been best characterized for the case of es- 1975, 1976) in their investigations of estrogen tradiol acting in the ventromedial nucleus of the receptor binding in Ringed Turtle-Doves. The rat (Pfaff 1980). Though the actions of steroids method was subsequently applied to a number on neurochemical functioning have been much of other species representing several orders (Ar- better documented in mammals than in birds, nold et al. 1976, Zigmond et al. 1980, Watson many of the major ways in which steroidsmodify and Adkins-Regan 1989). The results indicate neurotransmission have also been demonstrated that in hypothalamic and preoptic areas the pat- in birds in studies of copulatory and vocal be- tern of steroid receptor binding in birds corre- havior. For example, steroids profoundly affect spondsto the general vertebrate pattern (Morrell the activity of the monoamine neurotransmit- and Pfaff 198 1). The one exception to this rule ters, as measured by steroid-induced changesin is in the uniquely avian, steroid sensitive, sex- transmitter “turnover.” ually dimorphic complex of nuclei involved in Turnover refers to the overall rate at which neuro- the motor control of song that appearsto be spe- transmitters or neuropeptidesstored in a given tissue cific to members of the oscine suborder (Arnold are replaced. It can thus provide an index to the func- REVIEW 973 tioning of catecholamine neurotransmitters such as communication processthat can be modified by norepinephrine,epinephrine, and dopamine. Turnover steroidsto change neurotransmitter activity and for a catecholamine such as norepinephrine can be ultimately behavior is the availability of recep- detected by measuring the disappearancerate of en- tors for a particular transmitter. The density of dogenousnorepinephrine after the inhibition of cate- neurotransmitter receptorshas been found to be cholamine synthesisby an inhibitor ofthe rate limiting enzyme tyrosine hydroxylase (see Cooper et al. 1986 different between the sexes (Ball et al. 1989b) for discussionof this method). The transmitter level and to be modulated by steroids(Balthazart and itself can be measured by a variety of methods. One Ball 1989) in certain discrete brain regions as of the most sensitive approachesallows for the detec- determined by the method of quantitative au- tion of transmitter levels in microdissected“punches” toradiography. of tissue from individual brain nuclei using sensitive Quantitative autoradiographyfor neurotransmitterre- high performanceliquid chromatographywith electro- ceptorsis broadly similar to the autoradiographicpro- chemical detection methods (see Renner and Luine ceduresdescribed above for steroid hormone uptake. 1984 for a detailed description). However, in this casein vitro labelling proceduresare A modification in turnover can be achieved employed and a computer assisted image analysis is by a variety of different mechanisms. One such used to analyze the isotope sensitive film allowing for pathway is through the enzymes ultimately re- the quantification of the density of receptorsin a given sponsiblefor the amount of transmitter available brain area as well as for a description of their local- ization (see Kuhar 1985 for description of method). to the neuron. Steroids can also influence the receptors available for the neurotransmitter in- In the JapaneseQuail it has recently been dem- teraction. In birds relatively few studies have onstrated that in certain steroid-concentrating examined changesin neurotransmitter turnover, brain nuclei there is a sexdifference in the density either in response to a hormone cue or in re- of the alpha, receptor subtype for the neurotrans- sponseto an environmental stimulus suchas light. mitters norepinephrine and epinephrine (Ball et Ottinger and Balthazart (1987) as part of a series al. 1989b). For example, the density of receptors of studies designedto establish how testosterone is higher in males than in females in the dorsal induces copulatory behavior in the male Jap- portion of the infundibulum, an area implicated anese Quail have found that the removal of en- in the regulation of LH and in the midbrain nu- dogenous testosterone by castration leads to a cleus intercollicularis, an area important in the decreasein the turnover rate of norepinephrine control of vocal behavior (Ball et al. 1989b). In that is restoredto intact levels by testosteronein the dorsal infundibulum, castration leads to an males while opposite effectsare observed in fe- increase in the density of alpha, adrenergic re- males. Similarly, steroids have been shown to ceptorsand testosteronereverses this effect (Bal- induce changesin neurotransmitter functioning thazart and Ball 1989). The sex difference in in the song system of Zebra Finches, Poephila receptorbinding in this region and the regulation guttata (Barclay and Harding 1988a). Steroids of thesereceptors by testosteroneparallel the sit- were found to induce different changesin cate- uation for pituitary hormone LH, in that there cholamine turnover in the various vocal control are highercirculating plasma levels of LH in males and hypothalamic nuclei (Barclay and Harding than in females (Urbanski and Follett 1982) and 1988b). levels increase after castration and are reduced Steroids are known to modify enzymes asso- by testosterone.These findings suggestthat alpha, ciated with the regulation of neurotransmitter in the dorsal infundibulum could play a role in functioning in birds (Luine et al. 1980). For ex- the steroid feedback control of LH. amnle. in Zebra Finches castration results in a Steroid modification of neurochemical activ- de&ease in the activity of two cholinergic en- ity appears to be limited to discrete areas of the zymes in peripheral nerves that innervate the brain. An advantageof analysis of the avian brain syrinx (Luine et al. 1980). Replacement with tes- is that a number of steroid-modified neurochem- tosterone reinstates the activity of one of these ical systems are found in nuclei that have well enzymes, suggestingthat testosterone may reg- defined behavioral functions. It should be noted ulate singing behavior in passerinebirds through that the most detailed information on how a ste- the induction of enzymatic proteins in testoster- roid action in the brain results in a behavioral one target neurons and muscles. responseis only available for estrogenic effects Another component in the neurochemical in the ventromedial nucleusofthe rat (Pfaff 1980). 914 RAE SILVER ANDGREGORY F. BALL

In all the avian systems described above, the thazart 1983). This effect of testosteronecannot cascadeof events from steroid binding to neu- be induced in females even after administration rotransmitter and enzyme systems and the be- of pharmacological dosesof testosterone.Inter- havior remain to be fully described. estingly, circulating levels of androgens are only Analysis ofthe stepsin developing the chain(s) slightly lower in female than in male quail, with of causal events among steroid hormone action, an occasionaloverlap in male and female values neurochemical functioning, and behavior has be- (Doi et al. 1980, Balthazart et al. 1986). The gun in birds. Several avian reproductive behav- activation of male sexual behavior by testoster- iors such as copulatory behavior and song have one requires that testosteronebe metabolized to well characterized neural loci in birds. There is estradiol by the enzyme aromatase. It appears a wealth of speciesdifferences among birds. The that this behavioral dimorphism in quail de- analysis of avian neural mechanisms mediating pends on differential properties of brain areas reproduction is a potentially valuable source of involved in the control of behavior. In particular comparative data on how variations in hormone aromatase is more active in males than in fe- action relate to variations in behavior. males throughout the hypothalamus and espe- cially in the preoptic area (Schumacher and Bal- thazart 1986). The enzyme SP-reductase,which THE NEURAL ENVIRONMENT MODIFIES is more active in females, metabolizes testoster- HORMONE EFFECTIVENESS one into a biologically inactive form of the hor- The manner in which a hormone such as testos- mone, SP-dihydrotestosterone. terone modifies neural function and behavior is The evidence described above indicates that profoundly influenced by the nature of the sub- sex differences in the ability of testosterone to strate on which it acts. Before steroid hormones induce copulatory behavior rests on a sex differ- exert their effects they may be metabolized to ence in the steroid metabolizing ability in the active or inactive forms. Thus the density of me- brain. An obvious question to pursue is whether tabolizing enzymes in a specifictissue at a given speciesdifferences in the effectivenessof steroids time will greatly determine whether or not a hor- in inducing reproductive responsesmight also lie mone has any effect at a particular target site. in brain enzyme differences.Thus in the Spotted The interaction of the brain enzyme environ- Sandpiper, it may be that the reversal of sex roles ment with steroidhormone effectivenesshas been in behavior (which is not accompanied by sex characterized more effectively in the avian brain differences in hormone levels) may instead be than in any other vertebrate system (Hutchison accompanied by sex differences in metabolizing and Steimer 1983). enzymes. The techniquefor measuringchanges in enzymeactiv- ity utilize indirect radioenzymaticassays (Schumacher SUMMARY and Balthazart 1987).Here tissueis sectioned(300 Km) In this review, we have tried to highlight some and the area of interest is microdissectedusing a meth- of the major recent advances in our understand- od called “punch.” Tissue is then homogenized and ing of the brain mechanisms regulating the re- incubated with radioactive testosterone.The radioac- tive steroidsare then extractedand purified. By count- productive cycle in birds. While it is true that in ing the amount of estradiol generatedfrom the testos- some areasof researchit is the study of mammals terone by the substrate,one can infer the amount of which has led to the development of new tech- aromatase (an enzyme that converts testosterone to niques and principles, it is often true that avian estradiol) present in the tissue. While useful for quan- forms provide the most dramatic examples of tification, these microdissectionsprovide only a crude environment-brain-hormone-behavior integra- index of the location of the enzyme. Development of tion. This derives in part from the availability antibodies(in the future) to the enzyme will allow more of information on environmental ecologyof many precise (immunohistochemical) localization of aro- different avian species,and on diverse behavioral matase enzyme. adaptations shown by birds. In birds, we can In quail, male copulatory behavior is androgen study the neural and endocrine basis of brood dependent. It disappearscompletely after castra- parasitism, monogamy, polygyny, polyandry, di- tion and is restored after androgen treatment verse breeding cycles, and their regulation by a (Adkins and Adler 1972, Schumacher and Bal- variety of environmental cues. REVIEW 915

Understanding how the brain mediates the re- BARCLAY,S. R., AND C. F. HARDING. 1988a. An- sponseof the endocrine system to the constantly drostenedione modulation of monoamine levels and turnover in hypothalamic and vocal control changing physical and social world is a major nuclei in the male zebra finch: Steroid effects on problem now facing and re- brain monoamines. Brain Res. 459:333-343. productive biology. We anticipate that the wealth BARCLAY,S. R., AND C. F. HARDING. 1988b. Differ- of information available on specific avian ad- ential effects of androgensand estrogenson cate- aptations will provide insight into general prin- cholamine levels and turnover in hypothalamic and vocal control nuclei in the male zebra finch. ciples of neuroendocrine processes. Sot. Neurosci. Abstr. 14:434. BENOIT,J. 1935. Stimulation par la lumi&re artifi- ACKNOWLEDGMENTS cielle du dCveloppementtesticulaire chez les ca- nards aveuglt‘s par section du nerf optique. C. R. We thank M. Morton and his students for delaying Stances Sot. Biol. Fil. 120:133-136. their Thanksgiving dinner to make comments on pre- BUNTIN,J. D., AND R. J. WALSH. 1988. In vivo au- vious drafts of this paper. We also thank Michael Nu- toradiographicanalysis of prolactinbinding in brain man for reading and commenting on the paper. The and choroid plexusof the domestic ring dove. Cell researchon Ringed Turtle-Doves from the laboratory Tissue Res. 25 1:105-109. of RS was supportedby NIMH grant 29380. The figure CASSONE.V. M.., ANDM. MENAKER. 1985. Is the avian was drawn by L. Laszlo Meszoly. circadian system a neuroendocrineloop? J. Exp. Zool. 2321539-549. LITERATURE CITED CHENG,M.-F. 1979. Progressand prospectsin ring dove research:A personal view, p. 97-129. In J. ADIUNS,E. K., AND N. ADLER. 1972. Hormonal con- S. Rosenblatt, R. A. Hinde, C. Beer, and M.-C. trol of behavior in the Japanesequail. J. Comp. Busnel [eds.], Advances in the study of behavior. Physiol. Psychol. 8 1:27-36. Vol. 9. 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