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See detailAromatase-Immunoreactive Cells in the Quail Brain: Effects of Testosterone and Sex Dimorphism
Foidart, Agnès ULg; de Clerck, A.; Harada, N. et al

in Physiology & Behavior (1994), 55(3), 453-64

We previously demonstrated that testosterone (T) increases aromatase activity (AA) and that AA is sexually dimorphic (males > females) in the quail preoptic area (POA). The precise anatomical localization ... [more ▼]

We previously demonstrated that testosterone (T) increases aromatase activity (AA) and that AA is sexually dimorphic (males > females) in the quail preoptic area (POA). The precise anatomical localization of these effects is, however, impossible to obtain by biochemical assays even when samples are dissected by the Palkovits punch technique. We were recently able to set up an immunocytochemical (ICC) procedure that permits visualization of aromatase-immunoreactive (ARO-ir) cells in the quail brain. This showed that the ARO-ir cells of the quail POA actually outline the sexually dimorphic medial preoptic nucleus (POM). This ICC technique was used here to analyze the sex dimorphism of the quail preoptic aromatase and the localization of T effects on ARO-ir cells. In Experiment 1, the number of ARO-ir cells was counted in one section every 100 microns throughout the rostral to caudal extent of the POM of castrated birds that had been treated with increasing doses of T (5, 10, or 20 mm long Silastic implants). These T-treatments produced a dose-related increase in the sexual behavior of the birds and they increased the number of ARO-ir cells in POM, in the septal regions, and in the bed nucleus of the stria terminalis (BNST). The effect had a particularly large amplitude in the part of the POM located under the anterior commissure (AC). In Experiment 2, the same procedure was used to reanalyze the sex difference of the preoptic aromatase system. This showed that the POM of adult males contains more stained cells than the POM of females but only in a restricted region located just under and rostral to the AC. No significant sex difference was observed in the septum or in the BNST. In Experiment 3, the number of ARO-ir cells was determined in the POM of males and females that had been gonadectomized and treated with a same dose of T (40 mm implants). No sex difference in the number of ARO-ir cells could be detected in these conditions. This suggests that the sex difference in AA that had been previously observed in T-treated birds results either from a difference in aromatase concentration or activity in a similar number of positive cells or from a difference in the number of ARO-ir cells that is very discrete from the anatomical point of view.(ABSTRACT TRUNCATED AT 400 WORDS) [less ▲]

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See detailEffects of Apomorphine on Sexual Behavior in Male Quail
Absil, Philippe ULg; Das, S.; Balthazart, Jacques ULg

in Pharmacology, Biochemistry & Behavior (1994), 47(1), 77-88

In the rat, dopamine (DA) facilitates male copulatory behavior. Indirect evidence based largely on neuroanatomical data suggest that in quail DA is also implicated in the control of male reproductive ... [more ▼]

In the rat, dopamine (DA) facilitates male copulatory behavior. Indirect evidence based largely on neuroanatomical data suggest that in quail DA is also implicated in the control of male reproductive behavior but there is no pharmacological evidence to support this conclusion. To test this idea, castrated testosterone (T)-treated male quail were injected with various doses of the dopaminergic agonist apomorphine (APO) in the range 1-10,000 micrograms/kg. The sexual behavior of birds was recorded starting 15 min after APO injection for a duration of 30 min. A dose-dependent inhibition of male reproductive behavior that lasted for the entire duration of the test was observed. In a second experiment, gonadectomized T-treated male Japanese quail were injected daily with APO (0, 10, or 1,000 micrograms/kg) during 8 days. Their sexual interactions with a partner were quantified either 24 h or 15 min after the last injection. No influence of the treatment on copulatory behavior was observed 24 h after the last injection, but a strong inhibition was present when the test was performed 15 min after. To research whether the inhibitory effects of APO were due to a preferential action on D2 presynaptic autoreceptors, male quail were pretreated with two different D2 antagonists (spiperone or pimozide; 0.5 or 2 mg/kg) before being injected with APO (100 micrograms or 1 mg/kg). Spiperone facilitated male sexual behavior but did not suppress the inhibitory effect of APO. No significant effect of pimozide was observed. These results support the notion that DA modulates male sexual activity in the Japanese quail. The specific role of the different dopaminergic receptor subtypes remains, however, to be elucidated. [less ▲]

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See detailDistribution and Regulation of Estrogen-2-Hydroxylase in the Quail Brain
Balthazart, Jacques ULg; Stoop, R.; Foidart, Agnès ULg et al

in Brain Research Bulletin (1994), 35(4), 339-45

The anatomical distribution and endocrine regulation of the estrogen-2-hydroxylase activity were investigated in the brain of adult male and female Japanese quail. Significant levels of enzymatic activity ... [more ▼]

The anatomical distribution and endocrine regulation of the estrogen-2-hydroxylase activity were investigated in the brain of adult male and female Japanese quail. Significant levels of enzymatic activity were detected in all brain regions that were studied, but the highest levels were observed in preoptic and hypothalamic brain nuclei that are known to contain high levels of aromatase activity. These data are consistent with previous results suggesting that the placental aromatase is also responsible for the estrogen-2-hydroxylase activity. However, there is a marked sex difference and a control by T of aromatase activity in the quail brain, and no such difference in 2-hydroxylase activity could generally be detected except in the VMN. Further studies will be needed to know whether the previously published conclusions concerning the human placenta also apply to the brain. The present data are consistent with the idea that estrogens formed locally in the brain by testosterone aromatization could affect reproduction by interfering with the catecholaminergic transmission after being metabolized into catechol-estrogens. [less ▲]

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See detailThe Catecholaminergic System of the Quail Brain: Immunocytochemical Studies of Dopamine Beta-Hydroxylase and Tyrosine Hydroxylase
Bailhache, T.; Balthazart, Jacques ULg

in Journal of Comparative Neurology (The) (1993), 329(2), 230-56

The distribution of dopamine beta-hydroxylase and tyrosine hydroxylase, two key enzymes in the biosynthesis of catecholamines, was investigated by immunocytochemistry in the brain of male and female ... [more ▼]

The distribution of dopamine beta-hydroxylase and tyrosine hydroxylase, two key enzymes in the biosynthesis of catecholamines, was investigated by immunocytochemistry in the brain of male and female Japanese quail. Cells or fibers showing dopamine beta-hydroxylase and tyrosine hydroxylase immunoreactivity were considered to be noradrenergic or adrenergic, while all structures showing only tyrosine hydroxylase immunoreactivity were tentatively considered to be dopaminergic. The major dopaminergic and noradrenergic cell groups that have been identified in the brain of mammals could be observed in the Japanese quail, with the exception of a tuberoinfundibular dopaminergic group. The dopamine beta-hydroxylase-immunoreactive cells were found exclusively in the pons (locus ceruleus and nucleus subceruleus ventralis) and in the medulla (area of the nucleus reticularis). The tyrosine hydroxylase-immunoreactive cells had a much wider distribution and extended from the preoptic area to the level of the medulla. They were, however, present in larger numbers in the area ventralis of Tsai and in the nucleus tegmenti pedunculo-pontinus, pars compacta, which respectively correspond to the ventral tegmental area and to the substantia nigra of mammals. A high density of dopamine beta-hydroxylase- and tyrosine hydroxylase-immunoreactive fibers and punctate structures was found in several steroid-sensitive brain regions that are implicated in the control of reproduction. In the preoptic area and in the region of the nucleus accumbens-nucleus stria terminalis, immunonegative perikarya were completely surrounded by immunoreactive fibers forming basket-like structures. Given that some of these cells contain the enzyme aromatase, these structures may represent the morphological substrate for a regulation of aromatase activity by catecholamines. The dopamine beta-hydroxylase-immunoreactive fibers were also present in a larger part of the preoptic area of females than in males. This sex difference in the noradrenergic innervation of the preoptic area presumably reflects the sex difference in norepinephrine content in this region. [less ▲]

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See detailBrain Aromatase and the Control of Male Sexual Behavior
Balthazart, Jacques ULg; Foidart, Agnès ULg

in Journal of Steroid Biochemistry & Molecular Biology (1993), 44(4-6), 521-40

The activational effects of testosterone (T) on male copulatory behavior are mediated by its aromatization into estradiol. In quail, we have shown by stereotaxic implantation of steroids and metabolism ... [more ▼]

The activational effects of testosterone (T) on male copulatory behavior are mediated by its aromatization into estradiol. In quail, we have shown by stereotaxic implantation of steroids and metabolism inhibitors and by electrolytic lesions that the action of T and its aromatization take place in the sexually dimorphic medial preoptic nucleus (POM). The distribution and regulation of brain aromatase was studied in this species by product-formation assays measuring aromatase activity (AA) in microdissected brain regions and by immunocytochemistry (ICC). Aromatase-immunoreactive (ARO-ir) neurons were found in four brain regions: the POM, the septal region, the bed nucleus of the stria terminals (BNST) and the tuberal hypothalamus. ARO-ir cells actually outline the POM boundaries. ARO-ir material is found not only in the perikarya of neurons but also in the full extension of their cellular processes including the axons and the presynaptic boutons. This is confirmed at the light level by the demonstration of immunoreactive fibers and punctate structures in brain regions that are sometimes fairly distant from the closest ARO-ir cells. A lot of ARO-ir cells in the POM and BNST do not contain immunoreactive estrogen receptors (ER-ir) as demonstrated by double label ICC. These morphological data suggest an unorthodox role for the enzyme or the locally formed estrogens. In parallel with copulatory behavior, the preoptic AA decreases after castration and is restored by T to levels seen in sexually mature males. This probably reflects a change in enzyme concentration rather than a modulation of the activity in a constant number of molecules since the maximum enzymatic velocity (Vmax) only is affected while the affinity (Km) remains unchanged. In addition, T increases the number of ARO-ir neurons in POM and other brain areas suggesting that the concentration of the antigen is actually increased. This probably involves the direct activation of aromatase transcription as demonstrated by RT-PCR studies showing that aromatase mRNA is increased following T treatment of castrates. These activating effects of T seem to result from a synergistic action of androgenic and estrogenic metabolites of the steroid. The anatomical substrate for these regulations remains unclear at present especially in POM where ARO-ir cells do not in general contain ER-ir while androgen receptors appear to be rare based on both [3H] dihydrotestosterone autoradiography and ICC. Transynaptic mechanisms of control may be considered. A modulation of brain aromatase by catecholamines is also suggested by a few pharmacological studies.(ABSTRACT TRUNCATED AT 400 WORDS) [less ▲]

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See detailEffects of Neurochemical Lesions of the Preoptic Area on Male Sexual Behavior in the Japanese Quail
Bailhache, T.; Surlemont, C.; Balthazart, Jacques ULg

in Brain Research Bulletin (1993), 32(3), 273-83

Two experiments were carried out during which the noradrenergic neurotoxin, 5-amino-2,4-dihydroxy-alpha-methylphenylethylamine (5-ADMP) was applied to the brain of quail in order to evaluate the role of ... [more ▼]

Two experiments were carried out during which the noradrenergic neurotoxin, 5-amino-2,4-dihydroxy-alpha-methylphenylethylamine (5-ADMP) was applied to the brain of quail in order to evaluate the role of the noradrenergic system in the control of male copulatory behavior. In the first experiment, the ICV injection of 5-ADMP slightly enhanced the sexual behavior observed in testosterone (T)-treated castrated male quail. This brings additional support to the notion that norepinephrine tonically inhibits male copulatory behavior in quail. In the second experiment, 5-ADMP implanted directly into the preoptic area disrupted the restoration by T of copulatory behavior in castrated quail and, at the same time, produced a brain lesion that partly destroyed the sexually dimorphic medial preoptic nucleus, a previously established site of T action on behavior. These lesions produced by a high (presumably too high) concentration of neurotoxin provided an independent confirmation of effects previously observed after electrolytic lesions. Correlation analyses also confirmed that the medial part of the POM just rostral to the anterior commissure is more closely associated with copulatory behavior and may, therefore, represent a key center for steroid action on this behavior. [less ▲]

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See detailBehavioral Demasculinization of Female Quail Is Induced by Estrogens: Studies with the New Aromatase Inhibitor, R76713
Balthazart, Jacques ULg; De Clerck, A.; Foidart, Agnès ULg

in Hormones & Behavior (1992), 26(2), 179-203

The injection before Day 12 of incubation of estradiol benzoate (EB) into Japanese quail eggs produces a complete behavioral demasculinization of adult males that will hatch from these eggs. These males ... [more ▼]

The injection before Day 12 of incubation of estradiol benzoate (EB) into Japanese quail eggs produces a complete behavioral demasculinization of adult males that will hatch from these eggs. These males never show copulatory behavior even after administration of high levels of exogenous testosterone (T). It is usually assumed that such a demasculinization normally takes place in female embryos under the influence of endogenous estrogens but few experimental data are available to confirm the validity of this model. A series of four experiments was performed during which R76713, a triazole derivative that specifically inhibits aromatase (estrogen synthetase) activity, was injected into quail eggs at different stages of incubation to prevent the production of endogenous estrogens. The consequences of these embryonic treatments on the T-activated sexual behavior in adults were then quantified. When injected before Day 12 of incubation, R76713 completely blocked the behavioral demasculinization of females without affecting the behavior of the males. After a treatment with T, almost all R76713-treated females showed as adults a masculine copulatory behavior that was undistinguishable from the behavior of intact males. This effect was fully reversed by the injection in egg of EB demonstrating that the effects of R76713 were specifically due to the suppression of endogenous estrogens. Injection of R76713 during the late phase of the incubation (Day 12 or Day 15) only maintained weak copulatory behavior in females which confirmed that the behavioral demasculinization in quail takes place mainly though not exclusively during the early stages of ontogeny. In a last experiment, we combined an early R76713 treatment with an injection of EB either on Day 9 or on Day 14 of incubation. This showed that the sensitivity to differentiating effects of estrogens varies with age in a sexually differentiated manner. The EB injection on Day 9 demasculinized both male and female embryos. If this injection was delayed until Day 14, it was no longer effective in males but still caused a partial demasculinization of females. This demonstrates that even if females are not yet behaviorally demasculinized on Day 9 of incubation (suppression of aromatase activity at that age will maintain the behavior), their sensitivity to estrogens is already different from that of males. [less ▲]

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See detailImmunocytochemical Localization of Androgen Receptors in the Male Songbird and Quail Brain
Balthazart, Jacques ULg; Foidart, Agnès ULg; Wilson, E. M. et al

in Journal of Comparative Neurology (The) (1992), 317(4), 407-20

The distribution of androgen receptors was studied in the brain of the Japanese quail (Coturnix japonica), the zebra finch (Taeniopygia guttata), and the canary (Serinus canaria) by immunocytochemistry ... [more ▼]

The distribution of androgen receptors was studied in the brain of the Japanese quail (Coturnix japonica), the zebra finch (Taeniopygia guttata), and the canary (Serinus canaria) by immunocytochemistry with a polyclonal antibody (AR32) raised in rabbit against a synthetic peptide corresponding to a sequence located at the N-terminus of the androgen receptor molecule. In quail, androgen receptor-immunoreactive cells were observed in the nucleus intercollicularis and in various nuclei of the preoptic-hypothalamic complex, namely, the nucleus preopticus medialis, the ventral part of the nucleus anterior medialis hypothalami, the nucleus paraventricularis magnocellularis, the nucleus ventromedialis hypothalami, and the tuberal hypothalamus. In the two songbird species, labeled cells were also observed in various nuclei in the preoptic-hypothalamic region, in the nucleus taeniae, and in the nucleus intercollicularis. Additional androgen receptor-immunoreactive cells were present in the androgen-sensitive telencephalic nuclei that are part of the song control system. These immunoreactive cells filled and outlined the boundaries of the hyperstriatum ventrale, pars caudalis, nucleus magnocellularis neostriatalis anterioris (both in the lateral and medial subdivisions), and nucleus robustus archistriatalis. The immunoreactive material was primarily present in cell nuclei but a low level of immunoreactivity was also clearly detected in cytoplasm in some brain areas. These studies demonstrate, for the first time, that androgen receptors can be detected by immunocytochemistry in the avian brain and the results are in general agreement with the binding data obtained by autoradiography with tritiated dihydrotestosterone. Immunocytochemical methods offer several advantages over autoradiography and their use for the study of the androgen receptor will greatly facilitate the analysis of steroid-sensitive systems in the avian brain. [less ▲]

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See detailNeuroanatomical Specificity in the Autoregulation of Aromatase-Immunoreactive Neurons by Androgens and Estrogens: An Immunocytochemical Study
Balthazart, Jacques ULg; Foidart, Agnès ULg; Surlemont, C. et al

in Brain Research (1992), 574(1-2), 280-90

Testosterone (T) increases brain aromatase activity (AA) in quail and other avian and mammalian species. It was shown both in quail and in rat that this enzymatic induction results from a synergistic ... [more ▼]

Testosterone (T) increases brain aromatase activity (AA) in quail and other avian and mammalian species. It was shown both in quail and in rat that this enzymatic induction results from a synergistic action of androgens and estrogens. These studies provide little information on possible anatomical or cellular specificity of the effect. Using a polyclonal antiserum against human placental aromatase, we have previously identified aromatase-immunoreactive (ARO-ir) neurons in the quail brain and demonstrated that T increases the number of ARO-ir cells in the quail preoptic area (POA) supporting previous evidence that T increases AA in the brain. However, which T metabolites are involved, the actual mechanism of regulation and the possibility of anatomical specificity for these effects are not yet clear. In the present study, we disassociated the effects of androgens and estrogens in aromatase induction by comparing ARO-ir neurons of quail treated with T alone or T in the presence of a potent aromatase inhibitor (R76713), which has been shown to depress AA levels and to suppress T-activated copulatory behavior. T increased the number of ARO-ir cells in POA, bed nucleus striae terminalis (BNST) and tuberal hypothalamus (Tu). The T effect was inhibited by concurrent treatment with aromatase inhibitor in Tu, but not in POA and BNST. This differential effect of the aromatase inhibitor fits in very well with our previous studies of the co-localization of aromatase and estrogen receptors. The T effect was blocked by R76713 in areas where ARO-ir and estrogen receptor-ir are generally co-localized (Tu) and was not affected in areas with mainly ARO-ir positive, estrogen receptor-ir negative cells (POA, BNST). This suggests anatomical differences in the expression or clearance of aromatase which may be differentially sensitive to androgens and estrogens and dependent upon the presence of sex steroid receptors. [less ▲]

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See detailAromatase as a Cellular Marker of Testosterone Action in the Preoptic Area
Balthazart, Jacques ULg; Surlemont, C.; Harada, N.

in Physiology & Behavior (1992), 51(2), 395-409

We recently showed, using a new immunocytochemical technique, that aromatase-immunoreactive neurons are a specific marker for the sexually dimorphic medial preoptic nucleus (POM) in quail and that the ... [more ▼]

We recently showed, using a new immunocytochemical technique, that aromatase-immunoreactive neurons are a specific marker for the sexually dimorphic medial preoptic nucleus (POM) in quail and that the number of these immunoreactive cells is markedly increased by a systemic treatment with testosterone (T). Since the POM is a key site for the activation of copulatory behavior by T and this androgen must be converted into estrogen by local aromatization within the POM before it can exert its behavioral effects, we used aromatase immunocytochemistry to map, at a cellular level of resolution, the areas that are destroyed by electrolytic lesions or that are stimulated by the stereotaxic implantation of T in the preoptic area (POA). These measures of the cellular action of T in the preoptic area were then correlated with the behavior of the animals to identify the parts of the POA that are critical in the activation of behavior. The electrolytic lesions of the POA disrupted the activation of male sexual behavior by T only if they destroyed a significant part of the POM. All lesions reduced the volume of the dimorphic nucleus and the absolute number of its aromatase-immunoreactive neurons, but the density of these cells in the remaining POM was not affected, suggesting that the volume change in the nucleus reflected a centripetal displacement of its boundaries rather than an overall shrinkage of the structure. Stereotaxic T implants in or close to POM activated male copulatory behavior and increased the volume of the POM and the number of its aromatase-immunoreactive cells. These neuroanatomical effects were more prominent on the side of the implant, but they were also detected on the contralateral side. Correlative analyses suggested that a part of the POM just rostral to the anterior commissure is critical for the activation of copulatory behavior. The best correlations between the behavioral deficits induced by electrolytic lesions and the size of the lesions were indeed observed in this area. In addition, high correlations were also observed between the behavior activated by T implants and the POM size or number of aromatase-immunoreactive cells that were induced by T in this area. Aromatase immunocytochemistry therefore appears as a useful tool to map the brain areas in which T action is presumably critical for the activation of male sexual behavior. It has allowed us to identify in the present studies a small part of the sexually dimorphic POM that is closely associated with behavior.(ABSTRACT TRUNCATED AT 400 WORDS) [less ▲]

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See detailEffects of Alpha-Methyl-Para-Tyrosine on Monoamine Levels in the Japanese Quail: Sex Differences and Testosterone Effects
Balthazart, Jacques ULg; Foidart, Agnès ULg; Sante, P. et al

in Brain Research Bulletin (1992), 28(2), 275-88

Experiments were performed to obtain more information on the regulation by steroids of catecholaminergic systems in the brain of Japanese quail. Dose-response and time-response experiments were first ... [more ▼]

Experiments were performed to obtain more information on the regulation by steroids of catecholaminergic systems in the brain of Japanese quail. Dose-response and time-response experiments were first performed to determine optimal conditions for measuring turnover in the quail brain. The norepinephrine and dopamine turnover were then estimated in microdissected brain nuclei of birds that were either sexually mature or gonadectomized or gonadectomized and treated with testosterone. Two major facts that bear direct relationship with the control of masculine reproductive behavior were demonstrated. On one hand, the dopamine turnover in the medial preoptic nucleus, a sexually dimorphic brain structure which is critically implicated in the control of copulatory behavior was much higher in male than in female quail irrespective of the hormonal condition of the birds. On the other hand, norepinephrine concentrations appeared to be higher in several nuclei of the female brain by comparison with males. These sex differences might represent part of the causal factors that underlie the sex dimorphism in reproductive behavior in quail. [less ▲]

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See detailTestosterone Effects on Staining Density and Autoradiographic Investigations of the Alpha 2-Adrenergic Receptor in the Medial Preoptic Nucleus of the Japanese Quail: Relationship to the Activation of Reproductive Behavior
Balthazart, Jacques ULg; Sante, P.; Ball, G. F.

in Archives Internationales de Physiologie, de Biochimie et de Biophysique (1991), 99(6), 385-92

The effects of gonadectomy combined or not with testosterone (T) therapy on the sexual behavior, cloacal gland area, staining density and alpha 2-adrenergic receptors in the medial preoptic nucleus (POM ... [more ▼]

The effects of gonadectomy combined or not with testosterone (T) therapy on the sexual behavior, cloacal gland area, staining density and alpha 2-adrenergic receptors in the medial preoptic nucleus (POM) were studied in male and female Japanese quail. In castrated males, T increased the cloacal gland size and activated copulatory behavior. In agreement with previous reports, these effects were sexually differentiated: under the influence of T, cloacal gland growth was smaller and this treatment did not activate masculine sexual behavior in females. The optical density of the medial preoptic nucleus stained with thionein blue (Nissl stain) was decreased by castration and increased by T in both males and females. This suggests that T has a profound effect on the synthesis of proteins in the POM and, since POM is a critical site in the activation by T of masculine sexual behavior, these induced proteins are, in all probability, a part of the causal chain of biochemical events giving rise to copulatory behavior. The treatment with T had, by contrast, no clear effect on the number of binding sites and on the affinity of the alpha 2-adrenergic receptors in the POM. Therefore, if the noradrenergic transmission is involved in the induction by T of copulatory behavior, it is probably acting either through the alteration of another adrenergic receptor subtype or through the modulation of the baseline levels or turnover of the transmitter itself. [less ▲]

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See detailNeuroanatomical Specificity in the Co-Localization of Aromatase and Estrogen Receptors
Balthazart, Jacques ULg; Foidart, Agnès ULg; Surlemont, C. et al

in Journal of Neurobiology (1991), 22(2), 143-57

The relative distributions of aromatase and of estrogen receptors were studied in the brain of the Japanese quail by a double-label immunocytochemical technique. Aromatase immunoreactive cells (ARO-ir ... [more ▼]

The relative distributions of aromatase and of estrogen receptors were studied in the brain of the Japanese quail by a double-label immunocytochemical technique. Aromatase immunoreactive cells (ARO-ir) were found in the medial preoptic nucleus, in the septal region, and in a large cell cluster extending from the dorso-lateral aspect of the ventromedial nucleus of the hypothalamus to the tuber at the level of the nucleus inferioris hypothalami. Immunoreactive estrogen receptors (ER) were also found in each of these brain areas but their distribution was much broader and included larger parts of the preoptic, septal, and tuberal regions. In the ventromedial and tuberal hypothalamus, the majority of the ARO-ir cells (over 75%) also contained immunoreactive ER. By contrast, very few of the ARO-ir cells were double-labeled in the preoptic area and in the septum. More than 80% of the aromatase-containing cells contained no ER in these regions. This suggests that the estrogens, which are formed centrally by aromatization of testosterone, might not exert their biological effects through binding with the classical nuclear ER. The fact that significant amounts of aromatase activity are found in synaptosomes purified by differential centrifugation and that aromatase immunoreactivity is observed at the electron microscope level in synaptic boutons suggests that aromatase might produce estrogens that act at the synaptic level as neurohormones or neuromodulators. [less ▲]

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See detailDistribution of Aromatase-Immunoreactive Cells in the Mouse Forebrain
Balthazart, Jacques ULg; Foidart, Agnès ULg; Surlemont, C. et al

in Cell & Tissue Research (1991), 263(1), 71-9

The distribution of aromatase-immunoreactive cells was studied by immunocytochemistry in the mouse forebrain using a purified polyclonal antibody raised against human placental aromatase. Labeled ... [more ▼]

The distribution of aromatase-immunoreactive cells was studied by immunocytochemistry in the mouse forebrain using a purified polyclonal antibody raised against human placental aromatase. Labeled perikarya were found in the dorso-lateral parts of the medial and tuberal hypothalamus. Positive cells filled an area extending between the subincertal nucleus in the dorsal part, the ventromedial hypothalamic nucleus in the ventral part, and the internal capsule and the magnocellular nucleus of the lateral hypothalamus in the lateral part. The same distribution was seen in the two strains of mice that were studied (Jackson and Swiss), and the number of immunoreactive perikarya did not seem to be affected by castration or testosterone treatment. No immunoreactivity could be detected in the medial regions of the preoptic area and hypothalamus; these were expected to contain the enzyme based on assays of aromatase activity performed in rats and on indirect autoradiographic evidence in mice. Our data raise questions concerning the distribution of aromatase in the brain and the mode of action of the centrally produced estrogens. [less ▲]

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See detailTestosterone Metabolism in the Avian Hypothalamus
Balthazart, Jacques ULg

in Journal of Steroid Biochemistry & Molecular Biology (1991), 40(4-6), 557-70

Many central actions of testosterone (T) require the transformation of T into several metabolites including 5 alpha-dihydrotestosterone (5 alpha-DHT) and estradiol (E2). In birds as in mammals, 5 alpha ... [more ▼]

Many central actions of testosterone (T) require the transformation of T into several metabolites including 5 alpha-dihydrotestosterone (5 alpha-DHT) and estradiol (E2). In birds as in mammals, 5 alpha-DHT and E2, alone or in combination, mimic most behavioral effects of T. The avian brain is, in addition, able to transform T into 5 beta-DHT, a metabolite which seems to be devoid of any behavioral or physiological effects, at least in the context of reproduction. By in vitro product-formation assays, we have analyzed the distribution, sex differences and regulation by steroids of the 3 main T metabolizing enzymes (aromatase, 5 alpha- and 5 beta-reductases) in the brain of the Japanese quail (Coturnix c. japonica) and the zebra finch (Taeniopygia guttata castanotis). In the hypothalamus of quail and finches, aromatase activity is higher in males than in females. It is also decreased by castration and increased by T. The activity of the 5 alpha-reductase is not sexually differentiated nor controlled by T. The 5 beta-reductase activity is often higher in females than in males but this difference disappears in gonadectomized birds and no clear effect of T can be observed at this level. The zebra finch brain also contains a number of steroid-sensitive telencephalic nuclei [e.g. hyperstriatum ventrale, pars caudale (HVc) and robustus archistriatalis (RA)] which play a key role in the control of vocalizations. These nuclei also contain T-metabolizing enzymes but the regulation of their activity is substantially different from what has been observed in the hypothalamus. Aromatase activity is for example higher in females than in males in HVc and RA and the enzyme in these nuclei is not affected by castration nor T treatment. In these nuclei, the 5 alpha-reductase activity is higher in males than in females and the reverse is true for the 5 beta-reductase. These sex differences in activity are not sensitive to gonadectomy and T treatment and might therefore be organized by neonatal steroids. We have been recently able to localize aromatase-immunoreactive (AR-ir) neurons by ICC in the brain of the quail and zebra finch. Positive cells are found in the preoptic area, ventromedial and tuberal hypothalamus. AR-ir material is found in the perikarya of cells and fills the entire cellular processes including axons. At the electron microscope level, immunoreactive material can clearly be observed in the synaptic boutons. This observation raises questions concerning the mode of action of estrogens produced by central aromatization of T. [less ▲]

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See detailEffects of the Nonsteroidal Inhibitor R76713 on Testosterone-Induced Sexual Behavior in the Japanese Quail (Coturnix Coturnix Japonica)
Balthazart, Jacques ULg; Evrard, L.; Surlemont, C.

in Hormones & Behavior (1990), 24(4), 510-31

A new triazole derivative, R76713 (6-[4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-methyl-1H- benzotriazole), was recently shown to inhibit aromatase selectively without affecting other steroid ... [more ▼]

A new triazole derivative, R76713 (6-[4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-methyl-1H- benzotriazole), was recently shown to inhibit aromatase selectively without affecting other steroid-metabolizing enzymes and without interacting with estrogen, progestin, or androgen receptors. This compound was tested for its capacity to intefere with the induction of copulatory behavior by testosterone (T) in castrated Japanese quail (Coturnix coturnix japonica). In a first experiment, R76713 inhibited (range 0.01 to 1 mg/kg) the activation of sexual behavior by T silastic implants and hypothalamic aromatase activity in castrated male quail in a dose-dependent manner. The 5 alpha- and 5 beta- reductases of T were not systematically affected. Stereotaxic implantation of R76713 in the medial preoptic area similarly blocked the behavior activated by systemic treatment with T, demonstrating that central aromatization of androgen is implicated in the activation of behavior. These inhibiting effects of R76713 on behavior were observed when implants were placed in the medial part of the nucleus preopticus medialis, confirming the implication of this brain area in the control of male copulatory behavior. Finally, the behavioral inhibition produced by R76713 could be reversed by simultaneous treatment with a dose of estradiol, which was not behaviorally effective by itself. This suggests that the behavioral deficit induced by the inhibitor was specifically due to the suppression of estrogen production. This also shows that the activation of copulatory behavior probably results from the interaction of androgens and estrogens at the brain level, as the two treatments separately providing these hormonal stimuli (T with the aromatase inhibitor on one hand and a low dose of estradiol on the other hand) had almost no behavioral effects but they synergized to activate copulation when given concurrently. These data confirm the critical role of preoptic aromatase in the activation of reproductive behavior and demonstrate that R76713 is a useful tool for the in vivo study of estrogen-dependent processes. [less ▲]

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See detailDistribution of Aromatase in the Brain of the Japanese Quail, Ring Dove, and Zebra Finch: An Immunocytochemical Study
Balthazart, Jacques ULg; Foidart, Agnès ULg; Surlemont, C. et al

in Journal of Comparative Neurology (1990), 301(2), 276-88

An immunocytochemical peroxidase-antiperoxidase procedure using a purified polyclonal antibody raised against human placental aromatase was used to localize aromatase-containing cells in the brain of ... [more ▼]

An immunocytochemical peroxidase-antiperoxidase procedure 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. [less ▲]

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See detailAndrogen and Estrogen Action in the Preoptic Area and Activation of Copulatory Behavior in Quail
Balthazart, Jacques ULg; Surlemont, C.

in Physiology & Behavior (1990), 48(5), 599-609

The sites of androgen and estrogen action on sexual behavior were studied in the preoptic area of castrated male Japanese quail by stereotaxic implantation of hormones, antihormones and metabolism ... [more ▼]

The sites of androgen and estrogen action on sexual behavior were studied in the preoptic area of castrated male Japanese quail by stereotaxic implantation of hormones, antihormones and metabolism inhibitors. The first experiment demonstrated that bilateral implantation of the aromatase inhibitor, androstatrienedione (ATD), in the sexually dimorphic nucleus (POM) of the preoptic area can completely suppress the behavioral activation produced by a systemic treatment with testosterone. The effects of ATD were only observed if the implants were located in the POM. In the second experiment, implants in the POM of the synthetic estrogen, diethylstilbestrol, restored copulatory behavior in castrated males while implants of the synthetic nonaromatizable androgen, methyltrienolone, were almost ineffective. During the third experiment, the activating effects of a systemic treatment with testosterone were blocked by stereotaxic implants in the POM of the antiestrogen, tamoxifen, or the antiandrogen, flutamide. The effects of tamoxifen were more pronounced than those of flutamide. In addition, tamoxifen was active in all parts of the POM while a behavioral inhibition was observed only for flutamide implants which were located in the caudal part of the nucleus. Taken together, these results demonstrate that the sexually dimorphic POM is the area where the behaviorally active estrogenic metabolites of T have to be produced. The estradiol derived from T aromatization presumably acts within the POM to activate copulation as demonstrated by the effectiveness of DES implanted in this region. Androgens also have a direct action on sexual behavior as suggested by the partial inhibition observed in flutamide-treated birds. It is, however, suggested that androgens and estrogens do not act in the same brain area to activate behavior. [less ▲]

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See detailCopulatory Behavior Is Controlled by the Sexually Dimorphic Nucleus of the Quail Poa
Balthazart, Jacques ULg; Surlemont, C.

in Brain Research Bulletin (1990), 25(1), 7-14

The medial preoptic nucleus (POM) of the quail preoptic area is sexually dimorphic and testosterone sensitive. Stereotaxic implantation of needles filled with crystalline testosterone demonstrated that ... [more ▼]

The medial preoptic nucleus (POM) of the quail preoptic area is sexually dimorphic and testosterone sensitive. Stereotaxic implantation of needles filled with crystalline testosterone demonstrated that the POM is a critical site of steroid action in the control of copulatory behavior. Only implants located in the POM reliably restored the behavior in castrated birds. Implants around the nucleus weakly activated the behavior; those which were distant by more than 200 microns were totally inactive. Electrolytic lesions confirmed the role of the POM in the control of copulatory behavior. The percentage of the POM which was lesioned was highly correlated to the behavioral deficit while the absolute size of the lesion was not. Electrolytic lesions in or around POM also significantly decreased the volume of the nucleus suggesting that the afferents and efferents of the nucleus are required for its full development. The total volume of the POM was correlated with the sexual behavior of the birds. The morphological changes in POM observed following exposure to testosterone probably represent the signature of the behavioral effects of the steroid. The sexually dimorphic testosterone-sensitive POM is therefore an excellent animal model to study the brain-steroid interactions which mediate the activation of male reproductive behavior. [less ▲]

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See detailEffects of Castration and Testosterone Treatment on the Activity of Testosterone-Metabolizing Enzymes in the Brain of Male and Female Zebra Finches
Vockel, A.; Prove, E.; Balthazart, Jacques ULg

in Journal of Neurobiology (1990), 21(5), 808-25

Recently, we described the distribution of testosterone-metabolizing enzymes (i.e., aromatase, 5 alpha- and 5 beta-reductases) in the zebra finch (Taeniopygia guttata) brain using a sensitive radioenzyme ... [more ▼]

Recently, we described the distribution of testosterone-metabolizing enzymes (i.e., aromatase, 5 alpha- and 5 beta-reductases) in the zebra finch (Taeniopygia guttata) brain using a sensitive radioenzyme assay combined to the Palkovits punch method. A number of sex-differences in the activity of these enzymes were observed especially in nuclei of the song-control system. The hormonal controls of these differences have now been analyzed by gonadectomizing birds of both sexes and by giving them a replacement therapy with silastic implants of testosterone (T). Five nuclei of the song system (Area X [X], nucleus magnocellularis of the anterior neostriatum [MAN], nucleus robustus archistriatalis [RA], nucleus intercollicularis [ICo], hyperstriatum ventrale, pars caudalis [HVc]) and three preoptic-hypothalamic areas (preoptic anterior [POA], periventricular magnocellular nucleus [PVM], and posterior medial hypothalamic nucleus [PMH]) were studied as well as other limbic and control non-steroid-sensitive areas. The activity of the 5 alpha-reductase was higher in males than in females for the five song-control nuclei and was not affected by the hormonal treatments. The overall activity of this enzyme was not sexually dimorphic in POA and PVM. It was higher in males than in females in intact birds only, and was reduced by gonadectomy and enhanced by T. The activity of the 5 beta-reductase was higher in females than in males in all nuclei of the song system and in POA, but was not influenced by the changes in T level. Both sex and treatment effects were observed in the control of aromatase. The production of estrogens was dimorphic (females greater than males) in RA and PMH. It was increased by T in POA, PVM, and PMH, and also in RA. These data show that some of the sex differences in T-metabolizing enzymes result from the exposure to different levels of T in adulthood (e.g., 5 alpha-reductase in POA and PVM or aromatase in PVM), whereas others persist even if birds are exposed to the same hormonal conditions. These are presumably the result of organizational effects of steroids. The steroid modulation of the aromatase might be related directly to the activation of sexual, aggressive, and nest-building behaviors, whereas the stable dimorphism in 5 alpha- and 5 beta-reductase observed in the nuclei of the song system might be one of the neurochemical bases of the sex differences in the vocal behavior of the zebra finch. [less ▲]

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