THE JOURNAL OF COMPARATWE NEUROLOGY 301:27&-288 (1990)

Distribution of Ammatase in the Brain of the Japanese &wail, Ring Dove, and Zebra Finch Anli-n.mmocytochemicdStudy JACQUES BALlXUZAFiT, AGNES FOIDART, CHANTAL SURLEMONT, ANGELA VOCKEL, AND NOBUHIRO HARADA Laboratory of General and Comparative Biochemistry, University of Liege, B-4020 Liege, Belgium (J.B., A.F., G.S., A.V.); Molecular Genetics, School of Medicine, Fujita-Gakuen Health University, Toyoake, AICHI 470-11, Japan (N.H.)

ABSTRACT

An immunocytochemicalperoxidase-antiperoxidaseprocedure using a purified polyclonal antibody raised against human placental aromatase was used to localize aromatase-containing cells in the brain of three avian species: the Japanese quail, the ring dove, and the zebra finch. In quail and dove, immunoreactive cells were found only in the preoptic area and hypothalamus, with a high density of positive cells being present in the medial preoptic area, in the septal area above the anterior commissure, in the ventromedial nucleus of the hypothalamus, and in rostral part of the infundibulum. Immunoreactivity was weaker in zebra finches, and no signal could therefore be detected in the ventromedial and tuberal hypothalamus. The positive material was localized in the perikarya and in adjacent cytoplasmic processes, including the full length of axons always leaving a clear unstained cell nucleus. These features could be observed in more detail on sections cut from perfused brains and stained with an alkaline phosphatase procedure. The distribution of aromatase immunoreactivity was similar in the three species although minor differences were observed in the preoptic area. The localization of labelled neurons coincided with the distribution of aromatase activity as studied by in vitro radioenzyme assays on brain nuclei dissected by the Palkovits punch method. There was one striking exception to this rule: no immunoreactivity was detected in the zebra finch telencephalon, while assays had shown the presence of an active enzyme in several nuclei such as the robustus archistriatalis, the hyperstriatum ventrale pars caudale, and the hippocampus and area parahippocampalis. The origins of this discrepancy and the functional role of the aromatase observed in the axons are discussed. Key words: aromatase immunocytochemistry,brain aromatase, preoptic area

The Japanese quail (Coturnixcoturnixjaponica), the ring dove or barbary dove (Streptopelia risoria), and the zebra finch (Taeniopygia guttata castanotis) have been widely used as avian model species in the research on behavioral endocrinology during the last two decades. In all three species, behavioral experiments have demonstrated that some of the effects of testosterone (T) on reproductive behavior are mediated, at least in part, through the central aromatization of the steroid into estradiol (EJ. This has been established in experiments demonstrating that T effects on behavior can be mimicked by estrogen such as E,, estradiol benzoate, or diethylstilbestrol but not by nonaromatizable androgens such as 5a-dihydrotestosterone (Adkins et al., '80; Adkins-Regan, '81; Alexandre and Balthazart, '86; Harding et al., '83). More importantly it was also shown that aromatase inhibitors such as androstatrienedione (ATD) and R76713 are able to block the behav-

o 1990 WILEY-LISS, INC.

ioral effects of T (Adkins et al., '80; Balthazart et al., '90e; Evrard et al., '89; Harding et al., '83). Direct experimental evidence has also been collected demonstrating that it is the aromatization of T in the brain which i s critical in the activation of these behaviors (Balthazart et al., '90e; Evrard et al., '89; Hutchison et al., '86; Watson and Adkins-Regan, '89a). By combining topographical microdissections with radioenzyme assays, it has been possible to confirm the presence of aromatase activity in the brain of these three species and to determine its anatomical distribution (Schumacher and Balthazart, '84, '86, '87; Steimer and Hutchison, '80, '81; Vockel et al., '90a,b). In the quail and zebra Accepted July 27,1990 Address reprint requests to Dr. J. Balthazart, University of LiBge, Laboratory of General and Comparative Biochemistry (Bat. L l ) , 17 place Delcour, B-4020 Liege, Belgium.

IMMUNOCYTOCHEMISTRY OF BRAIN AROMATASE IN BIRDS finch, aromatase activity was even measured on brain nuclei dissected by the Palkovits punch technique (Palkovits, '73; Palkovits and Brownstein, '83) which provides the highest possible anatomical definition which can be expected by this technical approach (Balthazart et al., '9Oc; Schumacher and Balthazart, '87; Vockel et al., '90a, b). Finally, physiological experiments have shown that in all three species, the activity of the preoptic andlor hypothalamic aromatase is sexually differentiated (higher in males than in females) and strongly controlled by T (decrease followingcastration and increase after replacement therapy with the steroid: Balthazart, '89; Balthazart et al., '86; Balthazart et al., '90a; Hutchison et al., '89a; Schumacher and Balthazart, '86; Steimer and Hutchison, '81; Vockel et al., '90a,b). The effects of T on aromatase activity probably explain the sex difference mentioned above: males usually have higher plasma T levels than females (Balthazart et al., '87; Vockel et al., 'gob), and their higher level of enzyme activity might therefore result only from a higher activation by T. The aromatase of the quail preoptic region might, however, be an exception to this rule, as a higher induction of aromatase activity has been observed in castrated males treated with T than in ovariectomizedfemales receiving the same treatment (Balthazart, '89; Balthazart et al., '86, '90a; Schumacher and Balthazart, '86). These measures of aromatase activity on microdissected brain regions do not permit us to answer many questions concerning the cellular and subcellular distribution of the enzyme (precise anatomical distribution, presence in neurons or glial cells, co-localization with other enzymes, with transmitters and peptides). This could only be achieved if the enzyme was detected by immunocytochemistry (ICC). Purifications of placental aromatase have been reported and purified antigens have been used to generate polyclonal or monoclonal antibodies (Bellino et al., '87; Harada, '88; Mendelson et al., '85). With these antibodies, aromatase has been localized in the ovary and placenta (FournetDulguerov et al., '87; Inkster and Brodie, '89; Sasano et al., '89a, '89b) but until recently, it had been impossible to apply this technique to the brain. Using an antibody raised against human placental antigen X-P, (hPAX-P,), Shinoda and collaborators presented studies of "aromatase-contain-

ing neurons" in the rat and monkey brain (Shinoda et al., '89a,b,c,). According to these authors, hPAX-P, is associated with aromatase, and the antibody against this antigen can partially inhibit the activity of the enzyme in the placenta but "the possibility cannot be excluded that the antibody also recognizes other substances common to estrogen biosynthetic organs." This suspicion is enhanced by the fact that these authors only found strongly immunoreactive neurons in brain areas which do not contain high levels of aromatase activity and conversely found few or no immunoreactive neurons in the preoptic area, hypothalamus and medial amygdala in which enzyme assays localized very high aromatase activity (compare Shinoda et al., '89a,b,c with Roselli et al., '85; Roselli and Resko, '89). With a purified polyclonal antibody raised in rabbit against human placental aromatase (Harada, '88), we were recently able to demonstrate immunoreactive neurons in the quail brain (Balthazart and Foidart, '89; Balthazart et al., '90b,d). We are very confident that this immunocytochemical procedure specifically labels the aromatase for several reasons: the antibody completely and specifically inhibits the enzyme activity in the quail brain, it detects positive cells only in brain areas which are known to contain high levels of enzyme activity, no signal is obtained if the antibody is preabsorbed with purified human aromatase, the immunoreactive material is exclusively located in the cytoplasm of the cells and finally the intensity of the immunoreactive signal in the preoptic area is increased by testosterone as the enzyme activity (Balthazart et al., '9Oa,b,d; Schumacher and Balthazart, '86). In this paper, we describe the distribution of the aromatase immunoreactivity as observed by this procedure in two other avian species, the ring dove and the zebra finch, and we compare these results with those previously published for quail.

MATEXIALSANDMETHODS Animals Male and female Japanese quail (Coturnixcoturnix japonica) were obtained from a local breeder at the age of 3 weeks. Adult male ring doves (Streptopelia risoria) and

Abbreviations

AA AC AM

AP APH AR-ir ATD CA Cb CG

co

CPa DMA DS E E, FPL GLv HA HP

Hv

HVC ICC ICO

archistriatum anterior nucleus accumbens nucleus anterior medialis hypothalami alkaline phosphatase area parahippocampalis aromatase-immunoreactive androstatrienedione commissura anterior (anterior commissure) cerebellum central gray chiasma opticum commissura pallii nucleus dorsomedialis anterior thalami decussatio supraoptica ectostriatum estradiol fasciculus prosencephali lateralis (lateral forebrain bundle) nucleus geniculatus lateralis, pars ventralis hyperstriatum accessorium hippocampus hyperstriatum ventrale hyperstriatum ventrale, pars caudale immunocytochemistry nucleus intercollicularis

277

IH LHY MLD N NC

nucleus inferioris hypothalami regio lateralis hypothalami (lateral hypothalamic area) nucleus mesencephalicus lateralis, pars dorsalis neostriatum neostriatum caudale ov nucleus ovoidalis PA paleostriatum augmentatum PAP peroxidase-antiperoxidase nucleus preopticus medialis (medial preoptic nucleus) POM PP paleostriatum primitivum PVN (PVM) nucleus paraventricularis magnocellularis nucleus robustus archistriatalis RA nucleus rotundus ROT (RT) nucleus ruber RU SL nucleus septalis lateralis nucleus septalis medialis SM so nucleus supraopticus nucleus subrotundus SRt T testosterone TN nucleus taeniae tractus septomesencephalicus TSM tuber TU nucleus ventromedialis hypothalami VMN

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zebra finches (Teaniopygiaguttata castanotis) were bought from local dealers. Throughout their life in the laboratory, all birds were exposed to a photoperiod of 16 hours of light and 8 hours of dark per day. They always had food (egg laying hen flour for quail, specific mixtures of grains for the other two species) and water available ad libitum. A total of 6 ring dove and 8 zebra finch brains were systematically analyzed in these experiments. More than 20 Japanese quail brains have now been stained for aromatase in our laboratory and the results presented here for comparative purposes are based on all these birds. As in preliminary experiments, a very weak immunoreactivity only had been observed in the zebra finch brain, all zebra finches which were subsequently studied were implanted 10 days before sacrifice with silastic capsules filled with testosterone. The capsules were made of silastic tubing (Dow Corning tubing, number 602-175; 8 mm in length, 0.76 mm i.d.; 1.65 mm 0.d.) filled with crystalline testosterone (Sigma, T-1500). They were implanted subcutaneously in the flank region. We have shown recently that these capsules significantly increase plasma T levels in castrated zebra finches and increase the aromatase activity in the preoptic area and hypothalamus (Vockel et al., '90b). It was hoped that as shown recently in quail, the increase in enzyme activity would be paralleled by an increase in the immunocytochemical signal which would make it more easily identifiable. In view of the success encountered, a similar procedure was also used for two of the ring doves (implantation of two T-filled capsules 20 mm long each; Dow Corning tubing, number 602-252; i.d. 1.57 mm, 0.d. 2.41 mm). Birds were killed by decapitation, and the brain was rapidly dissected out of the skull and frozen on powdered dry ice. Brains were stored in a deep freezer at - 70°C for no more than 2-3 days until they were processed. In a number of quail (n = 4), aromatase was also revealed in perfused brains by immunocytochemistry using a different procedure with alkaline phosphatase as enzyme and fast blue as chromagen. In these cases, quail were deeply anesthetized with Hypnodil (Janssen Pharmaceutica, Belgium; 50 m g k g body weight). They were then perfused with about 50 ml saline solution (0.15 M) followed by 200 ml of paraformaldehyde (4% in phosphate buffer-saline 0.1 M, pH 7.2 [PBSI). Brains were then dissected and placed overnight in a 20% sucrose solution in 0.1 M PBS.

Immunocytochemistry A standard peroxidase-antiperoxidase procedure using diaminobenzidine as the chromagen was used unless otherwise stated. To study the neuroanatomical distribution of the aromatase in all three species, the frozen brains were cut in 20-30 pm coronal sections on a cryostat. Sections were collected every 100 pm in the POA and every 300 pm in the rest of the hypothalamus. Alternate sections (40 pm) were also saved and stained with toluidine blue to confirm neuroanatomical localizations. The neuroanatomical nomenclature used in this paper is based on previously published work on the quail, chicken, dove, pigeon, and canary brain (Bay16 et al., '74; Berk and Butler, '81; Karten and Hodos, '67; Kuenzel and VanTienhoven, '82; Kuenzel and Masson, '88; Stokes et al., '74; Vowles et al., '75; Viglietti-Panzica et al., '86). Sections were fixed for 30 minutes to 3 hours in 4% paraformaldehyde in PBST (phosphate buffer: 0,025 or

0.01 M, pH 7.2; NaC1: 0.125 M, Triton X-100: 0.1%) at room temperature. We have demonstrated that, within these limits, the duration of the fixation has no influence on the intensity of the immunocytochemical signal. Endogenous peroxidase was blocked by immersing the sections in a solution of 0.6% hydrogen peroxide in methanol for 20 minutes. In some cases, this was followed by a 20 minute incubation in normal goat serum (1/5) in order to reduce nonspecific binding. Sections were incubated overnight at 4°C with the primary antibody diluted at 1/1,000 in PBST (see below). On the next day, sections were processed according to the peroxidase-antiperoxidase(PAP) technique. The goat antirabbit (dilution 1/60 for 30 minutes) and PAP complex (1/300 for 30 minutes) were both diluted in PBST. Extensive rinses in PBST were made between each step. The peroxidase was finally revealed by immersing slides for 6 minutes in a solution of diaminobenzidine (DAB; 20 mg in 50 ml PBST containing 20 pl of hydrogen peroxide at 30%). Some of the slides were osmicated (3 minutes in OsO, at a dilution of 1/1,000) to enhance the contrast. They were then dehydrated and mounted with Eukitt. In the alkaline phosphatase (AP)technique, the perfused brains were cut on the cryostat at 20-30 km. Sections were either mounted on gelatin-coated slides or stained as freefloating sections. They were rinsed several times in a tris buffer-saline (Tris buffer: 0.05 M, pH 7.6; NaCl: 0.15 M [TBSI), immersed for 20 minutes in Triton X-100 (l%), rinsed again, and then incubated overnight with the primary antibody diluted 1/1,000 in TBST (TBS plus Triton X-100: 0.5%). On the next day, they were incubated for 6 hours in alkaline phosphatase-conjugated swine anti-rabbit immunoglobulins (Dakopatts D-306) diluted 1/20 in TBST. Extensive rinses in TBS were made between each step. Following incubations, the alkaline phosphatase was finally revealed for 15 minutes in a solution of Fast Blue (Fast Blue BB salt: 10 mgin 10 ml TBS 0.1 M, pH 8.2) containing 2 mg Naphtol AS-MS phosphate, 0.2 ml N,N-dimethylformamide, and 0.01 ml Levamisole 1M) which was filtered onto the slides. They were mounted in an aqueous gelatin jelly as the blue chromagen is soluble in alcohol.

Antibodyandcontrol The primary antibody which was used is a polyclonal antibody raised in rabbit against human placental aromatase and purified by ammonium sulfate fractionation and affinity chromatography (Harada, '88). This antibody appears to be monospecific as determined in classical biochemical and immunological tests (Harada, '88). It has also been demonstrated that this antibody completely inhibits aromatase activity in the quail brain without affecting the activity of other testosterone-metabolizing enzymes (Balthazart et al., '90b). Additional controls including the omission of the primary, secondary, or tertiary antibody and the use of a pre-immune rabbit serum in place of the primary antibody have all demonstrated that no residual signal is observed in these conditions. Finally, we have shown that when the primary anti-aromatase antibody is pre-absorbed with a n excess of purified human placental aromatase (see Harada, '88 for the preparation and characteristics of this preparation) no immunoreactivity is observed in the quail brain.

IMMUNOCYTOCHEMISTRY OF BRAIN AROMATASE IN BIRDS

RESULTS By the PAP technique, we had previously identified aromatase-immunoreactive (AR-ir) neurons in 4 regions of the quail brain: the sexually dimorphic Wiglietti-Panzica et al., '86) medial preoptic nucleus (POM), a diagonal band extending from the level of the nucleus paraventricularis to the septal region dorsal to the anterior commissure and the lateral forebrain bundle in the area of the nucleus accumbens and more caudally of the nucleus striae terminalis, the dorso-lateral edges of the ventromedial nucleus of the hypothalamus (VMN), and a diagonal band of cells in the rostral tuber (TU) at the level of the nucleus inferioris hypothalami (IH) (Balthazart et al., '90b,d). The same general pattern of distribution has now been confirmed in more than 20 different birds of both sexes. No qualitative difference in this distribution was ever seen between males and females. Drawings of the labelled cells in the preoptic region made at the camera lucida are shown in Figure 1to provide an exact description of their distribution in that region. The AP technique confirmed the pattern of distribution of AR-ir cells in the quail brain. There is almost no background staining with this method, so that positive cells are always identified without any ambiguity. In addition, as this staining method was used on perfused tissues, a much better preservation of the sections was obtained. This allowed to better distinguish the distribution of immunoreactive material in the cells (see Fig. 2). The blue chromagen was localized exclusively in the cyt,oplasmof cells, while nuclei remained completely clear. The immunoreactive aromatase apparently was filling the entire cell including relatively long cell processes. Some of these very long and thin processes are axons without any doubt (see Fig. 2B). This has actually been confirmed by

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electron microscopy: axons labelled for aromatase can be observed at that level and immunoreactive material is actually found in the presynaptic boutons (Naftolin et al., '90). By this technique, AR-ir boutons were also found forming synapses with other AR-ir neurons. This spatial arrangement was also suggested at the optic level: in several instances, immunoreactive cellular processes were found in close proximity of other immunoreactive neurons (Fig. 2). A very similar distribution of AR-ir cells was found in the ring dove brain (see Fig. 3). Heavily labelled neurons were seen in the medial preoptic area (Fig. 4B,D), the septal region dorsal to the anterior commissure and lateral forebrain bundle (Fig. 4D), and in the rostral tuber at the level of the nucleus inferioris hypothalami (Fig. 4E). Cells showing a weaker but still clearly positive response were seen in the PVN and in a region dorsal and lateral to the VMN overlapping partly with this nucleus as observed in Nissl-stained material (Fig. 4C). Although the general pattern of distribution was similar in quail and dove, a clear-cut difference was detected in the medial preoptic area. Distribution of labelled cells in this region was more extensive in dove than in quail as shown in Figure 1 (middle panel, top). AR-ir cells were found in a more rostral position in the dove than in the quail. In dove, they were clearly labelling a rostro-ventral part of the preoptic area identified as the nucleus preopticus, pars suprachiasmatica (see Fig. 4A), in the study of MartinezVargas et al., (Martinez-Vargas et al., '76). While in the quail preoptic area, the AFLir neurons were confined to the POM (see Fig. 6A), labelled cells showed a more extensive distribution in doves and extended ventrally in the nucleus supraopticus (Figs. 1 and 4A,B). The extension of the

/I\ / \

ZEBRA FINCH

Fig. 1. Camera lucida drawings of aromatase-immunoreactive cells in the preoptic area of the quail, dove, and zebra finch. In each panel, three coronal sections of the preoptic area are arranged from top to bottom in a rostral to caudal order. In these drawings, each dot represents one immunoreactive cell.

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Fig. 2. Aromatase-immunoreactivecells in the medial preoptic nucleus (POM) of the quail preoptic area as observed on tissue perfused with paraformaldehyde after localization of the bound antibody by the alkaline phosphatase technique. Final magnification is x 1,000; calibration bar is 20 km.

labelled cells in the dorso-ventral direction was therefore greater in the dove, in which they formed a long diagonal band situated in a juxta-ventricular position. In the caudal part of the preoptic area at the level of the anterior commissure, labelled cells were confined, like in quail, to an ovoid area which appears to be equivalent to the POM (Fig. 4D). More caudally, this ovoid area merged with the

diagonal band of labelled cells extending from the nucleus paraventricualris (PVN) to the septal region above the lateral forebrain bundle (same distribution as in quail, see Fig. 1). At a higher magnification (Fig. 51, the intracellular distribution of the AR-ir material was identical to what had been seen in quail. The positive material filled the cyto-

IMMUNOCYTOCHEMISTRY OF BRAIN AROMATASE IN BIRDS

28 1

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500 urn

3500 urn

800 ,urn

3800 ,urn

1100 ,urn

4100 IJ m

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Fig. 3. Distribution of aromatase-immunoreactive cells (dots) in the brain of the ring dove. Schematic representations of coronal sections in the brain are presented in the rostral to caudal order from top to bottom and from left to right. Numbers at the left side of the sections represent distances in microns from the level of the tractus septomesencephalicus.

plasm of the cells, including processes extending for several group of elements at the level and caudal to the anterior cell diameters away from the cell nucleus. These long commissure. At the most rostral level (not shown on figure), immunoimmunoreactive processes were especially noticeable in the POM and the septal region. They were rare or absent in the reactive cells were also seen in the POM under the tractus other labelled areas. AR-ir cells in the tuber were also septo-mesencephalicus (plate A 2.6 in Stokes et al., '74). No smaller than in the POM. The cell nuclei were always clear immunoreactivity was detectable in the tuberal hypothalafrom immunoreactive material. mus in regions which are homologous to those which were TU). In addition, no In the zebra finch our attempts to localize AR-ir cells labelled in the other two species (VMN, were first unsuccessful. An extremely weak label was trace of immunoreactivity could be observed in the zebra observed in some cells of the preoptic region, but it was finch telencephalon and in particular in the hippocampus impossible to ascertain that the label was specific. As (Hp), the area parahippocampalis (APH),and several nuclei testosterone is known to increase aromatase activity in the of the song control system (nucleus robustus archistriatalis hyperstriatum ventrale pars caudale [HVcl) in which zebra finch preoptic area (Vockelet al., '90b) and as such an [RA], increase was in quail associated with an intensification of very high levels of aromatase activity have been detected by the immunoreactivity (Balthazart et al., 'gob), additional radioenzyme assays (Vockelet al., '90a,b). immunocytochemicalexperiments were performed on brains coming from testosterone-treated zebra finches. Clearly DISCUSSION labelled cells could, in this way, be identified in the preoptic aredanterior hypothalamus (see Fig. 1,right panel). In this During the last two decades, aromatase activity has been region, the immunoreactive cells appeared to be localized identified in the brain of a large number of vertebrate mainly in the nucleus paraventricularis magnocellularis species following its first identification in the brain of (FVM) through its rostral to caudal extent as described in mammals during the early seventies (Naftolin et al., '75). the canary brain (Stokes et al., '74; plates A 2.4 to A 1.0). The activity of the enzyme has been measured in a variety This included a round mass of cells at the most rostral level of physiological conditions (see above), but it has been (see Fig. 6B), which progressively turned into a V-shaped impossible, until recently, to define its precise neuroanatom-

282

Fig. 4. Aromatase immunoreactive cells in the preoptic area and hypothalamus of the ring dove. A Rostral preoptic area (level of the nucleus preopticus, pars suprachiasmatica). B:Medial preoptic nucleus (POM). C: Rostral part of the paraventricular nucleus. D: Caudal part

J. BALTHAZART ET AL.

of the POM and septal area above the anterior commissure. E:Tuberal hypothalamus at the level of the nucleus inferioris hypothalami. Final magnification is x55 in A-C; x 4 0 in D; x35 in E. Calibration bar is ,100 pm in each case.

IMMUNOCYTOCHEMISTRY OF BRAIN AROMATASE IN BIRDS

283

Fig. 5. Aromatase-immunoreactive cells in the preoptic area, septum, and hypothalamus of the ring dove as obsel-ved on fresh frozen tissue stained by the PAP technique. A, B: Medial preoptic nucleus. C, D: Septal region above the anterior commissure. E: Nucleus paraventricularis. F: Nucleus inferioris hypothalami. Final magnification is x 1,000 calibration bar is 20 pm.

ical localization by immunocytochemistry because no specific antibody was available. Reports were published during last year which analyzed in the rat and monkey brain the distribution of the antigen hPAX-P,, which is thought to be specific to estrogen-biosynthetic organs (Shinoda et al.,

'89a-c). This antigen might be functionally associated with aromatase activity, but its distribution in the brain does not clearly match the distribution of the aromatase activity as studied by radioenzyme assays on nuclei dissected by the Palkovits punch technique (Roselli et al., '85; Roselli and

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Fig, 6 . Aromatase-immunoreactive cells in the nucleus preopticus medialis (POM) of the quail (A) and the rostral part of the nucleus paraventricularis magnocellularis (PVM)of the zebra finch (B). Final magnification is x 50 in A and x 100 in B; calibration bar is 400 pm in A and 200 pm in B.

Resko, '89). This cast doubts on the usefulness of the antibody directed against hPAX-P, for the study of the neuroanatomical distribution of aromatase. Using an antibody directed against purified human placental aromatase, we have recently established a PAP immunocytochemical procedure which permits us to specifically localize AR-ir cells in the quail brain (Balthazart et al., '90b,d). The specificity of this identification has been established by classical control procedures in immunocytochemistry (e.g., omission or pre-absorption of the primary antibody). It is also supported by several lines of biological evidence, such as the good matching between the distribution of immunoreactive cells and aromatase activity (with the important exception of the zebra finch telencephalon; see below), the similar effects of testosterone on the enzyme activity and immunoreactivity, and finally the demonstration that the antibody specifically inhibits aromatase activity in classical radioenzyme assays. In the present study, we demonstrate that this immunocytochemical procedure is able to identify AR-ir cells in the brain of two other avian species, the ring dove and the zebra finch. By introducing a modification of our original procedure (use of perfused tissue and of alkaline phosphate as enzyme marker), we also provide more detail on the distribution of the enzyme at the cellular level in quail. A very similar distribution of AR-ir cells was observed in the three avian species that were studied. Most of the positive cells appeared to be neurons, but at this stage it cannot be excluded that a few glial cells are also labelled and double label immunocytochemistry with specific neuronal and glial markers should be used to settle this question. The most intense label was observed in the preoptic area/ anterior hypothalamus of all species. This always included the medial preoptic nucleus (POM), which in quail is larger in males than in females (Panzica et al., '90; VigliettiPanzica et al., '86) and testosterone-sensitive (Panzica et al., '87, '90). Another V-shaped region extending from the level of the paraventricular nucleus to the lateral septum above the anterior commissure and the lateral forebrain bundle was also heavily labelled in quail, dove, and zebra finches (Figs. 1,3,4D).This area containing AR-ir cells does not correspond exactly to any of the classical nuclei defined in Nissl-stained material. This points to an important

problem in neuroanatomy which is especially crucial when dealing with the hypothalamus: the cell clusters which are defined by neurochemical markers do not always match those which received names based on classical histology techniques. In addition, there are discrepancies in the nomenclature of nuclei across species and even within species when observed by different authors (Crosby and Showers, '69; Huber and Crosby, '29; Kuenzel and VanTienhoven, '82; Oksche and Farner, '74; Panzica et al., '90). Additional studies using different neurochemical markers and/or defining the afferent and efferent connections of these areas should be carried out to establish more precisely the nomenclature of this brain region. The position of the immunoreactive cells at the dorsolateral edge of the VMN and in the rostral tuber at the level of the nucleus inferior hypothalami was also extremely similar in the quail and dove. No label could be detected in these two areas in the zebra finch, but this probably only reflects a sensitivity problem. The signal observed in the preoptic area of this species was clearly weaker than in the other two. It is reasonable to assume that aromatase is also present in the VMN and TU of this species but simply cannot be detected by the present procedure. With the exception of the broader distribution of aromatase-immunoreactive neurons in the rostral preoptic area of the dove (see below), it appears that the localization of the enzyme in the three species is very similar. Immunoreactive cells define cellular groups which are in all probability homologous in these species. This represents an additional illustration of the fact that certain aspects of sex hormones interactions with the brain have a widespread phylogenic validity. The binding sites of sex steroids in the preoptic area-hypothalamus have, for example, remained fairly stable from fishes to mammals (Kelleyand Pfaff, '78). The functional significance of these groups of aromataseimmunoreactive cells is not completely clear at present. If it is known that the transformation of androgens into estrogens controls several aspects of reproduction in each species (see above), the exact site where aromatization has to take place in the brain in order to modulate a given behavior is generally unknown. The quail makes an exception to this rule: it was recently demonstrated that aromatization in the preoptic area and most probably in the POM is specifi-

IMMUNOCYTOCHEMISTRY OF BRAIN AROMATASE IN BIRDS cally linked to the activation of copulatory behavior (Balthazart et al., '90e; Watson and Adkins-Regan, '89a). Such a conclusion is also likely to be valid for doves and zebra finches, but this has not been established so far. The biological significance of the other groups of aromataseimmunoreactive cells (dorsal to PVN and to VMN,tuber) is not known at present and future experiments including stereotaxic implantation of aromatase inhibitors should investigate the specific role of the enzyme in each of these brain nuclei. Besides these brain regions in which the distribution of aromatase-immunoreactive cells seems to be constant across species, one clear-cut species difference needs to be mentioned here. In the rostral preoptic area of the dove, the labelled cells extended much more ventrally and rostrally than in the quail. This difference corresponds with the distribution of dense neuronal populations in Nissl-stained sections of the preoptic area in the two species. In each case, the AR-ir cells coincide with the dense medial populations described as nucleus preopticus medialis (POM) in quail (Panzica et al., '87, '90; Viglietti-Panzica et al., '86) and in dove (Martinez-Vargas et al., '76; Vowles et al., '75). In the latter species, this nucleus is, however, much larger and includes a rostral part that was considered equivalent to the nucleus preopticus, pars suprachiasmatica of the rat (Martinez-Vargas et al., '76). The distribution of aromatase activity as measured by radioenzyme assays in the quail, dove, and zebra finch has been previously reported. In two species (quail, zebra finch), assays were performed on brain nuclei which had been dissected by the Palkovits punch technique from 200 pm thick cryostat sections, therefore providing a maximal anatomical resolution for this type of approach (Balthazart et al., '9Oc; Schumacher and Balthazart, '87; Vockel et al., '90a,b). In doves, brain samples were obtained by a topographical dissection providing less information on the exact position of the active sites (Steimer and Hutchison, '80, %l),but this nevertheless provided useful information which can be compared with the present immunocytochemical data. In the three avian species which were studied here, there was a close correlation between the distribution of the enzyme activity and of the immunoreactive cells observed with the antibody raised against human placental aromatase. The areas of the quail and zebra finch brain demonstrating immunopositive cells for aromatase (in quail: POM, VMN, TU; in zebra finches: POM, PVM) were previously shown to contain aromatase activity by product formation assays performed on brain nuclei microdissected by the "Palkovits punch technique" (Palkovits, '73; Palkovits and Brownstein, '83). Nuclei which were devoid of enzyme activity contained no immunoreactive cells (Balthazart et al., '9Oc; Schumacher and Balthazart, '87; Vockel et al., '90a,b). Such a close matching cannot be made in the dove, but it was shown that the highest levels of aromatase activity are located in the medial preoptic area (Steimer and Hutchison, '80) and this fully agrees with the present irnmunocytochemical identification. This coexistence between enzyme activity and immunoreactive cells is a further proof for the chemical specificity of the antibody that we used. The fact that treatments with testosterone (TI, which are known to increase aromatase activity in both doves and zebra finches (Hutchison and Steimer, '86; Hutchison et al., '89a; Steimer and Hutchison, '89; Vockel et al., '90b) enhanced the immunoreactivity of preoptic neurons also supports this notion (see also Balthazart et al.,

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'90b,d, for a similar argument in quail). In addition, this strongly suggests that the increased aromatase activity which is observed after a treatment with T in the three species results from a real increase in enzyme concentration. Alternative explanations involving conformational changes in the molecule remain possible but unlikely, as we used a polyclonal antibody and it would be surprising that one conformation of the enzyme masks all the immunoreactive sites. There is no clear interpretation at present to the fact that a weak immunoreactivity only was detected in zebra finches even after a treatment with testosterone. This might of course be explained by a lower enzyme concentration in this species. The maximal levels of aromatase activity that we observed in the zebra finch PVM are in the range 500-1,000 fmollmg proteinlhour (Vockel et al., '90a,b), while levels up to 3,000 fmoVmg proteinhour have been measured in the quail POM (Schumacher and Balthazart, '87; Balthazart et al., '9Oc) and similar high levels of activity are reported for the dove preoptic area (Steimer and Hutchison, '81). However, AR-ir neurons have also been clearly labelled in the quail VMN or TU, where enzyme activity is in the same range as in the zebra finch. It is therefore also possible that the zebra finch aromatase does not cross-react as well with our antibody as the quail or dove enzyme. This possibility could only be tested if pure preparations of the enzyme were available for these species. Another very puzzling observation needs to be mentioned concerning the zebra finch aromatase. In our previous studies using the radioenzyme assay technique, we found that significant levels of aromatase activity are present not only in preoptic and hypothalamic nuclei (POA, PVM) but also in a number of telencephalic structures such as nuclei of the song system (RA, HVc) and the hippocampal and parahippocampal region. Enzyme activity was in fact much higher in Hp-APH than in any other brain area, including the PVM (Vockel et al., '90a,b). It is therefore very surprising that no immunoreactivity was observed here in the telencephalon, while a weak but clearly positive signal was seen in the PVM. Two interpretations could take this discrepancy into account. It is possible that in the preoptichypothalamic areas, the aromatase is concentrated in a few cells, while it would be present in low concentration in most neurons of the positive areas of the telencephalon. This would result in the observed pattern of higher activity in Hp-AF'H than in PVM, while no immunoreactivity could still be detected in the telencephalic areas because not enough antigen would be present in any given cell. Alternatively, one might speculate that the structure of the telencephalic enzyme is slightly different and that therefore it does not cross-react (as well) with the antibody that was used here. This suggestion is indirectly supported by our recent finding that the hormonal regulations of aromatase activity are quite different in the hypothalamus and telencephalon in this species. While the enzyme activity of the PVM showed regulations similar to those which were previously observed in quail or doves (higher activity in males than in females, decrease after castration and increase following testosterone treatment), a very different pattern was observed in the song control nuclei and in the Hp-APH where some "reversed" sex differences were detected (e.g., activity higher in females than in males in RA and HVc) and no effect of castration or testosterone replacement therapy was visible (in RA,HVc, Hp, and APH; see Vockel et al., '90a,b). These differential regulations of the

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enzyme activity might just be an epiphenomenon but alternatively they could be the result of a significant difference in enzyme structure and therefore explain the difference in immunoreactivity. Additional studies should be performed to characterize independently the aromatase in the hypothalamus and telencephalon of this species. By using an alternative method for the revelation of bound antibody (alkaline phosphatase) on brain tissue that was fixed by perfusion, it has been possible here to obtain a better cellular localization of aromatase in the quail brain. This clearly confirmed the presence of immunoreactive material in the cytoplasm and its absence in the cell nuclei. The entire perikarya were clearly labelled by this procedure, including the full length of dendritic trees. Very thin cell processes which can be identified as axons were also clearly labelled. This has been recently confirmed by studies combining a pre-embedding immunostaining with electron microscopy: aromatase-immunoreactive material is found in the full length of axons, including the synaptic boutons (Naftolin et al., '90). These observations confirm two earlier papers which had identified by biochemical methods high levels of aromatase activity in synaptosomes prepared by differential centrifugation of quail or rat brain (Schlinger and Callard, '89; Steimer, '88). The presence of aromatase at the synaptic level raises important questions regarding the role played by estrogens produced by central aromatization of androgens. It is known that, in several model systems, estrogens have rapid membrane effects which do not appear to be mediated by an interaction with the classical nuclear receptors (see Blaustein and Olster, '89, for review). Future studies will have to determine whether estrogens formed in the central nervous system act, at least in part, through these non-genomic mechanisms to modulate behavioral and physiological aspects of reproduction. The availability of an immunocytochemical method to identify aromatase-containingneurons will now permit the study of the relationships between this enzyme and estrogen receptors in the brain. Ultimately this analysis will have to be performed by a double immunocytochemical technique labelling both antigens on the same sections, and this work is presently in progress in our laboratory. However, at present it is already possible to compare the distribution of the aromatase and of estrogen receptors as they have been separately revealed by autoradiography (in quail: Watson and Adkins-Regan, '89b; in dove: MartinezVargas et al., '75, '76; in zebra finch: Nordeen et al., '87) or by immunocytochemistry using the monoclonal antibody H222 (Greene et al., '84) raised against human estrogen receptors isolated from a mammary tumor (in quail: Balthazart et al., '89; in dove: Hutchison et al., '89b; in zebra finch: Gahr et al., '87; Gahr and Konishi, '88). In general, it can be stated that, in the three species which are considered here, aromatase-containingcells were found in brain areas which are known to be involved in the control by steroids of reproductive processes, namely, the preoptic area, the septum, the ventro-medial, and the tuberal hypothalamus. All these areas have been shown to contain estrogen receptors. There is a fairly close matching between the distribution of estrogen receptors and aromatase in the hypothalamic regions of the quail, dove and zebra finch. When a close analysis can be performed, some subtle differences in distribution are nevertheless present as recently shown for the quail POM. Estrogen receptors are preferentially found in the medial part of this nucleus, while AR-ir neurons are essentially present at its periphery

J. BALTHAZART ET AL. (Balthazart et al., '90d). Whether similar situations also exist in other species and for other brain nuclei can now be examined. On a broad scale, it can be concluded that estrogen receptors are always present in the areas containing AR-ir neurons. The reverse is, however, not true and estrogen binding sites are present in brain areas that contain no aromatase such as the nucleus intercollicularis or the nucleus taeniae in quail. These are presumably influenced directly by estrogens formed in the ovary. It is well established that the central aromatization of androgens is a critical step in the activation by testosterone of reproductive behavior (see above). It was assumed that estrogens produced centrally exerted their action by binding to nuclear estrogen receptors and promoting the synthesis of new proteins. The fact that estrogen receptors are present in the same brain areas as aromatase is certainly consistent with this idea. However, the finding that aromatase immunoreactivity is present in the synaptic boutons (see above) raises questions concerning the role of estrogens produced by central aromatization. Double-label experiments in the nuclei which contain both aromatase and estrogen receptors should now indicate whether they are co-localized in the same cells.

ACKNOWLEDGMENTS We are indebted to Professor E. Schoffeniels for his continued interest in our research. This work was supported by grants from the National Institutes of Health, Bethesda, MD (HD 22064); the Belgian Fonds National de la Recherche Scientifique (Crbdits aux Chercheurs); the Medical School of Liege, the University of Liege (Fonds Special pour la Recherche); and the EEC (SC1-0230-C/TT) to J. Balthazart and by a grant from the Belgian Fonds de la Recherche Fondamentale Collective (nbr 2.4518.80) to Professor Schoffeniels.

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