The medial preoptic nucleus receives vasotocinergic inputs in male quail: a tract-tracing and immunocytochemical study

Journal of Chemical Neuroanatomy 24 (2002) 27 /39 www.elsevier.com/locate/jchemneu

The medial preoptic nucleus receives vasotocinergic inputs in male quail: a tract-tracing and immunocytochemical study Philippe Absil a,1, Monica Papello b, Carla Viglietti-Panzica b, Jacques Balthazart a, GianCarlo Panzica b,* a

Center for Cellular and Molecular Neurobiology, Research Group in Behavioral Neuroendocrinology, University of Lie`ge, 17 Place Delcour, B-4020 Liege, Belgium b Department of Anatomy, Pharmacology and Forensic Medicine, University of Torino, Rita Levi Montalcini Center for Brain Repair, Laboratory of Neuroendocrinology, c.so M. D’Azeglio 52, I-10126 Torino, Italy Received 22 January 2001; received in revised form 3 December 2001; accepted 26 March 2002

Abstract The sexually dimorphic testosterone-sensitive medial preoptic nucleus (POM) of quail can be identified by the presence of a dense network of vasotocinergic fibers. This innervation is sexually differentiated (present in males only) and testosterone sensitive. The origin of these fibers has never been formally identified although their steroid sensitivity suggests that they originate in parvocellular vasotocinergic neurons that are found in quail only in the medial part of the bed nucleus striae terminalis (BSTm) and in smaller numbers within the POM itself. We report here that following injections of a retrograde tracer into the POM of male quail, large populations of retrogradely labeled cells can be identified in the BSTm. The POM also receives afferent projections from magnocellular vasotocinergic nuclei, the supraoptic and paraventricular nuclei. Double labeling for vasotocin immunoreactivity of the retrogradely labeled sections failed however to clearly identify magnocellular vasotocin-immunoreactive cells that were retrogradely labeled from POM. In contrast a substantial population of vasotocin-immunoreactive neurons in the BSTm contained tracer retrogradely transported from the POM. These data therefore demonstrate that a significant part of the vasotocinergic innervation of the quail POM originates in the medial part of the BST. An intrinsic innervation could however also contribute to this network. This interaction between BSTm and POM could play a key role in the control of male-typical sexual behavior and in its sex dimorphism in quail. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Bed nucleus striae terminalis; Retrograde tracing; Sexual behavior; Preoptic area; Sex difference; Japanese quail; Coturnix japonica

1. Introduction In birds and mammals, neurons expressing the nonapeptide vasotocin (VT; birds) or vasopressin (VP; mammals) can be divided in two main groups. One group of large neurons (magnocellular) is clustered in the various subdivisions of the paraventricular and supraoptic nuclei and is the origin of the hypothalamo-hypophyseal tract that is formed by thick fibers innervating the neurohypophysis. These neurons are

* Corresponding author. Tel.:
/39-11-670-7970; fax:
/39-11-6707732. E-mail address: [email protected] (G. Panzica). 1 Present address: Department of Biology, UIA, University of Antwerp, B-2610 Wilrijk, Belgium.

mainly implicated in osmoregulation. Another group of smaller (parvocellular) VT/VP-immunoreactive (ir) neurons are located in the hypothalamic and limbic systems and are supposed to be at the origin of the thin VT/VP-ir fibers that have been described in a diversity of brain areas (Panzica et al., 2001). The anatomical organization of the parvocellular part of this peptidergic system, its sexual dimorphism and its regulation by steroids have been the focus of a lot of attention in several animal models, and a number of species-specific differences have been identified (de Vries, 1990; de Vries et al., 1992, 1994; de Vries and Miller, 1998; Jurkevich et al., 1997, 1999; Aste et al., 1998; Moore and Lowry, 1998; Panzica et al., 2001). In rat, a prominent VP-ir innervation is described mainly in the lateral septum and is claimed to have its origin in the

0891-0618/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 8 9 1 - 0 6 1 8 ( 0 2 ) 0 0 0 1 7 - 0

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VP-ir neurons located in the bed nucleus of the stria terminalis (BST) and medial amygdala (MeA) (Caffe´ et al., 1987). This conclusion is supported by the fact that both these cells and fibers are sexually dimorphic and exquisitely sensitive to steroid treatments in adulthood: they essentially disappear in castrated animals and are restored to intact level by treatments with testosterone (T) or its estrogenic metabolite, 17b-estradiol. The medial preoptic area is also innervated by thin VP-ir fibers that are supposed to originate in the suprachiasmatic nucleus. This projection system is not known to be sensitive to gonadal hormones (de Vries et al., 1985, 1992, 1994). The origin of these VP-ir fibers in rat has however never been confirmed by tract tracing experiments combined with immunocytochemical visualization of the peptide. In hamsters, a sex dimorphism in the organization of the vasopressinergic system has been identified in the magnocellular cell groups. In contrast to rats, no sexually dimorphic population of parvocellular neurons can however be detected in the BST and MeA (Delville et al., 1994; Ferris et al., 1995). As a consequence, it has been assumed that the weak VP-ir innervation of the septum originates in magnocellular VP-ir neurons in this species, but again this hypothesis has never been formally tested. Data available so far clearly suggest that the weak innervation of the septum in hamster may not be homologous to the much denser VT-ir innervation of this brain region in rats. In gerbils, major sex differences in the vasopressinergic innervation have been detected both in the lateral septum and the dimorphic part of the preoptic area. Interestingly, this innervation is highly sensitive to the level of gonadal steroids in the lateral septum, but not in the medial preoptic area (complete disappearance of this innervation in the former case, no effect in the latter case) (Crenshaw et al., 1992). The origin of these fibers has not been investigated. In quail, a sexually dimorphic T-sensitive vasotocinergic innervation has been identified in a number of brain nuclei, including the lateral septum, the medial part of the BST (BSTm), and the medial preoptic nucleus (POM) (Viglietti-Panzica et al., 1992, 1994; Aste et al., 1998; Panzica et al., 1998, 2001). In addition to the VT-ir magnocellular neurons that are located mainly in the paraventricular and supraoptic nuclei and are not affected in detectable manner by testosterone, small populations of parvocellular T-sensitive VT-ir neurons have been identified in BSTm and to a lesser extent in POM. No VT-ir cell has ever been detected in the regions of the quail or chicken brain that are homologous to the mammalian amygdala (Zeier and Karten, 1971; Davies et al., 1997; Thompson et al., 1998), i.e. the ventral part of the archistriatum and more specifically the nucleus taeniae (Aste et al., 1996, 1998; Panzica et al., 1999). Because: (1) VT-ir magnocellular

neurons are not apparently affected by T (VigliettiPanzica et al., 1994, 2001; Panzica et al., 1999); (2) no VT-ir neurons can be detected in the quail archistriatum (the avian homologue of the mammalian amygdala, Aste et al., 1996); and (3) the only T-sensitive VT neurons are found in the BSTm and to some extent POM (Viglietti-Panzica et al., 1994; Panzica et al., 1999), it has been assumed that these neurons are at the origin of the steroid-sensitive VT-ir innervation of the POM. This conclusion has however not be experimentally tested. We report here that following injections of a retrograde tracer into the POM of male quail, large populations of retrogradely labeled cells can be identified in the BSTm. Immunocytochemistry confirmed that some of these retrogradely labeled cells at least also express VT. Taken together, these data demonstrate that a part at least of the vasotocinergic innervation of the quail POM originates in the medial part of the BST.

2. Material and methods 2.1. Subjects Experiments reported in this paper were performed on 14 male Japanese quail (Coturnix japonica ) that were bought from a local breeder in Belgium (Degros-Louppe farm, Rechrival) at the age of about 7 weeks. They were kept for about 2 weeks in individual cages in the laboratory before being injected with retrograde tracer. During their life in the breeding colony and in the laboratory, birds were housed under a controlled photoperiod (16L:8D) simulating long days and they always had food and water available ad libitum. All birds were housed, manipulated and sacrificed according to the Principle of Laboratory for Animal Care (NIH) and to the relevant Belgian laws on the protection of animals. The protocols had been previously approved by the Ethical Committee for Use of Animals at the University of Lie`ge. 2.2. Stereotaxic injections When birds were approximately 9 weeks old, retrograde tracer was injected stereotaxically into their medial preoptic nucleus under deep anesthesia (HypnodilTM 20 mg kg1 body weight; Janssen Pharmaceutica, Beerse, Belgium). The stereotaxic coordinates were determined based on the stereotaxic atlas of the quail brain (Bayle´ et al., 1974) and adjusted to the larger size of our birds. Targets were approached from the contralateral side with an angle (a) equal to 108 in the left-right axis to avoid leakage of the tracer in the adjacent areas as well as in the dorsal and occipital sinuses that are located dorsally along the midline.

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Coordinates were based on previous studies in which electrolytic lesions or retrograde tracers had been placed into the POM (Balthazart and Absil, 1997; Balthazart et al., 1998), namely: anterior (A) //5.10 mm, vertical (V) /
/2.35 mm, lateral (L) /(yo?/yo) sin a9/0.71 mm (five subjects were injected in the left POM, five others in the right POM) with yo? corresponding to the vertical coordinate at the surface of the brain and yo corresponding to the true zero point of the stereotaxic frame. Pressure injections were made unilaterally through a glass micropipette (tip diameter of about 50 mm, firepolished) connected to a PV800 pneumatic picopump (WPI, New Haven, CT) tuned at 20 PSI and 5 ms. This permitted the delivery of about 100 nl of the retrograde tracer that consisted of carboxylate-modified red latex Fluospheres# (RLF; Ø 0.04 mm; Excitation 580 nm/ Emission 605 nm; Molecular Probes, Eugene, OR; cat.nbr.F-8793) diluted twice to obtain a solution containing 2.5% vol of solids. The efficacy of the injection was assessed by observing under a binocular microscope the lowering of the liquid level in the micropipette. 2.3. Sacrifice Five days after the injection of tracer, birds were injected i.v. with heparin (10 mg kg1 body weight; cat.nbr. H-7005, Sigma, St. Louis, MO) and deeply anesthetized (HypnodilTM 20 mg kg1 body weight; Janssen Pharmaceutica, Beerse, Belgium). Their brain was fixed by intra-cardiac perfusion with about 300 ml of 9˜ saline (0.15 M NaCl) followed by 400 ml of fixative (4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.2). The brain was dissected out of the skull, cryoprotected overnight at 4 8C in 0.1 M PB containing 20% saccharose, frozen on dry ice and stored at /75 8C. 2.4. VT immunoreactivity Brains were cut in the coronal plane with a cryostat at 50 mm thickness and two series of alternate sections were collected in PB 0.01 M pH 7.2 /7.4. In the first ten birds used in this experiment, one set of sections was mounted on microscopic slides and coverslipped. They were then used to localize the position of retrogradely labeled cells in the entire brain. The second set of sections was stained for VT by immunofluorescence by the freefloating procedure. Briefly, the sections were collected in multidish wells in phosphate buffered saline (PBS, 0.01 M phosphate buffer, NaCl 150 mM, pH 7.2 /7.4), washed several times, covered by diluted (1:10) goat normal serum for 30 min at room temperature, and finally put for 48 h at 4 8C in the primary antibody antiVT (a generous gift of D.A. Gray, Bad Nauheim,

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Germany) diluted 1:2000 with PBS containing 0.2% of Triton X100 (PBST). After extensive washing with PBS, the sections were collected on cleaned slides and incubated with a rabbit anti-IgG serum coupled to fluorescein (Chemicon, Temecula, CA) diluted 1:400 in PBST for 60 min at room temperature. Slides were then washed in PBS and coverslipped using as a mounting medium a mixture of PBS-glycerol (1:1). All procedures were performed in the dark to avoid fading of the retrograde tracer and of the fluorescein. Because the immunofluorescence staining failed to identify VT-ir cell bodies in the POM and BSTm (contrary to our previous studies using immunocytochemical visualization by the avidin /biotin technique) coverslips were removed from the first set of sections that had been used to locate retrogradely labeled cells and these sections were stained for VT by the classical avidin /biotin technique. To block endogenous peroxidase activity, sections were immersed for 30 min in a solution of methanol/hydrogen peroxide (Streefkerke, 1972). The floating sections were incubated overnight at room temperature with the anti-VT serum at a dilution of 1:8000 in PBST. A biotinilated anti-rabbit serum (Vector, Burlingame, USA) was then used at a dilution of 1:200. The antigen-antibody reaction was revealed by a biotin /avidin system (BAS, Vectastain Elite Kit, Vector, Burlingame, USA). The peroxidase activity was visualized with a solution containing 0.15 mg ml 1 3,3-diamino-benzidine (DAB) and 0.025% hydrogen peroxide in 0.05 M Tris /HCl buffer pH 7.6. Sections were mounted on chromalum coated slides, air dried, washed in xylene and coverslipped with Entellan (Merck, Darmstadt, Germany). In the last four birds, brains were also cut in the coronal plane at 50 mm thickness as described above and two series of alternate sections throughout the forebrain were collected. The first set of sections was stained by the avidin /biotin technique with DAB as chromogen as described above. The second set was then stained by a three-step fluorescent procedure that was expected to provide a higher sensitivity than the two-step procedure original used (see above). Briefly these sections were successively incubated in the primary antibody diluted at 1:2000 (48 h at 4 8C as described above) and then successively for 90 min at room temperature in a biotinylated goat anti-rabbit antibody (Dakopatts A/S, Denmark) diluted 1:400 and for 90 min at room temperature in streptavidin coupled to FITC (Dakopatts A/S, Denmark) diluted 1:400. All other steps and rinses between incubations were performed as described in the other fluorescent procedure described above. 2.5. Antibody specificity The primary primary antibody against VT (gift of D.A. Gray, Bad Nauheim, Germany) was raised in

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Fig. 1

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Fig. 1 (Continued)

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rabbit against mammalian arginine-vasopressin (Gray and Simon, 1983). It was shown by radioimmunoassay that this antibody does not cross-react with related peptides such as mesotocin, oxytocin and angiotensin II (Gray and Simon, 1983). In addition, the specificity of this antibody for staining VT in the quail brain has been demonstrated by replacing the primary antibody by buffer or by antibody preadsorbed with pure antigen (Viglietti-Panzica et al., 1994). No immunoreactivity is detected in the quail brain in these control conditions. We also showed that the antibody does not cross-react with mesotocin in quail specifically (Viglietti-Panzica et al., 1994). 2.6. Data analysis Before observing sections under the fluorescence microscope, their boundaries and major neuroanatomical landmarks (ventricles, main fiber tracts) were drawn with the help of a camera lucida attached to a Leitz microscope (Laborlux 12). The distribution of the fluorescent tracer was then observed and recorded on the drawings with the aid of a Leica DMRE microscope using the filter for rhodamine. The double-stained sections were observed on a Leica DMRB microscope either with a double band filter (for fluorescein and rhodamine; tracer plus fluorescence immunocytochemistry) or with the corresponding individual single wave filters or by combining fluorescence illumination (fluorescein filter) and light transmission at very low levels (sections stained by DAB). Pictures of representative sections were recorded on slides (Kodak Ektachrome Professional film) and digitized with a Nikon slide scanner or were directly digitized on a digital still recorder (Sony DKR-700) using a 3CCD color video camera (Sony DXC-950P) coupled to the

microscope. The digitized images were then transferred to a micro-computer and prepared for printing with the Adobe Photoshop 4.0 software.

3. Results 3.1. Distribution of the tracer All brains were sectioned and the distribution of retrogradely labeled cells and of the injection site were recorded on drawings. In the first set on ten birds that were studied, four subjects only were found to have an injection of tracer that was confined to the intermediate region of the medial preoptic nucleus. In two other birds the injection site was slightly too rostral, two subjects had received the injection into the third ventricle, and finally in the last two birds, the injection was too lateral and had reached the lateral preoptic area. A very similar distribution of retrogradely labeled cells was observed in the four birds that had received an injection confined into the POM and were therefore selected for further studies. The distribution of these retrogradely labeled cells is summarized in Fig. 1. In agreement with previous tract tracing studies performed in quail (Balthazart et al., 1994; Balthazart and Absil, 1997), large numbers of labeled cells were observed in limbic, preoptic, and anterior hypothalamic regions. In the lateral septum (SL) positive elements were observed from rostral to caudal levels. The BSTm (as defined in the study of Aste et al., 1998) was also densely labeled from its more rostral aspect that is dorsal to the anterior commissure to the V-shaped region located caudally with respect to the anterior commissure (Figs. 2 and 3B). Labeled elements were also present within the nucleus of the pallial commis-

Fig. 1. Schematic representation of serial sections through the quail brain in the coronal plane illustrating the distribution of cells that were retrogradely labeled following an injection of Red Latex Fluospheres# in the POM. In panel C, the hatched area corresponds to the extension of the injection site. Drawings at the top illustrate a sagittal section of the quail brain showing the location of coronal sections. These distribution maps were established based on the results collected in four different birds. Abbreviations : AA, archistriatum anterior; AL, ansa lenticularis; AM, nucleus anterior medialis hypothalami; AV, archistriatum ventralis; AVT, area ventralis tegmentalis; BSTl, nucleus striae terminalis, pars lateralis; BSTm, nucleus striae terminalis, pars medialis; CA, commissura anterior; Cb, cerebellum; CO, chiasma opticum; CP, commissura posterior; DLAl, nucleus dorsolateralis thalami, pars lateralis; DLAm, nucleus dorsolateralis thalami, pars medialis; DMP, nucleus dorsomedialis posterior thalami; DS, decussatio supraoptica; E, ectostriatum; EW, nucleus of Edinger /Westphal; FLM, fasciculus longitodinalis medialis; FPL, fasciculus prosencephali lateralis; GCt, substantia grisea centralis; GLdp, nucleus geniculatus lateralis, pars dorsalis principalis; GLv, nucleus geniculatus lateralis, pars ventralis; HA, hyperstriatum accessorium; HIP, tractus habenulointerpeduncularis; HL, nucleus habenularis lateralis; HM, nucleus habenularis medialis; Hp, hippocampus; HV, hyperstriatum ventrale; ICo, nucleus intercollicularis; Imc, nucleus isthmi, pars magnocellularis; IPS, nucleus interstitio-pretecto-subpretectalis; LA, nucleus lateralis anterior thalami; LFSM, lamina frontalis suprema; LH, lamina hyperstriatica; LMD, lamina medullaris dorsalis; LMmc, nucleus lentiformis mesencephali, pars magnocellularis; LMpc, nucleus lentiformis mesencephali, pars parvocellularis; LPO, lobus paraolfactorius; ME, eminentia mediana; MLD, nucleus mesencephalicus laterodorsalis; N, neostriatum; nCPa, nucleus commissura pallii; nIII, nucleus oculomotorius; NIII, nervus oculomotorius; OM, tractus occipitomesencephalicus; PA, paleostriatum augmentatum; POA, nucleus preopticus anterior; POM, nucleus preopticus medialis; PP, paleostriatum principalis; PPC, nucleus principalis precommissuralis; PVN, nucleus paraventricularis magnocellularis; QF, tractus quintofrontalis; ROT, nucleus rotundus; Ru, nucleus ruber; SCNm, nucleus suprachiasmaticus, pars medialis; SL, nucleus septalis lateralis; SM, nucleus septalis medialis; SN, substantia nigra; SON, nucleus supraopticus; SP, nucleus subpretectalis; Spl, nucleus spiriformis lateralis; TeO, tectum opticum; Tn, nucleus taeniae; TO, tuberculus olfactorius; TrO, tractus opticus; TSM, tractus septomesencephalicus; Tu, nucleus tuberis; VLT, nucleus ventralis lateralis thalami; VMN, nucleus ventromedialis hypothalami.

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Fig. 2. Photomicrographs illustrating the presence in the caudal portion of the bed nucleus striae terminalis of cells that were retrogradely labeled by an injection of Fluospheres in the medial preoptic nucleus. (A) Low power enlargement showing the limits of the group of retrogradely labeled cells that corresponds to the bed nucleus striae terminalis. (B) High power enlargement illustrating the presence of Fluospheres in neurons of the BST. BSTm, bed nucleus striae terminalis, pars medialis; nCPA, nucleus of the pallial commissure; OM, tractus occipitomesencephalicus; SL, nucleus septalis lateralis. Magnification bar: 200 mm in A and 50 mm in B.

sure, scattered cells were observed in the medial septum and in the nucleus taeniae. Numerous labeled cells were observed in the anterior preoptic nucleus, whereas scattered elements containing fluospheres were present in the very rostral portion of the supraoptic nucleus (SON). Moving caudally, a large number of labeled cells was observed within the boundaries of the medial anterior hypothalamic nucleus (AM), while scattered cells were observed within the suprachiasmatic nucleus, pars medialis (SCNm) and in the region of the paraventricular nucleus (PVN). Scattered retrogradely labeled cells were also present in numerous caudal regions: the dorsomedial thalamus, the ventromedial nucleus, the raphe region and the mesencephalic central gray in its most rostral part.

3.2. Double staining vasotocin-tracer Among the regions where retrogradely labeled cells were detected, only three have been previously found to contain groups of VT-ir neurons: the SON, the PVN, and the BSTm. These regions were hence carefully examined for the presence VT-immunoreactive perikarya both in sections stained by the two immunofluorescent and the avidin /biotin techniques. Labeling of the VT-system by a two-step immunofluorescence technique successfully identified VT-ir cells in the magnocellular neuronal groups (SON and PVN), but this staining was weak in the parvocellular cells groups and in particular, no immunofluorescent cell could be detected within the BSTm. Networks of VT-ir

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Fig. 3. (A) Bright field photomicrographs illustrating the VT-ir cells and fibers in the medial bed nucleus striae terminalis (BSTm). (B) Photomicrograph of the same section in fluorescent illumination demonstrating the presence in the exact same location of red Fluospheres retrogradely transported from the POM. (C) Higher magnification of a section through the BSTm that had been labeled by the avidin /biotin technique with diaminobenzidine as a chromogen and illustrating the presence of weakly immunoreactive vasotocin neurons. (D) Photomicrograph of the same section in fluorescent illumination demonstrating the co-localization in four VT-ir cells (white arrows) of retrogradely transported fluorescent beads. OM, tractus occipitomesencephalicus. Magnification bar: 200 mm in A /B, 50 mm in C /D.

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Fig. 4. Photomicrographs of sections stained by the three-step immunofluoresence technique demonstrating in the same section the presence of retrogradely transported Fluospheres (red signal) and of VT-ir cells (green fluoresence). (A /B) Scattered VT-ir cells located periventricularly at the level of the nucleus anterior medialis hypothalami photographed separately under the two different wavelength and illustrating the more ventral position of VT-ir cells by comparisons with the retrogradely labeled cells. The arrow indicates a putative double labeled element but this weak signal probably results from a bleed-through of the intense green signal in the red filter (see text). (C) In the nucleus paraventricularis (photographed here under double wavelength illumination, the cells retrogradely labeled from POM are more lateral than the VT-ir cells (the asterisk indicated the third ventricle). (D) The three-step immunofluoresence technique allows visualization of vasotocin-immunoreactive cells in the BSTm. (E /F) Demonstration of the co-localization in one cell of the BSTm of vasotocin-ummunoreactive material (green) and of fluorescent beads (red). Magnification bar: 100 mm in A,B,D, 50 mm in C,E,F.

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fibers were however present in the BSTm but they were not labeled as densely as in previous studies based on the avidin /biotin or peroxidase-anti-peroxidase technique (Aste et al., 1998; Panzica et al., 1998). In the SON, the retrogradely labeled cells were rare and located more dorsally than the large VT immunofluorescent neurons. In the PVN, large VT-ir neurons are densely clustered in the medial part of the nucleus close to the ventricular wall but scattered in more lateral regions. Cells located within the PVN that were retrogradely labeled with the tracer were clearly smaller that the VT immunofluorescent cells. A co-localization of retrogradely transported tracer in the large VT-ir elements was never observed. The periventricular population of VT-ir elements located in the PVN also extends ventrally, along the ventricular wall. At this level the spatial differentiation between the population of retrogradely labeled cells and of VT-ir cells becomes even more distinct than in the PVN; cells retrogradely labeled with microbeads are clearly located more medially and dorsally than VT-ir cells. The retrogradely labeled cells are at this level located within the nucleus AM. In both the SL and BSTm numerous cells containing retrogradely transported fluorescent microbeads were observed in close contacts with VT-ir fibers, but no immunofluorescent VT cells could be detected. The sections stained for VT by the more sensitive avidin /biotin/DAB immunocytochemical method confirmed these results but in addition revealed a large number of positive fibers and cells in the BSTm region. The extension of these VT-ir elements matched perfectly the distribution of retrogradely labeled cells (Fig. 3C / D). Observation of these sections with a double illumination by transmission light at low level and fluorescent light demonstrated that the weakly stained VT-ir perikarya of the BSTm (appearing as dark cell bodies) also contain the retrograde tracer (fluorescent dots). This could also be confirmed by a close examination of the digitized images. By superimposing the visible image showing the VT-ir elements with the fluorescent image showing the retrogradely transported beads and then varying the opacity of this second image, the specific overlap of the two signal could be fully ascertained. This co-localization is illustrated in Fig. 3C /D that shows a number of VT-ir cells that contained fluorescent retrogradely transported beads indicated by arrows. The colocalization was regularly observed within the BSTm but was never clearly demonstrated in other regions of the VT-ir system (periventricular elements, paraventricular or supraoptic nuclei). These results were also confirmed in sections that had been stained by the sensitive three-step immunofluorescence method. This material confirmed that in the periventricular and in the magnocellular VT-ir cell groups, the cells bodies that display VT immunoreactivity are clearly located in a position that is distinct from

the position of the large majority of the retrogradely labeled cells. VT-ir cells are more medial in the paraventricular and more ventral in the supraoptic nucleus than the cells containing retrogradely transported beads. The possibility of cellular colocalization of these two markers is thus very low in these nuclei and no clear colocalization was indeed observed (see Fig. 4A /C) which fully confirms the conclusions obtained in DAB stained sections. In a few cases (arrow in Fig. 4A /B) scattered weakly immunofluorescent magnocellular or periventricular elements appeared to be double labeled, but this weak staining was presumably due to the partial bleedthrough of the very intense green fluorescence in the RITC red filter. This interpretation is clearly confirmed, when sections are directly observed under the microscope, by the fact that the green fluorescence (and its weak image due to bleed through in the red filter) is filling homogeneously the perikaryon of positive neurons whereas the red retrograde label is attached to micro-beads that can be individually discriminated under high magnification. This technique also successfully visualized the weakly immunoreactive VT elements that are located in the BSTm (Fig. 4D) and allowed us to confirm in double fluorescence that VT-ir elements of the BSTm contain fluorescent beads that had been retrogradely transported from the POM (see Fig. 4E / F).

4. Discussion The present study first confirmed that the POM in quail receives complex afferent inputs originating in a diversity of brain regions. The different nuclei that were found here to sent projections to the POM are in good agreement with previous results obtained by in vitro tracing with DiI (Balthazart et al., 1994) or by in vivo tracing with cholera toxin B or with red latex Fluospheres# (Balthazart and Absil, 1997). In particular the POM receives afferent fibers from the SL, BSTm, AM, anterior preoptic region, and from the magnocellular nuclei (SON and PVN). Several of these nuclei are known to contain cells that express VT (BSTm, SON, PVN) (Viglietti-Panzica, 1986; Sa´nchez et al., 1991; Viglietti-Panzica et al., 1992, 1994; Balthazart et al., 1997; Panzica et al. 2001) and could therefore theoretically be at the origin of the VT-ir innervation of the POM. Labeling for VT by a two-step immunofluorescence procedure confirmed the presence of VT-ir elements in the magnocellular nuclei of the quail brain (SON, PVN) but failed to reveal any immunoreactive cell in the BSTm. This result, although surprising at first sight, is easily explained by the facts that an immunofluorescence staining in two steps (fluorescein directly coupled to the secondary antibody) is clearly less sensitive than

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the three steps avidin /biotin technique and the density of VT immunoreactive material in the cells of the BSTm is definitely lower that in the magnocellular neurons. The same differential visualization has in fact already been reported when VT was stained in paraffin sections collected through the quail brain: no immunoreactive neurons were in this way detected in the BSTm while they were quite abundant in the SON and PVN (Viglietti-Panzica, 1986; Viglietti-Panzica et al., 1992). The double immunofluorescence technique demonstrated that the VT immunoreactivity observed in periventricular and magnocellular neurons (in SON and PVN) is rarely or never found in cells that project to the POM. Given that the quail brain does not, contrary to mammals (de Vries et al., 1992, 1994), contain VT-ir cells in the brain areas homologous to the amygdala (the ventral archistriatum and nucleus taeniae) (Viglietti-Panzica, 1986; Sa´nchez et al., 1991; Viglietti-Panzica et al., 1992, 1994; Aste et al., 1996; Balthazart et al., 1997), it could already be concluded by an exclusion process that the VT-ir fibers have to originate in the BSTm. However, due to the relatively low sensitivity of the VT detection by immunofluorescence, this conclusion could not be confirmed by the examination of the sections stained by double immunofluorescence. In additional studies, sections labeled for VT by the avidin /biotin /diaminobenzidine technique and examined under double illumination (visible light and fluorescence) or stained by a more sensitive three-step fluorescence technique were shown to contain VT-ir elements in the BSTm and unambiguously supported the notion that a substantial part of the VT-ir fibers seen in the POM originate in VT-ir neurons located in the BSTm and that no significant vasotocinergic input to the POM originates in the SON or PVN. The BSTm clearly contains a substantial population of vasotocinergic cells that can be retrogradely filled by tracer injected in the POM. These cells should be the origin of most if not all VT-ir fibers observed in the POM. No other nucleus expressing VT is indeed known to project to the POM. It must however be mentioned that under optimal staining conditions, a small number of weakly VT immunoreactive cells can be observed within the POM itself (Balthazart et al., 1997; Panzica et al., 1998) and the presence of these VT expressing cells has been confirmed in this nucleus by in situ hybridization (Aste et al., 1998; Panzica et al., 1999). A part of the vasotocinergic innervation of the POM could therefore be intrinsic but because VT-ir cells are by far more numerous in the BSTm than in the POM, we assume that this intrinsic innervation should be minimal. It must be noted that the present identification of specific vasotocinergic projections from the BSTm to the POM fits in well with previous studies that analyzed the

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steroid-dependence of these vasotocinergic systems. It has indeed been demonstrated that testosterone increases the VT immunoreactivity and VT mRNA concentration in both the BSTm and POM (VigliettiPanzica et al., 1994; Panzica et al., 1998, 1999), that this effect is mostly mediated in both nuclei by estrogens presumably derived from the central aromatization of testosterone whereas pure no-aromatizable androgens such as 5a-dihydrotestosterone have no effect (VigliettiPanzica et al., 2001) and finally that the VT immunoreactivity in both BSTm and POM differentiates sexually during ontogeny under the influence of estrogens (Panzica et al., 1998). Given these similarities in steroid dependence, it is not unexpected that the VT-ir fibers of the POM originate in VT-ir cell bodies located in the BSTm. The facts that: (a) beads retrogradely transported from the POM are usually not found in the same subregions of the PVN and SON as the VT-ir elements; and (b) these two markers are very rarely or never colocalized with magnocellular elements even when very sensitive immunocytochemical techniques are used to visualize VT are fully consistent with this conclusion. Based on work in rodents (de Vries et al., 1992, 1994) and on the comparison of the previously established distribution of VT-ir cells in galliforms (Viglietti-Panzica, 1986; Sa´nchez et al., 1991; Viglietti-Panzica et al., 1992, 1994; Balthazart et al., 1997; Panzica et al., 2001) and of the origin of the afferent inputs to the quail POM (Balthazart et al., 1994; Balthazart and Absil, 1997), the outcome of the present study was not unexpected and we had actually hypothesized in previous reports that the vasotocinergic innervation of the POM has its origin in the BSTm (Aste et al., 1997, 1998; Panzica et al., 1999). The present study is however the first one that identifies, in an avian species, by retrograde tract-tracing combined with immunocytochemistry for the peptide, the origin of the testosterone sensitive VT-ir innervation of a specific nucleus. It has been clearly established that the same treatment with T activates male-typical copulatory behavior in castrated male quail but that this treatment has no effect in females (Adkins, 1975; Adkins-Regan, 1983; Balthazart et al., 1983). This different behavioral responsiveness to T is organized by embryonic estrogens: females are demasculinized by their ovarian estrogens before the 12th day of incubation (Adkins, 1979; Adkins-Regan, 1983; Balthazart and Ball, 1995). These effects can be prevented in females by injection of an aromatase inhibitor (Balthazart et al., 1992) or mimicked in males by an injection of exogenous estrogen during the first half of the incubation period (Adkins, 1979). The brain mechanisms that differentiate during ontogeny and that are responsible for this differential responsiveness to testosterone in males and females have not been identified to this date. A number of neuroanatomical and neurochemical differences have been identified

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P. Absil et al. / Journal of Chemical Neuroanatomy 24 (2002) 27 /39

between male and female quail (Balthazart and Foidart, 1993; Balthazart et al., 1996) but none of them appears to be sufficient to explain the behavioral dimorphism (Balthazart et al., 1996). Most of these brain differences disappear when males and females are placed in similar endocrine conditions (e.g. are gonadectomized and treated with equal amount of T). These differences are therefore activational in nature and cannot be invoked to explain the behavioral sex dimorphism which is organizational in nature (the difference is organized by neonatal steroids and cannot be overridden by adult hormonal treatments). It is in this context interesting to note that the density of VT-ir structures in the POM and BSTm is sexually differentiated and that this difference is under the sequential control of organizational effects of estrogens during embryonic life and then activational effects of testosterone in adulthood (Viglietti-Panzica et al., 1994; Panzica et al., 1996, 1998, 1999). The sex difference in VT expression in these nuclei therefore correlates very well with the behavioral sex differences. In addition, stereotaxic lesion experiments have shown that both the POM and the BSTm are implicated in the control of male sexual behavior in quail (Balthazart et al., 1998). It is therefore likely that VT plays an important role in the control of male sexual behavior although the specific contribution of the neuropeptide remains somewhat unclear at present (see Castagna et al., 1998; Panzica et al., 2001 for discussion of this specific topic). Because the VT-ir innervation of the POM is sexually differentiated and this innervation originates mostly or exclusively in the BSTm, it can be speculated that the number of VT expressing cells in the BSTm is also sexually differentiated as should be the vasotocinergic projection between these two nuclei. This hodological sex difference may play a significant role in the differential activation in males and females of maletypical sexual behavior in quail. A similar sex difference in connectivity has previously been reported in rats where the number of cells projecting from the anteroventral periventricular (AVPv) nucleus to the arcuate nucleus of the hypothalamus is reported to be 6 times higher in females than in males (Gu and Simerly, 1994, 1997). This sex difference is thought to play a critical role in the sexually differentiated control of the sex steroid feedback on hypothalamic gonadotrophin hormone releasing hormone neurons (Simerly, 1998). A major sex difference in the projection from the BST to AVPv has also been described in rat (Hutton et al., 1998). However, with a few exceptions, neuroanatomical and neurochemical studies have so far largely failed to identify the critical aspects of the brain organization that are responsible for behavioral and physiological sex differences (see Balthazart et al., 1996 for a more detailed discussion of this topic). The analysis of brain connectivity has been largely neglected in this context.

The parvocellular testosterone-sensitive vasotocinergic system of the quail forebrain may provide a good model for this type of studies.

Acknowledgements This work was supported by grants from University of Torino to C.V., MURST (National Relevance Projects, COFIN 2000) to G.C.P., and CNR to C.V. It was also supported by grants from the NIMH (MH 50388), the Belgian FRFC (Nbr. 2.4555.01), the French Community of Belgium (ARC 99/04-241) and the University of Lie`ge (Cre´dits Spe´ciaux) to J.B.P.A. is post-doctoral researcher with the FWO.

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